Culture medium

Teratology, 1992 Nov, 46(5), 473 - 83
Development of rat embryos in culture media containing different concentrations of normal and diabetic rat serum; Styrud J et al.; In vitro culture of rodent embryos has been extensively used in the search for teratologic agents, with possible relevance to diabetic pregnancy . However, the high concentrations of rat serum added to the culture medium (approximately 75%) have raised concern that the teratogenic effects of some compounds may be attenuated or masked in this culture system and thereby forced the addition of pharmacological concentrations of the compounds (e.g., D-glucose and beta-hydroxybutyrate) to the medium . This issue has been examined in the present study where the effects of different concentrations of rat serum on growth and differentiation of rat embryos were recorded in cultures supplemented with increased concentrations of D-glucose and beta-hydroxybutyrate . The embryonic development was also evaluated after culture in medium supplied with serum from diabetic rats . Compared with normal rat serum, the diabetic serum had an elevated glucose concentration as well as markedly increased levels of triglycerides and branched amino acids, indicating a potentially rich supply of major nutrients for the cultured embryos . Lowering the serum concentration in the culture medium from 80% to 50% yielded progressively retarded embryonic growth but no increased rate of other morphological malformations . At 40% serum concentration, however, there was a sharp rise in the incidence of somatic malformations, in addition to the prevailing growth retardation . When the embryonic growth and development were compared at 50% and 80% serum concentrations, increased D-glucose or beta-hydroxybutyrate concentrations caused similar degrees of embryonic dysmorphogenesis . Also, the uptake of each compound by the embryos exposed to elevated levels of the two agents were similar in 50% and 80% serum cultures . There was, therefore, no protection against the teratogenic and growth-retarding effects of increased D-glucose or beta-hydroxybutyrate offered by high serum concentrations in the culture medium (i.e., 80% vs . 50%) . Embryos cultured in 50% or 80% diabetic rat serum at 30 mmol/L or 50 mmol/L D-glucose concentration showed similar rates of somatic malformations as did embryos exposed to the same proportion of normal rat serum at similar glucose concentrations . By contrast, the diabetic rat serum amplified the general retarding effects of high D-glucose levels, yielding lower protein levels and somite numbers in embryos from diabetic serum culture than in embryos cultured in normal rat serum.(ABSTRACT TRUNCATED AT 400 WORDS)

Am J Obstet Gynecol, 1992 Nov, 167(5), 1417 - 22
Chemotactic factor in the pregnant rabbit uterine cervix; Uchiyama T et al.; OBJECTIVE: Neutrophil accumulation is one of the characteristic changes observed in uterine cervical stroma at term pregnancy, but chemotactic activity in the tissue is obscure . Our study examined the existence and production of chemotactic factor in the rabbit uterine cervix . STUDY DESIGN: Uterine cervical explants of rabbits at term pregnancy and nonpregnant rabbits were cultured with and without interleukin-1 alpha . Rat neutrophilic chemotaxis in culture media was evaluated with a Boyden chamber . RESULTS: Tissue extract from the pregnant rabbit uterine cervix at term pregnancy contained more chemoattractive activity than the nonpregnant cervix . Production of chemoattractant from cultured rabbit cervical explants at term pregnancy was also higher than that from nonpregnant explants . The addition of interleukin-1 alpha to the culture system promoted its production . This chemoattractant was characterized as a true chemotactic factor and heat-stable and trypsin-sensitive protein with an apparent relative molecular mass of 16,200 . So far, these properties are very similar to those of the interleukin-8 family . Rabbit uterine cervical fibroblast is characterized as a chemotactic factor-producing cell in the rabbit uterine cervix . CONCLUSION: These results indicate that interleukin-8-like chemotactic factor participates in the cervical ripening at term pregnancy and that the production of this factor is controlled effectively by interleukin-1.

Arch Biochem Biophys, 1992 Nov 1, 298(2), 538 - 43
Purification of a tumor-specific PNA-binding glycoprotein, gp200, from a human embryonal carcinoma cell line; Schopperle WM et al.; A 200-kDa peanut agglutinin (PNA)-binding glycoprotein, gp200, has been purified and partially characterized from the human embryonal carcinoma cell line, HT-E (833k) . Tissue distribution analysis of this molecule by lectin blotting with PNA of detergent-extracted proteins from human cell lines and tissues demonstrated expression limited to nonseminomatous germ cell tumors . The 200-kDa protein was purified with lectin affinity and gel filtration chromatography . Purification to apparent homogeneity was demonstrated by one- and two-dimensional gel electrophoresis . Characterization of gp200 revealed it to be a surface integral membrane glycoprotein; however, gp200 could also be purified from the culture media of EC cells, suggesting gp200 has an extracellular role . The carbohydrate groups of gp200 are N-linked and partially sialylated and contain terminal galactose residues . These initial studies suggest that the PNA-defined glycoprotein, gp200, is a candidate for a nonseminomatous germ cell tumor marker.

Obstet Gynecol, 1992 Nov, 80(5), 888 - 91
Natural oocyte retrieval with intravaginal fertilization: a simplified approach to in vitro fertilization; Taymor ML et al.; To simplify in vitro fertilization (IVF), we have combined natural-cycle oocyte retrieval with intravaginal fertilization . Our subjects ranged in age from 28-40 years and were monitored by ultrasound and steroid hormone levels . Oocyte retrieval was carried out under vaginal ultrasound-guided aspiration 32-36 hours after the onset of the LH surge . The oocyte was identified and placed in a sealed capsule containing culture media and sperm . The capsule, in a sealed cryoflex envelope, was placed in the woman's vagina and removed 42-48 hours later . The embryo was then isolated and transferred to the woman's uterus . Fifty-one retrieval cycles were attempted in 45 patients . At least one oocyte was retrieved in 88% of cycles, and fertilization was achieved in 84% of oocytes . Of the five clinical pregnancies (10%), four have delivered and one is ongoing . The cost of this procedure is approximately one-third that of standard IVF . The advantages of our method are the elimination of the use of gonadotropins, the simplicity of monitoring and oocyte retrieval, and the lack of need for expensive laboratory equipment . Natural oocyte retrieval with intravaginal fertilization may prove appropriate for those women requiring IVF who fear multiple pregnancies, have side effects from controlled ovarian hyperstimulation, or cannot afford standard IVF.

Circ Res, 1992 Nov, 71(5), 1039 - 48
Cellular mechanisms for synthesis and secretion of atrial natriuretic peptide and brain natriuretic peptide in cultured rat atrial cells; Suzuki E et al.; To investigate the cellular mechanism for the synthesis and secretion of atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP), we examined the effects of vasoactive agents on the secretion rates and gene expression of ANP and BNP in cultured rat atrial cells . Endothelin (10(-7) M, +61%), 12-O-tetradecanoylphorbol 13-acetate (TPA, 10(-6) M, +62%), the calcium ionophore A23187 (10(-6) M, +95%), and Bay K 8644 (10(-6) M, +34%) (p < 0.05 each) all increased the secretion of ANP into the culture media in a dose-dependent fashion . On the other hand, endothelin (10(-7) M, +57%) and TPA (10(-6) M, +55%) (p < 0.01 each) increased the secretion of BNP in a dose-dependent manner, whereas A23187 (10(-6) M, -45%, p < 0.001) suppressed the secretion of BNP in a dose-dependent manner, and Bay K 8644 caused no significant effects on BNP secretion . The molecular forms of intracellular ANP were exclusively gamma-ANP, whereas those of BNP were gamma-BNP and its carboxy terminal 45-amino-acid peptide, BNP-45 . The ratio of media to cell contents was much higher in BNP than in ANP . Northern blot analysis revealed that both ANP mRNA and BNP mRNA levels were significantly increased by 10(-7) M endothelin (ANP mRNA, +52%; BNP mRNA, +36%; p < 0.05 each) and 5 x 10(-5) M 1-oleoyl-2-acetylglycerol (ANP mRNA, +296%; BNP mRNA, +133%; p < 0.01 each) but not by 10(-6) M A23187 . Thus, the secretion of ANP is stimulated by both the elevation of {Ca2+}i and the activation of protein kinase C, whereas its synthesis is increased mainly by the activation of protein kinase C . The synthesis and secretion of BNP are augmented by the activation of protein kinase C rather than the elevation of {Ca2+}i . Furthermore, the processing and secretion of ANP and BNP may be regulated in different manners.

Pigment Cell Res, 1992 Nov, 5(5 Pt 1), 253 - 62
Intrinsic pigment-cell stimulating activity in the catfish integument; Zuasti A et al.; In keeping with the concept that local factors in the vertebrate integument affect the expression of pigment cells, the present study was directed toward demonstrating the existence of such factors in the skin of the channel catfish, Ictalurus punctatus . This species has a dark dorsal surface in marked contrast to an almost white midventral surface . Pieces of skin from these two surfaces were used to condition culture media, which were in turn bioassayed using the Xenopus neural tube explant system (Fukuzawa and Ide, 1988, Dev . Biol . 129:25) . A certain number of neural crest cells grow out from the explant, and many of these are melanized in a culture medium of Steinberg's basic salt solution (BSS) . When the BSS was conditioned with either dorsal or ventral skin, a profound increase in both the number of crest cells emigrated from the neural tubes and the percentage of melanized cells was observed . The effects of dorsal skin were stronger than those of ventral skin and were evident on a dose/response basis . Initial fractionation of conditioned BSS with DEAE ion exchange chromatography produced fractions of particular potency in the stimulation of melanogenesis . A similarly conditioned medium based upon Leibovitz's L-15 was used in the primary culture of mature chromatophores, namely, melanophores, iridophores, and xanthophores from tadpoles of Rana pipiens . Both dorsal and ventral conditioned media stimulated iridophores and xanthophores, but seemed to have little or no effect on tadpole melanophores . A melanization inhibiting factor (MIF) from the ventral surface of adult frogs has been suggested as the basis for the light colored ventrum of amphibians, and although the present experiments were not designed to study catfish MIF, the possible existence of such a factor in this species was supported by the results . The total results of this investigation are discussed in the light of the possible presence of a melanization inhibiting factor (MIF) of greater prevalence in the ventrum and a melanization stimulatory factor (MSF) of greater prevalence in the dorsal integument . It is suggested that the light-colored ventral surface of the catfish and other poikilotherms may result from the presence of higher levels of MIF than MSF . Thus, the expression of melanophores is inhibited while that of iridophores is enhanced . In contrast, higher levels of MSF over MIF in the dark dorsal surface would result in melanophore stimulation and inhibition of iridophore expression.

J Endocrinol, 1992 Nov, 135(2), 229 - 37
Regulation of adrenal steroidogenesis by adrenaline: expression of cytochrome P450 genes; Guse-Behling H et al.; The effect of adrenaline on the secretion of cortisol and cyclic AMP (cAMP) and on the accumulation of four different mRNAs encoding cholesterol side-chain cleavage cytochrome P450 (P450scc), 17 alpha-hydroxylase cytochrome P450 (P450(17 alpha)), 21-hydroxylase cytochrome P450 (P450c21) and 11 beta-hydroxylase cytochrome P450 (P450(11 beta)) was studied in bovine adrenocortical cells in primary culture and compared with the effects of ACTH . Treatment of cultured cells with adrenaline (1-100 mumol/l) showed a biphasic response in cortisol release over 1-24 h . Concentration of cAMP in the culture media increased from a basal level of < 0.06 pmol/dish to a maximal level of 40.14 +/- 8.9 pmol/dish with a half-maximal release of 20.07 pmol cAMP/dish in the medium reached 1.2 h after treatment with 10 mumol adrenaline/l . This stimulation resulted in an uniform increase in the levels of all four P450 mRNAs as revealed by Northern blot analysis . Increasing doses of adrenaline produced a maximal mRNA accumulation at a concentration of 10 mumol adrenaline/l . Incubation of the cells with 10 mumol adrenaline/l for 1-24 h produced a biphasic time-course with a half-maximal stimulation after about 5-6 h . Maximal stimulation with ACTH (100 nmol/l) caused different accumulations of the four mRNAs: P450sec mRNA increased twice as much and P450(17 alpha) mRNA six times as much as the accumulation of P450c21 mRNA and P450(11 beta) mRNA, which was about ten-fold over basal values . Propranolol totally blocked the stimulatory effect of adrenaline but not the effect of ACTH.(ABSTRACT TRUNCATED AT 250 WORDS)

Exp Cell Res, 1992 Nov, 203(1), 214 - 21
Characterization of a panel of rat ventral prostate epithelial cell lines immortalized in the presence or absence of androgens; Rundlett SE et al.; We have transfected rat ventral prostate (RVP) epithelial cells with a plasmid containing the SV40 large T-antigen in an attempt to establish a panel of cell lines that will be useful in molecular genetic studies of prostate cell function . Since the distribution of cell types in the RVP is dramatically affected by androgen withdrawal and replacement, cells isolated from normal, castrated, or castrated rats that were given daily injections of testosterone were used in these experiments . Cell lines were established in media that were supplemented or depleted of androgens to accommodate the possible requirements of different prostate cell types . Numerous cell lines were isolated which retain characteristics of RVP epithelial cells and five of these cell lines were studied in detail . All five cell lines express the SV40 large T-antigen, supporting the role of this viral protein in immortalization . The RVP cell lines were shown to contain high levels of functional glucocorticoid receptors, but very low levels of androgen binding activity even though androgen receptor RNA could be detected . It was determined that the decreased androgen receptor activity in the RVP cells was apparently due to low receptor expression based on the results of transient transfection assays using androgen receptor cDNA . Taken together, the biochemical, cytological, and morphological characterizations of the RVP cell lines suggest that they may all have been derived from basal prostate epithelial cells despite the initial differences in androgen status of the animal and the level of androgens in the culture media.

Hum Reprod, 1992 Nov, 7(10), 1435 - 9
Platelet activating factor in culture media as an indicator of human embryonic development after in-vitro fertilization; Nakatsuka M et al.; Although human chorionic gonadotrophin can detect trophoblast after implantation of the conceptus, there is a need to detect the conceptus before implantation . We have investigated whether human embryo-derived platelet activating factor is formed during embryonic development after in-vitro fertilization . A total of 99 ova from 12 patients were cultured and the 54 media were analysed . Platelet activating factor was also measured by radioimmunoassay after extraction . Fertilization increased the amount of platelet activating factor 4-fold over non-fertilized ova to a level of 4 ng/ml . This increase was also dependent on the degree of embryonic development with a maximum level of platelet activating factor of 7 ng/ml at the 2-cell stage . The follicular inducing agent used to treat the patient also had an effect on platelet activating factor; buserelin treatment gave embryos with a higher level than did clomiphene citrate treatment . These results indicate that platelet activating factor may have a role in embryonic development before implantation and may serve as a useful marker for fertilization and the developmental stage of the embryo.

Brain Res, 1992 Oct 30, 594(2), 197 - 204
cAMP-mediated expression of inwardly rectifying potassium channels in cultured mouse Schwann cells; Konishi T; Voltage-gated ionic currents were recorded from freshly dissociated or cultured mouse Schwann cells obtained from neonatal sciatic nerves by the whole-cell variation of the patch-clamp technique . Schwann cells virtually lost inwardly rectifying potassium (Kir) currents within 2 days after nerve transection or in culture conditions of neonatal sciatic nerves confirming the previous results that axonal signals were suggested to play an important role in the expression of functional Kir channels . To see the effects of adenosine 3',5'-monophosphate (cAMP) analogues or forskolin on the expression of Kir channels in cultured Schwann cells, these agents were added to the culture medium 4 days after the start of the culture, when Kir currents were almost eliminated from cultured Schwann cells . Cultured Schwann cells restored the expression of Kir currents by co-culture with agents which elevate intracellular cAMP level . The dose-response of 8-(4-chlorophenylthio) (CPT) cAMP for the incidence of the expression of Kir currents showed a steep increase in the percentage of cells with Kir currents between 0.02 and 0.1 mM of external CPT cAMP and approximately two thirds of cells had Kir currents in higher concentrations of more than 0.1 mM of CPT cAMP after 4 days of incubation . After removal of CPT cAMP from the culture media after 4 days of incubation, Kir currents disappeared from cells within 2 days . The simultaneous application of cycloheximide (1 microgram/ml), an inhibitor of protein synthesis, with CPT cAMP suppressed the expression of Kir currents for up to 6 days of incubation.(ABSTRACT TRUNCATED AT 250 WORDS)

Cancer, 1992 Oct 15, 70(8), 2137 - 42
Fibronectin is an immunosuppressive substance associated with epithelial ovarian cancer; Olt G et al.; BACKGROUND . Although the ascites of patients with ovarian cancer has been reported to contain immunosuppressive factors, the identity and source of this activity has not been determined . Previously, the authors showed that conditioned media from two of four epithelial ovarian cancer cell lines inhibits proliferation of mitogen-stimulated human lymphocytes . The physical characteristics of the inhibitory substance are unlike those of peptide growth factors but closely resemble those of fibronectin . METHODS AND RESULTS . In the current study, it was found that the two ovarian cancer cell lines that produce the inhibitory substance have more fibronectin on the cell surface and secrete significantly more immunoreactive fibronectin into their culture media than the other two ovarian cancer cell lines . In addition, the immunosuppressive activity was bound to a gelatin-Sepharose affinity column, known to bind fibronectin . Finally, in ascites from 20 patients with advanced epithelial ovarian cancer, fibronectin levels correlated with the ability to inhibit proliferation of lectin-stimulated lymphocytes (P < 0.001) . CONCLUSIONS . Fibronectin is produced by some ovarian cancer cell lines and acts to inhibit proliferation of mitogen-stimulated lymphocytes . Additional studies are needed to clarify the role of fibronectin in ovarian cancer.

J Immunol Methods, 1992 Oct 19, 155(1), 121 - 7
Restoring antibody activity of a monoclonal anti-RNP antibody by dissociative HPLC . Demonstration of blocking antibody binding sites with antigen released from effete hybridoma cells; Ma J et al.; In biomedical research, monoclonal anti-nuclear antibodies have a number of advantages over polyclonal antibodies in terms of both specificity and reproducibility . However, there are some potential problems in the preparation of monoclonal antibodies . A well characterized mouse monoclonal anti-ribonucleoprotein antibody (anti-RNP antibody, 2.73) known to function in Western blotting was found to lose this activity when produced in vitro from long term hybridoma cell culture . Whilst it could no longer detect RNP antigen by Western blotting, it could still function effectively in affinity purification of RNP antigen . Further studies suggested that this was due to blocking of antibody binding sites by RNP antigen released from effete hybridoma cells in culture . The activity of the antibody in affinity purification was retained because the antigen was stripped away by repeated elutions with 6 M urea . HPLC gel filtration in the presence of 6 M guanidine was able to restore the antibody activity of the protein A purified monoclonal antibody . This finding has important general consequences for the preparation of monoclonal antibodies against antigens present in hybridoma cell culture media.

Neurosci Lett, 1992 Oct 12, 145(2), 234 - 5
Quinolinic acid in culture media used for in vitro neurotoxicology studies; Heyes MP; Serum from several species is frequently added to the incubation media of cells in vitro . The excitotoxin and N-methyl-D-aspartate receptor agonist quinolinic acid was found to be present in serum in concentrations ranging from 59 to 4895 nM . Some neuronal systems are reported to be particularly sensitive to the neurotoxic effects of quinolinic acid . Therefore, quinolinic acid in serum should be considered as a potentially confounding variable in neurotoxicology studies in vitro.

Eur J Cell Biol, 1992 Oct, 59(1), 92 - 105
The trans-Golgi network can be dissected structurally and functionally from the cisternae of the Golgi complex by brefeldin A; Ladinsky MS et al.; TGN38, a transmembrane glycoprotein predominantly localized to the trans-Golgi network, is utilized to study both the structure and function of the trans-Golgi network (TGN) . The effects of brefeldin A (BFA) on the TGN were studied in comparison to its documented effects on the Golgi cisternae . During the first 30 min of BFA treatment, the TGN loses its cisternal structure and extends as tubules throughout the cytoplasm . By 60 min, it condenses into a stable structure surrounding the microtubule-organizing center . By electron microscopy, this structure appears as a population of large vesicles, and by immunolabeling, most of these vesicles contain TGN38 . TGN38 cycles to the plasma membrane and back, which is shown by addition of TGN38 luminal domain antibodies directly to cell culture media . This results in rapid uptake of antibodies which label the TGN within 30 min, both in its native and BFA-induced conformation . A number of transmembrane proteins have been shown to take this cycling pathway, but TGN38 is unique in that it is the only one predominantly localized to the TGN . To investigate the cycling of TGN38, the endocytic pathway was labeled by internalization of Lucifer Yellow, and in the presence of BFA there was partial colocalization with TGN38 . Further studies were carried out in which microtubules were depolymerized, resulting in dispersal of Golgi elements and inhibition of transport from endosomes to lysosomes . TGN38 cycling continues in the absence of microtubules . Taken together, these studies indicate that TGN38 returns from the plasma membrane via the endocytic pathway . We conclude that the TGN is structurally and functionally distinct from the Golgi cisternae, indicating that different molecules control membrane traffic from the Golgi cisternae and from the TGN.

Zentralbl Veterinarmed B, 1992 Oct, 39(8), 553 - 62
Prevalence of mycoplasmas in the respiratory tracts of pneumonic calves; ter Laak EA et al.; The prevalence of mycoplasmas in the respiratory tracts of 148 pneumonic calves originating from 25 herds in the Netherlands is reported . Four types of culture media were used to isolate mycoplasmas: solid modified Edward medium, 2 types of Friis media, and A7B differential agar medium . Mycoplasmas were isolated both from nasal swab specimens and lung lavage fluids collected from live calves and from nasal mucosa and lung tissue specimens collected post mortem . All of the mycoplasma strains isolated could be identified as either Ureaplasma diversum (isolated from 80% of 25 herds), Mycoplasma dispar (92%), M . bovirhinis (88%), M . bovis (20%), M . bovigenitalium (4%), M . arginini (16%), or M . canis (12%) . Isolation rates of M . dispar and U . diversum were considerably higher from lung lavage fluids than from nasal swab specimens . M . bovis was detected only in fattening herds and not in dairy herds . The respiratory tracts of 75% of the calves examined contained at least 2 mycoplasma species . In total, 25 different combinations of mycoplasma species were detected in specimens collected from noses and lungs . The pathogenic species U . diversum and M . dispar had not been isolated before in the Netherlands.

Poult Sci, 1992 Oct, 71(10), 1762 - 7
Effect of feed allowance during rearing and breeding on female broiler breeders . 3 . Ovarian steroidogenesis; Yu MW et al.; The influence of feed allowance during rearing (4 to 18 wk) and breeding (18 to 62 wk) on in vitro follicular steroidogenesis was examined . Four feeding programs were imposed: FF, consumed feed ad libitum throughout; FR, provided ad libitum access from 0 to 18 wk of age and restricted thereafter; RF, provided ad libitum access to feed from 0 to 4 wk, restricted from 4 to 18 wk, and consumed feed ad libitum thereafter; RR, consumed feed ad libitum from 0 to 4 wk of age and restricted thereafter . All birds received a starter diet (2,739 kcal ME/kg, 19.1% CP) from 0 to 3 wk of age, a grower diet (2,729 kcal ME/kg, 15.5% CP) from 3 to 22 wk of age, and a layer diet (2,889 kcal ME/kg, 14.6% CP) from 22 to 62 wk of age . Restricted feeding was based on commercial guidelines . At 33 wk of age, 10 to 12 (FF = 10, FR = 12, RF = 10, and RR = 11) birds from each feeding regimen were killed by cervical dislocation and the follicles removed for in vitro steroidogenesis . Feed allowance during breeding (18 to 62 wk) had a significant effect on in vitro follicular steroidogenesis at 33 wk of age . In broiler breeders fed ad libitum, some of the F2 (second largest) follicles had an endocrine profile characteristic of the F1 (largest) preovulatory follicles, secreting very little androstenedione and large amounts of progesterone . In feed-restricted birds, only the F1 follicles, which were destined to ovulate next, secreted any significant amount of progesterone into the culture media . Compared with restricted feeding, ad libitum feeding significantly increased the production of androstenedione in small white follicles (less than 1 mm).(ABSTRACT TRUNCATED AT 250 WORDS)

Mikrobiyol Bul, 1992 Oct, 26(4), 367 - 72
{Comparative evaluation of the isolation of dermatophytes by direct laboratory evidence and MSDA with MDTM culture media}; Yavuzdemir S; In this study, the hair, skin and nail examples taken from 225 patients suspected dermatophytoses clinically were investigated with direct microscopic examination and cultivated methods . Direct microscopic examination were positive in 135 examples (60%) and dermatophytes were isolated from 109 samples (48.4%) . In the isolation of dermatophytes, the effectiveness of modified Sabouraud Dextrose Agar (mSDA) was 93.5% (102/109) and the effectiveness of modified Dermatophyte Test Medium (mDTM) was 95.4% (104/109) . It was detected that there were no significant difference between these media for the isolation rate.

J Vet Med Sci, 1992 Oct, 54(5), 1043 - 6
In vitro study on the increase of trypsin resistance in the rat testicular sperm head; Matsuzawa T et al.; The alteration of trypsin resistance in rat sperm head was investigated after the sperm were incubated in the culture media (199-Earele, 1% BSA) containing the inhibitors of protein synthesis . The head of non-incubated testicular sperm was easily digested by trypsin (1 mg/ml) while the head of the incubated sperm showed resistance to trypsin digestion after 3 hr-incubation . Trypsin resistance in epididymal sperm head did not change after the sperm was incubated . Neither cycloheximide (100 micrograms/ml) nor chloramphenicol (100 micrograms/ml) affected the trypsin resistance in the testicular sperm head.

Biol Chem Hoppe Seyler, 1992 Oct, 373(10), 989 - 99
An investigation into the glycolipid metabolism of alpha-N-acetylgalactosaminidase-deficient fibroblasts using native and artificial glycolipids; Klima B et al.; Deficient activity of lysosomal alpha-N-acetylgalactosaminidase represents a recently recognized lysosomal disorder whose neurologic manifestation in infancy is infantile neuroaxonal dystrophy . The lysosomal enzyme defect, inherited as an autosomal recessive trait, was first identified in the two brothers, GD and BD . Metabolic modification of glycolipids with terminal alpha-GalNAc was studied in fibroblasts from these patients . {Ceramide-3H}Forssman-glycosphingolipid (GSL), the fluorescent C6-NBD-lyso-Forssman-glycolipid (GL) and a 14C-labelled neoglycolipid containing the blood group A trisaccharide were synthesized and used as probes in degradation studies with cell homogenates and with cells in culture . Assays of each of these substrates with fibroblast homogenates of the patients demonstrated the profound deficiency of alpha-N-acetylgalactosaminidase activity compared with controls . Residual activities in the patients' fibroblast homogenates were detected with all glycolipid substrates; those amounted to 6.3 +/- 3.7% (BD) and 12.8 +/- 6.3% (GD) of the mean activity in controls for {3H}Forssman-GSL, and to 2.2 +/- 0.8% (BD) and 3.6 +/- 1.8% (GD) for C6-NBD-lyso-Forssman GL, respectively . alpha-N-Acetylgalactosaminidase deficiency in intact cells was confirmed by TLC analyses, which showed impaired glycolipid modification in cell extracts obtained following addition of {3H}Forssman GSL and C6-NBD-lyso-Forssman GL to the culture media of fibroblasts from the patients.

J Clin Invest, 1992 Oct, 90(4), 1628 - 33
Epiligrin, the major human keratinocyte integrin ligand, is a target in both an acquired autoimmune and an inherited subepidermal blistering skin disease; Domloge-Hultsch N et al.; Epiligrin, the major component of human keratinocyte extracellular matrix, serves as the preferred integrin ligand for alpha 3 beta 1 in plasma membranes and focal adhesions, and colocalizes with alpha 6 beta 4 in hemidesmosomes . In human skin, epiligrin is found in the lamina lucida subregion of epidermal basement membrane, where it is thought to be associated with anchoring filaments . We have identified three patients with an acquired mucosal predominant subepidermal blistering disease who have IgG anti-basement membrane autoantibodies that bind the lamina lucida/lamina densa interface of epidermal basement membrane, stain cultured human keratinocyte extracellular matrix, and immunoprecipitate disulfide linked polypeptides of 170, 145, 125, and 95 kD in human keratinocyte culture media in a pattern identical to that of P1E1, a murine monoclonal antiepiligrin antibody . Comparative immunoprecipitation studies of patient sera, P1E1, and GB3 monoclonal antibody show that epiligrin is identical to the antigen (i.e., BM600 or GB3 antigen) previously reported to be absent from the skin of patients with lethal junctional epidermolysis bullosa, an inherited subepidermal blistering disease . Moreover, skin from a fetus with this disease shows no evidence of reactivity to patient antiepiligrin autoantibodies or P1E1 . These studies show that antiepiligrin autoantibodies are a specific marker for a novel autoimmune blistering disease and that the epidermal basement membrane antigen absent in patients with lethal junctional epidermolysis bullosa is epiligrin.

J Clin Invest, 1992 Oct, 90(4), 1379 - 85
Spontaneous production of transforming growth factor-beta 2 by primary cultures of bronchial epithelial cells . Effects on cell behavior in vitro; Sacco O et al.; The ability of airway epithelial cells to produce transforming growth factor-beta (TGF-beta) may be an important mechanism for the control of growth, differentiation, and repair of the airway epithelium . To determine whether airway epithelial cells are capable of producing TGF-beta, we examined primary cultures of bovine bronchial epithelial cells . Using a bioassay, TGF-beta activity was detected readily in media conditioned by bovine bronchial epithelial cells . Neutralizing antisera to TGF-beta 1 and TGF-beta 2 were used to demonstrate that the majority of the activity was of the TGF-beta 2 isoform . Interestingly, some of the TGF-beta activity was present in the conditioned media as "active" TGF-beta, not requiring acid activation . The production of TGF-beta was variable, depending on cell density and the presence of retinoic acid . The presence of endogenously produced active TGF-beta in the culture media was shown to modulate the behavior of the cell cultures as evidenced by the effects of TGF-beta-neutralizing antisera on cell size and fibronectin production . Our results suggest that active TGF-beta produced by airway epithelial cells may function in an autocrine or paracrine manner to modulate epithelial cell behavior.

J Clin Invest, 1992 Oct, 90(4), 1205 - 18
Mannose-induced dysmorphogenesis of metanephric kidney . Role of proteoglycans and adenosine triphosphate; Liu ZZ et al.; Because various fetal anomalies are seen in diabetic offspring, we examined the effects of sugars on proteoglycans (PGs): extracellular matrix (ECM) macromolecules modulating morphogenesis . 13-d-old mouse metanephric kidney explants were exposed to mannose for 7 d and labeled with {35S}sulfate, {35S}-methionine, or {3H}thymidine . Mannose exposure caused reduction in kidney size and disorganization of ureteric bud branches with inhibition of glomerulogenesis . Tissue autoradiographic and immunofluorescence studies indicated decreased expression of sulfated PGs in ECMs . Helix pomatia lectin binding to D-GalNAc residues of glomerular epithelial cells was also reduced . Biochemical studies revealed decreased synthesis of sulfated PGs . PGs were of lower molecular weight with reduced charge density and increased chondroitin/heparan sulfate ratio . Immunoprecipitation of {35S}methionine-labeled proteins confirmed the reduction of PG core peptides . Intracellular ATP levels were reduced . The addition of 0.1 mM ATP to culture media restored kidney size, the population of glomeruli, and the synthesis and characteristics of PGs to almost normal, with no detectable effect on the replication of cells as determined by {3H}thymidine incorporation . The effect of ATP could be partially blocked by the P2y-purinoreceptor, i.e., reactive blue-2 . Data suggest that mannose causes energy depletion by cellular ATP consumption and thus selectively alters the synthesis of heavily glycosylated proteins with rapid turnover, such as PGs, resulting in renal dysmorphogenesis.

Biol Reprod, 1992 Oct, 47(4), 648 - 55
Follicular heterogeneity and oocyte maturation in vitro in pigs; Ding J et al.; Experiments were conducted to confirm that co-culture of follicular shells and immature pig oocytes improved the rate of male pronuclear (MPN) formation and to test the hypothesis that the maturational state of the follicle used in co-culture would significantly affect the quality of oocytes matured in vitro . In a preliminary experiment, co-culture of oocyte complexes with follicular shells did not affect nuclear maturation, slightly inhibited penetrability (p = 0.04) but greatly enhanced (p = 0.0004) MPN rate . In the main experiment, oocyte complexes (10-15 per dish), obtained from follicles 36 h after eCG injection of prepubertal gilts, were co-cultured with single 36-h small (3.5-5.0 mm), 36-h large (6-9 mm), 72-h small (4-7 mm), or 72-h large (7.5-11.0 mm) follicular shells prior to insemination . MPN formation in penetrated oocytes was significantly affected by follicular size (small: 60.14% vs . large: 72.08%, p = 0.014) but not by time of recovery (36 h: 72.32% vs . 72 h: 59.90%, p = 0.39) . Overall, MPN formation rate was significantly correlated with follicular diameter (r = 0.45, p = 0.005), follicular fluid progesterone (r = 0.37, p = 0.02), and estradiol (r = 0.40, p = 0.01), and to the ratios of follicular fluid progesterone:testosterone (r = 0.39, p = 0.018) and follicular fluid estradiol:testosterone (r = 0.43, p = 0.007) . There were no simple correlations between rate of MPN formation and steroid concentrations and their ratios in culture media collected at the completion of culture.(ABSTRACT TRUNCATED AT 250 WORDS)

Mol Ecol, 1992 Oct, 1(3), 183 - 90
Influence of DNA supercoiling on the loss of culturability of Escherichia coli cells incubated in seawater; Gauthier MJ et al.; The relationship between the loss of culturability of Escherichia coli cells in seawater and the DNA supercoiling level of a reporter plasmid (pUC8) have been studied under different experimental conditions . Transfer to seawater of cells grown at low osmolarity decreased their ability to grow without apparent modification of the plasmid supercoiling . We found that E . coli cells could be protected against seawater-induced loss of culturability by increasing their DNA-negative supercoiling in response to environmental factors: either a growth at high osmolarity before the transfer to seawater, or addition of organic matter (50-mg/l peptone) in seawater . We further found conditions where a DNA-induced relaxation was accompanied by an increase in seawater sensitivity . Indeed, inactivation of either one of the subunits A and B of DNA gyrase, which leads to important DNA relaxation, was accompanied in both cases by an increased loss of culturability of conditional mutants after transfer to seawater which could not be explained uniquely by the increase in the temperature required to inactivate the gyrase . Similarly, a strain harbouring a mutation in topoisomerase I, compensated by another mutation in subunit B of the gyrase, was more sensitive to seawater than the isogenic wild-type cell and this greater sensitivity was correlated to a relaxation of plasmid DNA . Again, in these different cases, a previous growth at high osmolarity protected against this seawater sensitivity . We thus propose that the ability of E . coli cells to survive in seawater and maintain their ability to grow on culture media could be linked, at least in part, to the topological state of their DNA.(ABSTRACT TRUNCATED AT 250 WORDS)

Comp Immunol Microbiol Infect Dis, 1992 Oct, 15(4), 249 - 59
Effects of pseudorabies virus infection upon cytotoxicity and antiviral activities of porcine alveolar macrophages; Iglesias G et al.; Alveolar macrophages (AM) infected with Pseudorabies virus (PRV) were compared to noninfected AM for cytotoxicity against foreign or transformed cells and production of interferon (IFN) . Five PRV strains were used to infect AM including strains that are known to be highly virulent for pigs, i.e . strain 4892 and strain S-62 as well as strains that are regarded as mild or nonvirulent, i.e . BUK and Bartha . The multiplicity of infection ranged from 0.005 to 0.05 TCID50/cell . The target cells in the cytotoxicity assays were either chicken red blood cells, PRV-infected vero cells, or human myeloblastoma cells (K562 cell line) . For the production of IFN, AM cultures were treated with polyinosinic:polycytidylic acid (Poly I:C) diluted in tissue culture media at a concentration of 5 micrograms/10(6) cells . Culture supernatants were collected at various times poststimulation and tested for antiviral activity using the Vesicular Stomatitis Virus replication inhibition test . Swine AM were able to lyse chicken red blood cells in an antibody-independent way but not in an antibody-dependent way, whereas lysis of PRV-infected vero cells was accomplished both ways . The cytotoxicity against chicken red blood cells was reduced in the PRV-infected AM as compared to noninfected cells, particularly in AM infected with virulent PRV strains . Specific 51Cr release values for AM infected with S-62 and 4892 strains were 14 and 19, while the noninfected AM had values of 36 . Similarly, in the antibody-dependent cytotoxicity assay against PRV-infected vero cells there was no activity of AM against K562 cells . The production of IFN was readily stimulated with Poly I:C . The optimal time for supernatant collection was between 12 and 16 h poststimulation . The antiviral activity was abrogated by treatment of the supernatant with antiserum against human leukocyte IFN; it was therefore considered to be due to interferon-alpha (IFN alpha) released from the macrophages . The antiviral activity present in supernatants of PRV-infected AM was reduced compared to noninfected AM . The difference between AM cultures infected with virulent strains of PRV and noninfected AM cultures was statistically significant at P < or = 0.025 . The results provide support to the premise that the role of AM in lung defense can be compromised by PRV infection.

Zhonghua Liu Xing Bing Xue Za Zhi, 1992 Oct, 13(5), 300 - 3
{Study on the medium (BALM) for isolation of Legionella pneumophila}; Shu Q; L . pneumophila was apparently using blue algae (cyanobacteria) extracellular products as carbon and energy sources for its proliferation . Based on this observation, a medium (BALM) in which all chemicals and reagents were made in China for isolation of L . pneumophila was developed . The recoveries of L . pneumophila serogroups 1 and 6 standard strains from contaminated air-conditional water and infected guinea pig spleens were evaluated by using two culture media: BALM and BCYE (buffered charcoal yeast extract agar) . Recoveries of standard strains of L . pneumophila serogroup 1 were similar on both media, while those of L . pneumophila serogroup 6 were more efficient on BALM than on BCYE . In the process of isolation of L . pneumophila NANJ-1 strain, which was obtained from the material of tracheal lavage of a pneumonic patient in one of hospitals in Nanjing City, the recovery of this strain was also more efficient on BALM than on BCYE and other common used media . The results suggested that the use of BALM medium in place of BCYE may improve the recover of L . pneumophila from clinical and environmental specimens.

Ann Trop Med Parasitol, 1992 Oct, 86(5), 487 - 502
Axenic cultivation of amastigotes of Leishmania donovani and Leishmania major and their infectivity; al-Bashir NT et al.; Two clones of promastigotes, one of Leishmania donovani and one of L . major, and an uncloned stock of L . major were axenically transformed to heat-shock amastigotes, at 35 and 37 degrees C, respectively . Of the four different culture media tested, a relatively cheap, liquid medium, RBLM, was found to be the best, both for the transformation of the promastigotes and the serial, axenic cultivation of the amastigotes . In an experiment of 30 days duration, serial cultivation, in an atmosphere with 5% CO2, was possible by subculturing every three days . There were significant differences in virulence in BALB/c mice between axenically-cultured amastigotes and promastigotes, both in terms of the weights, lengths and parasite burdens of the spleens of mice infected intraperitoneally (ip) with L . donovani or L . major and of the appearance, type and size of the cutaneous lesions which developed in mice given L . major by intradermal inoculation.

J Cell Sci, 1992 Oct, 103 ( Pt 2), 511 - 9
Proliferation and differentiation of fetal rat intestinal epithelial cells in primary serum-free culture; Fukamachi H; It has been a subject of controversy whether fibroblastic cells are necessary for the proliferation of intestinal epithelial cells in primary culture . To answer this question, we have developed a serum-free primary culture system which allows reproducible and quantitative assays of proliferation and differentiation of fetal rat intestinal epithelial cells in the absence of fibroblastic cells . Pure intestinal epithelial tissues were obtained from 16.5-day fetal rats without contamination of mesenchymal cells, and were successfully cultured on a collagen gel in a medium consisting of Ham's F12, bovine serum albumin, epidermal growth factor (EGF), insulin, cholera toxin, transferrin and hydrocortisone . The epithelial nature of the cultured cells was confirmed by the presence of cytokeratin in the cells . Under optimal culture conditions, intestinal epithelial cells readily attached to the substratum in a day, and proliferated rapidly in vitro, increasing their number about 10 times in the first 5 days . EGF, insulin, cholera toxin, transferrin and hydrocortisone synergistically induced the epithelial proliferation, and lack of any one of them resulted in a significant reduction of the proliferation . In contrast, fetal bovine or horse serums, which have been widely used to supplement culture media, severely inhibited the epithelial proliferation . Histological examination showed that the epithelial cells formed simple cuboidal epithelia with basally-located nuclei when cultured on collagen gels . The intestinal epithelial nature of the cells was affirmed by the presence of villin on their luminal surface . Ultrastructurally, cells were connected by tight junctions and desmosomes at the subluminal region, and microvilli were projecting on the luminal surface, indicating that the cells in primary culture retained some characteristics of absorptive epithelial cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Otolaryngol Head Neck Surg, 1992 Oct, 107(4), 553 - 7
Effect of substance P on ciliary beat frequency in human adenoid explants; Staskowski PA et al.; This study investigated the direct effects of substance P (SP) on ciliary beat frequency of human upper airway mucosa . Human adenoid explant tissue was maintained in serum free culture media, MCDB153 . Ciliated epithelial cells were observed with phase-contrast microscopy and ciliary activity was measured using a photometric technique . Oscillations in transmitted light caused by ciliary beating were recorded and modal ciliary beat frequency was determined by fast Fourier transformation . Specimens were treated with SP at concentrations of 10(-4), 10(-5), 10(-6), and 10(-7) mol/L and with equal molar solutions of SP and (D-Pro2,D-Trp7,9)-SP, a SP antagonist . Substance P was found to increase ciliary beat frequency in a dose-dependent manner with a maximum increase of 12.1% . This effect was not seen with solutions containing (D-Pro2,D-Trp7,9)-SP . This suggests that SP exerts a direct stimulatory effect on ciliated cells of the upper airway . Because SP is known to be released in the upper airway in response to chemical irritation, it is presumed that the stimulatory effect of SP on mucosal cells provides a protective mechanism against inhaled irritants.

FEBS Lett, 1992 Sep 21, 310(1), 17 - 21
Stimulation of receptor-coupled phospholipase A2 by interferon-gamma; Ponzoni M et al.; The biomolecular mechanisms that mediate signal transduction by type II (gamma) interferon (IFN) are poorly understood . IFN-gamma is a potent growth inhibitory cytokine also endowed with antiviral, immunomodulatory, and differentiating activities on various cell targets, including neural cells . IFN-gamma induced a rapid and transient activation of phospholipase A2 in LAN-5, a human neuroblastoma cell line . A consequence of phospholipase A2 activation was the release of arachidonic acid and the generation of lysophospholipids from membrane phospholipids . Treatment of pre-labeled LAN-5 cells with a receptor-saturating concentration of IFN-gamma led to a time-dependent release of {3H}arachidonic acid into the culture media and generation of {32P}lysophosphatidylcholine . Pretreatment of cultures with the phospholipase A2 inhibitor, bromophenacyl bromide, markedly inhibited both {3H}arachidonic acid release and lysophosphatidylcholine production induced by IFN-gamma treatment . Pretreatment of LAN-5 cells with nordihydroguaiaretic acid, a lipoxygenase inhibitor, or with indomethacin, a cyclooxygenase inhibitor, amplified the release of {3H}arachidonic acid and production of lysophosphatidylcholine induced by non-saturating concentrations of IFN-gamma . In parallel, and with the same time-dependent effect, a significant decrease in phosphatidylcholine labeling was observed in IFN-gamma-treated cells, further indicating that a potential signal transduction mechanism of IFN-gamma is the hydrolysis of membrane phosphatidylcholine by phospholipase A2.

Biochem Biophys Res Commun, 1992 Sep 16, 187(2), 1193 - 9
Incomplete process of recombinant human platelet-derived growth factor produced in yeast and its effect on the biological activity; Calderon-Cacia M et al.; Partially purified recombinant human Platelet-derived Growth Factor BB homodimer isolated from yeast culture media contains variable amounts of unprocessed PDGF-BB . This unprocessed PDGF-BB is found as a result of incomplete cleavage of the precursor to form the mature protein . Although the signal peptide is efficiently removed, a fraction of the PDGF secreted has an extended sequence corresponding to the truncated yeast alpha-factor leader . The data suggest that it is the amino acid chain from the truncated a-factor leader and not the sugar moiety attached to it that is responsible for the higher mitogenic activity found in this unprocessed molecule compared to highly purified PDGF-BB.

Am J Trop Med Hyg, 1992 Sep, 47(3), 372 - 7
Mefloquine-halofantrine cross-resistance in Plasmodium falciparum induced by intermittent mefloquine pressure; Rojas-Rivero L et al.; The study was designed to evaluate how exposure of Plasmodium falciparum to mefloquine modifies the sensitivity of the parasite to four major antimalarial drugs . A recently culture-adapted strain of P . falciparum was subjected to intermittent drug pressure at three different mefloquine concentrations (2.34, 4.68, and 9.37 ng/ml) . Growth was monitored by daily evaluation of parasitemia on thin smears . Drug sensitivity tests were done weekly, using a radioisotope microdilution method . Mefloquine was removed from culture media when decreasing parasitemia was observed, and reintroduced when multiplication reoccurred . Parasite survival was inversely proportional to drug concentrations . The parasites tolerated progressively higher concentrations of mefloquine with prolonged exposure to the drug . Throughout this adaptation, the 50% inhibitory concentration for chloroquine and quinine showed no modification, but it increased considerably for mefloquine, exceeding known levels of resistance . Furthermore, a parallel increased resistance to halofantrine was observed, surpassing the normal range of sensitivity . Cross-resistance between mefloquine and halofantrine shown in this study has now been confirmed by epidemiologic in vitro surveys and clone analysis . These findings may have important in vivo consequences and eventually affect the choice of antimalarial therapy.

J Protozool, 1992 Sep-Oct, 39(5), 605 - 8
Continuous cultivation and drug susceptibility testing of Plasmodium falciparum in a malaria endemic area; Oduola AM et al.; Isolates (UCH-23 and OM) and cloned strains of Plasmodium falciparum (Clones W-2 and D-6) were maintained in continuous culture for 28 to 150 days using culture media supplemented with 10% (v/v) heat inactivated semi-immune human plasma . Microscopic appearance and growth rates (R) of the parasites in media supplemented with semi-immune human plasma {R = 1.13 (W-2), 0.92 (D-6), 0.75 (OM) and 0.84 (UCH-23)} were comparable to those of parallel cultures maintained in media supplemented with 10% (v/v) heat inactivated non-immune human plasma {R = 1.42 (W-2), 0.83 (D-6), 0.66 (OM) and 0.89 (UCH-23)} . In addition, IC50 for chloroquine and mefloquine against the two cloned strains of P . falciparum maintained in culture media supplemented with either non-immune human plasma or semi-immune human plasma were identical . Although growth rates of new isolates (UCH-23 and OM) fluctuated over time, they stabilized between the 12th and 19th day of adaptation to culture . This fluctuation in growth rates of the new isolates underscores the influence of population dynamics during adaptation of P . falciparum to continuous culture . Sixty-eight percent of the primary isolates (170 of 250) obtained from patients in Ibadan were successfully adapted and maintained in continuous culture using semi-immune human plasma . The results of these studies indicate that semi-immune human plasma is a suitable supplement for continuous cultivation and drug susceptibility testing of P . falciparum.(ABSTRACT TRUNCATED AT 250 WORDS)

J Lab Clin Med, 1992 Sep, 120(3), 459 - 64
Effects of complement activation on platelet-activating factor and eicosanoid synthesis in rat mesangial cells; Lianos EA et al.; The effects of in vitro complement activation and of isolated complement components (C3a, C5a, C3b) on the activity of the microsomal enzyme acetyl coenzyme A: 1-0-alkyl-glycero-3-phosphocholine acetyl transferase (AcTr) in cultured mesangial cells and on the synthesis of prostaglandin E2 (PGE2) were assessed . In vitro complement activation induced by the introduction of purified cobra venom factor (CVF) in culture media containing human serum enhanced mesangial cell PGE2 synthesis and had no effect on microsomal AcTr activity . When media containing C6-deficient serum were used, the stimulatory effect of CVF on PGE2 synthesis was abolished . Introduction of CVF in these media enhanced mesangial cell AcTr activity . These effects were partially reversed when the C6-deficient serum was supplemented with C7-deficient serum to allow formation of the C5b-9 complex . Isolated C3a, C5a, and C3b had opposite effects on PGE2 synthesis and on AcTr activity . Specifically, all components enhanced mesangial cell PGE2 synthesis . In contrast, AcTr activity was enhanced by C3a alone; C5a had no effect, whereas C3b had an inhibitory effect . The observations indicate that in response to complement activation and specific anaphylatoxin agonists, mesangial cell eicosanoid synthesis is not coupled with changes in the activity of AcTr and PAF synthesis.

J Clin Endocrinol Metab, 1992 Sep, 75(3), 756 - 61
Activin-A inhibits progesterone production by macaque luteal cells in culture; Brannian JD et al.; Since inhibin is produced during the luteal phase of the menstrual cycle in women and nonhuman primates, the primate corpus luteum (CL) may be a local site of inhibin/activin action . This study was designed to determine whether inhibin or activin altered steroidogenesis by macaque luteal cells in vitro . Luteal cells were obtained by enzymatic dispersion of CL from rhesus monkeys at midluteal phase of the menstrual cycle . Cells (2 x 10(4)/0.2 mL) were cultured in wells coated with extracellular matrix from bovine corneal endothelial cells in Dulbecco's modified Eagle's medium/F-12 medium (1:1 vol/vol) + insulin (2 ng/mL), transferrin (5 ng/mL), H2SeO3 (0.25 nmol), and aprotinin (10 micrograms/mL) . Various concentrations (0-400 ng/mL) of recombinant human-inhibin-A, recombinant human-activin-A or human CG (hCG) (100 ng/mL; CR123) alone or in combination with inhibin or activin were added to the culture media (n = 5 Exp) . Media were changed daily for 4 days and progesterone (P) concentrations were determined by RIA . Inhibin exposure did not alter P levels compared to that of control (untreated) cultures . In contrast, activin (10-400 ng/mL) suppressed P production (P less than 0.05) below controls and inhibin-treated cultures by days 3 and 4 . Exposure to hCG increased P levels throughout culture (9 x control levels by day 4; P less than 0.05) . hCG-stimulated P production was unaltered by inhibin, whereas activin (50-400 ng/mL) reduced (maximal inhibition of 40%; P less than 0.05) hCG-stimulated P production by day 4 of culture . Cell number on day 4 was not altered by any dose of inhibin or activin, but the number of cells staining for 3 beta-hydroxysteroid dehydrogenase was reduced (P less than 0.05) by 32.9 +/- 2.6% in activin-treated cultures . Since P levels declined during culture in all treatment groups, in a second series of experiments (n = 4), luteal cells were cultured for 4 days with or without hCG (100 ng/mL) and low density lipoprotein (LDL; 100 micrograms/mL) +/- 0-400 ng activin/mL . P production in the presence of hCG+LDL was greatly enhanced compared to other treatment groups and was sustained during days 2-4 of culture . Activin at doses of 50-400 ng/mL suppressed (maximal inhibition of approximately 35%; P less than 0.05) hCG+LDL-stimulated P production on days 3 and 4 . These results suggest that the primate CL is a target for activin action to suppress luteal cell activities, including gonadotropin-regulated, lipoprotein-mediated steroidogenesis.

J Cell Physiol, 1992 Sep, 152(3), 632 - 8
Relationship between culture conditions and the dependency on mitochondrial function of mammalian cell proliferation; van den Bogert C et al.; In cultured mammalian cells, the relationship was investigated between mitochondrial function and proliferation under various culture conditions . Continuous inhibition of the expression of the mitochondrial genome was used to reduce the activity of enzymes involved in oxidative phosphorylation by 50% at every cell division . Under these conditions, culturing in relatively poor media resulted in arrest of the proliferation of most cell lines after 1 cell division . This was preceded by decreasing levels of ATP and increasing levels of ADP, suggesting that the ATP-generating capacity of the cells was limiting . Culturing in richer media led to arrest of the proliferation after 5 to 6 divisions, but accumulation of ADP was not observed . Addition of pyruvate to rich culture media and, at least for 1 cell line, increasing the CO2 levels, completely prevented proliferation arrest . Inability to synthesise metabolic precursors via mitochondrial intermediary metabolism probably explains growth arrest of cells cultured in rich media . Pyruvate and CO2 were, however, without effect on the proliferation arrest of cells cultured in relatively poor media . Therefore, pyruvate dependency for growth of cells without functional mitochondria holds true only under culture conditions where the ATP-generating capacity of the cells is not limiting.

Photochem Photobiol, 1992 Sep, 56(3), 413 - 6
Dihydroxy-carotenoid liposomes inhibit phototoxicity in Paramecium caudatum; Rich MR et al.; The phototoxic effects of hematoporphyrin derivative, using Paramecium caudatum as a model system, are significantly reduced in the presence of carotenoid-containing liposomes . Multilammelar large or small unilammelar vesicles, containing specific carotenoids, were effective in protecting the organism, whether administered exogenously in the bathing solution, or via incubation of paramecia in starved culture media containing carotenoid liposomes . The effectiveness of the carotenoids as inhibitors of phototoxic effects was found to depend on the mode of administration, with small unilammelar being more effective than multilammelar large vesicles for all carotenoids tested . Small unilammelar vesicles containing the dihydroxy-carotenoids zeaxanthin or astaxanthin afforded the greatest protection in both exogenous and endogenous studies . The results of this study suggest that carotenoid efficacy may be determined, in part, by the environment of the carotenoid molecules.

Prostaglandins Leukot Essent Fatty Acids, 1992 Sep, 47(1), 77 - 81
The regulation of prostaglandin biosynthesis in cells derived from human gestational tissues: effects of fetal calf serum; Edwin SS et al.; We have evaluated the production of prostaglandin E2 (PGE2) and its regulation in amnion, chorion, and decidual cells in the presence and absence of fetal calf serum (FCS), and in the absence of FCS but with supplementation with substrate arachidonic acid (AA) . Basal rates of PGE2 biosynthesis in amnion, chorion and decidual cell cultures were significantly reduced in the absence of FCS . The magnitudes of the responses to various stimulatory agents were different between cells from different tissues and the different culture media . We conclude that these different experimental conditions must be taken into account when interpreting the results of such in vitro experiments.

J Reprod Fertil, 1992 Sep, 96(1), 61 - 71
Biosynthesis of platelet-activating factor by two-cell mouse embryos; Wells XE et al.; Incubation of two-cell mouse embryos with a range of radiolabelled compounds resulted in the incorporation of label into platelet-activating factor (PAF; 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) in the culture media . The demonstration that known precursors ({1-14C}hexadecanol, {1-3H}hexadecanol, 1-O-{alkyl-1'2'-3H}lyso-PAF, 1-O-{alkyl-1'2'-3H}acetyl-glycerol and {methyl-3H}choline chloride) were incorporated into PAF showed that embryo-derived PAF biosynthesis occurred via pathways present in other PAF-producing cells . The enzyme responsible for the formation of the ether linkage of the PAF molecule, alkyl-dihydroxyacetone-phosphate synthase, was present in the preimplantation embryo as {1-3H}hexadecanol was incorporated into PAF . Incorporation of label from alkylacetyl-glycerol and choline chloride into lyso-PAF was also observed, suggesting a role for lyso-PAF in the metabolism of embryo-derived PAF . Incubation of embryos with each of three {14C}carbohydrate energy substrates resulted in the incorporation of label into PAF in culture media, indicating that the composition of embryo culture media is important in the synthesis of PAF precursors . Incorporation of label from {2-14C}pyruvate was greatest and is consistent with the suggestion that pyruvate is the major energy source at the two-cell stage of development . L-{U-14C}Lactate was also incorporated into embryo-derived PAF, but the mean amount incorporated relative to the concentration of labelled substrate in the medium was 40 times less . The incorporation of D-{U-14C}glucose into PAF was 2405 times less than that from pyruvate, relative to the concentration in the medium.

Int Ophthalmol, 1992 Sep, 16(4-5), 381 - 5
Intraoperative and post operative treatment with 5-fluorouracil and mitomycin-c: long term effects in vivo on subconjunctival and scleral fibroblasts; Khaw PT et al.; Rabbits undergoing full thickness glaucoma filtering surgery were exposed to one of 4 treatments . Group 1 received intraoperative distilled water, group 2 received intraoperative mitomycin-c 0.2 mg/ml for 5 minutes, group 3 received 5 post operative injections of 5-Fluorouracil (5-FU) 5 mg in 0.1 ml, and group 4 received intraoperative 5-FU 50 mg/ml followed by 5 post operative injections of 5-FU . 30 days after the operation tissue biopsies were taken from the subconjunctival and scleral tissue at the treated area and 90 and 180 degrees from the treated area . The biopsies were then placed in tissue culture media and the outgrowths quantitated . The fibroblast outgrowths from all areas did not differ significantly from each other except for the outgrowths from the areas directly treated with mitomycin 0.2 mg/ml which were significantly smaller . In addition then cells were morphologically abnormal although there were foci of normal cells which appeared to be growing from localised areas in the tissue biopsies . The outgrowths from the areas 90 and 180 degrees from the treated area were normal . Intraoperative treatments with mitomycin-c result in long term inhibition of fibroblast proliferation limited to the treated area, when compared with intraoperative and postoperative treatment with 5-FU . Failure of filtration surgery in eyes treated with intraoperative mitomycin may in part be due to unaffected cells reproliferating.

Cytokine, 1992 Sep, 4(5), 361 - 8
Induction of inflammatory cytokine release from cultured human monocytes by C-reactive protein; Ballou SP et al.; The human acute phase protein, C-reactive protein (CRP), is capable of specifically binding to and modulating the function of mononuclear phagocytes . To investigate whether CRP can also affect the capacity of these cells to produce inflammatory cytokines, enzyme immunoassays and Western blot techniques were used to quantitate interleukin 1 beta (IL-1 beta), interleukin 6 (IL-6) and tumor necrosis factor alpha (TNF-alpha) produced by freshly-isolated normal human monocytes . CRP induced the rapid release of each cytokine, with significantly elevated levels in culture supernatants at 4 hours and maximal levels of TNF-alpha at 8 hours, and of IL-1 beta and IL-6 at 16 hours of culture . The effects of CRP were dose-dependent; greater than 10-fold increases of each cytokine were observed following culture with greater than or equal to 50 micrograms/ml CRP, concentrations which are often found in the presence of moderate to severe inflammation or tissue injury . The induction of cytokine release by CRP was unaffected by inclusion of 25 micrograms/ml polymyxin-B in culture media, but was completely abrogated by prior boiling of the CRP, a procedure which had no effect on induction of monocyte cytokine release by lipopolysaccharide . The dose-dependent induction of inflammatory cytokines by CRP provides further support for the hypothesis that interaction with mononuclear phagocytes constitutes an important biological role for this acute phase protein.

J S Afr Vet Assoc, 1992 Sep, 63(3), 128 - 31
The diagnosis and treatment of bovine genital ureaplasmosis: a case study; Gummow B et al.; An outbreak of granular vulvitis and ulcerative posthitis in 24-month-old virgin Bonsmara heifers and bulls is reported . Ureaplasma was isolated locally from all the clinically infected cattle . There was marked clinical improvement within 3 d of the commencement of a 5 d course of tylosin administered intramuscularly at 10 mg kg-1 . Ureaplasma could not be cultured from the external genitalia of either heifers or bulls following clinical recovery . Details of the Ureaplasma culture media are given.

J Anim Sci, 1992 Sep, 70(9), 2779 - 86
The effect of alkaloids and seed extracts of endophyte-infected tall fescue on prolactin secretion in an in vitro rat pituitary perfusion system; Strickland JR et al.; The objective of this research was to measure the effects of endophyte-infected tall fescue seed extract and various alkaloids associated with the endophyte on in vitro prolactin secretion by rat hemipituitaries . Rat anterior pituitaries (AP) were dissected into halves and placed in temperature-controlled culture chambers (37 degrees C) . The tissue was perfused with culture media at a flow rate of 12 mL/h . After perfusion for at least 90 min with control media, AP halves were exposed to their respective treatments for 15 min before they were returned to the control media . The treatments for Exp . 1 were .01 micrograms of alpha-ergocryptine/mL of culture medium, .01 microgram of ergonovine/mL of culture medium, .01 gram-equivalents of endophyte-infected tall fescue seed/mL of culture medium, and .01 gram-equivalents of endophyte free tall fescue seed/mL of culture medium . Treatments for Exp . 2 consisted of 10(-4), 10(-6), and 10(-8) M concentrations of perloline, N-formyl loline, N-acetyl loline, N-methyl loline, and alpha-ergocryptine . alpha-Ergocryptine suppressed (P less than .10) prolactin secretion in both experiments . Ergonovine and perloline both stimulated (P less than .10) prolactin secretion . The loline alkaloids (N-formyl loline, N-acetyl loline, N-methyl loline) had no effect on prolactin secretion . The endophyte-infected seed extract treatment suppressed (P less than .10) prolactin secretion . The endophyte-free seed extract treatment had no effect on prolactin secretion . In Exp . 2, prolactin secretion from AP responded to alpha-ergocryptine treatment in a dose-dependent fashion.(ABSTRACT TRUNCATED AT 250 WORDS)

Hokkaido Igaku Zasshi, 1992 Sep, 67(5), 623 - 37
{Experimental allergic encephalomyelitis: immunopathological analysis of antigenic reactivity and loss of encephalitogenicity}; Tokuchi F; Experimental allergic encephalomyelitis (EAE) is an autoimmune demyelinating disease of the central nervous system (CNS) . EAE can be induced by immunization with myelin basic protein (MBP) or passive transfer of MBP-reactive T cell lines and clones . We established several T-cell clones from SJL/J mice by immunization with whole rat MBP or a synthetic peptide encompassing guinea pig MBP 89-101 which contains the encephalitogenic determinant for SJL/J mice . One clone was found to have lost its encephalitogenicity during long-term passages in vitro, although this clone maintains its specific reactivity to the encephalitogenic determinant . To clarify the difference between the encephalitogenic T cell clone (4b . 14a) and the non-encephalitogenic T-cell clone (4b . 14a/n), we examined the suppressive activity of 4b . 14a/n on the reactivity to antigen of 4b . 14a, various lymphokine production and adhesion molecules expression of 4b . 14a and 4b . 14a/n . The culture fluid of the both 4b . 14a/n and 4b . 14a revealed a suppressive effect on the proliferation of 4b . 14a stimulated by MBP 89-101, and the effect was not different between these clones . In lymphokine production, the activities of lymphotoxin, interferon or interleukin-2 were not different between encephalitogenic clones (4b.14a and TNT-1) and 4b . 14a/n, whereas the activity of tumor necrosis factor-alpha, passively secreted by antigen presenting cell, was higher in culture media of 4b . 14a/n . Examination of adhesion molecule expression of 4b.14a/n failed to show any differences in expression of lymphocyte function-associated antigen-1 (LFA-1) alpha and CD2 in the comparison with 4b . 14a . However, LFA-1 beta expression of 4b . 14a/n was always less than of 4b . 14a . The present studies indicated that the lack of encephalitogenicity of T-cell clones which were responsive to an encephalitogenic determinant depends not on the difference in major lymphokines production but partially on adhesion molecules expression which was decreased in non-encephalitogenic T-cell clone.

Rev Esp Fisiol, 1992 Sep, 48(3), 167 - 70
R5020 enhances PGE2 stimulated steroidogenesis in cultured rat granulosa cells; Fanjul LF et al.; Progesterone biosynthesis and metabolization to 20 alpha-hydroxyprogesterone was stimulated in granulosa cells cultured in the presence of 20 ng/ml of follicle stimulating hormone (FSH) or increasing concentrations of PGE2 (10(-9)-10(-7)M) . Concurrent treatment with the synthetic progestin R5020 (10(-6) M) enhanced the FSH or PGE2 stimulated progesterone and 20 alpha-hydroxyprogesterone accumulation in culture media, as well as delta 5-3 beta-hydroxysteroid dehydrogenase activity in granulosa cell homogenates . These findings may represent another example of an autocrine control mechanism in which the steroidogenic product of the granulosa cell exerts an ultra-short loop regulation of its own production.

Food Addit Contam, 1992 Sep-Oct, 9(5), 551 - 60
In vitro model for the evaluation of toxicity and antinutritional effects of sulphites; Stammati A et al.; The food preservatives, sulphur dioxide and its salts, are known to present some toxic, mutagenic and antinutritional effects; in fact they interact with a number of nutrients, e.g . some vitamins, notably thiamine (Th) and folic acid (FA) . The effect of different concentrations of sodium bisulphite in cell culture media has been studied in vitro on a human cell line, HEp-2, deriving from a carcinoma of the larynx . Moreover, the sulphites have been tested with different levels of Th and FA with the aim of elucidating how much the cellular response depended on either the anti-nutritional effect or the toxicity of sulphites . Cell growth has been taken as an index of cytotoxicity and measured both as total protein content and as colony-forming ability . With no Th and FA in the culture medium, a clear decrease of cell growth was observed either with or without addition of sodium bisulphite . A dose-dependent reduction of protein content was detected in cells treated with 10, 50, 100, 200, 250 or 500 microM sodium bisulphite . Moreover, when the cells were treated with 10 or 100 microM of this compound, the colony-forming ability was reduced both in number and colony size . As far as the interaction of the two vitamins with sodium bisulphite is concerned, when these nutrients were present in the medium at 0.5, 1.0, 1.5, 2.0 or 2.5 mg/l, a similar growth profile, determined from their concentration, was observed in treated and control cells, the growth levels being affected by the sodium bisulphite contents . At higher levels of Th and FA, the growth index was still increasing only in treated cells, this phenomenon being particularly evident in cultures treated with 200 microM sodium bisulphite . The colony-forming ability was reduced in controls but still increased in treated cells at the highest concentration of vitamins.

Biochem Biophys Res Commun, 1992 Aug 31, 187(1), 51 - 7
Okadaic acid is a potent inducer of AP-1, NF-kappa B, and tumor necrosis factor-alpha in human B lymphocytes; Rieckmann P et al.; Treatment of human B lymphocytes with an optimal concentration of okadaic acid, an inhibitor of phosphatases 1 and 2A, resulted in the induction of the transcription factor, AP-1 and a marked increase in NF-kappa B levels . In contrast, no effect on the levels of the octamer binding proteins, Oct-1 or Oct-2, were found . Since both AP-1 and NF-kappa B have been reported to be important in the induction of the tumor necrosis factor-alpha (TNF-alpha) gene we examined the effects of okadaic acid on TNF-alpha mRNA levels . Treatment with okadaic acid resulted in a striking increase in TNF-alpha mRNA transcripts within 1 h of stimulation and large amounts of TNF-alpha were released into the culture media . Although okadaic acid provides a potent inductive signal for AP-1 and NF-kappa B it did not induce either B cell proliferation or immunoglobulin secretion.

J Biol Chem, 1992 Aug 25, 267(24), 17241 - 7
Expression and function of GLUT-1 and GLUT-2 glucose transporter isoforms in cells of cultured rat pancreatic islets; Tal M et al.; We have previously investigated glucose induction of glucokinase, glucose usage and insulin release in isolated cultured rat pancreatic islets (Liang, Y., Najafit, H., Smith, R . M., Zimmerman, E . C., Magnuson, M . A., Tal, M., and Mastchinsky, F . M . (1992) Diabetes (1992) 41, 792-806) . Here we studied the expression and function of GLUT-1 and GLUT-2 glucose transporter isoforms, using the same system, i.e . isolated pancreatic rat islets immediately after isolation or cultured in the presence of 3 or 30 mM glucose for as long as 10 days . We found by immunofluorescence microscopy and Western and Northern blot analysis of islet extracts that GLUT-1 expression was induced in islet beta-cells in tissue culture both with low or high glucose present . The induction of GLUT-1 was specific to beta-cells but was not present in all beta-cells and was not detected in alpha-cells . GLUT-2 expression was also specific for beta-cells and was not observed in all beta-cells . Some beta-cells in culture coexpressed GLUT-1 and GLUT-2 . The expression of the two glucose transporters was regulated in the opposite direction in response to glucose concentration in the culture medium . GLUT-1 was more effectively induced when glucose was low, and GLUT-2 expression was more pronounced when glucose was high in the culture media . Another difference between the two glucose transporters was that GLUT-2 expression was increased while GLUT-1 expression was decreased as culturing continued as long as 7 days . Thus, after 7 days of culture GLUT-2 expression in beta-cells was nearly the same at low and high glucose, whereas GLUT-1 was practically absent no matter what the glucose level was . In attempts to correlate GLUT-1 and GLUT-2 expression to beta-cell function glucose uptake and glucose-stimulated insulin release in fresh and cultured islets were measured . In freshly isolated islet glucose uptake was estimated to be 100-fold in excess of actual glucose use . Glucose uptake was reduced by 7-day culture to about one-third of that observed in freshly isolated islets no matter what the glucose concentration of the culture media . We conclude that in the present experimental system GLUT-1 and GLUT-2 expression and function are not closely associated with glucose usage rates or the secretory function of beta-cells.

Brain Res Dev Brain Res, 1992 Aug 21, 68(2), 149 - 62
Expression of inherent neuronal shape characteristics after transient sensitivity to epigenetic factors; Stichel CC et al.; We investigated effects of different substrates and culture media on the early morphological differentiation of rat neocortical neurons in culture . In particular, we examined the effects of homotypic astrocytes, the adhesive glycoprotein laminin and the polycationic substrate poly-L-lysine, as well as diffusible astrocyte-derived conditioned medium factors and serum on (1) soma area, (2) total neuron area and (3) primary neurite number . To assess variations in morphological reactions of neurons with a defined neurotransmitter phenotype, we analyzed the differentiation of GABAergic neurons . The morphology of young neocortical neurons was dramatically affected by both substrate and culture medium . Replacement of the astrocytic monolayer or the astrocyte-conditioned medium by other substrates or non-conditioned medium, respectively, was accompanied by (1) spreading and flattening of neuronal somata, (2) a marked decrease in total neuron area and (3) an increase in the number of primary neurites . The various morphological parameters studied exhibited different sensitivities to changes of these external factors . Moreover, the influences of epigenetic factors on the generation of primary neurites depended on the transmitter phenotype of the neuron . The induced morphological alterations were transient . At the end of the first week in culture, the surviving neurons underwent substantial remodeling of their morphology leading to an expression of in vivo shape characteristics . These observations suggest that despite an early, transient sensitivity to environmental influences, the neuronal differentiation with respect to the morphological parameters studied in culture is to a large degree determined by intrinsic factors.

J Immunol, 1992 Aug 15, 149(4), 1348 - 55
Biosynthesis and secretion of complement component (C3) by activated human polymorphonuclear leukocytes; Botto M et al.; We tested the hypothesis that human polymorphonuclear leukocytes (PMN), bearing complement receptors CR1 and CR3, might also synthesize C3, particularly when activated by LPS or cytokines . Northern blot analysis of total RNA, obtained from purified PMN stimulated overnight with LPS or cytokines (IFN-gamma, TNF-alpha, and IL-1) showed the 5.3-kb RNA transcript reported for C3 in hepatocytes and monocytes . No transcripts for C4 and factor B were detected . Time course studies of C3 mRNA expression in PMN treated with LPS or TNF-alpha demonstrated a steady increase with a plateau at 24 h that correlated with secretion of C3, determined by ELISA . In contrast, IFN-gamma and IL-1 induced a transient increase in C3 transcript with a peak around 8 h after stimulation, which was not reflected in an increased rate of C3 secretion . The content of C3 protein in PMN culture media, measured by ELISA, was about 4 ng/ml/10(7) cells after overnight stimulation with LPS or TNF-alpha . A very small amount of C3 (about 0.7 ng/ml/10(7) cells) was detected in supernatants from unstimulated and IFN-gamma- or IL-1-induced PMN . Immunoprecipitation with a polyclonal anti-human C3, followed by SDS-PAGE analysis, from {35S}methionine labeled PMN, revealed the presence in culture supernatants of three major bands at 185, 115 and 70 kDa, corresponding to pro-C3, alpha and beta chains, respectively . Analysis of {14C}methylamine incorporation and of autolytic cleavage showed that the C3 produced in tissue culture by PMN contained an intact thiolester bond . The capacity of PMN to secrete functional C3 in response to LPS and TNF-alpha might be an important mechanism of host defense at sites of inflammation.

Eur J Biochem, 1992 Aug 15, 208(1), 23 - 30
Control of human coagulation by recombinant serine proteases . Blood clotting is activated by recombinant factor XII deleted of five regulatory domains; Citarella F et al.; The availability of engineered serine proteases allows one to study the activation, substrate specificity and regulation of human coagulation and fibrinolytic activities . Human coagulation factor XII is composed of the protease catalytic region at the C-terminus, a hinge proline-rich region and regulatory domains at the N-terminus . From cDNA clones coding for factor XII, two DNA molecules were constructed, one being full length and the other being deleted of exons coding for the regulatory domains . Engineered factor-XII cDNA species were inserted by a homologous recombination technique into vaccinia viruses, which were used to infect the human hepatoma cell line HepG2 . Two recombinant proteins were prepared from the culture media and identified by their antigenic properties and electrophoretic mobilities . The recombinant protein of larger size was identified as the full-length factor XII of 80 kDa and its specific activities and activation patterns, determined both by the coagulation and the amidolytic assays, are very similar to these of native human factor XII . The recombinant protein of smaller size was identified as a 319-amino-acid-deleted factor-XII protein of 32 kDa, containing only the entire protease region and part of the proline-rich hinge . This protein was expected to be the 'minimal' portion of factor XII able to sustain protease but unable to recognize substrates and surfaces necessary to activate the contact phase of coagulation . However, this 'minimal' factor-XII protein displays a marked protease activity and, although lacking five regulatory domains of factor XII, is bound and activated by negative charges and promotes coagulation with high efficiency.

Plast Reconstr Surg, 1992 Aug, 90(2), 289 - 94
Full-thickness skin wound explants in tissue culture: a mechanical evaluation of healing; Greenwald DP et al.; This study was designed to evaluate biomechanically defined wound healing in full-thickness skin explants in tissue culture . The requirement for preculture incubation of wounds in situ was characterized . Full-thickness skin incisions were made in 44 rats and closed immediately . Wounds were incubated in situ for 0, 12, 24, 36, 48, 72, or 96 hours before harvesting and placement into tissue culture media for 6 weeks . Healing was evaluated by biomechanical criteria: tensiometric distraction to wound rupture generated true stress and energy absorption data . Burst-strength (maximum true stress) and toughness (energy absorption) were five times higher in the 48-hour group than in any other group; other groups were not different from each other . This study demonstrates long-term survival of full-thickness skin in culture and shows that full-thickness skin explants heal in tissue culture . Possible explanations for the narrow window of opportunity for harvest (48 hours, no more and no less) are discussed.

Fundam Appl Toxicol, 1992 Aug, 19(2), 268 - 74
Toxicology of maternally ingested trichloroethylene (TCE) on embryonal and fetal development in mice and of TCE metabolites on in vitro fertilization; Cosby NC et al.; Trichloroethylene (TCE), an industrial solvent, is a soil and ground water contaminant found across the United States . The metabolism and carcinogenic potential of TCE have been studied extensively in the past 15 years yet there is little information on the chemical's possible effects on reproduction . No reference to the reproductive effects in mice of TCE by oral administration exists in the literature . In this study, female B6D2F1 mice were gavaged from Days 1 to 5, 6 to 10, or 11 to 15 (Day 1 = vaginal plug) with TCE in corn oil at 0, 1/10, and 1/100 of the oral LD50 . Weights of mice were recorded and the livers and kidneys were weighed and preserved in 10% buffered formalin . Litters were counted, sexed, weighed, and measured for crown-rump length until weaning on Day 21 and some animals were allowed to develop to 6 weeks of age . At this time, a minimum of two litters from each dose were killed and gonads removed, weighed, and preserved in Bouin's fixative . Litters were also assessed for developmental abnormalities . No maternal or reproductive effect of TCE was seen at either dose level . TCE, administered consecutively on Days 1 to 5, 6 to 10, and 11 to 15 of pregnancy, does not appear to be a potential reproductive toxicant up to 1/10 the oral LD50 . In a second series of studies, TCE and its metabolites dichloroacetic acid (DCA), trichloroacetic acid (TCAA), and trichloroethanol (TCOH) were added to culture media to assess the toxic effects on in vitro fertilization (IVF) in mice.(ABSTRACT TRUNCATED AT 250 WORDS)

Am J Physiol, 1992 Aug, 263(2 Pt 1), E374 - 82
Apolipoprotein expression and cellular differentiation in Caco-2 intestinal cells; Wagner RD et al.; Caco-2 cells, cultured for 18 days on porous filter supports and conventional plastic culture dishes, were used to study the effects of cellular differentiation on the expression of apolipoprotein (apo) genes . Media of filter-grown cells accumulated more apo B as apo B-48 and contained three times the amount of edited apo B mRNA compared with plastic-grown cells . The accumulation of apo A-I by media of plastic-grown cells was higher than accumulation by filter-grown cells, despite similar concentrations of apo A-I mRNA . The apo A-IV was detectable in the culture media earlier with filter-grown cells compared with plastic-grown cells, despite similar apo A-IV mRNA concentrations . Plastic-grown cells contained more apo E mRNA, and their media accumulated more apo E than filter-grown cells . With the exception of apo A-I, apo gene expression changed with Caco-2 cell differentiation to resemble more closely the patterns seen in adult enterocytes . There were no effects or minimal effects of added retinoic acid, 1,25-dihydroxyvitamin D3 {1,25(OH)2D3}, or thyroid hormone on apo accumulation in media of filter-grown cultures of Caco-2 cells . However, 1,25(OH)2D3 and thyroid hormone increased apo B, apo A-IV, and apo A-I mRNA concentrations, retinoic acid increased apo B mRNA concentrations alone, and all three reduced apo E mRNA concentrations . Ratios of edited to unedited apo B mRNA were unaffected . In conclusion, culture substratum importantly influences Caco-2 cell differentiation . Soluble factors that influence cellular differentiation may affect apo gene expression over and above effects mediated by the culture substratum.(ABSTRACT TRUNCATED AT 250 WORDS)

Mol Gen Genet, 1992 Aug, 234(2), 332 - 6
Isolation and characterization of mutants of Aspergillus niger deficient in extracellular proteases; Mattern IE et al.; In the present study, the extracellular protease activity in a strain of the filamentous fungus Aspergillus niger was investigated and mutant strains deficient in the production of extracellular proteases were isolated . The major protease, which is responsible for 80-85% of the total activity, is aspergillopepsin A, a protein of ca . 43 kDa, the activity of which is inhibited by pepstatin . In addition, a second protease, aspergillopepsin B, is produced, which is much less sensitive to inhibition by pepstatin . Several protease-deficient mutants were obtained by in vivo UV mutagenesis . In addition, a mutant lacking aspergillopepsin A was constructed by an in vitro gene replacement strategy . In this mutant, AB1.1, the entire coding region of the gene for aspergillopepsin A (pepA) is deleted . In three UV-induced mutants, aspergillopepsin A is also missing . One of these mutants, AB1.18, is mutated in the pepA gene, which is located on chromosome I . One of the other mutants, AB1.13, which has only 1-2% of the extracellular protease activity in the parent strain, is deficient in both aspergillopepsin A and aspergillopepsin B . The mutation involved, prt-13, has been localized to chromosome VI, and is probably a mutation in a regulatory gene . Another mutation involved in loss of protease function, prt-39, is located on chromosome VIII . Degradation of various heterologous proteins in culture media of the mutants is reduced but, even in strain AB1.13, not completely abolished.

J Reprod Immunol, 1992 Aug, 22(2), 185 - 95
Relative immunosuppressive activity of human seminal prostaglandins; Skibinski G et al.; Human seminal plasma contains uniquely high concentrations of prostaglandins of the E series which are believed to contribute to its immunosuppressive effects in vivo . In order to obtain further insight into their activity we have compared the immunosuppressive properties in vitro of PGE1, PGE2 and 19-OH PGE using three immunological systems known to be modulated by prostaglandins, namely, mitogen induced lymphocyte proliferation, IL-2 and transferrin receptor expression and NK-cell mediated cytotoxicity . These studies revealed that PGE1 and PGE2 exerted a greater immunosuppressive effect than 19-OH PGE, but considerably higher levels of 19-OH PGE in semen might contribute the majority of immunosuppressive activity in vivo . Our studies also show that the lower stability of 19-OH PGE in culture media may be responsible for its lower immunosuppressive effect observed in vitro.

Indian J Exp Biol, 1992 Aug, 30(8), 751 - 3
Relative ranking of culture media based on proliferation kinetics and spontaneous chromosomal aberrations in PHA-responsive human peripheral blood lymphocytes; Jha AN; Proliferation kinetics and spontaneous yield of chromosomal aberrations phytohemagglutinin (PHA)-responsive peripheral blood lymphocytes were studied from blood samples collected from 45 individuals in 4 different synthetic media . Except for a significant difference for Eagle's MEM and RPMI 1640, the other media did not show difference for the yield of chromatid or chromosome type of aberrations . Differences were however noticed in the proliferation kinetics (mitotic and proliferative rate indices) of cells among the media used . The study indicated that (i) the intrinsic properties of media which influence proliferation rate and yield of chromosomal aberrations are independent of each other as higher proportion of first division cells do not correspond with higher frequency of chromosomal aberrations, (ii) the amount of free-radical scavengers present in the medium, apart from the genetic make-up of the individuals, may contribute to the spontaneous yield of chromosomal aberrations and (iii) RPMI 1640 medium, which showed higher transformation and faster cycling rate for the lymphocytes, may be considered as medium of choice for analysing two main cytogenetic end-points, chromosomal aberrations and sister chromatid exchanges (SCEs).

Parasitology, 1992 Aug, 105 ( Pt 1), 55 - 62
Adhesion of Trichomonas vaginalis to plastic surfaces: requirement for energy and serum constituents; Gold D et al.; The ability of Trichomonas vaginalis to adhere to plastic surfaces in the presence of various agents and under different growth conditions was examined in wells of microtitre plates containing unsupplemented TYI medium or the same, with various supplements . Following incubation, the wells were thoroughly washed and adhesion was determined by microscopic counting of the adherent organisms . There was no detectable adhesion in the absence of both serum and carbohydrate . Optimal adhesion (about 10-20% of the total number of parasites) was obtained throughout the growth curve in culture media supplemented with either serum or serum Cohn fractions IV-I (rich in alpha-globulin) or IV-4 (rich in alpha + beta-globulin) and 25 mM glucose, maltose or fructose, but not in plates pre-coated with the Cohn fractions . Cohn fraction II + III (rich in beta + gamma-globulin) moderately enhanced adhesion while Cohn fractions II (rich in gamma-globulin) or V (albumin), fibronectin, Tamm-Horsfall glycoproteins and polylysine were without effect . Non-metabolizable sugars (methyl derivatives of glucose, mannose or fucose) did not support growth, but, surprisingly, enhanced adhesion . At 4 degrees C, the trichomonads were not able to adhere and pre-adherent organisms detached from the plastic surface . Optimal adhesion was obtained at a pH range of 6.5-7.5 but was already detectable at pH 5.5 . Cytochalasin E markedly suppressed adhesion.(ABSTRACT TRUNCATED AT 250 WORDS)

J Neurobiol, 1992 Aug, 23(6), 720 - 38
Transient expression of adheron molecules during chick retinal development; Tsui HC et al.; Neuritogenesis and synapse formation are transient phenomena mediated in part by filopodial attachments (Tsui, Lankford, and Klein, Proc . Natl . Acad . Sci . 82:8256-8260 1985) . The attachments can be labeled by antisera against adherons, adhesive microparticles isolated from cell culture media (Tsui, Schubert, and Klein, J . Cell Biol . 106:2095-2108 1988) . Here, two monoclonal antibodies raised against adherons have been found to recognize transiently expressed membrane antigens of developing avian retina . Early in development, monoclonal antibody (mAb) AD1 stained antigens that spanned the entire tissue . With time, immunoreactivity became restricted to optic fiber, ganglion cell, and inner plexiform layers . Immunoblots of embryonic day (E) 13 retina showed a broad band at 66-72 kD for particulate fractions and a fine band at 70 kD for soluble fractions . The particulate forms disappeared as retinas matured, but the soluble form did not . mAb AD2 initially labeled retina antigens of optic fiber, ganglion cell, and inner plexiform layers (IPL) . Labeling in the plexiform layer showed discrete lamina . Immunoreactivity first appeared at E9, peaked at E15, and then disappeared shortly after hatching . In isolated cells, AD2 labeled small cell surface aggregates . Cytoarchitectural studies, using whole-mount transmission electron microscopy, showed AD2 antigen in cell surface microfilaments, including some that joined filopodia together . The adheron antigens recognized by mAbs AD1 and AD2 thus were (1) topographically restricted; (2) associated with cell surfaces; and (3) developmentally down-regulated . This pattern suggests a role in developmentally transient cell surface phenomena, such as neurite extension or junction biogenesis.

Protein Expr Purif, 1992 Aug, 3(4), 313 - 21
Production, purification, and characterization of human recombinant IL-8 from the eucaryotic vector expression system baculovirus; Kang XQ et al.; The cDNA for the human chemotactic interleukin, IL-8, was subcloned from a bacterial source into the eucaryotic vector expression system baculovirus . Recombinant human IL-8 (rhIL-8) was synthesized and secreted from Sf9 cells derived from Spodoptera frugiperda following infection of a recombinant virus harboring the full-length IL-8 structural gene . Infected Sf9 cells produced rhIL-8 in a range from 0.5 to 2.0 mg of rhIL-8/liter of postinfection cell culture media . The recombinant interleukin was purified (greater than 600-fold) to homogeneity using preparative HPLC . rhIL-8 retained all of the physical, immunological, and biochemical properties observed for the natural product, monocyte-derived IL-8 . rhIL-8 was assessed for biological efficacy by three criteria: (a) ability to induce chemotaxis in human neutrophils, (b) ability to induce oxygen burst metabolism, and (c) ability to be recognized by purified rabbit antibody generated against monocyte-derived IL-8 . Antibody generated against monocyte-derived IL-8 recognized rhIL-8 isolated during all stages of the purification protocol . rhIL-8 was strongly chemotactic for human neutrophils and exhibited a chemotactic index comparable to that reported for other strong chemotactic peptides . rhIL-8 was identified following sodium dodecyl sulfate-polyacrylamide gel electrophoresis as a single silver-stained band having an estimated molecular weight of 9.2 kDa and displayed amino acid residue molar abundance homology predicted for the mature form of the interleukin . Baculovirus vector expression coupled to preparative HPLC proved to be a very efficient method for large-scale recombinant interleukin production.

Angew Parasitol, 1992 Aug, 33(3), 161 - 7
{The improvement of polyxenic cultivation of Entamoeba histolytica type HK 9}; Luckner G et al.; The serological diagnosis of extraintestinal infections with E . histolytica by indirect immunofluorescence (IFAT) requires a corpuscular antigen . This is produced in our laboratory via polyxenic cultivation of E . histolytica strain HK 9 in a nutrient medium containing a simple salt mixture (Resembling the Ringer-mixture), calf serum and rice starch . It was the aim of the experiments described in this paper to search for a useful medium in which the amebae grow in high density but without substances disturbing the antigen production like debris or rice starch granules . It was shown that the cell culture media of EAGLE MEM and PARKER were easy to handle (contrary to the worldwide used DIAMOND medium TYI-S-33) and brought good results . After a period of 10 subcultures both media were suitable for the cultivation of E . histolytica for making an antigen reasonable for IFAT . As a side effect it was noticed that there was an adaptation phase during which the amebae grew slowly when the medium was changed to another which was completely different (from a cell culture medium to a Ringer-like solution).

Virus Genes, 1992 Aug, 6(3), 273 - 80
Neutralizing monoclonal antibodies against alpha and beta subunits of the Ustilago maydis virus encoded toxin; Ginzberg I et al.; The toxins secreted by Ustilago maydis are encoded by dsRNA viruses . The KP6 toxin encoded by subtype P6 consists of two polypeptides alpha and beta, which are not covalently bound . Neutralizing monoclonal antibodies (MoAbs) were raised against each subunit . Some of the anti-beta MoAbs identify different epitopes in the antigen . The MoAbs were used to affinity purify alpha and beta polypeptides from culture media and to detect the precursor of the mature toxin.

J Reprod Fertil, 1992 Aug, 95(3), 841 - 54
Origin of oestrus-associated glycoproteins in bovine oviductal fluid; Wegner CC et al.; The aim of the present study was to determine whether the synthesis of an oestrus-associated protein found in bovine oviductal fluid varies with oviductal region, stage of cycle or day of pregnancy . Explant culture was performed using oviducts recovered from naturally cycling animals either at oestrus or 12-14 days after oestrus . Three oviductal regions, the preampulla, ampulla and isthmus, were cultured individually in the presence of 20 muCi {35S}methionine in serum-free medium for 6 h at 37 degrees C . Synthesis of oestrus-associated protein was assessed by one-dimensional SDS-PAGE, fluorography and densitometry of radiolabelled bands . Significantly more oestrus-associated protein was synthesized by the ampullar region of the oviduct, although it was detected in explant culture media from both the isthmic and preampullar regions . A polyclonal antibody produced against oestrus-associated protein was used to localize the protein in paraffin-embedded sections of oviductal explant cultures and other bovine tissues . Localization of the protein in oviductal tissue sections varied with stage of cycle (oestrus > luteal > pregnant) and region of oviduct (ampulla > preampulla/isthmus) . These findings indicate an effect of oviductal region and hormonal state (cycling versus pregnant) on the synthesis and secretion of the oestrus-associated protein . Lectin affinity studies indicated that galactosyl(beta 1,3)N-acetylgalactosamine and N-acetylglucosamine residues are associated with the oestrus-associated protein.

J Endocrinol, 1992 Aug, 134(2), 307 - 12
Receptor binding and growth-promoting activity of insulin-like growth factor-I in a bovine mammary epithelial cell line (MAC-T3); Zhao X et al.; Insulin-like growth factor-I (IGF-I) has been known to be mitogenic to a variety of cell types, although a growth-regulatory role for IGF-I on bovine mammary epithelial cells has not been fully investigated . In the present study, we examined the receptor binding of IGF-I and its effect on growth in a bovine mammary epithelial cell line (MAC-T3) . Specific receptors for IGF-I were detected on cultured bovine mammary epithelial cells . Competitive binding revealed that half-maximal inhibition of 125I-labelled IGF-I binding by IGF-I was approximately 3 micrograms/l . Dissociation rate constant of the IGF-I receptor was 3.10 +/- 0.06 nmol/l (S.E.M.) with a receptor site concentration of 366 +/- 8 fmol/mg protein for the average of three experiments . IGF-I exerted a positive mitogenic effect on MAC-T3 cells according to both direct DNA assay and thymidine incorporation assay . Moreover, the mitogenic effect of IGF-I on MAC-T3 cells was enhanced by the addition of fetal calf serum in the culture media . The present results suggest that the bovine mammary epithelial cell line (MAC-T3) provides a useful model system with which to study the biological actions of insulin-like growth factors on the bovine mammary secretory tissue in vitro.

Nippon Sanka Fujinka Gakkai Zasshi, 1992 Aug, 44(8), 949 - 59
{Implantation model in vitro}; Negami AI; Implantation is thought to be interactive and synchronized process between mother and embryo, however, the mechanisms of the early implantation process remain unelucidated . Therefore, the effectiveness of in vitro fertilization and embryo transfer is still poor . As a result; immunohistochemically, type IV collagen and other extracellular matrix components were detected mainly below the epithelial layer . Type I and III collagens were detected diffusely in the stromal layer . In the lower stromal layer and superficial myometrial layer, type V collagen was detected . Human endometrial epithelial cells performed the re-epithelialization following glandular formation . Rabbit endometrial cells performed also re-epithelialization following the fold formation . The stromal cells were invaded into the inner layer of the folding . Estrogen added to the culture media stimulated the glandular formation . Progesterone administration after estrogen priming did not affect the glandular formation, however, the proliferation of the superficial epithelium and the re-epithelialized area were increased . Rabbit blastocysts successfully attached and implanted into the reconstructed endometrium . The development of the implanted embryos was morphologically normal . Human cultured trophoblast cells attached, invaded and penetrated into the extracellular matrix components . On the other hand, using type V collagen coated dishes, trophoblast cells could invade the stromal layer, however, type V collagen layer did not permit the trophoblast cells to invade into the collagen layer . In vivo, type V collagen, expressed in the lower stromal layer and the surface of the myometrium, may play a role to maintain the early embryo in the decidual compartment . EGTA inhibited the attachment of the blastocysts . Anti-laminin antibody and RGDS peptide, attachment domain of laminin, also inhibited the implantation . These findings suggested that the Ca2+ dependent process was necessary for the attachment between the trophoblasts and the endometrial cells, and then the implantation process was triggered after the attachment to the laminin in basement membrane . The endometrial tissue, obtained from the infertile patient, was cultured on the basement membrane extract with serially obtained maternal serum . Addition of the maternal serum after proper administration of pFSH, high estrogen conditions were made in the culture dishes . These conditions increased the height of the glandular structure and relatively decreased the area of the surface epithelium . Decreased the area of surface epithelium affected the rate of the attachment . Endometrial cell culture system associated with functional and morphological characteristics was established . Serial observation of the endometrial cells in this system revealed the rabbit and the human implantation process, and the embryo-endometrial interaction.(ABSTRACT TRUNCATED AT 400 WORDS)

J Pharmacol Toxicol Methods, 1992 Aug, 28(1), 9 - 14
Quantitative colorimetric assay for basic fibroblast growth factor using bovine endothelial cells and heparin; Kajio T et al.; An accurate and reproducible colorimetric assay was established to determine the concentration of basic fibroblast growth factor (bFGF) or bFGF-like activity in culture media and biological fluids . Fetal bovine heart endothelial cell line ATCC CRL 1395 was used as the bFGF-dependent cell line . The proliferation-stimulating activity of bFGF was determined by measuring the amount of formazan formed by the mitochondrial enzymes from a tetrazolium salt, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), instead of counting the viable cell numbers or measuring the incorporation of {3H}-thymidine . The addition of 250 ng/mL of heparin to the culture medium resulted in about a tenfold increase in the proliferation-stimulating activity of bFGF and allowed the detection of as low as 10 pg/mL of bFGF . Heparin also resulted in much smaller inter- and intraassay variations . The bFGF concentrations determined by this colorimetric assay correlated well with those determined by both the {3H}-thymidine incorporation assay using BALB/c 3T3 fibroblast cells (r = 0.998) and the cell number count assay (r = 0.996) . This assay can be adapted to quantify bFGF or bFGF-like activity in tissue culture media and biological fluids such as plasma and organ extracts.

Comp Biochem Physiol B, 1992 Aug, 102(4), 897 - 904
The biosynthesis of polyunsaturated fatty acids by rat sertoli cells; Oulhaj H et al.; 1 . The biosynthesis of polyunsaturated fatty acids (PUFA) of the n-6 and n-3 series was investigated in cultured Sertoli cells . 18:2n-6, 18:3n-6, 20:2n-6, 18:3n-3 and 20:3n-3 were added individually at a concentration of 20 mumol to culture media . 2 . Maximum incorporation of 20- and 22-carbon PUFA into membrane lipids was observed after 72 hr of incubation with all the exogenous substrates used . 3 . As reported in other cell systems, the delta 6 desaturation was the first rate-limiting step; the major factor regulating this activity was the concentration of linoleic acid or alpha-linolenic acid in the medium . 4 . Our data show that the delta 5-desaturation represents a second regulatory step in PUFA biosynthesis . 5 . The sum of n-6 and n-3 PUFA of the 22 carbon chain length constantly represented between 11 and 12% of total fatty acids, regardless of the exogenous substrate used . 6 . Our kinetic studies of the incorporation of PUFA of the n-6 and n-3 series did not permit detection of a delta 8 desaturase activity.

Br J Pharmacol, 1992 Aug, 106(4), 931 - 6
Cultured astrocytoma cells generate a nitric oxide-like factor from endogenous L-arginine and glyceryl trinitrate: effect of E . coli lipopolysaccharide; Salvemini D et al.; 1 . The inhibitory activity of astrocytoma cells (0.25-3 x 10(5)) treated with indomethacin (10 microM) on platelet aggregation was enhanced by incubating the cells with E . coli lipopolysaccharide (LPS, 0.5 micrograms ml-1) for 18 h . This effect was attenuated when cycloheximide (10 micrograms ml-1) was incubated together with LPS . The inhibition of platelet aggregation by cells treated with LPS was potentiated by superoxide dismutase (60 u ml-1) and ablated by oxyhaemoglobin (oxyHb, 10 microM) or NG-monomethyl-L-arginine (L-NMMA, 30-300 microM) . The effects of L-NMMA were reversed by co-incubation with L-arginine (L-Arg, 100 microM) but not D-arginine (D-Arg, 100 microM) . LPS also increased the levels of nitrite in the culture media and this increase was ablated by co-incubation with L-NMMA (300 microM) or cycloheximide (10 micrograms ml-1) . 2 . Astrocytoma cells (0.5 x 10(5)) treated with indomethacin (10 microM) enhanced the platelet inhibitory activity of glyceryl trinitrate (GTN, 11-352 microM) but not that of sodium nitroprusside (4 microM) . Furthermore, when incubated with GTN (200 microM) a 4 fold increase in the levels of guanosine 3':5'-cyclic monophosphate (cyclic GMP) was observed . These effects were abrogated by co-incubation with oxyHb (10 microM) but not with L-NMMA (300 microM) . Treatment of the cells with LPS (0.5 micrograms ml-1) for 18 h did not enhance their capacity to form NO from GTN . 3 . Thus, in cultured astrocytoma cells, LPS enhances the formation of nitric oxide from endogenous L-arginine.(ABSTRACT TRUNCATED AT 250 WORDS)

Brain Res Mol Brain Res, 1992 Aug, 14(4), 293 - 301
The effect of active serum albumin on PC12 cells: I . Neurite retraction and activation of the phosphoinositide second messenger system; Dyer D et al.; Vertebrate blood sera contain a factor that triggers oscillatory chloride currents in Xenopus oocytes through activation of the phosphoinositide/Ca2+ second system . The active serum component consists of lipids bound to an isoform of serum albumin that we have named active serum albumin (ASA) . In undifferentiated PC12 cells, micromolar concentrations of ASA inhibit the early morphological changes induced by NGF, whereas in differentiated PC12 cells ASA caused a rapid withdrawal of neurites, which was reversible and dependent upon culture age . In contrast to normal serum, plasma and thrombin did not cause neurite retraction . Preincubation of ASA with monospecific antibodies to serum albumin suppressed its ability to induce neurite retraction in a dose dependent fashion . As in the oocyte, ASA activated the phosphatidylinositol second messenger system of PC12 cells, causing a several fold increase in Ins1,4,5P3 levels within minutes of application . The Ins1,4,5P3 increase was also blocked, in a titratable fashion, when ASA was preincubated with monospecific antibodies to serum albumin . This suggests that ASA-induced neurite retraction in PC12 cells may depend, at least in part, on activation of the phosphatidylinositol second messenger system . Results involving albumin-depleted sera show that ASA is the main factor responsible for serum vulnerability of neurites in PC12 cells . These findings point to some limitations in the use of serum in culture media, and raise the possibility that the serum factor may impair neuronal plasticity in disorders that are accompanied by the activation of blood coagulation together with a breakdown of the blood-brain barrier.

Biochem Biophys Res Commun, 1992 Jul 31, 186(2), 1020 - 4
Replication-defective virus infection of feather buds produces a localized region of beta-galactosidase activity; Widelitz RB et al.; We are interested in using retroviral vectors to trace cell lineage and to introduce exogenous genes in chicken skin explant cultures . Here the LZ10 virus carrying the gene encoding beta-galactosidase was introduced to the skin explants by two different means: a) the virus was added to the media or b) the virus was microinjected into regions of the developing feather buds . Infection by microinjection led to localized expression of beta-galactosidase in the developing feather bud, while, surprisingly, infection by adding the virus to the culture media led to localized band of beta-galactosidase expression in the middle of the feather filament . The significance of this finding in skin morphogenesis and as a tool for experimental embryology is discussed.

J Biol Chem, 1992 Jul 25, 267(21), 14839 - 45
Lipoprotein assembly . Apolipoprotein B size determines lipoprotein core circumference; Spring DJ et al.; Apolipoprotein B (apoB) is an essential structural protein for the two triglyceride-rich lipoproteins synthesized by humans: chylomicrons and very low density lipoproteins . Although much is known about the role of apoB in clearance of lipoproteins from the circulation, relatively little is known about its role in the assembly of nascent lipoproteins . Therefore, we have investigated the relationship between the length of various N-terminal apoB fragments and the characteristics of the lipoproteins with which these fragments were associated . After the addition of puromycin, HepG2 cells secreted a discrete series of C-terminally truncated apoB fragments on lipoprotein particles including apoB25, apoB29, apoB31, apoB33, apoB36, apoB38, apoB42, apoB45, apoB49, apoB51, apoB55, apoB70, and apoB80 . Also, using plasmids encoding apoB26, apoB33, apoB37, apoB42, and apoB48, C-terminally truncated apoB fragments were expressed and secreted after transient transfection of HepG2 cells . Lipoproteins bearing the metabolically labeled apoB fragments were isolated from the cell culture media and characterized in terms of size, density, flotation coefficient, and composition . Lipoprotein radii, calculated from their flotation coefficients and buoyant densities, were used to derive the circumference of the non-polar core of each lipoprotein species . When plotted as a function of apoB size, core circumference defined a straight line of near-zero intercept . The slope of this line was approximately 1 A of core circumference/1 kDa of apoB molecular mass . A model for the mechanism of lipoprotein assembly in HepG2 cells, consistent with the concept that apoB size determines lipoprotein core circumference, is proposed.

Cancer Genet Cytogenet, 1992 Jul 15, 61(2), 126 - 30
The use of giant cell tumor conditioned media in cytogenetic studies of hematologic malignancies; Wason D et al.; The use of conditioned media produced from solid tumor cell lines has been beneficial in the study of hematologic malignancies . Conditioned media from giant cell tumors (GCT), human lung adenocarcinoma, and human bladder carcinoma express growth factors that have been used to stimulate growth of bone marrow cells and improve the quality of the preparations . It has been reported that addition of Lu-CSF1-conditioned media from a lung adenocarcinoma cell line masks abnormalities in cases of acute leukemia {1.} Because we routinely use GCT-CM in bone marrow and leukemic blood cultures for chromosome analysis in our lab, we investigated this potential effect on our case analysis . We have performed a serial study of a 100 cases of hematologic malignancies received for analysis in our lab to determine the effect of the addition of GCT-CM to our culture media with respect to 1) mitotic index, 2) quality of preparation, and 3) differential selection of either chromosomally normal or abnormal cell lines . Our results indicate that the mitotic index and quality of metaphases is enhanced with the addition of GCT media and that there is no difference in the rate of abnormality detection with or without the addition of GCT media.

Biochem J, 1992 Jul 15, 285 ( Pt 2), 655 - 60
Triacylglycerol accumulation and secretion in hepatocyte cultures . Effects of insulin, albumin and Triton WR 1339; Emmison N et al.; We have investigated the possibility that the apparent inhibition of very-low-density lipoprotein (VLDL)-triacylglycerol secretion by the addition of insulin to rat hepatocyte cultures may result from insulin-mediated enhancement of hepatic lipase secretion and, consequently, of extracellular triacylglycerol hydrolysis . We have, therefore, studied the effects of the inhibitor of lipase activity, Triton WR 1339, on the secretion of triacylglycerol by cultured rat hepatocytes . Incubation of hepatocyte cultures with increasing concentrations of Triton WR 1339 increased the accumulation of acylglycerol in the medium, suggesting that, in normal incubations, a substantial rate of degradation of secreted triacylglycerol does occur and that it results in an under-estimation of the rate of triacylglycerol secretion . However, Triton did not counteract the inhibitory effects of insulin, suggesting that the observed increased activity of hepatic lipase induced by the hormone cannot account for the inhibition of acylglycerol accumulation in the medium that occurred in the presence of insulin . BSA increased the accumulation of triacylglycerol in culture media by about 2-fold and also decreased the activity of hepatic lipase by 80% . A causative relationship between these two effects was supported by the further observation that Triton abolished the effects of BSA on triacylglycerol accumulation in the medium . The implications of these data for the validity of the use of Triton for the study of hepatic rates of triacylglycerol production in vivo and of secretion by hepatocytes in vitro are discussed.

Biochem J, 1992 Jul 15, 285 ( Pt 2), 603 - 11
Purification and characterization of matrix metalloproteinase 9 from U937 monocytic leukaemia and HT1080 fibrosarcoma cells; Morodomi T et al.; The precursor of matrix metalloproteinase 9 (proMMP-9), also known as '92 kDa progelatinase/type IV procollagenase', was purified from the conditioned medium of U937 monocytic leukaemia and HT1080 fibrosarcoma cell lines stimulated with phorbol 12-myristate 13-acetate . ProMMP-9 in these culture media is non-covalently complexed with the 29 kDa tissue inhibitor of metalloproteinases (TIMP), but free proMMP-9 was separated from the TIMP-proMMP-9 complex by chromatography on Green A Dyematrex gel . The final product was homogeneous on SDS/PAGE, with a molecular mass of 88 kDa without reduction and 92 kDa with reduction . Treatment of proMMP-9 with 4-aminophenylmercuric acetate converted the 88 kDa precursor into 80 kDa and 68 kDa forms . Gelatin-containing zymographic analysis showed zones of lysis associated with all three species . However, only the 68 kDa species was shown to be catalytically active by its ability to bind to alpha 2-macroglobulin . In the presence of an equimolar amount of TIMP, only the 80 kDa species was generated by treatment with 4-aminophenylmercuric acetate, but no enzyme activity was detected . This indicates that TIMP binds to the 80 kDa intermediate and inhibits the generation of the active 68 kDa species . Eight endopeptidases (trypsin, chymotrypsin, plasmin, plasma kallikrein, thrombin, cathepsin G, neutrophil elastase and thermolysin) were tested for their ability to activate proMMP-9 . Of them, trypsin was the most effective activator of proMMP-9 . Only partial activation (10-30%) was observed with plasmin, cathepsin G and chymotrypsin . The active forms generated by trypsin were identified as 80 kDa, 74 kDa and 66 kDa by their abilities to bind to alpha 2-macroglobulin . In the presence of an equimolar amount of TIMP, proMMP-9 was also converted into the same molecular-mass species by trypsin, but they were not proteolytically active . This suggests activated MMP-9 is inhibited by TIMP . Activated MMP-9 digested gelatin, type-V collagen, reduced carboxymethylated transferrin and, to a lesser extent, type-IV collagen and laminin A chain . The specific activity against gelatin was estimated to be 15,000 units/mg (1 unit = 1 microgram of gelatin degraded/min at 37 degrees C) by titration with alpha 2-macroglobulin . Comparative studies on digestion of gelatin and collagen types IV and V by MMP-9 and MMP-2 indicated that both enzymes degrade these substrates into similar fragments . However, the susceptibilities of laminin, fibronectin and reduced carboxymethylated transferrin to these two MMPs were sufficiently different to indicate differences in substrate specificities between these two closely related proteinases.

Curr Opin Ophthalmol, 1992 Aug, 3(4), 473 - 81
Corneal transplantation; Bigar F et al.; Corneal transplantation is the most widely practiced form of clinical transplantation . This was made possible by the development of donor handling and preservation techniques, such as cooled culture media and organ-culture systems, that guarantee a sufficient supply of donor tissue . Corneal grafting is performed to improve visual function, to preserve the integrity of the eye, or to reduce pain . Patients with visual disability who present with keratoconus or dystrophy have a good prognosis for retaining a thin, transparent graft on a long-term basis . In this patient group the limiting factor for a gratifying visual outcome is high residual postkeratoplasty astigmatism, a still-too-frequent occurrence . Refinement in trephination techniques should help overcome this pitfall . Patients with vascularization or regrafting who are in the high-risk category may benefit from HLA matching or the use of cyclosporine and other immunosuppressive agents that are currently being tested in experimental models for reducing the impact of allograft rejection.

J Clin Invest, 1992 Jul, 90(1), 282 - 7
Human hepatic lipocytes synthesize tissue inhibitor of metalloproteinases-1 . Implications for regulation of matrix degradation in liver; Iredale JP et al.; Hepatic lipocytes play a central role in the pathogenesis of liver fibrosis, both via production of extracellular matrix proteins and through secretion of matrix metalloproteinases . In this study, we have characterized lipocyte expression and release of tissue inhibitor of metalloproteinases-1 (TIMP-1), an important inhibitor of metalloproteinase activity, whose role in liver has not previously been examined . TIMP-1 was immunolocalized to human lipocytes, and secretion of TIMP-1 was confirmed by ELISA of culture media; (mean +/- SD) 159 +/- 79 ng of TIMP-1/10(6) cells per 24 h . Evidence for functional inhibitory activity of released TIMP-1 was obtained by (a) reverse zymography that demonstrated a single inhibitor band, M(r) 28 kD, that co-migrated with a TIMP-1-positive control sample; and (b) unmasking of inhibited gelatinase activity in lipocyte medium by separating it from TIMP-1 using gelatin sepharose chromatography; gelatinase activity in chromatographed medium increased more than 20-fold, compared with unfractionated medium, and could be reinhibited by adding back fractions that contained inhibitor . By Northern analysis, freshly isolated human lipocytes exhibited low levels of mRNA expression for TIMP-1, but this increased markedly relative to beta-actin expression with lipocyte activation during cell culture . We conclude that human hepatic lipocytes synthesize TIMP-1, a potent metalloproteinase inhibitor, and that TIMP-1 expression increases with lipocyte activation . These data indicate that hepatic lipocytes can regulate matrix degradation in the liver, and suggest that expression of TIMP-1 by activated lipocytes may contribute to the progression of liver fibrosis.

Fertil Steril, 1992 Jul, 58(1), 101 - 4
Human embryos produce transforming growth factors alpha activity and insulin-like growth factors II; Hemmings R et al.; OBJECTIVE: To assess whether growth factors are produced by early human embryos in culture . DESIGN: We studied various growth factors in the culture media of human embryos (n = 6) cultured from days 3 to 8 after fertilization . MAIN OUTCOME MEASURES: Four growth factors were measured: Insulin growth factors I and II (IGF-I and IGF-II), epidermal growth factor (EGF) and transforming growth factor alpha (TGF alpha) activity . RESULTS: Nonconditioned INRA Menezo B2 (Biomerieux, S.A., Paris, France) culture medium contained significant levels of TGF alpha activity (5.2 ng/mL) and low levels of IGF-I (1.02 ng/mL) and IGF-II (2.8 ng/mL), whereas EGF was below detection of our assay . With human embryo, the culture media contained lower TGF alpha activity on days 3 and 4 after fertilization (2.5 ng/mL and 2.8 ng/mL, P less than 0.05) . From days 5 to 8 after fertilization, a significant increase in TGF alpha activity and IGF-II was detected (TGF alpha activity: day 5: 3.7 ng/mL; day 6: 4.4 ng/mL; day 7: 6.4 ng/mL; day 8: 8.4 ng/mL) (IGF-II: day 5: 3.4 ng/mL; day 6: 3.1 ng/mL; day 7: 4.1 ng/mL; day 8: 4.2 ng/mL) . Epidermal growth factor was undetectable, and IGF-I did not vary significantly . CONCLUSION: Transforming growth factor alpha activity and IGF-II are produced by human embryos in culture at a time when they could play a role in morula to blastocyst transformation.

Recenti Prog Med, 1992 Jul-Aug, 83(7-8), 470 - 2
{Collagen-induced platelet aggregation in K562 neoplastic cells culture media}; Nubile G et al.; The evidence suggests that the propagation of neoplastic cells by solid tumors, seem the platelets have a protective role on the neoplastic cells respect to immunocompetent system . In this study, the AA . valued the variation of platelet aggregation induced by collagen when PRP was incubated with liquid culture of neoplastic cells K562 . The preliminary results, show the platelet aggregation induced by collagen, increase alter interaction with this liquid culture.

J Vet Diagn Invest, 1992 Jul, 4(3), 264 - 9
Effect of culture media and incubation temperature on growth of selected strains of Francisella tularensis; Payne MP et al.; The rate and amount of growth of 4 field isolates and reference strain ATCC 6223 of Francisella tularensis were evaluated on isolation media with 2 different agar bases and with different supplements and incubated at 25 C, 35 C, and 42 C . Biochemical reactions on conventional differential media with and without cysteine were evaluated . Two of the field isolates and the reference strain were F . tularensis subspecies tularensis (formerly biovar tularensis or Type A), and 2 isolates were subspecies holarctica (formerly subspecies palaearctica or Type B) . Bacto cystine heart blood agar supplemented with 1% hemoglobin, glucose cystine heart blood agar, and brain-heart infusion blood agar supported good growth of all 4 field strains, with the most luxuriant growth occurring on Bacto cystine heart blood agar with hemoglobin . Heart infusion blood agar and trypticase soy blood agar supported growth of the field isolates, although growth was diminished and delayed . Strain 6223 was distinctly fastidious and failed to grow on heart infusion or trypticase soy blood agars . Growth of strain 6223 was best on Bacto cystine heart blood agar with hemoglobin . The agar base did not affect growth unless the supplements became limiting, in which case Bacto agar base generally supported growth better than BiTek agar base . Incubation at 35 C was optimum for all 5 strains . Growth at 42 C was slow, with the greatest decrease in the rate and amount of growth occurring with field isolates of F . tularensis subspecies tularensis . Strain 6223 did not grow at 25 C, and the 4 field isolates grew slowly at the lower temperature.(ABSTRACT TRUNCATED AT 250 WORDS)

Cell Biol Toxicol, 1992 Jul-Sep, 8(3), 9 - 17
Hormonogenesis in thyroid cells cultured on porous bottom chambers; Chabaud O et al.; Different processes implied in thyroid hormonogenesis (thyroglobulin, thyroperoxidase and hydrogen peroxide generating system expressions) and their regulation by TSH and iodide have been studied using porcine thyroid cells cultured in porous bottomed chambers . This system allowed to reproduce the functional bipolarity . Cells form a tight and polarized monolayer . Both apical and basolateral poles of epithelial cells were independently accessible and the cell layer separated two compartments which can contain different media . A major polarized secretion of thyroglobulin into the apical compartment was observed; it was increased in the presence of TSH as well as the thyroglobulin synthesis and mRNA level . These TSH effects were the consequence of adenylcyclase stimulation . Active transport of iodide, iodination of thyroglobulin and hormonosynthesis took place only in the presence of TSH . These steps occurred at the apical pole of cells . In the culture chamber system, thyroglobulin was weakly iodinated (6 atoms of iodide per mole of thyroglobulin; in vivo up to 40 atoms per mole) but hormonogenesis efficiency was close to this one observed in vivo (40%) . Iodide concentrations higher than 0.5 microM daily added to the basal medium inhibited iodination of thyroglobulin and hormonosynthesis . Some components contained in culture media were inhibitors for iodination when they were present in the apical medium such as vitamins, amino acids and phenol red . The culture system appears to be interesting for pharmacological and toxicological studies.

Am J Obstet Gynecol, 1992 Jul, 167(1), 265 - 70
Comparison of cytokine levels and embryo toxicity in peritoneal fluid in infertile women with untreated or treated endometriosis; Taketani Y et al.; OBJECTIVE: Our aim was to examine the relationship between the levels of cytokines in peritoneal fluid and its embryo toxicity . STUDY DESIGN: The levels of interleukin-1 and tumor necrosis factor were measured in peritoneal fluid from infertile women who did not have endometriosis (n = 21), who had untreated endometriosis (n = 19), and who had undergone medical treatment for endometriosis (n = 10) . Embryo toxicity was investigated in mouse two-cell embryos cocultured with the oviducts in culture media that contained various concentrations of peritoneal fluid . RESULTS: The levels of cytokines were significantly higher in the peritoneal fluid from women who had untreated endometriosis than in women who did not have endometriosis, but they were extremely low in women who had undergone medical treatment with either danazol or buserelin . The peritoneal fluid from women who had untreated endometriosis adversely affected the cleavage of mouse two-cell embryos . After medical treatment the embryo toxicity of the peritoneal fluid was almost undetectable . CONCLUSION: These results offer some theoretic bases in support of medical treatment to improve reproductive performance in infertile women who have endometriosis.

Am J Otol, 1992 Jul, 13(4), 303 - 7
Hyaluronan synthesis by in vitro cultured endolymphatic sac cells; Amoils CP et al.; The endolymphatic sac (ELS) has been the subject of investigation for many years and yet its overall function remains unclear . It is believed mainly to be involved in the regulation of endolymph through fluid resorption and secretion of osmotically active substances . The present study was performed using in vitro cultured, fetal ELSs of 18 to 19 day gestational mice, to assess whether the ELS cells can synthesize the osmotically active polysaccharide, hyaluronan (HA) . The ELS and portions of the membranous labyrinth were dissected from whole otocyst specimens and placed in 14C glucose-enhanced tissue culture media . A light microscopic (LM), autoradiographic study was performed to assess whether 14C glucose could be incorporated by the tissue into HA . Both the ELS cells and the adjacent cartilage demonstrated radiolabel incorporation within 4 hours of incubation in tissue culture medium, with increased radiolabel density in ELS cells after 24 hours of incubation . HA-specific hyaluronidase (HAase) resulted in removal of HAase-sensitive compounds in the ELS in both 4-hour and 24-hour cultured specimens when compared to adjacent cartilage cells (p = 0.001) . Approximately 43 percent of the radiolabel was incorporated into HA in ELS specimens, as compared to a 22 percent HA synthesis in the adjacent cartilage tissue, suggesting preferential synthesis by ELS cells . The dissected murine otocysts demonstrate viability in vitro as measured by their ability to incorporate 14C glucose from tissue culture medium . Under these conditions the cultured ELS demonstrates an ability to synthesize HA . A theory of ELS function is proposed.

Rev Alerg, 1992 Jul-Aug, 39(4), 69 - 73
{Anemophilous fungi: viability and allergenicity}; Gomez Echevarria AH et al.; Three strains of anemophilous fungi (Alternaria, Rhizopus and Monilia) from different genera were studied . We found that the liquid Sabouraud and culture media used in this study lost nutrients after seven days of strain growth, without affecting their viability . This was proven when the fungi were reseeded at 15 and 60 days . Extracts from supposedly sensible patients were developed using intradermoreaction skin tests, finding no significant differences among the results obtained from these tests . We recommended further studies with more prolonged growth periods (90, 120, 180 or more days) to see whether this incides on the viability and allergenicity of the anemophilous fungi.

J Dermatol Sci, 1992 Jul, 4(1), 11 - 7
Determination of the action spectrum for UV-induced plasminogen activator synthesis in mouse keratinocytes in vitro; Takashima A et al.; Mouse epidermal keratinocyte-derived Pam 212 cells were irradiated with UV light, and the culture media were examined for plasminogen activator (PA) activity by measuring the capacity to convert exogenous plasminogen into plasmin . Exposure of cells to a broad spectrum of light in the UVB range induced a significant elevation of PA activity at 16 h after irradiation . A dose-response study revealed that a maximal enhancement, 15-fold higher than non-irradiated controls, was induced at a sublethal UVB dose of 100 J/m2, which significantly inhibited cell proliferation without affecting cell viability . Addition of 5 micrograms/ml of cycloheximide lowered the UV-induced elevation of PA activity, suggesting that protein synthesis is required for this phenomenon . Action spectra for PA synthesis were obtained by irradiating cells with monochromatic light ranging from 250 to 360 mm, and the data demonstrated that the action spectrum was 250-320 nm in length with a peak between 260 and 280 nm . The results suggest that UV exposure is an important physiological trigger for modulating PA synthesis in the epidermis.

Cancer Genet Cytogenet, 1992 Jul 1, 61(1), 46 - 9
Impact of polydonor mixed lymphocyte culture media on quantity and quality of myeloid metaphases; Taylor N et al.; A novel mitotic stimulator of myeloid hematopoietic cells was prospectively evaluated to detect enhanced quantity and quality of metaphases from bone marrow (BM) cultures for cytogenetic analysis . This polydonor mixed lymphocyte conditioned (PMLC) media decreased the culture failure rate, improved detection of chromosomal aberrations, and reduced technologists' analysis time . The conditioned media is recommended as a highly effective culture system for normal and neoplastic cells of myeloid lineage.

Fertil Steril, 1992 Jul, 58(1), 105 - 13
Laminin in the human embryo implantation: analogy to the invasion by malignant cells; Turpeenniemi-Hujanen T et al.; OBJECTIVE: To explore the possible similarities between the biochemical processes of embryo implantation and malignant invasion . DESIGN: The expression of a basement membrane (BM) glycoprotein laminin, a matrix binding cell surface receptor protein beta 1-integrin, and a BM collagen degrading metalloproteinase type IV collagenase, was studied in cultured human in vitro fertilized embryos . PATIENTS: Eight healthy women suffering from tubal infertility were participating in the IVF program in the Department of Obstetrics and Gynecology in University Hospital of Oulu . Twenty oocytes and 110 pre-embryos that were not transferred for the fertilizations were used in this study . MAIN OUTCOME MEASURES: Fibronectin, laminin, beta 1-integrin, and type IV collagenase immunoreactive proteins were studied in embryos by immunoperoxidase staining, and type IV collagen degrading activity was measured from the culture media of the embryos . RESULTS: Laminin and beta 1-integrin were expressed in the early human embryos before the time of implantation . Type IV collagen degrading activity and the 72 kd-type IV collagenase immunoreactive protein were expressed at the time of implantation . Laminin supported the expression of type IV collagenase . CONCLUSIONS: The expression of laminin, beta 1-integrin, and type IV collagenase in vitro are temporally in good correlation with the time of the implantation in vivo . Laminin and beta 1-integrin can relate to the attachment of the embryos to the uterine BM and type IV collagenase to the degradation of the BM collagen during the implantation . Laminin can augment the process locally.

Development, 1992 Jul, 115(3), 839 - 52
Mechanism of skin morphogenesis . II . Retinoic acid modulates axis orientation and phenotypes of skin appendages; Chuong CM et al.; The factors that determine the axial orientation and phenotypes of skin appendages were analyzed by studying the effect of retinoic acid (RA) on embryonic chicken skin explant cultures . With RA uniformly distributed in the culture media, the feather buds became smaller, were disoriented or were transformed into scale-like structures in a concentration-dependent manner (from 0.05-2.5 microM) . With RA distributed as a gradient created by a RA-soaked anion exchange bead, a radial zone of inhibition with a rim of disoriented buds was observed . The new axis of the disoriented buds appeared to be determined by a combination of the original feather axis determining force and a new axial force pointing centrifugally away from the RA source . This observed result can be simulated with a computer model using a vectorial sum of different feather axial determination forces . The size of the inhibited zone is linearly correlated to the RA concentration and may be used to quantify the morphogenetic activity of retinoids . These effects are specific to developmental stages (Hamburg and Hamilton stage 31-34) . Both all-trans and 13-cis RA have morphogenetic activity . Retinol has no effect and retinal has a small inhibitory effect but neither phenotypic transformation nor axial disorientation were observed . The antero-posterior gradient of homeoprotein XlHbox 1 in feather buds became diffusive after RA treatment . RA dissolves dermal condensations and the distribution of N-CAM is altered from an anterior localized pattern to a diffusive presence in the bud cores . Endogenous retinoids in developing skins show developmental stage-dependent changes both quantitatively and qualitatively . The results suggest that RA either is or can modulate the endogenous morphogen(s) that determine the orientation and phenotype of skin appendages, and that this morphogenetic pathway involves Hox genes and adhesion molecules.

Can J Vet Res, 1992 Jul, 56(3), 189 - 93
A pathogenesis study of foot-and-mouth disease in cattle, using in situ hybridization; Brown CC et al.; Eight calves were exposed in an aerosol chamber to nebulized foot-and-mouth disease virus . Two control animals were exposed in a similar manner to cell culture media only . Animals were euthanized at intervals and various tissues examined by in situ hybridization using a biotinylated RNA probe corresponding to a portion of the viral gene coding for the polymerase enzyme . By this technique large amounts of viral nucleic acid were found in coronary band, interdigital cleft and tongue as early as six hours postexposure, indicating a very rapid delivery from the portal of entry to the predilection sites for lesion development . This occurred well before the onset of viremia which by virus isolation was not detectable until 30 hours postexposure . The in situ hybridization signal in these tissues decreased in intensity and extent with time to focally positive areas, occasionally surrounding a vesicle . Other epidermal sites not normally thought of as sites for foot-and-mouth lesion development, such as carpus and eyelid, also had some viral nucleic acid detectable at various time intervals . In the lung by in situ hybridization, alveolar septa had viral nucleic acid early in infection (6-18 h postexposure) while later (36-96 h postexposure), the in situ hybridization signal was prominent in alveolar macrophages.

Int J Biochem, 1992 Jul, 24(7), 1081 - 6
Increase in superoxide production by heat-shocked cells of Neurospora crassa, demonstrated by a fluorometric assay; Lin WS et al.; 1 . Increase in superoxide production by heat-shocked cells of Neurospora crassa was demonstrated by a fluorometric assay . 2 . A sensitive fluorometric assay for the estimation of superoxide anion radical--based on the liberation of 4-methylumbelliferone from 4-methyl-beta-D-umbelliferyl glucopyranoside--is described . 3 . Using this system the level of superoxide in the medium of heat-shocked Neurospora crassa cells was found to be consistently higher, in comparison with that of non-shocked cells, cultured at the normal growth temperature of 28 degrees C . 4 . Addition of superoxide dismutase to the culture media suppressed the production of 4-methylumbelliferone.

J Allergy Clin Immunol, 1992 Jul, 90(1), 76 - 84
Protective effect of nedocromil sodium on the interleukin-1-induced production of interleukin-8 in human bronchial epithelial cells; Vittori E et al.; Cellular constituents of the bronchial mucosa may participate in the recruitment of polymorphonuclear leukocytes to the inflamed airways through the generation of the neutrophil chemotactic peptide, interleukin-8 (IL-8) . One important aspect of this interaction could be represented by the ability of pulmonary monokines, particularly IL-1, to induce gene expression of IL-8 in bronchial epithelial cells . In this study, we have evaluated the constitutive and IL-1-induced production of IL-8 by cultured human bronchial epithelial cells and examined the ability of the anti-inflammatory drug, nedocromil sodium, to inhibit the cytokine synthesis and release . Northern blot analysis demonstrated that IL-1 beta-treated human bronchial epithelial cells expressed appreciable levels of IL-8 mRNA during a period of 8 hours . This finding was associated with an increased release of immunoreactive IL-8 to the culture media, as assessed by specific ELISA . The bronchial epithelial cell-derived IL-8 demonstrated specific biologic activity, since the supernatants of stimulated bronchial epithelial cells possessed neutrophil chemotactic activity that could be inhibited by an antibody against human IL-8 . Nedocromil sodium reduced the IL-1-induced release of IL-8 in a dose-dependent manner, but concentrations of the compounds, up to 10(-5) mol/L, did not affect the constitutive production of this cytokine.

Biochem Biophys Res Commun, 1992 Jun 30, 185(3), 1022 - 33
Functional expression of soluble ICAM-1 by baculovirus-infected Sf9 cells; Cobb RR et al.; Inflammation, metastasis and ischemia are processes that require lymphocyte or leukocyte cell recognition and adherence to endothelial counter receptors such as ICAM-1 . Mapping the sites of interaction of ICAM-1 with LFA-1, the receptor for ICAM-1 on lymphocytes, may lead to the design of novel inhibitors of inflammation or metastasis . To this end, recombinant soluble ICAM-1 cDNA was engineered into the baculovirus expression system, which is capable of expressing large amounts of proteins . These constructs were designed to contain a protein leader sequence so that the transfected insect cells would secrete the recombinant polypeptide into the culture media for ease of isolation . We engineered four constructs of ICAM-1 into the baculovirus system and obtained relatively high expression of two soluble forms of ICAM-1, a two domain and a five domain form . These truncated proteins were isolated and shown to promote adherence of HL-60 cells and Molt-4 cells . These recombinant soluble proteins also inhibited cell adherence to purified intact ICAM-1 isolated from K562 cells.

J Biol Chem, 1992 Jun 25, 267(18), 12692 - 9
Characterization of recombinant human insulin-like growth factor binding proteins 4, 5, and 6 produced in yeast; Kiefer MC et al.; The insulin-like growth factor binding protein (IGFBP) family comprises six structurally distinct, but highly homologous proteins . They have been identified in serum and other biological fluids, tissue extracts, and cell culture media . We have recently cloned cDNAs encoding human IGFBP-4, -5, and -6 and have now expressed these BPs in yeast as ubiquitin (Ub)-IGFBP fusion proteins . Western ligand blotting with 125I-IGF II under nonreducing conditions of recombinant human (rh) IGFBP-containing yeast lysates revealed specific binding bands for IGFBP-4, -5, and -6 at apparent molecular masses of 24-26, 30-32, and 24-26 kDa, respectively, indicating processing of the fusion proteins . High-performance liquid chromatography-purified rhIGFBPs had virually the same amino acid composition, amino acid number, and NH2-terminal sequences as the native BPs . Except for the affinity of rhIGFBP-6 for IGF I (Ka = 8.5 x 10(8) M-1), the affinity constants of the three IGFBPs for IGF I and II lie between 1.7 and 3.3 x 10(10) M-1, i.e . 25-100 times higher than the IGF I and II affinities of the type I IGF receptor . When present in excess, rhIGFBP-4, -5, and -6 inhibited IGF I- and II-stimulated DNA and glycogen synthesis in human osteoblastic cells, but rhIGFBP-6 had only a weak inhibitory effect on IGF I in agreement with its relatively lower IGF I affinity constant . The results of this study show that the primary effect of the three rhIGFBPs is the attenuation of IGF activity and suggest that IGFBPs contribute to the control of IGF-mediated cell growth and metabolism.

J Biol Chem, 1992 Jun 5, 267(16), 11196 - 201
Spontaneous cytosolic calcium oscillations driven by inositol trisphosphate occur during in vitro maturation of mouse oocytes; Carroll J et al.; Immature mouse oocytes undergo spontaneous meiotic maturation when released from antral follicles into culture media . The first sign of meiotic resumption is germinal vesicle breakdown (GVB) . Cytosolic free Ca2+ was measured in mouse oocytes during spontaneous maturation by monitoring fluorescence of indo-1 or fluo-3 . The majority of oocytes showed a series of Ca2+ oscillations that continued for 1-3 h . Repetitive Ca2+ increases occurred every 1-3 min and lasted for 10-60 s . The Ca2+ oscillations appeared to be caused by an increase in inositol 1,4,5-trisphosphate (InsP3) because once they ceased, similar oscillations were triggered by injection of exogenous InsP3 . Also, injection of the InsP3 receptor antagonist heparin (final concentration, 100 micrograms/ml) blocked the spontaneous Ca2+ oscillations . In contrast, Ca2+ oscillations induced by thimerosal were not inhibited by heparin . Treating oocytes with media containing 20 microM BAPTA/AM abolished Ca2+ oscillations in oocytes but did not affect the rate of GVB . The data show that cytosolic Ca2+ oscillations apparently caused by polyphosphoinositide turnover occur during mammalian oocyte maturation . However, the spontaneous oscillations do not appear to trigger GVB . Also, the data indicate that there are two separate Ca2+ release mechanisms in mouse oocytes, one sensitive to InsP3, the other to thimerosal.

J Biol Chem, 1992 Jun 5, 267(16), 11424 - 30
Induction of 103-kDa gelatinase/type IV collagenase by acidic culture conditions in mouse metastatic melanoma cell lines; Kato Y et al.; Gelatinases/type IV collagenases have been shown to be involved in tumor invasion and metastasis . In this study, we examined the effect of culture medium pH on the secretion of the gelatinases from mouse B16 melanoma cell lines and human tumor cell lines using zymography analysis . The highly metastatic clone F10 of B16 melanoma did not secrete any gelatinase in neutral culture media (pH 7.1-7.3), whereas it secreted a high level of a 103-kDa gelatinase in an initial pH range of 5.4-6.1 . The addition of an excess amount of glucose into a neutral culture medium also induced the gelatinase secretion from the cells by decreasing the medium pH during incubation . The extent of the acid-induced gelatinase secretion by the B16 melanoma cell lines was in the order of BL6 greater than F10 greater than F1 much greater than the parent B16 line, in good agreement with the order of their metastatic potentials . Two human cell lines (A549 and HT1080) secreted a higher level of a 90-kDa gelatinase at pH 6.8 compared with pH 7.3 . The acid-induced gelatinase secretion from B16-F10 cells was blocked by cycloheximide, indicating that the enzyme induction was due to de novo synthesis . When in vitro tumor cell invasion was assayed in Boyden chambers, B16-F10 cells incubated in an acidic medium exerted a more active migration through type IV collagen gel than those in a neutral medium . These results suggest that the acidic environment formed around tumor tissues may be an important factor in invasion and metastasis of some types of tumors.

Leuk Res, 1992 Jun-Jul, 16(6-7), 693 - 701
Effects of a fibroblast-derived inhibitor on the growth of normal marrow and leukemic clonogenic cells; Wang H et al.; Blood cells develop in the bone marrow, controlled by a network of regulatory factors, some of which originate in the stroma . Previously, we found that most fibroblastoid (FB) cells growing in primary cultures of rat marrow bear surface antigens different from those found on FB of certain other tissues . As determined by two monoclonal antibodies ("ST3" and "ST4"), the "marrow type" is ST3+/ST4- and releases predominantly a colony-stimulating activity (CSA) into its culture media (CM), whereas the "peripheral type" (e.g . lung) is predominantly ST3-/ST4+ and produces inhibitory activity in excess of CSA . The studies described here show that this inhibitor also is active on rat leukemic myeloblasts (the BNML cell line), but not on eight other cell lines derived from rat tumors of various origins or on the human-derived leukemic cell lines tested . It was produced without exogenous stimulation, was labile to heat and acid, was not neutralized by antisera to transforming growth factor-beta, beta-interferon, or ferritin, and had an apparent mol wt in the range of 100-120 kD (peak of activity by gel filtration) . From the results obtained at this time, we are not able to ascribe this fibroblast-derived activity to any known inhibitor molecule.

Ann Surg, 1992 Jun, 215(6), 553 - 9; discussion 559-60
Prostaglandin E2 production during hepatic regeneration downregulates Kupffer cell IL-6 production; Goss JA et al.; The liver possesses the remarkable ability to regenerate to its original size after a 70% partial hepatectomy . There has been little effort to characterize the Kupffer cells' role in this unique mammalian reparative physiologic phenomenon . The capacity of rat Kupffer cells (KC) isolated at specific intervals after partial hepatectomy to produce interleukin-6 (IL-6) and prostaglandin E2 (PGE2) in response to endotoxin was evaluated in standard RPMI-1640 (1200 microM L-arginine) and arginine-depleted RPMI-1640 (10 microM L-arginine) media . Regenerating liver KC 48 to 120 hours after partial hepatectomy responded to endotoxin stimulation with a significantly greater (p less than 0.05) production of IL-6 in standard RPMI-1640 . Because Kupffer cells function in an environment where high arginase activity results in negligible L-arginine levels, the 10 microM L-arginine RPMI-1640 was used to simulate the true hepatic microenvironment . Production of IL-6 by regenerating liver KC was further increased (p less than 0.05) by placing these same KC in 10 microM L-arginine RPMI-1640 tissue culture media . During the same period, regenerating liver KC produced significantly (p less than 0.01) more PGE2 than sham-operated KC in both standard and low-arginine media . When the cyclo-oxygenase inhibitor indomethacin (1 x 10(-5) M) was added to cultures, the PGE2 production was inhibited, and IL-6 production was upregulated (p less than 0.05) in arginine-depleted cultures . The authors conclude that during hepatic regeneration KC IL-6 production is elevated but controlled in an autoregulatory fashion by KC PGE2 production.

Nippon Ganka Gakkai Zasshi, 1992 Jun, 96(6), 715 - 20
{The synthesis of cytokines by human lens epithelial cells--interleukin 1 (IL-1), tumor necrosis factor (TNF) interleukin 6 (IL-6), and epidermal growth factor (EGF)}; Nishi K et al.; We proposed the hypothesis that pseudophakic inflammation, including the fibrin reaction, may be caused by cytokines and/or prostaglandins, synthesized by residual lens epithelial cells (LEC) . To test our hypothesis, we measured IL-1 alpha, TNF-alpha, IL-6 and EGF in the culture media of human LEC, obtained by capsulotomy during cataract surgery, by ELISA . IL-1 alpha was detected in one of the two pools of 2-week cultures (20.7 pg/10(5) cells), in two of the three pools of 3-week cultures (12.0 pg/10(5) cells and 13.9 pg/10(5) cells), and in one pool of 4-week cultures (11.1 pg/10(5) cells) . IL-6 was detected in 1-week culture (195 pg/10(5) cells) and in 7-week culture (81.6 pg/10(5) cells) . TNF-alpha and EGF were not detected . During culture, the cells proliferated and underwent fibroblast-like changes on exposure to the plastic wells . IL-1 and IL-6 may be also produced in vivo by residual LEC contacting with posterior chamber lens after cataract surgery, and these mediators may play a role in postoperative inflammation including fibrin reaction.

Appl Environ Microbiol, 1992 Jun, 58(6), 1975 - 9
Characterization of form variants of Xenorhabdus luminescens; Gerritsen LJ et al.; From Xenorhabdus luminescens XE-87.3 four variants were isolated . One, which produced a red pigment and antibiotics, was luminescent, and could take up dye from culture media, was considered the primary form (XE-red) . A pink-pigmented variant (XE-pink) differed from the primary form only in pigmentation and uptake of dye . Of the two other variants, one produced a yellow pigment and fewer antibiotics (XE-yellow), while the other did not produce a pigment or antibiotics (XE-white) . Both were less luminescent, did not take up dye, and had small cell and colony sizes . These two variants were very unstable and shifted to the primary form after 3 to 5 days . It was not possible to separate the primary form and the white variant completely; subcultures of one colony always contained a few colonies of the other variant . The white variant was also found in several other X . luminescens strains . DNA fingerprints showed that all four variants are genetically identical and are therefore derivatives of the same parent . Protein patterns revealed a few differences among the four variants . None of the variants could be considered the secondary form . The pathogenicity of the variants decreased in the following order: XE-red, XE-pink, XE-yellow, and XE-white . The mechanism and function of this variability are discussed.

APMIS, 1992 Jun, 100(6), 490 - 7
Ultrastructure of human osteoblasts and associated matrix in culture; Kassem M et al.; The ultrastructure, as visualized by transmission electron microscopy, of cells obtained from human bone explants and subsequently cultured is described along with the electron microscopic appearance of the associated intercellular matrix . The cells were characterized as osteoblasts on the basis of immunohistochemical, enzymatic, and functional criteria . Although the osteoblasts could be cultured in standard culture media and always appeared singly, not forming syncytia, the cultures were eventually confluent and formed multilayers . The cells were fusiform or cuboidal with diameters ranging between 10-15 microns . The cytoplasm was characterized by numerous large mitochondria, and especially by a very prominent RER . The intercellular matrix was woven with collagen fibres surrounding large numbers of matrix vesicles . In areas with matrix vesicles, evidence for osteoblast activity, i.e . mineralization related to matrix vesicles, could be observed after incubation with beta-glycerophosphate . In conclusion, we provide evidence that human osteoblasts cultured in vitro synthesize collagen and produce a matrix with vesicles capable of initiating mineralization processes.

Invest Ophthalmol Vis Sci, 1992 Jun, 33(7), 2262 - 8
Whole-cell currents from noncultured human lens epithelium; Rae JL et al.; Perforated patch techniques were used to measure whole-cell ionic currents in freshly dissociated human lens epithelial cells that had not been subjected to culture media or serum . With a 150 mmol/l K+ internal solution, the cells had resting voltages of -27.4 +/- 4.7 mV (mean +/- standard deviation {SD}) and capacitances of 10.4 +/- 2.8 pF (mean +/- SD) . The input resistance of the cells was 1.6 +/- 0.7 G omega (mean +/- SD) at large negative voltages . A delayed outwardly rectifying K+ current was found in most cells studied . Current magnitudes of 1-2 nA at +80 mV were common . The current had selectivities, activation time constants, deactivation time constants, open probability versus voltage relationships, and inactivations similar to those of the delayed rectifying K+ current found in many cell types and studied previously in cultured human lens epithelium . These results verify the existence, at high density, of these currents in noncultured human epithelial cells.

Lab Invest, 1992 Jun, 66(6), 680 - 90
Localization of matrix metalloproteinase 3 (stromelysin) in osteoarthritic cartilage and synovium; Okada Y et al.; Degradation of proteoglycans is an initial change in osteoarthritic cartilage . Matrix metalloproteinase-3 (MMP-3; stromelysin) capable of degrading cartilage proteoglycans and type IX collagen was immunolocalized in osteoarthritic and normal cartilage . Immunohistochemical studies showed MMP-3 in chondrocytes of the superficial and transition zones in approximately 90% of osteoarthritic cartilage (60 of 67 samples) and in 31% of those of the superficial zone in some normal cartilage (4 of 13 samples) . MMP-3 staining correlated directly with the histological histochemical scores of Mankin and with proteoglycan depletion, up to a certain grade of severity . Chondrocytes in the deep radial zone, clusters, and osteophytes were immunostained only when proteoglycan depletion and fissures affected them . Culture media from osteoarthritic cartilage contained significantly higher levels of metalloproteinase activity that was identified as MMP-3 by immunoblotting and lower amounts of tissue inhibitor of metalloproteinases compared with those in the control samples . MMP-3 was also immunolocalized in the lining cells of most osteoarthritic synovium (20 of 23 specimens, 87%) with a direct correlation with scores of inflammatory cell infiltration in the synovium, but it was not detected in the normal synovium . Light and electron microscopic studies demonstrated that MMP-3 digests proteoglycan aggregates in human articular cartilage . Treatment of normal and osteoarthritic cartilage slices with tumor necrosis factor-alpha and/or interleukin-1 alpha increased the number of MMP-3-immunoreactive chondrocytes and the intensity of the staining . These data suggest that MMP-3 produced by the chondrocytes and synovial lining cells under stimulation with these cytokines may be important in proteoglycan degradation in human ostoearthritic cartilage.

J Bacteriol, 1992 Jun, 174(11), 3445 - 9
Improved bacterial baby machine: application to Escherichia coli K-12; Helmstetter CE et al.; Exponentially growing derivatives of Escherichia coli K-12 were immobilized onto the surfaces of nitrocellulose membrane filters which had been coated with poly-D-lysine . The cells attached firmly to the surfaces, and when flushed with culture medium, the immobilized cells continued to divide and newborn cells were released into the effluent . Cell cycle parameters were examined with the technique, and it was found that K-12 derivatives possessed differing values for interdivision times, C, D, and average cell sizes when grown in the same culture media . It was also found that the cells released from immobilized populations of one culture consisted of two predominant size classes: newborn cells of unit size with single nucleoids and newborn cells of double this unit size . The results demonstrated that K-12 derivatives can be used in the baby machine culture technique to examine all aspects of the cell cycle of this organism . Furthermore, the yield of newborn cells was about fivefold greater than that obtained previously with cultures of strain B/r immobilized onto uncoated membranes.

Exp Cell Res, 1992 Jun, 200(2), 351 - 7
Prostaglandins A and J arrest the cell cycle of cultured vascular smooth muscle cells without suppression of c-myc expression; Sasaguri T et al.; The effects of prostaglandins (PGs) A and J, which are anti-tumor eicosanoids, on the proliferation of cultured vascular smooth muscle cells were investigated . Serum-stimulated DNA synthesis was potently inhibited by PGA1, PGA2, PGJ2, and delta 12-PGJ2 in similar dose-dependent fashions . The effects of PGA1 and PGA2 were reversible when they were removed from the culture media, whereas recoveries were only partial in the cells treated with PGJ2 and delta 12-PGJ2 . PGs were effective even if they were added immediately before entry into S phase . Inhibition of DNA synthesis was sustained when hydroxyurea, which blocks cell cycle at the G1/S border, was added after the removal of PGA2, and vice versa; PGs blocked DNA synthesis when they were added after the removal of hydroxyurea . Levels of c-myc mRNA formed two peaks during the G1 phase, at 1-2 h and at 8-12 h . The PGs did not affect the first elevation, but enhanced the second and sustained it up to 18-24 h, whereas in controls, c-myc mRNA decreased quickly after entry into S phase . The rate of degradation of c-myc mRNA was much smaller in PG-treated cells than in nontreated cells . We conclude, therefore, that PGA and PGJ inhibit a crucial event(s) in the cell cycle occurring at the G1/S border, but that this inhibition is not accompanied by the reduction in c-myc gene expression in contrast with some types of tumor cells treated with PGs.

Calcif Tissue Int, 1992 Jun, 50(6), 533 - 40
A phorbol ester induces secretion of alkaline phosphatase activity in human osteosarcoma cells; Ringbom-Anderson T et al.; The phorbol ester 12-O-tetradecanoyl-13-acetate (TPA) blocked the growth of, and induced the appearance of processes in the human osteosarcoma cell line U-2 OS . The phorbol ester decreased the intracellular level of alkaline phosphatase (APase) activity (as measured per mg cell protein) and caused a marked increase in the APase activity secreted from the cells into the culture medium . The secretion of APase appeared after a lag period of 4-6 hours of TPA treatment, and it could also be visualized with histological staining . Differential ultracentrifugation of the culture media showed that the APase was released to the media in the form of vesicles . The vesicles were studied by electron microscopy and appeared similar to matrix vesicles isolated from cartilage and chondrocytes . It is thus concluded that TPA is able to induce the primary steps of mineralization in these cells.

Nippon Sanka Fujinka Gakkai Zasshi, 1992 Jun, 44(6), 710 - 6
{Production of CA125 in cell lines derived from human ovarian carcinoma: in relation to the cell cycle}; Suzuki M et al.; The association of the production of CA125 with the cell cycle was investigated in two cell lines derived from human ovarian cancer, one from a serous cystadenocarcinoma (HTOA) and the other from a mucinous cystadenocarcinoma (RMUG-s) . HTOA and RMUG-s cells secreted CA125 at about 50 and 30U/ml/10(5) cell/24hr, respectively, in the logarithmic growth phase and at about 75 and 100U/ml/10(5) cell/24hr in the steady phase . Analysis by FCM revealed that cultures of both cell lines cultured for 7 days contained more cells in the G0/G1 phase and less cells in the S phase than those cultured for 3 days . The positive rate of immunologically stained DNA polymerase alpha was 31% in HTOA cells and 39% in RMUG-s cells after cultivation of the cells for 3 days . The addition of EGF at 0.01, 0.1 or 1.0nM did not affect the production of CA125 in HTOA or RMUG-s cells while the addition of NaBT at 1, 3 and 5mM raised production in both cell lines as the dose rose . With RMUG-s cells, the addition of EGF at 0.01nM to the culture media accelerated both logarithmic and steady phase growth without a significant change in the production of CA125 . In contrast, the addition of NaBT at 1mM suppressed growth, but tended to increase the production of CA125 per cell . With the effect of EGF on the cell cycle of both cell lines, cells in the S phase increased by about 20% as compared with the control, 48 hours after its addition at 0.01nM . In contrast, after cultivation for 48 hours in the presence of 1mM NaBT, cells in the S phase were decreased while those in the G0/G1 phase increased . The results presented above suggested the possibility that some factors other than the cell cycle were involved in the production of CA125 . There also is close correlation between cells in the G0/G1 phase and the production of CA125 in the culture of human ovarian cancer cells.

Indian J Malariol, 1992 Jun, 29(2), 73 - 82
Effect of tissue culture media on multiplication of Plasmodium falciparum in vitro; Sutar NK et al.; To date RPMI-1640 has been the best medium for cultivation of Plasmodium falciparum in vitro . In addition to this medium, several alternative media, essentially the ones used in animal and plant tissue culture, were employed for the cultivation of P . falciparum . Only the media rich in glucose content, viz . Nitsch medium and White's medium S-3, supported the parasite multiplication.

J Dermatol, 1992 Jun, 19(6), 325 - 34
Optimization of murine keratinocyte culture for the production of graftable epidermal sheets; Rouabhia M et al.; The aim of the present study was to optimize murine epidermal cell cultures in order to obtain graftable sheets . Newborn (1-3 days old) Balb/c mice skin were used to optimize culture media and plating cell concentration, then epidermal sheet production, and grafting . Epidermal cells were plated at various concentrations in different culture media containing low (0.1 mM) or high (greater than 1 mM) Ca2+ levels . After a 3 day culture at the 10(4) cells/cm2 plating cell concentration, the percentage of differentiated cells was more than 80% in the high Ca2+ culture medium and less than 50% with bulky cells in the low Ca2+ culture medium . Under these conditions confluence was not obtained . At the 10(5) cells/cm2 seeding inoculum, the percentage of confluence increased to 95-100% during the first 72 h of culture in both high and low Ca2+ culture media . Three-day-old culture showed stratified multilayer epidermal sheets in the high calcium medium, and monolayer epidermal sheets were present in the low calcium medium after seeding keratinocytes in fibronectin precoated flasks . Seven days after plating, post confluent cultures were composed of a high percentage of differentiated cells (90%) with an increase in shedding cells in the medium . Considering the above morphological observations, sheets obtained with 10(5) cells/cm2 in MCDB-153 (A), DME-HAM (B) or GMEM (C) media after 3 days in culture were grafted . Twenty days after grafting, histological analysis of biopsies showed an epidermal structure and organization comparable to normal murine epidermis without hair follicles . Epidermal transplants showed a complete basement membrane, hemidesmosomes, and tonofilament bundles . Sheets obtained after seven day culture in all media showed lower coverage of the wound bed . These studies point out the importance of the plating cell and Ca2+ concentrations, and the culture time for murine keratinocyte confluence and differentiation to obtain graftable epidermal sheets.

Lymphokine Cytokine Res, 1992 Jun, 11(3), 167 - 73
Use of a modified Transwell culture to quantitate the contribution of non-IL-7 substances to growth of an IL-7-dependent pre-B cell clone; Muirhead M et al.; Clone 3 is a mouse pre-B cell line that cannot grow in standard tissue culture media but is immortalized by coculture with a bone marrow-derived feeder layer . In addition, clone 3 cells can be passaged indefinitely in recombinant interleukin-7 (IL-7) in the absence of a feeder layer if maintained at high cell density . Using monoclonal antibody to IL-7 and Transwells ligated to dialysis membranes, we have examined the relative contributions of IL-7, both endogenous and exogenous, and of "non-IL-7" feeder layer factors to growth promotion of clone 3 cells . There is synergy between feeder layers and exogenous IL-7 that is most marked when the latter is present in suboptimal concentrations . The synergizing activity is not neutralized by antibody to IL-7 and appears to be freely dialyzable . This "non-IL-7" effect is common to two different feeder layers, the one derived from bone marrow (3E) being an IL-7 producer, and the other, 3T3 fibroblasts, making no detectable IL-7 . These experiments reveal a substantial contribution of the dialyzable moiety to the total feeder layer effect, and are the first to demonstrate cytokine-dependent low-molecular-weight synergy in a coculture . This demonstration is possible because the synergizing cofactor(s) can cross a semipermeable membrane whereas the cytokine and its neutralizing antibody cannot.

Jpn J Cancer Res, 1992 Jun, 83(6), 631 - 7
Enhancing effect of pokeweed mitogen on the proliferation and the cytotoxicity of lymphokine-activated killer cells; Kimoto Y et al.; In order to obtain more potent lymphokine-activated killer (LAK) cells for use in adoptive immunotherapy, pokeweed mitogen (PWM) was added to the culture medium for the initial 24-48 h of culturing . The proliferation rate of PWM-stimulated LAK cells reached about 1000-fold after 3-week culture . This rate was nearly the same as that of LAK cells stimulated by 10 ng/ml of OKT3, the mouse anti-CD3 monoclonal antibody . However, the cytotoxicity of PWM-stimulated LAK cells was significantly more potent than that of OKT3-stimulated LAK cells . Phenotypic analysis revealed that PWM-stimulated LAK cells were CD3+CD56(+)-dominant while OKT3-stimulated LAK cells were CD3+CD56(-)-dominant . About half of CD3+CD56+ PWM-stimulated LAK cells was CD8+ . These results suggest that more efficient adoptive immunotherapy is possible by using high-dose PWM-stimulated LAK cells with more potent cytotoxicity . Interleukin-1 beta and tumor necrosis factor alpha were significantly increased in the culture media after 24-h incubation with 1 micrograms/ml of PWM . Secretion of interferon-gamma was not enhanced by this concentration of PWM within 24 h . Therefore, PWM is considered to activate monocytes or macrophages to produce these cytokines in advance, influencing the proliferation and the cytotoxicity of LAK cells.

FASEB J, 1992 Jun, 6(9), 2726 - 34
Long-term culture of normal human colonic epithelial cells in vitro; Baten A et al.; Studies of normal cellular function as well as the understanding of cellular mechanisms of carcinogenesis and other diseases of the large intestine have been limited, particularly due to the lack of long-term culture of normal human large intestinal epithelial cells (NHLIEC) . Using the epithelia from surgically resected human colon, we have dissociated a sufficient number of viable NHLIEC and maintained them in in vitro culture for up to 5 months . Normal-appearing human large intestinal mucosal fragments (1 mm2) were treated with 0.01 mg/ml trypsin, 0.2 mg/ml collagenase + 0.1 mM EGTA or 0.1 mg/ml trypsin + 0.1 mM EGTA in a Stomacher laboratory blender to isolate the cells . Compared with other methods, the use of the Stomacher blender combined with low concentrations of proteolytic enzymes yielded greater numbers of cells per gram of tissue, with up to 84% viable cells . Primary and serially passaged NHLIEC were cultured in CMRL-1066, MEM with 5% serum, and serum-free KGM . These media were all supplemented with insulin, hydrocortisone, epithelial growth factor, and bovine pituitary extract . CMRL-1066 was found to be the best medium for NHLIEC . Contaminating fibroblasts were selectively removed by briefly allowing the cells to adhere to the culture vessel and adding 25 U/ml collagenase to the culture media at the first subculture treatment . The epithelial nature and secretory function of the established cells were confirmed by morphological criteria (light microscopy, phase contrast microscopy and electron microscopy), immunoreactivity to cytokeratin, and positive mucin cytochemistry . We propose that using this methodology for the culture and maintenance of NHLIEC for an extended period of time would serve as a valuable model for a variety of investigations.

Neurochem Int, 1992 Jun, 20(4), 493 - 9
Interaction of sialosyl cholesterol with the cell surface of rat astrocytes and its biological activities; Ito J et al.; The interaction between sialosyl cholesterol (alpha- or beta-D-N-acetyl neuraminyl cholesterol, alpha- or beta-SC) and the plasma membrane of astrocytes was investigated by the use of 14C-labeled alpha- or beta-SC . Both alpha- and beta-SC were dose-dependently and time-dependently bound to rat astrocytes . The Scatchard plot analyses showed that rat astrocytes bound apparently 9.69 x 10(9) molecules of both alpha-SC/cell (apparent Kd = 2.29 x 10(-5) M) and beta-SC/cell (apparent Kd = 5.39 x 10(-5) M) at 37 degrees C . Both the binding of alpha-SC to astrocytes and the subsequent inhibition of DNA synthesis were decreased at the low temperature (4 degrees C), and also suppressed by serum proteins including albumin . One molecule of bovine serum albumin (BSA) bound 2.3 molecules of alpha-SC with the slightly lower Kd-value (8.03 x 10(-6) M) than that for the binding site on astrocytes . BSA not only suppressed the alpha-SC-binding to astrocytes but also increased its release from the cells to the culture media . Gangliosides such as GM1 and GM3 unaffected the alpha-SC-binding, promoted the small release of alpha-SC from the cell surface, and inhibited the morphological changes of astrocytes induced by alpha-SC . The mechanism of alpha-SC-binding to cultured astrocytes with reference to the effects of serum or gangliosides is discussed.

Orv Hetil, 1992 May 31, 133(22), 1359 - 62
{The effect of low density lipoprotein (LDL) on NO formation and on arginase activity in mouse peritoneal macrophages}; Romics L et al.; A large body of the evidence is available to the causative relationship between the elevated blood plasma concentrations of LDL and the atherogenesis . The oxid-LDL (modified LDL) is internalized more rapidly by the macrophages, and there is now substantial evidence that the modified LDL is actually present in atherosclerotic lesions . Recently it has been proved that the endothel cells and monocyta/macrophages generate nitric oxide (NO) from arginine, and that the LDL inhibits the formation of NO in endothel cells . The authors found that the human LDL in vitro exerts an inhibitory effect on the formation of NO in murine PED (peritoneal exudate cells) and synchronously severalfold increasing of the arginase activity in the culture media . Both effects of LDL proved to be dose dependent and the oxid-LDL has been found to be more effective . The increased activity of arginase provides a very likely explanation for the reducing of NO production in macrophage treated by LDL . The reducing or blocking of NO-formation causes a local vasocontraction which induces clinical symptoms.

Biochem Biophys Res Commun, 1992 May 15, 184(3), 1250 - 5
Purification and properties of an iminopeptidase from culture media of Streptomyces plicatus; Ehrenfreund P et al.; The degradation of the prosequence of the secreted enzyme endo-beta-N-acetylglucosaminidase H from Streptomyces plicatus is not elucidated . Both the primary structure of this segment and the finding that the secreted species contain ragged aminoterminal ends of specific structure suggested that a dipeptidylaminopeptidase might mature this enzyme . Therefore, we tested the culture medium of Streptomyces plicatus for prolin-specific peptidases . Proline iminopeptidase was purified about 800-fold to homogeneity from the culture medium . Dipeptidylaminopeptidase, the enzyme that seemed most likely to process the prosequence of endo-beta-N-acetylglucosaminidase H, could not be detected.

Int J Cancer, 1992 May 8, 51(2), 244 - 9
In vivo anti-tumor activity of arginine deiminase purified from Mycoplasma arginini; Takaku H et al.; Arginine deiminase (EC 3.5.3.6) was purified to homogeneity from the cell extract of Mycoplasma arginini by molecular-sieve, anion-exchange and arginine-affinity chromatographies . The purified enzyme was composed of 2 identical sub-units with a molecular weight of 45.000 and had a pI of 4.7 . Its Vmax value and Km value for L-arginine were estimated to be 50 units/mg protein and 0.2 mM, respectively . It exerted maximal enzyme activity at pH 6.0-7.5 and at 50 degrees C . The arginine deiminase was stable at neutral pH . When injected i.v . into mice, the half-life of the arginine deiminase in blood was about 4 hr . In culture, the enzyme strongly inhibited the growth of 6 kinds of mouse tumor cell lines by depleting L-arginine in the culture media . When the in vivo growth-inhibitory activity of arginine deiminase was tested for the 6 tumor cell lines, i.p . administration of the purified enzyme effectively prolonged the survival time of the mice injected with all kinds of the tumor cell lines . Especially, the in vivo growth of a hepatoma cell line, MH134, was completely prevented by the daily administration at a dose of 0.2 mg/mouse for 14 days . These results raise the possibility of the use of the arginine deiminase derived from Mycoplasma arginini as a new anti-tumor drug.

Ann N Y Acad Sci, 1992 May 4, 651, 422 - 32
Human immunodeficiency virus activates c-myc and Epstein-Barr virus in human B lymphocytes; Astrin SM et al.; We have established a line of malignantly transformed human B cells by infecting purified primary B lymphocytes with human immunodeficiency virus type 1 (HIV-1) . This line, termed B-HIV1, may serve as a model system for a subset of AIDS-related B-cell lymphomas in which the transformed phenotype may be initiated and/or maintained through an HIV-1 gene product . The B-HIV1 line contains both Epstein-Barr virus (EBV) and HIV-1 genomes . In addition, the c-myc gene is expressed at levels 10 to 20 times those in normal B cells . Similarly, EBV sequences, including those for the latent membrane protein (LMP), are expressed at greatly enhanced levels relative to expression in normal, EBV-immortalized B cells . The upregulation of c-myc and of EBV gene expression can both be produced by infection of susceptible B cells (not already harboring HIV) with exogenous HIV-1 . The B-HIV1 line exhibits properties of malignantly transformed cells, in that it grows logarithmically in 1% serum, clones in soft agar, and produces invasive, malignant B-cell lymphomas in severe combined immunodeficient (SCID) mice . We have shown that HIV-1 has the ability to infect primary human B cells and to activate expression of EBV and c-myc . HIV activation of EBV has been documented previously in certain cell lines, here we note that such activation can occur in primary B cells and, under certain conditions, can result in outgrowth of immortalized cell lines . This phenomenon may contribute to the clinical manifestation of lymphadenopathy early after infection with HIV . In addition, we have demonstrated that HIV infection of primary B cells in vitro can result in appearance of a fully malignant phenotype . This phenotype is likely to be due, at least in part, to the activation of c-myc by HIV . Preliminary experiments indicate that Tat, the gene product of the transactivator of viral gene transcription tat, can upregulate c-myc transcription after addition to the culture media of certain B-cell lines . This raises the possibility that Tat can bind to target sequences in cellular RNA and enhance transcription as it does for HIV.(ABSTRACT TRUNCATED AT 400 WORDS)

Can J Microbiol, 1992 May, 38(5), 365 - 9
Solid-phase polymerase chain reaction: applications for direct detection of enteric pathogens in waters; Toranzos GA et al.; The techniques in current use for detection of pathogens in environmental samples are restricted to those organisms whose replication in either culture media or cell culture is feasible . These methods lack the selectivity and sensitivity necessary for their unequivocal detection and identification . We have developed an assay for the detection of bacterial cells in large volumes of water . Low concentrations of cells containing target sequences were concentrated on membrane filters and were subjected to amplification directly using a stepwise polymerase chain reaction . This procedure, together with nucleic acid probes, has enhanced the limit of detection to the level of a single bacterial cell . This technique could be used for the detection of any bacteria or virus in water or air.

J Environ Pathol Toxicol Oncol, 1992 May-Jun, 11(3), 177 - 89
Caffeine, theophylline, theobromine, and developmental growth of the mouse mammary gland; VanderPloeg LC et al.; The purpose of this study was to determine the comparative activities of three methylxanthines, i.e., 1,3,7-trimethylxanthine (caffeine), 1,3-dimethylxanthine (theophylline), and 3,7-dimethylxanthine (theobromine) on developmental growth of the mammary gland in ovarian-hormone treated, mature nulliparous female Balb/c mice . When caffeine or theophylline was administered daily (via drinking water, 500 mg/L) for 30 days to 17 beta-estradiol/progesterone-treated intact or ovariectomized mice, a significant (p less than 0.05) enhancement of hormone-induced mammary gland lobulo-alveolar differentiation was observed . Caffeine or theophylline thus accelerated and/or intensified mammae lobulo-alveolar differentiation induced by the ovarian steroids . In contrast, theobromine (500 mg/L drinking water) did not significantly modify this developmental process . The stimulatory effect of caffeine and theophylline on mammae development was comparable quantitatively . In an effort to determine whether or not the stimulatory effect of caffeine or theophylline was directly on the mammary gland, small slow-release Elvax-40P pellets containing these methylxanthines were implanted directly into the mammary gland of mice concurrently treated with estrogen and progesterone . No significant stimulatory effect of caffeine or theophylline (or theobromine) was observed . Furthermore, the addition of methylxanthines (caffeine, 100 microM) to the culture media of whole mouse mammary glands (organ cultures) did not enhance lobulo-alveolar differentiation induced by mammotrophic hormones . Thus, while a consistent significant stimulatory effect of caffeine and theophylline on mammary lobulo/alveolar differentiation was observed when the methylxanthines were consumed orally (drinking water), no direct effect of these methylxanthines, when placed directly into the mammary gland or in culture media, on mammae development was observed . These data demonstrate that certain methylxanthines (e.g., caffeine and theophylline) but not others (e.g., theobromine) can significantly enhance mammotrophic hormone-induced mammary lobulo-alveolar differentiation in female Balb/c mice, an effect that appears not to be manifested via a direct action of the methylxanthines on the mammary gland.

No Shinkei Geka, 1992 May, 20(5), 547 - 51
{Basic research for interferon gene therapy against malignant glioma}; Mizuno M et al.; In order to establish new interferon therapy against malignant glioma by selective transfection of its gene, we developed a novel transfection system using liposomes bearing positive charges on their surface and entrapping plasmids containing the HuIFN- beta gene (pSV2IFN-beta) . The liposomes were composed of N-(alpha -trimethylammonioacetyl) -didodecyl-D-glutamate chloride (TMAG), dilauroylphosphatidylcholine (DLPC), and dioleoylphosphatidylethanolamine (DOPE) as a molar ratio of 1:2:3 . The liposomes were not observed to have cytotoxicity for human glioma cells, when applied at a rate less than 15nmol/ml . This liposome-mediated transfection of the gene into the cultured glioma cells (U-251-MG) resulted in the secretion of HuIFN- beta into the medium . The HuIFN- beta level in the culture media of glioma cells reached 23 IU/ml after 96h of incubation . When the pSV2IFN-beta containing liposomes were coupled with a monoclonal antibody (G-22 MCA) against glioma-associated antigen (G-22), the level of HuIFN-beta in the medium was 181 IU/ml, resulting in a 7-fold increase . It was indicated that this transfection system was a safe and selective method in the case of TMAG/DLPC/DOPE liposomes.

In Vitro Cell Dev Biol, 1992 May, 28A(5), 337 - 47
Ultrastructural and autoradiographic study of preimplantation rabbit embryos grown in conventional or uterine flushing-supplemented culture media; Fischer B et al.; Rabbit morulae and blastocysts were cultured in conventional culture media {Ham's F10 or BSM II supplemented with bovine serum albumin (BSA) or serum} or in Ham's medium supplemented with synchronous or asynchronous uterine flushings, mostly for 2 days, and afterwards investigated by light and electron microscopy and by autoradiography . Ultrastructure and cell proliferation differed considerably between cultured embryos and noncultured controls . Cultured embryos displayed more dead cells . They were developmentally retarded (predominance of smooth endoplasmic reticulum rather than the age-specific rough endoplasmic reticulum, mitochondria still round to ovoid shaped) and showed nonspecific signs of cells damage (swollen mitochondria and Golgi complex vesicles, increased number of lysosomes) . All these features were also present in embryos grown in uterine flushing-supplemented media, but were less pronounced . Cell damage and impaired cell proliferation had affected trophoblast cells more than embryoblast cells . Endoderm could be differentiated only if culture had been started with blastocysts--not with morulae--and seems to require uterine secretions . No significant ultrastructural differences were observed between embryos cultured in synchronous or in asynchronous uterine flushings . Present results indicate that cultured preimplantation rabbit embryos deviate clearly from those grown in vivo and maintain, for some time, a better cellular structure--and probably function--in the presence of uterine flushings than in conventional culture media . Specific abnormal morphologic features related to a particular medium could not be identified.

Biol Reprod, 1992 May, 46(5), 829 - 45
In vitro prostaglandin release from and platelet-activating factor accumulation in isolated endometrial cells from pregnant and pseudopregnant rabbits; Kasamo M et al.; Prostaglandin (PG) release from and platelet-activating factor (PAF) accumulation by enzymatically isolated endometrial epithelial and stromal cells from Day 6 pregnant and Day 6 pseudopregnant rabbits were studied in vitro, using RIA for PG measurement and a platelet aggregation assay for PAF measurement . On the first day of culture in serum-free media, PGF release into the medium was significantly higher from epithelial cells from Day 6 of pregnancy than from stromal cells from Day 6 of pregnancy or pseudopregnancy . PGE release did not differ significantly among these cell types . The addition of indomethacin (10(-5) M) to similar cultures inhibited release of both PGs from both cell types, but to a much greater extent from stromal than from epithelial cells . Significant stimulation of PG release by A23187 was achieved under all conditions on the fifth day of culture; PGE release was significantly greater than PGF release from stromal cells from Day 6 of pregnancy and pseudopregnancy, and release of both PGs from stromal cells was significantly greater from Day 6 of pregnancy than from Day 6 of pseudopregnancy . PG release from similar cells, cultured in medium containing 10% calf serum, was highest on the first or second day of culture and then, especially for PGF, declined with continued culture . PGE release was significantly higher than PGF release from stromal cells on the third and fourth days of culture . The ratios of PGF/PGE release from epithelial cells were significantly higher than those from stromal cells over the 5-day culture period for both reproductive stages . These ratios indicate the differential release of PGE and PGF from rabbit endometrial cell subpopulations and indicate a preferential release of PGE from stromal and of PGF from epithelial cells . Under basal conditions, PAF was not detected in epithelial or stromal cells cultured for 2 or 4 days, or in the associated culture media . If PAF had been released into the medium, it would have rapidly metabolized . Short exposure to calcium ionophore A23187 (10(-5) M) was able to stimulate PAF accumulation in epithelial and stroma cells in serum-free media, probably via the remodeling pathway . PAF was not detected in the medium . Intracellular PAF accumulation after exposure to A23187 (10(-5) M) for 5 min was significantly greater on the second day of culture than on the fourth day in epithelial and stromal cells from Day 6 of pregnancy.(ABSTRACT TRUNCATED AT 400 WORDS)

Biol Reprod, 1992 May, 46(5), 793 - 801
Isolation of human Leydig cell mesenchymal precursors from patients with the androgen insensitivity syndrome: testosterone production and response to human chorionic gonadotropin stimulation in culture; Chemes H et al.; Mature Leydig cells, the main source of testicular testosterone in mammals, arise from immature mesenchymal precursors through an LH-dependent differentiation process . In order to study the steroidogenic potential of these precursors, undifferentiated mesenchymal cells were obtained from the testicular interstitium of two patients with androgen insensitivity syndrome . After double digestion with collagenase and separation of the suspensions in a Percoll density gradient, the cells were cultured in Ham's F12 medium: Dulbecco's Modified Eagle Medium (1:1) supplemented with antibiotics, transferrin, insulin, hydrocortisone, and vitamin E with or without 1 IU of hCG/ml . At 11 days in culture, samples were removed for morphological characterization and determination of 3 beta-hydroxysteroid dehydrogenase activity (3 beta-HSD) . Testosterone concentration was determined by RIA in the culture medium at different intervals . Cultured cells were mesenchymal in appearance, elongated in shape, with numerous processes running in different directions . No mature Leydig cells were present . In basal conditions, the percentages of 3 beta-HSD-positive cells at 11 days on patients 1 and 2 were 33% and 28%, respectively, and the testosterone concentrations in the culture media were 4.8 and 8.4 ng.10(6) cells.24 h, respectively . In cultures stimulated with hCG, there was an increase of histochemical reactivity (47% and 42% in patients 1 and 2, respectively) and in the amount of testosterone secreted (10.2 and 12.0 ng.10(6) cells, respectively) . Electron microscopic studies of cultures grown in the absence of hCG demonstrated a homogenous population of poorly differentiated, fibroblastic-type mesenchymal cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Arch Pathol Lab Med, 1992 May, 116(5), 537 - 9
Disseminated Nocardia asteroides diagnosed by blood culture in a patient with disseminated histoplasmosis; Vannier AM et al.; We describe herein a provocative case involving an immunosuppressed patient with disseminated Histoplasma capsulatum and also disseminated Nocardia asteroides, which was documented by multiple routine blood culture samples . Nocardia asteroides was isolated from 15 routine blood cultures using the nonradiometric blood culture system (Bactec NR-660, Becton Dickinson, Towson, Md) . Several different species of the genus Nocardia proved to thrive in blood culture bottles (Bactec) when experimental inoculations were performed . The paucity of reports in the literature of disseminated nocardiosis with positive blood cultures has led us to consider cause for the apparent poor recovery of these organisms from blood culture media . We suggest that the media (Bactec) can successfully support growth of Nocardia species and that other variables, such as incubation times and subculture, should be considered to optimize isolation of this pathogen in blood culture systems.

Dev Biol, 1992 May, 151(1), 48 - 54
Recombinant mouse ZP3 inhibits sperm binding and induces the acrosome reaction; Beebe SJ et al.; Mammalian fertilization involves interactions of sperm surface receptors with ligands of the zona pellucida, an extracellular matrix surrounding the ovulated egg . In mouse, the zona is composed of three glycoproteins . One of them, ZP3, participates in primary sperm binding and in the subsequent triggering of the sperm's acrosome reaction . Considerable evidence suggests that carbohydrate determinants of ZP3 are responsible for binding to sperm and may be important for acrosomal exocytosis . A full-length cDNA encoding mouse ZP3 was assembled and cloned into expression vectors that contained either a cytomegalovirus (CMV) or a vaccinia (P11) promoter . Mouse L-929 cells were stably transformed with the pZP3-CMV constructs, and green monkey CV-1 cells were infected with a recombinant vaccinia virus containing ZP3 . rZP3 was affinity purified from culture media and detected on Western blots as a single 60- to 70-kDa band, which differed in molecular weight from native ZP3 (mean, 83 kDa) . Nevertheless, rZP3 is biologically active . rZP3 decreases sperm-zona binding with a potency equivalent to that of native zona pellucida and, like native ZP3, rZP3 triggers acrosomal exocytosis in capacitated mouse sperm . Thus, rZP3 isolated from both rodent and primate cells appears to contain those carbohydrate and protein structures necessary for ZP3's dual role in fertilization.

J Invest Dermatol, 1992 May, 98(5), 753 - 7
Organ culture of psoriatic lesions: appearance of granular layers in vitamin A-free culture media; Kondo S et al.; Some morphologic changes of the epidermis of psoriatic skin were observed in organ culture in the presence of absence of vitamin A . Normal and uninvolved and involved psoriatic skin areas, punch biopsied in 3-4-mm diameter specimens, were put in serum-free medium containing no vitamin A with or without delipidized fetal calf serum (FCS) and rotation cultured at 60 rpm under an atmosphere consisting of 95% O2 + 5% CO2 . The involved psoriatic skin specimens showed well-developed granular layers after 1 d of culture . The values of labeling indices of 3H-thymidine (3H-TdR) incorporated into the epidermal layers of the cultured specimens of the psoriatic skin were nearly constant during culture for as long as 8 d, although the viable epidermal layer gradually became thinner . Addition of tretinoin to the culture caused uninvolved and involved psoriatic skin specimens to become parakeratotic at concentrations as low as about 2.0 x 10(-6) M and 4.0 x 10(-8) M, respectively, suggesting that the involved psoriatic epidermis is much more sensitive, in terms of keratinization, than uninvolved epidermis to the effect of tretinoin.

Exp Hematol, 1992 May, 20(4), 395 - 400
Acquisition of growth autonomy and tumorigenicity by an interleukin 6-dependent human myeloma cell line transfected with interleukin 6 cDNA; Okuno Y et al.; In human multiple myeloma, an autocrine growth mechanism through interleukin 6 (IL-6) has been advocated . However, growth of myeloma cells in vitro is poor except for established cell lines, and IL-6 autocrine growth is quite rare in myeloma cell lines . In the present study, we devised a model of IL-6 autocrine growth in vitro by transfecting IL-6 cDNA into a human myeloma cell line that had a proliferative response to IL-6 but did not produce IL-6 . After IL-6 transfection, the cells proliferated in culture media without IL-6, and their growth rate was elevated at higher cell densities . IL-6 was detected by enzyme-linked immunosorbent assay in the culture media of the transfectants . IL-6 mRNA was distinctly expressed in these cells when analyzed by Northern blotting . The growth of the transfectants was definitely inhibited by anti-IL-6 or anti-IL-6 receptor monoclonal antibodies . Furthermore, the transfectants were successfully transplanted to nude mice . These results indicate that the myeloma cells obtained growth autonomy in vitro through IL-6 and tumorigenicity in vivo, after IL-6 transfection.

J Pharmacobiodyn, 1992 May, 15(5), 239 - 46
Effect of benzylidene derivative (novel antirheumatic agent) on chondrocyte metabolism; Watanabe K et al.; The effects of protocatechualdehyde (PAL), one of the metabolites of 3,4-diacetoxy benzylidene diacetate (ACP), on proteoglycan metabolism and secretion of interleukin 1 (IL-1) like activity (lymphocyte activating factor; LAF activity) were studied using rabbit articular chondrocytes culture under phorbol myristate acetate (PMA) and Ca2+ ionophore A23187 (A23187) or IL-1 alpha stimulation . In IL-1 alpha (20 u/ml) or PMA (0.1 micrograms/ml) and A23187 (0.2 micrograms/ml) treated culture of rabbit articular chondrocytes, PAL significantly reduced the degradation of 35S-proteoglycan (35S-PG) from the cells and matrix layers into the culture media in a dose dependent fashion without affecting proteoglycan synthesis . Similarly, the secretion or production of matrix metalloproteinases which degrade proteoglycans was also inhibited to the same extent under IL-1 alpha stimulated condition . However, PAL caused no effect on the secretion of IL-1 like activity by chondrocytes . These results suggest that an attractive candidate for an anti-inflammatory drug, ACP, which is a prodrug of PAL, has also a favorable action on chondrocyte metabolism in terms of proteoglycan degradation via inhibition of matrix metalloproteinases secretion or production.

Nippon Seikeigeka Gakkai Zasshi, 1992 May, 66(5), 476 - 84
Antitumor effect of thermosensitive CDDP-liposomes on human osteosarcoma cells in culture; Hattori M et al.; We have administered cis-diamminedichloroplatinum (II) (CDDP) containing thermosensitive liposome and simultaneous hyperthermia into human osteosarcoma cells (OST) and studied the antitumor effects . Experiments were conducted under the following three different culture conditions as follows, (1) drug treatment alone, (2) hyperthermia after drug treatment, and (3) simultaneous hyperthermia and drug treatment . Antitumor effects were estimated by inhibition of DNA synthesis and decrease in cell growth rates . The antitumor action of simultaneous hyperthermia and drug treatments was the most dominant and synergistic among three experimental conditions . Accumulation of cellular platinum from thermosensitive CDDP-liposomes was markedly enhanced by hyperthermia . Flow cytometric analysis of the cell cycle indicated that CDDP-liposomes only under the simultaneous application of hyperthermia released free CDDP, and evoked cell accumulation in S and G 2/M phase . The results suggested that thermosensitive CDDP-liposomes were not taken up into cells by endocytosis, and released free CDDP into culture media under hyperthermia at 42 degrees C . From these, we have concluded that administration of thermosensitive CDDP-liposomes and simultaneous local hyperthermia could be a promising method for the treatment of osteosarcoma especially in the extremities.

J Androl, 1992 May-Jun, 13(3), 208 - 13
Basal and FSH-stimulated steady state levels of SGP-2, alpha 2-macroglobulin, and testibumin in culture media of rat seminiferous tubules at defined stages of the epithelial cycle; Kangasniemi M et al.; Production of several proteins by rat Sertoli cells is dependent on the stage of the cycle of the seminiferous epithelium . The authors have determined steady state levels and follicle-stimulating hormone responsiveness of three Sertoli cell products in culture media of rat seminiferous tubule segments at different stages of the epithelial cycle: SGP-2 (sulfated glycoprotein-2), alpha 2-macroglobulin, and testibumin . Basal SGP-2 levels were twofold higher in stages VII through VIII compared with stages XIII to I to VI (P less than 0.05) . Highest basal alpha 2-macroglobulin levels were found in stages II through VIII; this was about 35% greater than in stages XIII through I of the cycle (P less than 0.05) . Basal testibumin levels were twofold higher in stages II through VI compared with stages IX through XII of the cycle . Follicle-stimulating hormone had no effect on SGP-2, but by contrast it (50 mg/L) increased the level of alpha 2-macroglobulin significantly (P less than 0.05) in stages XIII through I . Follicle-stimulating hormone treatment (10 mg/L) elevated testibumin levels at each stage-pool by about 40% (P less than 0.05) . The current results using staged tubular segments in vitro demonstrate cyclic basal steady-state levels of the three proteins along the seminiferous tubules and follicle-stimulating hormone regulation of alpha 2-macroglobulin and testibumin.

J Toxicol Environ Health, 1992 May, 36(1), 27 - 41
Teratogenic and macromolecular synthesis inhibitory effects of trimethylamine on mouse embryos in culture; Guest I et al.; Trimethylamine (TMA) is an aliphatic amine, and its blood levels can increase after ingestion of certain foods, such as fish, and during disease states, such as chronic renal failure . We recently reported that TMA can inhibit fetal development in vivo and in vitro in mice . The present studies were done to find out if the inhibitory effects of TMA on embryonic development are caused by a decrease in macromolecular synthesis, using mouse embryo cultures as the experimental model . At a submaximally toxic concentration (0.75mM), TMA inhibited the growth of embryos to approximately 70% of control and caused neural-tube defects in 73% of embryos . By 42 h of culture, DNA, RNA, and protein content of TMA-treated embryos were approximately 50% of the control values . Embryotoxic effects of TMA were not caused by changes in pH and osmolarity of the culture media . The inhibitory effects of TMA on embryonic growth were time dependent and apparent at 2-4 h of culture . The inhibition of growth was accompanied by a decrease in the incorporation of tritium-labeled thymidine, uridine, and leucine into DNA, RNA, and proteins, respectively . Thiols (L- and D-cysteine, glutathione) and the antioxidant L-ascorbic acid did not cause significant antagonism of embryotoxic effects of TMA . It is concluded that TMA exerts teratogenic effects on mouse embryos in culture and inhibits their growth by reducing macromolecular synthesis; these effects may not involve glutathione depletion or generation of free radicals.

Endocrinology, 1992 May, 130(5), 2745 - 50
Cyclic adenosine 3',5'-monophosphate negatively regulates clusterin gene expression in Leydig tumor cell lines; Pignataro OP et al.; The clusterin protein and its messenger RNA were identified in many tissues including testis . In this report, we demonstrate the expression of clusterin gene in four Leydig tumor cell lines, including mouse MA-10 and I-10 and rat R2C and LC-540 . When the cells were incubated with 0.1 mM 8-bromo-cAMP or (Bu)2cAMP for 17 h, an unexpected, profound suppression of clusterin mRNA accumulation was observed . A 60-70% decrease in clusterin mRNA was observed in MA-10 and R2C cells, 10% in I-10 cells, and no apparent change in LC-540 cells . The inhibitory effect of cAMP was specific to the clusterin gene, since in the same cells cholesterol side-chain cleavage enzyme mRNA was drastically elevated in MA-10 and I-10 cells while alpha-tubulin mRNA levels were not changed in all four cell lines . The reduction could be detected as early as 4 h, and was evident at 17 h after cAMP administration . Removal of cAMP from culture media at 17 h prevented the decline of clusterin mRNA . The suppression of clusterin gene expression can also be demonstrated by treatment with human CG or forskolin, which were known to elevate intracellular cAMP levels . Our observations suggest: 1) cAMP negatively regulates clusterin gene expression in two Leydig tumor cell lines, MA-10 and R2C; 2) The inhibitory effect of cAMP on clusterin gene expression is probably acting through the protein kinase A pathway; and 3) The four Leydig tumor cell lines respond differently to cAMP in the expression of clusterin and side-chain cleavage genes.

Blood, 1992 May 1, 79(9), 2256 - 61
Hematopoietic growth factors released by marrow stromal cells from patients with aplastic anemia; Kojima S et al.; We studied the production of granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), and interleukin-6 (IL-6) by stromal cells from 33 patients with aplastic anemia (AA) . Complete, confluent stromal layers were produced by 29 of the 33 samples using the long-term bone marrow culture (LTBMC) system . The concentration of G-CSF, GM-CSF, and IL-6 in culture media with or without interleukin-1 (IL-1) stimulation was determined by an enzyme-linked immunoadsorbent assay (ELISA) . The spontaneous production of G-CSF, GM-CSF, and IL-6 did not differ significantly between normal controls and the patients with AA . The ability of stromal cells to release the three hematopoietic growth factors in response to IL-1 was either normal or elevated in all but one patient . We also studied the change in production of G-CSF, GM-CSF, and IL-6 by stromal cells before and after antilymphocyte globulin (ALG) therapy in 16 patients with AA . There was no correlation between the change in production of these cytokines and the response to ALG . In contrast to previous studies that showed a defect in the production of hematopoietic growth factors by stromal cells from patients with AA, the results indicated a normal or elevated production of G-CSF, GM-CSF, and IL-6 by marrow stromal cells in patients with AA.

Proc Soc Exp Biol Med, 1992 May, 200(1), 101 - 8
RNA/nucleotide enhances antibody production in vitro and is moderately mitogenic to murine spleen lymphocytes; Jyonouchi H et al.; Suppression of immune functions was demonstrated in both humans and animals when exogenous RNA was eliminated from the diet . However, direct actions of RNA/nucleotide on the immune system are virtually unknown . Thus, in this study, we explored effects of RNA and nucleotide on lymphocyte functions in vitro . Yeast whole RNA, which is free of endotoxin, was supplemented to culture media, and changes in mitogen responses, thymocyte proliferation, or in vitro antibody production by murine spleen lymphocytes were analyzed . Yeast whole RNA potentiated the proliferation of spleen lymphocytes and it also strikingly enhanced in vitro antibody production in response to sheep red blood cells at least 10-fold . However, it did not potentiate the proliferation of thymocytes (immature lymphocytes) . These enhancing activities of yeast RNA were significantly reduced by RNAse treatment, but not by treatments with DNAse or polymyxin B . Certain mononucleotides exhibited less, but similar, action on murine spleen lymphocytes . The whole yeast RNA employed was already degraded to small nucleotide (less than 1 kb) . Therefore, it may be suggested that certain components of RNA degraders can function as powerful immunomodulators, indicating that exogenous RNA or nucleotide may be important in facilitating immune responses under certain circumstances.

Invest Ophthalmol Vis Sci, 1992 May, 33(6), 1923 - 7
Gelatinolytic metalloproteinase secretion patterns in ocular melanoma; Cottam DW et al.; Fifteen posterior uveal melanoma cell lines were analyzed qualitatively for gelatinolytic and caseinolytic proteinase activity after one to five in vitro passages . All 15 cell lines secreted a gelatinolytic metalloproteinase, with an apparent molecular weight of 72 kD, into protein-free culture media; nine of these secreted an additional gelatinolytic metalloproteinase with an apparent molecular weight of 92 kD . Neither species had the ability to degrade casein . This approach may provide insight into the mechanisms of tumor metastasis in uveal melanoma.

Endocrinology, 1992 May, 130(5), 2789 - 94
Regulation of thyroid hormone synthesis in cultured ovine thyroid follicles; Becks GP et al.; Primary cultures of sheep thyroid follicles were used to study the regulatory control mechanisms of thyroid hormone production . When maintained under serum-free conditions in vitro these follicles exhibit hormone-dependent iodide transport, iodine organification, and physiological de novo thyroid hormone formation . In previous studies we have also shown that thyroid follicles condition their culture media with insulin-like growth factors (IGFs) and IGF-binding proteins which are of potential autocrine or paracrine significance in thyroid hormonogenesis . TSH (100 microU/ml) alone modestly stimulated iodine uptake and organification, which was further potentiated by pharmacological or physiological concentrations of insulin and by physiological concentrations of IGF-I or IGF-II . A combination of TSH and cortisol (10 nM) optimally stimulated iodine uptake and organification without additive or synergistic effects among combinations of cortisol with insulin or IGFs . Insulin, IGF-I, IGF-II, or cortisol alone were without effect on iodine uptake and organification . The effect of TSH was mimicked by forskolin or (Bu)2cAMP, and the synergistic effect of cortisol with TSH was duplicated in incubations of dexamethasone with TSH . In long term studies of the same experimental conditions, with 10(-6) M NaI added to the culture medium, an increase in radioimmunoassayable T4 and T3 in conditioned cell culture media and cell layer extracts was confirmed for all conditions, with the exception of physiological concentrations of insulin . IGF-I and IGF-II were equipotent in their stimulation of thyroid hormonogenesis in the presence of TSH . The effect of high concentrations of insulin may be explained by a combined action through insulin and type I IGF receptors . We have previously reported that the stimulation of iodine uptake and organification (de novo thyroid hormone formation here) by TSH and cortisol is inversely correlated with their inhibition of IGF-binding proteins released by the cells while IGF release is unchanged . Overall, these data suggest that the regulation of thyroid hormonogenesis involves the endocrine hormones TSH and cortisol, acting in synergy with locally produced IGFs.

J Biol Chem, 1992 Apr 25, 267(12), 8012 - 20
Comparative study of the sugar chains of factor VIII purified from human plasma and from the culture media of recombinant baby hamster kidney cells; Hironaka T et al.; The asparagine-linked sugar chains of blood coagulation factor VIII preparations purified from human plasma of blood group A donors and from the culture media of recombinant BHK cells were released as oligosaccharides by hydrazinolysis . These sugar chains were converted to radioactive oligosaccharides by reduction with sodium borotritide and separated into neutral and acidic fractions by paper electrophoresis . Most of the acidic oligosaccharides were converted to neutral ones by sialidase digestion, indicating that they are sialyl derivatives . The neutral and sialidase-treated acidic oligosaccharides were fractionated by serial chromatography on immobilized lectin columns and Bio-Gel P-4 column . Structural study of each oligosaccharide by sequential exo- and endoglycosidase digestion and by methylation analysis revealed that both factor VIII preparations contain mainly high mannose-type and bi-, tri-, and tetra-antennary complex-type sugar chains . Some of the biantennary complex-type sugar chains from human plasma factor VIII contain blood group A and/or H determinant, while those from recombinant product do not . Some of the bi-, tri- and tetra-antennary complex-type sugar chains of the recombinant factor VIII contain the Gal alpha 1----3Gal group . A small number of the triantennary complex-type sugar chains from both preparations was found to contain the Gal beta 1----4(Fuc alpha 1----3)GlcNAc beta 1----4 (Gal beta 1----4GlcNAc beta 1----2)Man group . Studies of pharmacokinetic parameters of the recombinant factor VIII infused into baboons revealed that its half-life in blood circulation is similar to that of plasma derived factor VIII, suggesting that the oligosaccharide structural differences between them do not affect the fate of factor VIII in vivo.

J Chromatogr, 1992 Apr 15, 576(1), 71 - 7
Single-step method for purification of human transferrin from a by-product of chromatographic fractionation of plasma; Rivat C et al.; A rapid, simple and convenient method is described for the isolation, on a pilot scale, of pure and functional human transferrin from an unexploited by-product of chromatographic fractionation of plasma . In a single chromatographic step on DEAE-Spherodex, 97% pure transferrin was obtained in 75% yield . A virus inactivation treatment was included in the preparative process in order to guarantee the safety of the final product, which could be used in culture media.

Eur J Biochem, 1992 Apr 15, 205(2), 561 - 7
Comparison of human tenascin expression in normal, simian-virus-40-transformed and tumor-derived cell lines; Carnemolla B et al.; Tenascin is a polymorphic high-molecular-mass extracellular-matrix glycoprotein composed of six similar subunits . Using two-domain-specific anti-tenascin monoclonal antibodies, we have studied the expression and distribution of tenascin in four cultured normal human fibroblasts, two simian-virus-40-(SV40)-transformed and three tumor-derived (melanoma, rhabdomyosarcoma and fibrosarcoma) cell lines . We found that (a) cultured normal human fibroblasts accumulate considerable amounts of tenascin and retain 60-90% in the extracellular matrix, while they release the remainder into the tissue-culture medium; (b) of the two SV40-transformed counterparts we have tested, the AG-280 cell line accumulates no detectable amounts of tenascin and the WI-38-VA cell line accumulates about 10-times less tenascin than its normal counterpart and releases about 90% of it into the culture medium; (c) some tumor-derived cell lines accumulate considerable amounts of tenascin, but in these cases, more than 90% is released into the culture media; (d) in normal human fibroblasts, two major tenascin isoforms, generated by alternative splicing of the mRNA precursor, are detectable (280 kDa and 190 kDa, respectively) and the lower-molecular-mass tenascin isoform is accumulated preferentially in the extracellular matrix; (e) in SV40-transformed or tumor-derived cell lines, only the higher-molecular-mass isoform is detectable and it is more sialylated than the tenascin produced by the normal human fibroblast cell lines.

Cell Immunol, 1992 Apr 15, 141(1), 169 - 81
Involvement of major histocompatibility complex class II antigen in Epstein-Barr virus-mediated B cell proliferation; Chan KH et al.; Five MHC class II monoclonal antibodies costimulated proliferation of cord blood leukocytes with Epstein-Barr virus . These agonistic antibodies were of different isotypes, but all of them were either specific for or cross-reacting with HLA-DR . The other MHC class II antibodies, including three that were specific for HLA-DQ and one that was specific for HLA-DP and also those that were specific for MHC class I or leukocyte common antigen, were not costimulatory . The agonistic effect of different MHC class II antibodies was additive, such that costimulation by different antibodies combined significantly exceeded that achieved by either of these antibodies alone . Spent culture media of B cell lines also costimulated B cell proliferation with the virus . Although MHC class II antibodies augmented the effects of suboptimal concentration of the conditioned media, their combined effects did not exceed the maximum costimulation achieved by either the antibodies or the spent culture media alone . These results raised the possibility that MHC class II antigen may contain distinct functional domains involved in the regulation of B cell progression.

Int J Parasitol, 1992 Apr, 22(2), 195 - 202
Comparative studies on the growth dynamics of two genetically distinct isolates of Giardia duodenalis in vitro; Binz N et al.; The in vitro growth behaviour of the intestinal protozoan Giardia duodenalis was studied in detail and comparisons were made between two genetically and biologically distinct cloned isolates . Replicates of each clone were grown at six different initial cell concentrations and in culture media at four different pH values . Significant differences in in vitro growth were found between the two isolates, BAH12 and P1 . BAH12 had a specific narrow pH requirement, with satisfactory growth only obtained at pH 6 . The mean generation time of BAH12 at pH 6 between days 1 and 3 was 10.8 h, compared to an average of 6 h for the same period for P1, both at pH 6 and pH 7 . Comparative health of cultures was assessed during both the pH and growth experiments using a suite of six variables . Consistent changes in the health of cultures over time were found to reflect growth behaviour over time . These results provide the first detailed evidence that genetically different isolates of Giardia may differ in such fundamental biological parameters as growth rate and pH requirements . These differences may have important epidemiological and taxonomic implications.

Immunol Invest, 1992 Apr, 21(2), 103 - 10
Regulatory role of interleukins 5 and 6 on immunoglobulin production in cultured rat salivary glands; Pockley AG et al.; The effects of interleukin-5 and interleukin-6 on immunoglobulin production in rat salivary glands were investigated using an in vitro tissue culture system . Detectable levels of IgA, IgG and IgM were observed in the culture media of unstimulated tissues of rat salivary glands after 7 days in culture . Incubation of the tissues with recombinant murine IL-5 (50 U/ml) enhanced the levels of IgA (205% of the control) and IgG (136% of the control), but had no effect on the levels of IgM . Similarly, incubation with recombinant human IL-6 (50 U/ml) enhanced IgA (224% of the control) and IgG (149% of the control) production, but had no effect on IgM . A combination of both IL-5 and IL-6 had no additional effect on the enhanced IgA levels than that seen with IL-5 or IL-6 alone . These data demonstrate a potential regulatory function of lymphokines in glandular mucosal tissue that differs from that previously noted in experiments with cultured rat lacrimal glands or isolated cells derived from both mucosal and non-mucosal tissues.

J Toxicol Environ Health, 1992 Apr, 35(4), 235 - 46
Exposure of human lung fibroblasts to ozone: cell mortality and hyaluronan metabolism; Mayer D et al.; Exposure of cultures of human lung fibroblasts to 0.5 ppm ozone for 20 h resulted in a significant increase in cellular mortality by 29%; after exposure to 2.5 ppm ozone for 4 h, the increase amounted to 74% . A marked difference in sensitivity to ozone was observed between fibroblast lines from different individuals . This variability in resistance to ozone was more evident after exposure to 0.5 ppm ozone for 20 h, when compared with 2.5 ppm ozone for 4 h . In one fibroblast line, synthesis of hyaluronan was enhanced by exposure to 0.5 ppm ozone for 20 h . The concentrations of hyaluronan in culture media increased in experiments using different fibroblast cell lines, a phenomenon that was obvious both if cell numbers and combined protein concentrations of cells and media are selected as references for hyaluronan concentrations.

Biol Reprod, 1992 Apr, 46(4), 715 - 20
Biosynthesis and release of oxytocin by granulosa cells derived from preovulatory bovine follicles: effects of forskolin and insulin-like growth factor-I; Meidan R et al.; Granulosa cells derived from preovulatory bovine follicles were cultured in the presence of insulin-like growth factor-I (IGF-I, 10-100 ng/ml), forskolin (10 microM), or a combination of the two agents . Forskolin alone was the most potent stimulator of both oxytocin (OT) and progesterone (P4) secretion . The two hormones had different patterns of secretion during the course of incubation . OT production peaked on Day 5 of culture and declined thereafter, whereas P4 rose gradually to a peak between Days 7 and 9 . The addition of IGF-I to forskolin did not augment OT release beyond that achieved with forskolin alone, but it did maintain higher levels of OT secretion beyond the Day-5 peak . Two antisera, (antiserum I and antiserum II) directed against OT and its C-terminally extended forms, respectively, were used to identify the OT forms in culture media and granulosa cell and corpus luteum extracts . Fully processed OT was detected only in small amounts (0.43 ng/mg protein) in granulosa cell extracts, whereas the corpus luteum extracts contained 6 ng/mg protein . However, granulosa cells that had been incubated with forskolin contained stores of the OT precursor oxytocin-neurophysin, which is found in young corpora lutea . These data indicate that forskolin (whose action probably mimics gonadotropin action) is an effective stimulator of OT biosynthesis and release in cultured bovine granulosa cells.

Am J Physiol, 1992 Apr, 262(4 Pt 2), F533 - 9
Metanephric transforming growth factor-alpha is required for renal organogenesis in vitro; Rogers SA et al.; The role of transforming growth factor-alpha (TGF-alpha) in metanephric development was examined . Metanephroi were removed from 13-day-old rat embryos and grown in organ culture for up to 6 days using serum-free chemically defined media . During this time the metanephroi increased in size and morphological complexity . Messenger RNA for TGF-alpha was present in the renal anlage . Immunoreactive TGF-alpha was produced by metanephroi in vitro and released into culture media . TGF-alpha of metanephric origin coeluted with recombinant human 125I-TGF-alpha after high-performance liquid chromatography of media . In contrast, epidermal growth factor was not detectable . Levels of TGF-alpha were relatively constant during 4 days in culture and averaged approximately 10(-10) M . Growth of the metanephric anlage, arborization of the ureteric bud, and tubulogenesis within the metanephric blastema were inhibited by the addition of anti-TGF-alpha antibodies to organ cultures . These data demonstrate production of TGF-alpha by developing rat metanephroi in organ culture . The peptide is necessary for growth and development in vitro . Our findings suggest a necessary role for TGF-alpha of metanephric origin as a promoter of renal organogenesis in vivo.

Arch Pathol Lab Med, 1992 Apr, 116(4), 364 - 72
In vitro enhancement of early-stage embryos with co-culture; Thibodeaux JK et al.; Over the years, a great deal of effort has been made to maintain the viability of mammalian embryos once they have been removed from the female's uterus . Early embryo research studies used the simplest of medium (eg, physiological saline) in an effort to maintain embryo homeostasis in vitro, but little progress on embryo viability was made . The addition of heat-treated serum, buffers, and essential amino acids to culture media have shown evidence that improvements could be made in culturing mammalian embryos in vitro; however, it has become clearly evident that this was not going to be a simple task . In recent years, the most notable progress made in this research area has been made with the use of "helper" cells to co-culture early-stage embryos to hatched blastocysts . This article reviews the classic works that have contributed to the development of embryo culture systems that are now used in genetic engineering research and commercial embryo transplant units in the livestock industry . The development of trophoblastic vesicle culture methods and uterine cell co-culture systems were basic contributors to the development of the mammalian embryo co-culture systems as we know them today . Recent studies with follicular granulosa cells and oviduct epithelial cells have added much to our understanding of embryo development in vitro . Novel co-culture systems, such as culturing mammalian embryos in the amnion of a developing chick embryo, certainly offer encouragement that research efforts should continue until an optimal culture system is developed for each mammalian species . The potential uses of embryo culture systems for both humans and animals have yet to be fully understood.

Am J Respir Cell Mol Biol, 1992 Apr, 6(4), 397 - 403
Tissue factor procoagulant expression by rat alveolar epithelial cells; Gross TJ et al.; Fibrin deposition in the alveolar space is characteristic of inflammatory lung injury . The formation of fibrin in the alveolus results from the coagulation of extravasated plasma . The cellular elements that promote intra-alveolar clotting have not been completely defined . We have investigated the capacity of alveolar epithelial cells (AEC) to promote coagulation through the expression of procoagulant activity (PCA) in tissue culture . Using a single-stage coagulation assay, rat AEC monolayers were found to contain 20,750 +/- 4,035 procoagulant units (PCU)/10(6) cells; 10- to 20-fold greater activity than that found in concomitantly isolated alveolar macrophages . The epithelial-derived procoagulant was shown to be tissue factor by a series of assays using clotting factor-deficient human plasmas . Freshly isolated AEC also possessed PCA (2,500 +/- 1,000 PCU/10(6) cells) and expressed a 2.1-kb mRNA that hybridized with a cDNA for murine tissue factor . Using a kinetic turbidometric assay of clot acceleration, PCA was found on the surface of unstimulated epithelial monolayers and could be increased to 170% of control by incubation with phorbol myristate acetate (PMA) . This response to PMA was accompanied by a parallel increase in the relative abundance of tissue factor mRNA . AEC shed particulate PCA into the culture media that displayed a specific activity similar to that recovered from alveolar lining fluid . Therefore, by expressing both cell surface and particulate PCA, the alveolar epithelium likely contributes significantly to the modulation of intra-alveolar coagulation.

Arch Biochem Biophys, 1992 Apr, 294(1), 184 - 7
Pulmonary microvascular endothelial cells constitutively release xanthine oxidase; Partridge CA et al.; The generation of oxidants in reperfused ischemic tissues by xanthine oxidase (XO) may contribute to tissue damage . We exposed bovine pulmonary microvascular endothelial (BPMVE) cells to hypoxia and subsequent reoxygenation and examined alterations in intracellular and extracellular XO activities . BPMVE cells incubated 24 h under hypoxic conditions (less than 1% O2) showed a twofold increase in intracellular xanthine dehydrogenase activity and a smaller increase in intracellular XO activity compared to normoxic BPMVE . Both normoxic and hypoxic BPMVE cells constitutively released XO activity into their culture media . Incubation of hypoxic or normoxic BPMVE cells with oxygenated medium (95% O2) stimulated the release of XO activity into the extracellular medium within 5 min . The XO activity could not be detected in the oxygenated medium after 60 min incubation with 95% O2 . These results indicate that endothelial cells in culture constitutively release XO and that oxygenation rapidly enhances XO release . The released XO activity may play an important role in generation of oxidants in the extracellular milieu during reperfusion.

Hum Reprod, 1992 Apr, 7(4), 545 - 9
Secretion of alpha-immunoreactive inhibin by human pre-embryos cultured in vitro; Phocas I et al.; alpha-Immunoreactive inhibin was measured using an enzyme immunoassay kit in the culture medium (Ham's F-10 medium supplemented with 14% heat-inactivated human serum) from day 3 or 4 to day 14 post-fertilization of 31 surplus pre-embryos from eight women participating in an in-vitro fertilization programme . Inhibition secretion was demonstrated in all of them from the fourth day after fertilization (mean +/- SEM: 3.0 +/- 0.7 U) and was independent of the morphological development of pre-embryos (2-4 cells, n = 4; 6-8 cells, n = 4; 8-10 cells, n = 9; 10-12 cells, n = 4; morulae, n = 5 and blastocysts, n = 4) . On days 7, 10, 13 and 14 post-fertilization, mean inhibin values +/- SEM for non-disintegrated pre-embryos were respectively: 6.5 +/- 0.9 U, 12.3 +/- 2.0 U, 16.8 +/- 3.2 U and 20.2 +/- 3.7 U; however, when disintegration was noted on days 10 and 13 after fertilization, inhibin mean values were 9.0 +/- 1.4 U and 8.4 +/- 1.7 U respectively . Inhibin levels were significantly correlated with human chorionic gonadotrophin levels in the same culture media only on day 13, while correlation with pregnancy specific beta 1-glycoprotein occurred on day 7 post-fertilization . In conclusion, early human pre-embryos secrete alpha-immunoreactive inhibin before the cytotrophoblast is formed . This secretion increases significantly with time when development is continued, while disintegration is followed by a net decline in the rate of inhibin release.

Zhonghua Bing Li Xue Za Zhi, 1992 Apr, 21(2), 73 - 6
{Effect of high density lipoprotein (HDL) on the clearance of intracellular lipids with special reference to the co-effect of nifedipine}; Duan HW; More than 90% of peritoneal macrophages collected from the diet-induced hyperlipidemic rabbits were noticed to be loaded with abundant lipids, and were morphologically similar to the foamy cells infiltrating in the atheromatous lesion . Data of this experiment showed that HDL could promote the clearance rate of intracellular cholesterol efflux and this effect could be strengthened in the progress of time that HDL could transport more than 30% of the accumulated lipids from the cultured cells in 96 hours . Data also indicated that nifedipine was able to promote the hydrolysis of cholesteryl esters in the cultured cells . Addition of nifedipine to the culture media originally containing only HDL promoted the ability of foamy cells in excreting intracellular cholesterol via a hitherto unknown mechanism . This study indicated that nifedipine enhanced the cholesterol efflux mediated by HDL.

Anal Biochem, 1992 Apr, 202(1), 40 - 5
Characterization of insulin-like growth factor binding proteins by two-dimensional polyacrylamide gel electrophoresis and ligand blot analysis; Fazleabas AT et al.; Insulin-like growth factor binding proteins (IGFBPs) in pregnant baboon serum and tissue culture media obtained following explant culture of uteri from pregnant baboons were characterized by two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (2D SDS-PAGE) followed by Western ligand blot analysis using 125I-labeled IGF-I . IGFBP-1 (Mr 30,000; pI 4-4.2), IGFBP-2 (Mr 34,000, pI 5.7-6.2), IGFBP-3 (doublet Mr 42-48,000; pI 6.2-6.8), and IGFBP-4 (Mr 24,000; pI 5.7-6.0) were clearly separated from one another . The authenticity of IGFBP-1, -2, and -3 was verified by immunoprecipitation using polyclonal antibodies followed by ligand blotting . Specificity of 125I-labeled IGF-I binding to IGFBPs was also determined by competitive binding studies using unlabeled IGF-I and -II . This technique allows for the identification of IGFBPs in complex biological fluids on the basis of their characteristic Mr and pI with or without the availability of specific antibodies and can be done rapidly using the mini 2D SDS-PAGE systems.

J Neurosci Res, 1992 Apr, 31(4), 662 - 9
Differentiation of PC12 cells with nerve growth factor is associated with induction of transin synthesis and release; Fillmore HL et al.; We have identified and characterized a calcium-dependent metalloproteinase which is induced in rat pheochromocytoma cells (PC12 cells) during differentiation with nerve growth factor (NGF) . Assays of proteolytic activity in media from differentiated PC12 cell cultures revealed a NGF-dependent increase in the activity of a proteinase which has a molecular weight of 62 kDa . Studies using serine, thiol, and metalloproteinase inhibitors demonstrated that the secreted enzyme is a metalloproteinase . Treatment of culture supernatants with aminophenylmercuric acid (APMA), a known activator of metalloproteinases, resulted in a decrease in the molecular weight of the proteinase . Western blot analysis of culture media from NGF-treated PC12 cells using an antibody directed against a synthetic peptide of rat transin identified this metalloproteinase as transin . Treatment of PC12 cells with acidic and basic fibroblast growth factor (FGF) resulted in distinct morphological changes as well as transin release . Incubation with epidermal growth factor (EGF) did not induce transin release . Dexamethasone inhibited the induction of transin release by NGF . 35S-methionine labeling and immunoprecipitation of newly synthesized proteins from culture supernatants confirmed that NGF induced the synthesis of this enzyme 8 hr after NGF treatment . The NGF-dependent induction of transin, a calcium-dependent metalloproteinase which degrades type IV collagen, laminin, and fibronectin suggests that transin may function to degrade the surrounding extracellular matrix during the invasive process of axonal elongation in neuronal development thereby allowing the movement of growth cones and axons toward specific targets.

J Steroid Biochem Mol Biol, 1992 Apr, 42(2), 201 - 10
Immunocytochemical determination of estrogen and progesterone receptors in human endometrial adenocarcinoma cells (Ishikawa cells); Hata H et al.; The regulation of both estrogen and progesterone receptor levels in human endometrial adenocarcinoma cells of the Ishikawa line was investigated immunocytochemically by using monoclonal antibodies . Positive staining for estrogen and progesterone receptors was observed in the nuclei of Ishikawa cells . Intercellular heterogeneity in receptor content was evident from the presence of receptor-positive or -negative cells and from differences in staining intensity of positive cells . Quantitative analysis was performed by scoring the staining intensity and the proportion of positively stained cells . The time and dose-dependent stimulatory effect of estradiol added to culture media on progesterone receptor levels was studied by applying both immunocytochemical and biochemical methods . Estradiol at 10 nM (optimal concentration) increased the intensity score for PR from an initial value of 10.1 to 78.3 after 72 h incubation, and the proportion of the positive staining cells from 6.7 to 42.7% . Promegestone (R5020) was effective at 1 microM concentration in decreasing the intensity score for ER from 31.1 to 14.6 after 72 h exposure and the proportion of positive cells from 19.0 to 11.4%.

Endocrinology, 1992 Apr, 130(4), 2426 - 8
Norepinephrine stimulates immunoreactive (ir) atrial natriuretic peptide (ANP) secretion and pro-ANP mRNA expression from rat hypothalamic neurons in culture: effects of alpha 2-adrenoceptors; Huang W et al.; Although ANP or its smaller congeners are produced and secreted from rat hypothalami, the role or roles of neurotransmitter(s) in regulating their release and production from the neurons remains unclear . We report here that norepinephrine or epinephrine (NE/EPI) facilitates irANP secretion and pro-ANP mRNA expression in long term cultures of rat hypothalamic neurons through their effects on alpha 2-adrenoceptors . Hypothalami of 3 day-old Sprague-Dawley rats were removed and digested with collagenase . The dispersed cells were plated on poly-D-lysine coated culture dishes (10(6) cells/well) in Hepes buffered Dulbecco's Modified Eagle Medium supplemented with 8% fetal calf serum . Six days after plating, media were replenished with serum free media and the cultures incubated for 4 more days with vehicle or various doses of NE, EPI, alpha- or beta-adrenoceptor agonists in the presence of absence of antagonists . Culture media were then extracted with C18 Sep-pak and the levels of irANP determined by a well characterised RIA for ANP . NE or EPI treatment significantly increased irANP secretion from the cultures in a dose related manner with ED50 and Emax of approximately 0.2 microM and 1 microM respectively . The stimulation effect of NE was blocked by yohimbine (alpha 2-antagonist), but not prazosin (alpha 1-antagonist) or propranolol (beta-antagonist) . Clonidine (alpha 2-agonist), but not phenylephrine (alpha 1-agonist) or isoprenaline (beta-agonist) mimicked the effects of NE or EPI . At the concentration of 0.1 microM, clonidine increased irANP release approximately 3 fold above that of control values (34.7 +/- 3.3; mean +/- SE, n = 4) . These changes were accompanied by corresponding increments in the abundance of pro-ANP mRNA in the cultures as examined by a colorimetric Northern blot analysis . Our results indicate that NE or EPI, acting through its alpha 2-adrenoceptors, may modulate the function of ANP neurons in rat hypothalami by regulating the secretion and production of the neuropeptide at the genomic level.

Biochim Biophys Acta, 1992 Mar 25, 1124(3), 262 - 72
Contributing factors in the trafficking of {3H}arachidonate between phospholipids; Blank ML et al.; Cultured human promyelocytic leukemia cells (HL-60), depleted of arachidonic acid by continued growth in serum-free media, were used as a model system to examine various factors that control the incorporation and distribution of {3H}arachidonic acid into classes and subclasses of cellular lipids . Increasing the culture media concentration of {3H}arachidonic acid from 1 x 10(-8) M to 1 x 10(-5) M caused a greater percentage of the cellular tritium to be distributed into triacylglycerols (from less than 1% at 1 x 10(-8) M to 38% at 1 x 10(-5) M) with a corresponding decrease in cellular {3H}diradylglycerophosphoethanolamine (from 53% at 1 x 10(-8) M to 12% at 1 x 10(-5) M) during 2 h incubations . A greater proportion of the tritium present in diradylglycerophosphoethanolamine and diradylglycerophosphocholine, at the higher media concentration of {3H}arachidonic acid (1 x 10(-5) M), was found in the diacyl subclasses of these two lipids than was observed at the lower concentrations (less than 1 x 10(-6) M) of {3H}arachidonic acid . Significant amounts of diarachidonoyl molecular species were found in the phosphatidylethanolamine (10%) and phosphatidylcholine (15%) of HL-60 cells that were labeled for 2 h with 1 x 10(-5) M {3H}arachidonic acid . This was the only molecular species of phosphatidylcholine to completely disappear when prelabeled cells were placed in arachidonate-free media for 22 h . Prelabeling-chase experiments with 1 x 10(-5) M {3H}arachidonic acid were consistent with movement of {3H}arachidonate from triacylglycerols into diradylglycerophosphatides and from diacylphospholipids into ether-linked phospholipids . Increasing the concentration of HL-60 cells in the incubations influenced the distribution of {3H}arachidonic acid in cellular lipid classes in a manner analogous to decreasing the concentration of {3H}arachidonic acid in the media . Increasing the endogenous level of cellular arachidonate in phospholipid classes with supplements of unlabeled arachidonic acid changed the subsequent lipid class distribution of a low concentration (1 x 10(-8) M) of {3H}arachidonic acid to resemble results obtained with a much higher mass level of {3H}arachidonate in arachidonate depleted cells . HL-60 cells differentiated into granulocytes by treatment with dimethyl sulfoxide incorporated less {3H}arachidonic acid but had a greater proportion associated with alkylacylglycerophosphocholine and alk-1-enylacylglycerophosphoethanolamine than undifferentiated HL-60 cells.

J Biol Chem, 1992 Mar 25, 267(9), 5739 - 42
Dynamics of interleukin-6 internalization and degradation in rat hepatocytes; Nesbitt JE et al.; We have investigated the dissociation, internalization, and degradation of 125I-interleukin-6 (125I-IL-6) by primary rat hepatocytes . Temperature shift experiments following saturation binding of 125I-IL-6 to cell surface receptors in hepatocytes showed a rapid loss of surface-bound 125I-IL-6 (t1/2 = 15 min), concomitant with a rapid rise in internalized radiolabeled ligand . After reaching a maximum by 30 min at 37 degrees C, the level of internalized 125I-IL-6 decreased with time and appeared in the culture media in a non-trichloroacetic acid-precipitable (degraded) state . The addition of the lysosomotropic agent chloroquine inhibited this receptor-mediated degradation of IL-6 without affecting ligand internalization . Polyacrylamide gel electrophoresis analysis of internalized 125I-IL-6 confirms these results . Additionally, we show that the IL-6.IL-6 receptor complex is stable, and dissociation of these two molecular species occurs at a pH below 5.0 . In contrast to published results, data presented in this study clearly indicate that IL-6 is rapidly internalized and degraded within hepatocytes by a receptor-mediated mechanism.

Eur J Pharmacol, 1992 Mar 3, 212(2-3), 129 - 36
Quisqualate neurotoxicity in rat cortical cultures: pharmacology and mechanisms; Zinkand WC et al.; Quisqualate is a potent neurotoxin in cortical cultures of the rat . Unlike N-methyl-D-aspartate (NMDA), the toxicity of quisqualate is due to overstimulation of a membrane receptor after the agonist has been removed . This receptor appears to be the 2-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor since 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo(F)quinoxaline (NBQX) and 6,7-dinitroquinoxaline-2,3-dione (DNQX) are potent antagonists when added to the post incubation media . NBQX and DNQX are ineffective when present only during quisqualate exposure, indicating the AMPA receptor is not involved in the initial event . Transfer of culture media 30 min after quisqualate exposure to either neuronal or non-neuronal cells was found to cause toxicity in previously untreated neuronal cells . This effect could not be reproduced with NMDA . The neurotoxic chain of events could be interrupted during quisqualate exposure by removal of sodium from the incubation media, suggesting the involvement of a sodium-dependent plasma membrane uptake mechanism . Quisqualate may be continually recycled by internalization and release, causing neurotoxicity by persistent stimulation of the AMPA receptor.

Eur J Pharmacol, 1992 Mar 3, 212(2-3), 151 - 8
Nitric oxide does not mediate the neurotrophic effects of excitatory amino acids in cultured cerebellar granule neurons; Boje KM et al.; Excitatory amino acids, such as N-methyl-D-aspartate (NMDA) and kainate, promote neuritogenesis and viability in primary cultures of cerebellar granule cells . In view of the recent demonstration that excitatory amino acids activate the synthesis of nitric oxide, the present study examined a potential role of nitric oxide in mediating the neurotrophic effects of excitatory amino acids . NMDA enhanced the viability of 8-day-old cerebellar granule cell cultures in a concentration-dependent fashion, whereas kainate showed a concentration-dependent biphasic effect . A specific inhibitor of nitric oxide synthase, NG-nitroarginine (0.5 mM), did not antagonize the neurotrophic effects of NMDA (0.5 mM) or kainate (0.05 mM) . The concentration of NG-nitroarginine was sufficient to inhibit NMDA or kainate stimulated nitric oxide synthesis and was stable in the culture media throughout the 8-day culture period . Using a specific chemiluminescence detection method, endogenous nitric oxide was directly measured in the headspace gas phase of homogenized cultured cerebellar granule cells, and was not detected when homogenates were incubated with NG-nitroarginine (0.5 mM) . Furthermore, addition of a nitric oxide precursor, S-nitroso-N-acetylpenicillamine (1-200 microM), was not neurotrophic, but rather, was neurotoxic in granule cells . These findings indicate that nitric oxide is not a neurotrophic factor in primary cultures of cerebellar granule cells.

Neurosci Lett, 1992 Mar 2, 136(2), 198 - 202
Effect of viscosity on neurite outgrowth and fractal dimension; Caserta F et al.; The growth mechanism by which neurons achieve their characteristic ramified morphology has long been of interest, but determining whether physical parameters, such as viscosity, are important has been difficult due to a lack of useful hypotheses and standard reproducible techniques . We have recently shown that neurons exhibit fractal behavior and that their fractal dimension (df) is consistent with a physical process called diffusion-limited aggregation (DLA) . We suggested that this DLA behavior might stem from viscosity differences, chemical gradients or electrical fields (Caserta et al., Phys . Rev . Lett., 64 (1990) 95-98) . DLA is a model for a large family of growth processes . In order for a process to fit the DLA model, the growth rate must be proportional to the gradient of a field at a point on the growing structure (Feder, Plenum, New York, 1988, Ch . 4) . Chemical, electrical, or fluid pressure fields can fit the model depending on the particular physical system under study . Here, we studied growth of retinal neurons from chick embryos in culture media of various fluid viscosities . Thus, we test whether DLA in this system was based on a fluid pressure field . As viscosity was increased from 1 to 4.3 cps, the number of neurite branches decreased 98% . However, there was no effect on df . Over this range of viscosities, total cellular protein synthesis decreased only 17% . The results indicate that, while differences in viscosity between the interior and exterior of the cell affect neurite outgrowth, they do not affect the fractal behavior of neurons . Thus, viscosity differences are not the basis for the DLA pattern of neuronal arborization.

Fertil Steril, 1992 Mar, 57(3), 641 - 7
An improved medium for long-term culture of human embryos overcomes the in vitro developmental block and increases blastocyst formation; FitzGerald L et al.; OBJECTIVE: To determine if a culture medium, designated CZB after the authors who first described it, which is supplemented with 0.11 mM ethylenediaminetetraacetic acid, 1.0 mM glutamine, 31.30 mM lactate, and 0.27 mM pyruvate and is lacking glucose for the initial stages of culture that overcomes the in vitro two-cell block of mouse embryos, can improve the rate of blastocyst formation of human embryos in long-term cultures and increase the pregnancy rate (PR) when used in a clinical in vitro fertilization (IVF) program . DESIGN: The study is in two parts . Initially, excess oocytes from IVF and gamete intrafallopian transfer patients were fertilized in vitro and then placed in long-term culture of either CZB plus 10% heat-inactivated human serum (32 zygotes) or Earle's balanced salt solution (EBSS) supplemented with 0.45 mM pyruvate plus 10% human serum (28 zygotes) to determine if CZB medium could enhance in vitro development and increase blastocyst formation when compared with EBSS . Subsequently, CZB or EBSS medium was used for short-term cultures of embryos in a clinical IVF program to determine if the use of CZB could increase the clinical IVF PR . SETTING: Private practice of one author (M.D.) . PATIENTS: The excess oocytes were donated by couples not wishing to have cryopreservation . In the clinical trial, 49 couples presenting with tubal or male factor infertility and who had three or more fertilized zygotes were randomly assigned to one of the culture media being used . RESULTS: In long-term cultures, embryos were observed at 42, 66, 90, 114, and 138 hours after fertilization and scored for blastomere number, degree of fragmentation, and developmental arrest . When CZB- and EBSS-cultured embryos were evaluated over 138 hours, there was a significant increase in the number of CZB-cultured embryos reaching the blastocyst stage (56% versus 20%; P less than 0.009) and less fragmentation of CZB-cultured embryos (18.8% versus 50%; P less than 0.01) . In short-term cultures, pronuclear stage embryos from patients undergoing IVF were cultured in CZB or EBSS for 24 hours, graded, and then used in embryo transfers . Of the 28 patients assigned to EBSS, 6 became pregnant (21.4%), and of the 21 assigned to CZB, 5 attained pregnancy (23.8%) . These results were not significantly different . CONCLUSIONS: The use of CZB for the long-term culture of human embryos is highly beneficial and increases the rate of blastocyst formation, but its use in an IVF program does not increase the clinical PR.

Fertil Steril, 1992 Mar, 57(3), 637 - 40
Immunosuppressive activity and alpha interferon concentrations in human embryo culture media as an index of potential for successful implantation; Jones KP et al.; OBJECTIVE: To evaluate potential correlations between establishment of pregnancy and immunosuppressive activity secreted by the preimplantation embryo . DESIGN: To evaluate immunosuppressive activity, supernatants from preimplantation embryos were assessed for their ability to inhibit lymphocyte proliferation . Additionally, alpha interferon concentrations were also measured in these supernatants . We compared these parameters from embryo culture supernatants of women who did and did not achieve pregnancy after in vitro fertilization (IVF) . Immunosuppression was assessed using a lymphocyte proliferation assay with concanavalin A (Con A) and phytohemagglutinin (PHA) as mitogens . SETTING: In vitro fertilization program at the University of Utah Medical Center . PARTICIPANTS: Couples less than 40 years of age, with normal semen quality and bilateral tubal obstruction . RESULTS: Immunosuppression calculated using the stimulation index (mean +/- SEM) in pregnant and nonpregnant women, respectively, were: Con A: 43.9 +/- 3.9 versus 19.1 +/- 10.1, P less than 0.04 . PHA: 23.6 +/- 5.6 versus 12.5 +/- 12.8, P less than 0.02 . Alpha interferon levels (mean +/- SD) in pregnant and nonpregnant women were not significantly different: 23.98 +/- 9.6 U/mL versus 24.79 +/- 2.5 U/mL . CONCLUSIONS: We conclude that pre-embryos with the capacity for successful implantation secrete greater amounts of immunosuppressive factors than those destined not to implant, as measured by Con A and PHA lymphocyte proliferation assays . Refinement of assay techniques and identification of the substances involved could have significant impact on IVF programs.

Fertil Steril, 1992 Mar, 57(3), 631 - 6
Discordant secretion of pregnancy specific beta 1-glycoprotein and human chorionic gonadotropin by human pre-embryos cultured in vitro; Dimitriadou F et al.; OBJECTIVE: To study and compare the secretion of pregnancy specific beta 1-glycoprotein (SP1) and human chorionic gonadotropin (hCG) by human pre-embryos, cultured in vitro, with their respective morphological development . DESIGN: Spare human pre-embryos from randomly selected women participating in a program of in vitro fertilization (IVF) were studied prospectively . SETTING: Pre-embryos were cultured, and hormone release was determined in academic research laboratories . PATIENTS, PARTICIPANTS: Pre-embryos (n = 108) cultured for 14 days after fertilization in Ham's F-10 medium (GIBCO Ltd., Paisley, Scotland) were observed, and hCG and SP1 were measured in the culture media at regular intervals . MAIN OUTCOME MEASURES: Discordant secretion of SP1 and hCG . RESULTS: Of the 98 bipronucleate pre-embryos, 53.6% formed blastocysts, 17.3% of which hatched . Human chorionic gonadotropin was detected from day 7 after fertilization concomitantly with blastocyst formation, thereafter showing a logarithmic increase (maximum 10,650 mIU) until the first signs of embryonic disintegration . Pregnancy-specific beta 1-glycoprotein release started 3 to 4 days after fertilization independently of the morphological development and the future production of hCG, thereafter displaying a nonlogarithmic increase (maximum 41 ng) . CONCLUSIONS: Hormone secretion and morphological development are unique for each pre-embryo . Human chorionic gonadotropin and SP1 seem to have different biochemical and physiological regulation.

Yakugaku Zasshi, 1992 Mar, 112(3), 183 - 92
{Pharmacological study on Naja naja kaouthia Lesson . III . Effects of 50% ethanolic extracts from liver and gall bladder on phagocytic activity of mouse reticuloendothelial system}; Matsuda H et al.; Effects of 50% ethanolic extracts from the liver and the gall bladder of Naja naja kaouthia Lesson (COB-L or COB-G) were studied on the phagocytic activity of a mouse reticuloendothelial system . The clearance-rate of carbon was shortened by the oral administration of COB-L or COB-G (50 or 200 mg/kg) . COB-L or COB-G promoted the phagocytosis of latex by peritoneal macrophages (M phi) . Furthermore, effects of these extracts on the peritoneal M phi were biochemically investigated using in vitro experimental models . The activities of two lysosomal enzymes, acid phosphatase and beta-glucuronidase, and that of lactate dehydrogenase in the peritoneal M phi were enhanced when the M phi were cultured with COB-L or COB-G (10-100 micrograms/ml) for 4 d . In addition, the consumption of glucose in the culture media by the M phi was also enhanced by culturing the media with COB-L or COB-G . When COB-L and COB-G were given orally immediately before and 16 h after the application of picryl chloride (PC) or sheep red blood cell (SRBC), these extracts at a dose of 200 mg/kg did not show any inhibitory effects on the swelling by picryl chloride-inducing contact dermatitis (PC-CD) and by sheep red blood cell delayed type hypersensitivity (SRBC-DTH) in mice . However, these extracts inhibited the immunosuppression with the SRBC 1 x 10(9) cells priming and with the frozen and dried ascites of Ehrlich carcinoma-bearing mice containing immunosupressive substances (EC-sup) . These results suggest that COB-L or COB-G biochemically activates the mouse peritoneal M phi, and promotes the phagocytic activity of the M phi and the interaction between M phi and T cell.

J Androl, 1992 Mar-Apr, 13(2), 125 - 30
Effects of insulin-like growth factor-I on androgen production by highly purified pubertal and adult rat Leydig cells; Gelber SJ et al.; Leydig cells were isolated and purified from adult and midpubertal rats to study the effects of insulin-like growth factor-I (IGF-I) on steroidogenesis . Androgen production, as measured in Leydig cell conditioned culture media, from four different treatment groups (1 = no hormone; 2 = 70 ng/ml IGF-I; 3 = 0.1 ng/ml LH; 4 = 70 ng/ml IGF-I + 0.1 ng/ml LH) were compared daily . After 3 days in culture, the cells were treated with a maximally stimulating dose of luteinizing hormone (LH) (100 ng/ml) for 3 hours . Androgen production was highest in the cells treated with both IGF-I and low concentrations of LH . In the presence of IGF-I, regardless of LH, cells derived from pubertal animals had a greater increase in steroidogenesis during the culture period than did cells from adult animals . Pretreatment with IGF-I prior to maximal LH stimulation induced a greater increase in androgen production in cells from pubertal rats than in cells from adult animals . It is concluded that IGF-I has a direct effect on Leydig cells and may act synergistically with LH to promote androgen synthesis . The greater response in pubertal cells raises the possibility that IGF-I is important in the maturing process of the testis.

J Gen Microbiol, 1992 Mar, 138 ( Pt 3), 619 - 25
Isolation and partial characterization of haemagglutinins from plasmodia of Physarum polycephalum; Morita M et al.; The soluble haemagglutinins produced by plasmodia of Physarum polycephalum were purified by chromatographic methods and resolved into haemagglutinins I and II . On SDS-PAGE, purified haemagglutinins I and II each gave a single band with an apparent molecular mass of 6 and 11 kDa, respectively . The results of gel-filtration chromatography suggested that both haemagglutinins were dimers of the respective subunits under non-denaturing conditions . Rabbit erythrocytes were preferentially agglutinated by both haemagglutinins . The human type A, B and O erythrocytes were agglutinated by haemagglutinin II to an equal degree but were not agglutinated by haemagglutinin I . Simple sugars failed to inhibit the activities of both haemagglutinins . The activities, however, were effectively inhibited by the addition of thyroglobulin . Other glycoproteins such as fetuin, orsomucoid and transferrin inhibited the activity of haemagglutinin I but not that of haemagglutinin II . These haemagglutinins were detected in a slime fraction obtained from the culture media of starved plasmodia, suggesting that they are released to the outside of the plasmalemma to become associated with the slime layer on the plasmodial surface.

J Steroid Biochem Mol Biol, 1992 Mar, 42(1), 113 - 20
Identification of 5 alpha-androstane-3 beta,17 beta-diol and 3 beta-hydroxy-5 alpha-androstan-17-one sulfates as quantitatively significant secretory products of porcine Leydig cells and their presence in testicular venous blood; Raeside JI et al.; By means of high performance liquid chromatography and gas chromatography-mass spectrometry it has been found that 5 alpha-androstane-3 beta,17 beta-diol sulfate and 3 beta-hydroxy-5 alpha-androstan-17-one sulfate (epiandrosterone) are major secretory steroids of the mature boar testes . These same compounds were similarly identified in culture media when porcine Leydig cells were incubated with androstenedione as substrate . In addition, they were seen as the principal secretory products when {3H}androstenedione and {3H}testosterone were used as substrates; and their presence was greatly reduced by an inhibitor of 5 alpha-reductase (N,N-diethyl,4-methyl-3-oxo-4-aza-5 alpha-androstane-17 beta-carboxamide) . Greater quantities of 5 alpha-androstanediol than epiandrosterone were noted in all instances . These findings provide further evidence of the versatile activity of the boar testes in steroidogenesis.

Mol Reprod Dev, 1992 Mar, 31(3), 189 - 94
Joint effects of sodium chloride, glutamine, and glucose in mouse preimplantation embryo culture media; Lawitts JA et al.; A new medium, SOM, produced by simplex optimization, has been used to study the joint effects of NaCl, glutamine, and glucose on the development of outbred CF1 mouse zygotes to the blastocyst stage . Contrary to previous reports, glucose has no significant inhibiting effect on development to the blastocyst stage in this medium . Even in the presence of 5 mM glucose, 70% of the embryos develop to at least four cells, and 60% reach the blastocyst stage . Raising the concentration of NaCl from 75 to 125 mM, in the absence of glutamine, progressively inhibits development . Moreover, the response to glutamine depends on the concentration of NaCl in the medium . When the NaCl concentration is low, glutamine inhibits development . In contrast, when the NaCl concentration is high, glutamine protects against the inhibitory effect of the salt . We propose that glutamine protects against high concentrations of NaCl in the medium by acting as an organic osmolyte.

J Invest Dermatol, 1992 Mar, 98(3), 379 - 83
Human keratinocytes and A-431 cells synthesize and secrete factor B, the major zymogen protease of the alternative complement pathway; Yancey KB et al.; Biosynthetic radiolabeling studies demonstrate that human keratinocytes and A-431 cells, a human epidermoid carcinoma cell line, synthesize and secrete factor B as a monomeric 105-kD protein . Epithelial cell-derived factor B comigrates in SDS-PAGE with that produced by HepG2 cells, a human hepatoma cell line traditionally employed in studies of complement component biosynthesis . Comparative pulse-chase studies in A-431 and HepG2 cells show that this alternative pathway complement component is produced as co-migrating 100-kD intracellular proteins that are processed in both cell types to 105-kD extracellular factor B . Quantitatively, immunoprecipitable factor B accounts for 0.05% of radiolabeled proteins in A-431 cell culture media . Treatment of biosynthetically radiolabeled A-431 cell culture media with cobra venom factor and factor D for 60 min at 37 degrees C produces the specific factor B cleavage products Ba and Bb . These fragments are not identifiable in control culture media subjected to similar treatment in the absence of alternative pathway activators . Northern blot analysis of total cellular RNA from human keratinocytes, A-431 cells, and HepG2 cells reveals qualitative identity of a 2.8-kb factor B mRNA species in these three cell types . The relative level of factor B mRNA expression in these cells parallels their level of factor B protein synthesis (i.e., HepG2 cells greater than A-431 cells greater than human keratinocytes) . Epithelial cell-derived factor B may play an important role in local inflammatory reactions and also directly interact with epithelial cell derived C3--a key classical and alternative pathway complement component recently shown to be produced by human keratinocytes.

Gastroenterology, 1992 Mar, 102(3), 949 - 55
Ontogeny of the receptor for polymeric immunoglobulins in rat hepatocytes; Buts JP et al.; Based on in vitro experiments measuring daily secretion rates in the culture media of rat hepatocytes and in vivo experiments using pulse labeling of intracellular precursors, the present study examines the ontogenic expression of the polymeric immunoglobulin receptor and secretory component by hepatocytes during growth . Our data indicate that hepatocytes from infant and suckling rats (day 5, 15) cultured in serum-free and hormone-free conditions only secreted trace amounts of secretory component . Beginning on day 20, basal secretion rate showed a marked upsurge with a 10-fold increase by day 35 . The addition of dexamethasone (10(-7) mol/L) to the culture media enhanced by 2.5-fold the basal secretion of secretory component by hepatocytes from 20-, 25-, and 35-day-old rats, while addition of insulin to the media had no effect . The response to dexamethasone was dose-dependent (10(-5), 10(-6), 10(-7) mol/L) and specific . In vivo pulse labeling of receptor precursors in hepatocytes from 40-day-old rats allowed the identification of three intracellular forms: a 105-kilodalton peptide and a 116-120-kilodalton mature doublet . In 13-day-old rats, three immature precursors were detected: a 105-kilodalton peptide and a high molecular weight doublet of 185-190 kilodaltons . Sucklings (13 days) treated with corticosterone showed a pattern of precursors similar to controls . These findings support the following conclusions: (a) hepatocytes from infant and suckling rats synthesize and process immature receptor precursors whose expression is unaffected by corticosterone treatment, and (b) active secretion of secretory component is initiated at weaning independently from humoral and hormonal factors while the magnitude of its production by the liver is under the control of glucocorticoids.

Endocrinology, 1992 Mar, 130(3), 1255 - 62
Angiotensin II stimulation of plasminogen activator inhibitor-1 gene expression in astroglial cells from the brain; Rydzewski B et al.; In this study, we investigated the mechanism of angiotensin II (Ang II) induced secretion of plasminogen activator inhibitor-1 (PAI-1) from astroglial cells prepared from 21-day-old rat brain . Competition-inhibition experiments with the use of selective antagonists for Ang II receptor subtypes indicated that astroglial cells contain chiefly Ang II type 1 (AT1) receptors . The interaction of Ang II with AT1 receptors resulted in a time- and concentration-dependent stimulation of PAI-1 gene expression . A maximal, 20-fold induction of PAI-1 messenger RNA (mRNA) steady-state levels was observed with 10 nM Ang II . This effect of Ang II was blocked by DuP753, an AT1 receptor antagonist, but not by PD123177, an AT2 receptor antagonist . Raise in PAI-1 mRNA levels was followed by an elevation in PAI-1 concentration in culture media reaching its maximum after 24 h . Interaction of Ang II with AT1 receptors also resulted in a time- and concentration-dependent stimulation of inositol phospholipid (IP) hydrolysis . A maximal, 3- to 5-fold stimulation of IP hydrolysis was observed with 10 nM Ang II . The time course experiments indicated that Ang II-induced stimulation of IP hydrolysis precedes the stimulation of PAI-1 mRNA . This suggested that activation of phospholipase C, IP hydrolysis system and possibly protein kinase C (PKC) may mediate Ang II's effect on PAI-1 mRNA . Direct stimulation of PKC by phorbol ester, phorbol 12,13-dibutyrate (PDB), resulted in a time- and concentration-dependent elevation of PAI-1 mRNA levels, similar to that caused by Ang II (maximal stimulation of 20-fold with 100 nM PDB for 4 h) . This effect was totally blocked by the protein kinase C inhibitor, H7 . In addition, Ang II stimulation of PAI-1 mRNA was also blocked by H7 . In contrast, Ang II did not elevate PAI-1 mRNA levels in astroglial cultures from neonatal rat brains . However, treatment of neonatal cultures with PDB increased levels of this mRNA species . These observations indicate that the coupling of AT1 receptors with IP hydrolysis and PKC activation may be important for Ang II stimulation of PAI-1 gene expression . The lack of Ang II's effect on PAI-1 mRNA in neonatal astroglia may be explained either by a low coupling efficiency between AT1 receptors and the second messenger system, or by a low AT1 to AT2 receptor level ratio.

Drug Metab Dispos, 1992 Mar-Apr, 20(2), 218 - 25
Correlations between conceptal concentrations of all-trans-retinoic acid and dysmorphogenesis after microinjections of all-trans-retinoic acid, 13-cis-retinoic acid, all-trans-retinoyl-beta-glucuronide, or retinol in cultured whole rat embryos; Kraft JC et al.; Retinol (4,000 ng/ml), all-trans-retinoyl-beta-glucuronide (4,000 ng/ml), and 13-cis-retinoic acid (1,500 ng/ml) each produced dysmorphogenic effects qualitatively similar to those elicited by 250 ng/ml of all-trans-retinoic acid after microinjections of the respective individual retinoids into the amniotic cavities of cultured whole rat embryos . Subsequent HPLC analyses of the cultured whole conceptuses, embryos proper, yolk sacs, and culture media (24 hr after microinjections) indicated that conceptal biotransformation of each of the retinoids had occurred during the culture period . All-trans-retinoic acid was present in the embryos proper at quantitatively similar concentrations (20-100 nM) after microinjections of the selected quantities of each of the microinjected retinoids: retinol, all-trans-retinoyl-beta-glucuronide, 13-cis-retinoic acid, or all-trans-retinoic acid . The results suggested that all-trans-retinoic acid acted as an ultimate dysmorphogen for the retinoids tested with respect to the anomalies monitored in the embryo culture system.

J Chromatogr, 1992 Feb 7, 574(1), 17 - 21
Monitoring of the uptake and metabolism of aminooxy analogues of polyamines in cultured cells by high-performance liquid chromatography; Hyvonen T et al.; A high-performance liquid chromatographic method for the determination of polyamines and their aminooxy analogues is described . Oxime derivatization with a ketone is used to protect the aminooxy group during post-column reaction with o-phthalaldehyde . The amount of the polyamines and of the oximes of their aminooxy analogues can be determined simultaneously in cultured cells and cell culture media . The limit of detection is 20-30 pmol, and the response of the fluorescence detection is linear up to 4 nmol . The separation of the aminooxy analogues from the naturally occurring polyamines can be varied by using different ketones for oxime formation . The method was used to measure the stability of aminooxy analogues of putrescine (1-aminooxy-3-aminopropane) and spermidine {N-(2-aminooxyethyl)-1,4-diaminobutane and 1-aminooxy-3-N-(3-aminopropyl)aminopropane} in cell culture media and the uptake into cultured baby hamster kidney (BHK21/C13) cells.

J Biol Chem, 1992 Feb 5, 267(4), 2798 - 801
Expression and polarized apical secretion in Madin-Darby canine kidney cells of a recombinant soluble form of neutral endopeptidase lacking the cytosolic and transmembrane domains; Corbeil D et al.; Neutral endopeptidase-24.11 (NEP; EC 3.4.24.11) is an abundant metalloendopeptidase of the brush border membrane of kidney proximal tubules . We have recently shown that NEP is delivered directly to the apical domain of the plasma membrane when expressed in polarized Madin-Darby canine kidney (MDCK) cells in culture (Jalal, F., Lemay, G., Zollinger, M., Berteloot, A., Boileau, G., and Crine, P . (1991) J . Biol . Chem . 266, 19826-19832) . Here, a soluble form of NEP consisting of the signal peptide of pro-opiomelanocortin fused in-frame with the ectodomain of NEP has been expressed in MDCK cells . Enzymatic assays performed on apical and basolateral culture media of MDCK cells grown on semi-permeable supports indicated that the recombinant enzyme was predominantly released at the apical surface . In contrast, when the chimeric protein was expressed in NIH 3T3 cells or when pro-opiomelanocortin was expressed in MDCK cells, non-polarized secretion was observed into both the apical and basolateral compartments of the culture chamber . Our results suggest that the ectodomain of NEP is sufficient for directing the targeting of this protein to the apical membrane of polarized MDCK epithelial cells.

Hybridoma, 1992 Feb, 11(1), 53 - 9
Isolation and characterization of a monoclonal anti CK-2 alpha subunit antibody of the IgG1 subclass; Schmidt-Spaniol I et al.; A monoclonal antibody was produced against the recombinant human alpha subunit of CK-2 . The antibody was of the IgG1 subclass and it was isolated from serum-free cell culture media and purified by affinity chromatography on Protein G Sepharose . The antibody can be used to detect specifically the CK-2 alpha subunit in immunoblots from tissue extracts . An ELISA detection test was also established which also allows the identification of the CK-2 alpha subunit.

J Cell Biol, 1992 Feb, 116(4), 1035 - 42
Induction of proliferation or hypertrophy of chondrocytes in serum-free culture: the role of insulin-like growth factor-I, insulin, or thyroxine; Bohme K et al.; In bone forming cartilage in vivo, cells undergo terminal differentiation, whereas most of the cells in normal articular cartilage do not . Chondrocyte hypertrophy can be induced also in vitro by diffusible signals . We have identified growth factors or hormones acting individually on 17-d chick embryo sternal chondrocytes cultured in agarose gels under strictly serum-free conditions . Insulin-like growth factor I or insulin triggered the first steps of chondrocyte maturation, i.e., cell proliferation and increased matrix deposition while the chondrocytic phenotype was maintained . However, cells did not progress to the hypertrophic stage . Proliferation and stimulated collagen production was preceded by a lag period, indicating that synthesis of other components was required before cells became responsive to insulin-like growth factor I or insulin . Very small amounts of FBS exerted effects similar to those of insulin-like growth factor I or insulin . However, FBS could act directly and elicited hypertrophy when constituting greater than 1% of the culture media . Basic FGF has been claimed to be the most potent chondrocyte mitogen, but had negligible effects under serum-free conditions . The same is true for PDGF, a major serum-mitogen . Under the direction of thyroxine, cells did not proliferate but became typical hypertrophic chondrocytes, extensively synthesizing collagen X and alkaline phosphatase.

Endocrinology, 1992 Feb, 130(2), 811 - 8
Prolactin variants in ram adenohypophyses vary with season; Stroud CM et al.; Secretion of PRL in sheep is affected by photoperiod being highest during the spring and summer, lowest in fall and winter . The objectives of this study were to determine if 1) the production of variant forms of PRL, and 2) immuno- and bioactivities of PRL (iPRL and bPRL) differ during times of the year selected to represent periods of low, transitional and high PRL secretion . Twelve mature rams were maintained on pasture and killed in October, December, and April (n = 4/month) . Individual pituitary glands were dispersed, cells obtained, and fixed for immunocytochemical flow cytometry, extracted with 0.01 N NaHCO3 or cultured in serum-free, defined media . The Mr of PRL extracted from cells immediately following dispersion ranged from 14-140K, with significantly more bands greater than 40K being detected from rams sacrificed in December than from those killed in October and April (P less than 0.01) . No bands of PRL greater than 25K were observed when samples were reduced with beta-mercaptoethanol prior to electrophoresis, indicating that the high Mr forms were disulfide-linked aggregates . Culture media from October and April contained variants of PRL that ranged from 22-40K but those greater than 25K were generally not observed from cells harvested during December . Extracts of cells after 24 h in culture contained fewer high Mr species during December than had been present in initial extracts from that month . In contrast, during April more high Mr intracellular forms were present after culture than had been detected prior to culture during that month . The percentage of lactotrophs averaged 50.0 +/- 2.5, 47.4 +/- 5.7, and 59.4 +/- 5.5 for October, December, and April, respectively . Initial lactotroph content (pg/lactotroph) of iPRL was higher (P = 0.06) in April (46.0 +/- 17.0) when compared to October and December (8.0 +/- 2.0 and 20.0 +/- 10.0, respectively) . In contrast, the bPRL content of initial extracts was higher (P = 0.05) in December (267.0 +/- 68.0) than in October (101.0 +/- 35.0), but not than in April (190.0 +/- 70.0) . Although iPRL and bPRL concentrations in culture media were similar for the 3 months, the intracellular iPRL (P less than 0.001) and bPRL (P less than 0.0001) content after culture was greatest during April . In summary, in addition to the well-documented seasonal changes in blood concentrations of PRL, different molecular forms of PRL were found within the pituitary at different times of the year and seasonal variations in iPRL and bPRL did not occur in parallel.

J Clin Endocrinol Metab, 1992 Feb, 74(2), 266 - 71
Calcium-regulated secretion of tissue plasminogen activator and parathyroid hormone from human parathyroid cells; Bansal DD et al.; The effects of Ca and other agents on secretion of plasminogen activator (PA) and PTH have been examined and compared, using parathyroid cells obtained from the glands of chronic renal patients . During 2 weeks culture at different {Ca}, the secretory rates of PA activity and PTH were parallel; steady-state secretion over 24-h periods was maximal at 0.5-0.9 mM Ca, minimal at 1.5-2.5 mM Ca, and the {Ca} at 50% suppression was 1.1 mM . At 2.5 mM Ca, two inhibitors of cellular proteolysis, 3-methyladenine and chloroquine, stimulated secretion of both PA activity and PTH . The results indicated that secretion of PA from human parathyroid cells is regulated similarly to that of PTH . The characteristics of human parathyroid PA were also examined using human parathyroid adenoma tissue . In homogenates, the highest specific activity of PA was in microsomal fractions . The Mr of PA from tissue and from culture media was 70 kilodalton by sodium dodecyl sulfate gel electrophoresis followed by zymography, or by Western blotting using antisera to human tissue PA (tPA) . Enzyme activity was inhibited by incubation with antisera to tPA but not to urokinase . In contrast to bovine parathyroid cells that secrete a urokinase, human parathyroids apparently contain and secrete tPA.

Zhonghua Bing Li Xue Za Zhi, 1992 Feb, 21(1), 8 - 10
{The protective effect of apolipoprotein C1 on endothelial cells injured by low density lipoprotein in vitro}; Liu QH; High serum low density lipoprotein (LDL) level is known injurious to endothelial cell (EC) and that high density lipoprotein (HDL) protects EC from such injury . The protective effect of HDL and apolipoprotein C1 (apo C1) on the endothelial cells (EC) isolated originally from human umbilical vein and injured by LDL was studied morphologically with phase-contrast and transmission electron microscope . Additionally, their functional changes were detected by measuring the amount of lactate dehydrogenase (LDH) and 6-keto-prostaglandin F1 alpha (PGF1 alpha) released . The EC were divided into four groups: control, HDL+LDL, apo C1 + IDL and LDL group . EC after being injured by LDL showed cell contraction, increased release of LDH and decreased secretion of PGF1 . Anyhow, they would be left normal on morphology, LDH release and PGF1 alpha synthesis if HDL or apo C1 had been added to the culture media before LDL injury . The results indicated that both HDL and apo C1 can resist the injurious effect of LDL on the cultured EC.

J Assist Reprod Genet, 1992 Feb, 9(1), 45 - 52
Effects of human sera and human serum albumin on mouse embryo culture; Leveille MC et al.; Human proteins normally used to supplement human in vitro fertilization-embryo transfer (IVF-ET) culture media were tested for their effects on mouse embryo development from the zygote stage . These proteins included follicular and luteal-phase maternal sera, fetal cord sera, and both human and bovine serum albumin . Our results revealed that both maternal and fetal cord sera did not permit mouse blastocyst formation . Furthermore, predialysis of the human maternal sera and removal of IgG by protein A column chromatography did not improve their support of mouse embryonic development to the blastocyst stage . Similar detrimental effects were observed with maternal sera from term-pregnant IVF-ET patients . Interestingly, these serum samples had supported the in vitro growth of the human zygotes which resulted in these patients' pregnancies . Only some batches of human serum albumin supported mouse blastocyst formation, whereas all sources of bovine serum albumin were effective in this regard . These results raise the question of the suitability of the mouse embryo culture system as a quality control for the testing of protein supplements for human IVF-ET.

Mol Cell Endocrinol, 1992 Feb, 83(2-3), 105 - 15
Testicular Leydig cells in vitro secrete only inhibin alpha-subunits, whereas Leydig cell tumors can secrete bioactive inhibin; de Winter JP et al.; The secretion of inhibin and inhibin-related proteins by testicular Leydig cells was studied by estimation of inhibin immunoreactivity and bioactivity in spent media of preparations of immature and mature rat Leydig cells and of tumor Leydig cells . Immature and mature rat Leydig cells expressed inhibin alpha-subunit mRNA and secreted immunoreactive inhibin . The immunoreactive material did not contain inhibin bioactivity as measured by an in vitro rat pituitary bioassay system . Results of pulse labeling with {35S}methionine followed by immunoprecipitation indicated that the inhibin-related proteins secreted by the immature Leydig cell preparations are 26 kDa and 44 kDa molecules . Mature rat Leydig cells only secreted the 44 kDa inhibin-related protein . Tumor Leydig cells (rat H540 and mouse MA10) secreted immunoreactive and bioactive inhibin, which could be immunoneutralized by an antibody against inhibin . In the culture medium of some H540 tumor Leydig cells 26 kDa and 42 kDa inhibin-related proteins and 30 kDa inhibin were detected . In culture medium of other H540 tumor Leydig cells, not secreting bioactive inhibin, only 26 kDa and 42 kDa inhibin-related proteins were found . No activin bioactivity was detected in culture media of immature rat Leydig cells, H540 and MA10 tumor Leydig cells . It is concluded that normal Leydig cells secrete inhibin alpha-subunits, while Leydig cell tumors can also secrete bioactive inhibin . Neither normal Leydig cells nor Leydig cell tumors produce activin.

Am J Respir Cell Mol Biol, 1992 Feb, 6(2), 158 - 67
Airway epithelial cell mucin release: immunologic quantitation and response to platelet-activating factor; Rieves RD et al.; Mucus production is an integral component of airway mucosal inflammation . Platelet-activating factor (PAF) is a phospholipid mediator implicated in the pathogenesis of many inflammatory processes, including airway inflammation . PAF functions as a mucus secretagogue when mucus is quantitated as radiolabeled glycoconjugates released from airway organ cultures . To more directly assess the interaction of PAF and airway epithelial mucous cell secretion, we used primary feline tracheal epithelial cell cultures and an immunoassay for a specific mucous cell secretory vesicle component . Cultured tracheal epithelial cells were shown to synthesize and secrete glycoconjugates with mucin characteristics . These mucin-type glycoconjugates were immunoreactive with a mucous cell-specific antibody . Localization of this antibody to components of the secretory vesicles of cultured epithelial cells was confirmed by electron microscopic immunogold labeling . Using this monoclonal antibody, an immunoassay was developed to quantitate release of immunoreactive material into cell culture media . Exposure of cultures to PAF produced a concentration-dependent, prompt release of immunoreactive material . Concentration-dependent inhibition of this effect was demonstrated by coincubation with the PAF receptor antagonists, WEB 2086 and Ro 19-3704 . A component of the signal transduction pathway for PAF effects was studied in cultured tracheal epithelial cells by coincubation of PAF with nordihydroguaiaretic acid (NDGA), a combined lipoxygenase and cyclooxygenase inhibitor, or p-bromophenacyl bromide (BPB), an inhibitor of cellular arachidonic acid release . Both NDGA and BPB blocked PAF-stimulated mucin release in a concentration-dependent manner . These studies demonstrate a direct airway epithelial mucous cell secretagogue effect that appears to be dependent upon airway epithelial PAF receptors and altered cellular lipid metabolism . These findings suggest a direct and potent mechanism for goblet cell secretion during airway inflammation.

J Biomech Eng, 1992 Feb, 114(1), 149 - 53
The effect of storage on the biomechanical behavior of articular cartilage--a large strain study; Kwan MK et al.; The transplantation of stored shell osteochondral allografts is a potentially useful alternative to total joint replacements for the treatment of joint ailments . The maintenance of normal cartilage properties of the osteochondral allografts during storage is important for the allograft to function properly and survive in the host joint . Since articular cartilage is normally under large physiological stresses, this study was conducted to investigate the biomechanical behavior under large strain conditions of cartilage tissue stored for various time periods (i.e., 3, 7, 28, and 60 days) in tissue culture media . A biphasic large strain theory developed for soft hydrated connective tissues was used to describe and determine the biomechanical properties of the stored cartilage . It was found that articular cartilage stored for up to 60 days maintained the ability to sustain large compressive strains of up to 40 percent or more, like normal articular cartilage . Moreover, the equilibrium stress-strain behavior and compressive modulus of the stored articular cartilage were unchanged after up to 60 days of storage.

J Virol Methods, 1992 Feb, 36(2), 129 - 40
Characterisation of visna virus reverse transcriptase: a micro scale reverse transcriptase assay adapted for use with an automated cell harvester; Sargan DR et al.; The reverse transcriptase of the sheep lentivirus visna/maedi virus has been characterised . Optima for magnesium ion concentration (5-10 mM), potassium ion concentration (150 mM) and pH (8.25) for this enzyme are very similar to those previously described for the human immunodeficiency viruses . The assay used for this work makes use of a cell harvester to speed up the processing of multiple samples . It is small scale, requiring 15 microliters of sample, is rapid, and is able to detect virus at titres below 10(3)/ml . Harvesting the assay onto either DEAE paper or using TCA and glass fibre mats make it suitable for use with either tissue culture media or infected cell lysates, but not with body fluids . It has been used to detect cell-associated reverse transcriptase in choroid plexus cells within 36 h of visna infection.

Lab Invest, 1992 Feb, 66(2), 143 - 51
Dynamic model of differentiation in Ewing's sarcoma cells . Comparative analysis of morphologic, immunocytochemical, and oncogene expression parameters; Noguera R et al.; We report the establishment of a model of neural differentiation in four well-characterized Ewing's sarcoma cell lines . This process was induced by serum-depleted medium (1% fetal bovine serum) and agents such as dibutyryl cyclic AMP and retinoic acid . The morphologic changes were characterized predominantly by the presence of neurite-like elongated processes showing varicosities and branching along their course with numerous internal filaments and electron-dense granules . Immunocytochemically, differentiation was accompanied by a considerable increase in reactivity for neural markers of several types: neuroblastic, neuroepithelial, neuroendocrine, Schwannian and even glial . In contrast, the tumor promoter, phorbol 12-myristate 13-acetate inhibited differentiation . Several morphologic changes were observed in phorbol 12-myristate 13-acetate-treated cells: the cells became smaller and rounder, were poorly adherent to substrate, by electron microscopy lacked cytoplasmic organelles, electron-dense granules or neural processes, and showed decreased expression of neural markers . Northern blot analysis was performed to establish whether there was any relationship between neural differentiation and degree of N-myc, c-myc and dbl oncogene expression . There was no N-myc oncogene expression in the mRNA of Ewing's sarcoma cells, even after neural induced differentiation . The degree of c-myc and dbl oncogene expression appeared heterogeneous, and varied with the culture condition . Based on these results, it may be inferred that Ewing's sarcoma cells in vitro display a variable neural phenotype, there being a variety of biologic responses to diverse culture media and various differentiation agents, but with no consistent effect on N-myc, c-myc and dbl oncogene expression.

Biochem Biophys Res Commun, 1992 Jan 31, 182(2), 644 - 50
Effect of extracellular phosphatidylinositol on c-myc gene-expressed human renal cancer cell line; Noguchi S et al.; Effect of exogenously added soybean phosphatidylinositol on c-myc gene expressed and unexpressed human cancer cell lines was investigated . When phosphatidylinositol liposomes were introduced into culture media, viability of c-myc unexpressed cells was reduced, while that of c-myc expressed cells was not . Death of c-myc unexpressed cells by phosphatidylinositol liposomes was found to be caused by abnormally accumulated intracellular Ca2+, and it seemed to be related to reduction of protein kinase C activity.

J Biol Chem, 1992 Jan 25, 267(3), 1576 - 83
Conversion of 1-O-alkyl-2-acyl-sn-glycero-3-phosphocholine to 1-O-alk-1'-enyl-2-acyl-sn-glycero-3-phosphoethanolamine . A novel pathway for the metabolism of ether-linked phosphoglycerides; Strum JC et al.; Madin Darby canine kidney (MDCK) cells convert 1-O-{3H}alkyl-2-acyl-sn-glycero-3-phosphocholine {( 3H}alkylacylGPC) to a product tentatively identified as an ethanolamine-containing phosphoglyceride (PE) (Daniel, L . W., Waite, B . M., and Wykle, R . L . (1986) J . Biol . Chem . 261, 9128-9132) . In the present study, analysis of the radiolabeled phosphoglycerides as diradylglycerobenzoate derivatives indicated that {3H} alkylacylGPC was initially converted to 1-O-{3H}alkyl-2-acyl-sn-glycero-3-phosphoethanolamine {( 3H}alkylacylGPE) which was subsequently desaturated to 1-O-{3H}alk-1'-enyl-2-acyl-sn-glycero-3-phosphoethanolamine {( 3H}alkenylacylGPE) . The conversion of {3H}/{32P}alkyl-lysoGPC to {3H}alkenylacylGPE indicated that base exchange enzymes were not involved in this pathway . A phosphono analog of alkyl-lysoGPC, resistant to phospholipase D hydrolysis and radiolabeled in the 1-O-alkyl chain was readily incorporated, acylated, and subsequently metabolized to {3H}alkylacylGPC and {3H}alkenylacylGPE . Therefore, the involvement of phospholipase D in the conversion pathway was ruled out . The conversion of {3H}alkylacylGPC or its phosphono analog to {3H}alkenylacylGPE was significantly enhanced by the addition of 100 microM ethanolamine to the culture media, suggesting that {3H}alkylacylglycerol is an intermediate in the cytidine-dependent pathway of PE synthesis . MDCK cell cytosol and microsomes contained no detectable phospholipase C activity . However, incubation of microsomes with CMP resulted in the degradation of {3H}alkylacylGPC and accumulation of {3H}alkylacylglycerol . Furthermore, the addition of CDP-ethanolamine to microsomes following preincubation with CMP, resulted in a decrease in {3H}alkylacylglycerol with a concomitant increase in {3H}alkenylacylGPE . Overall, these results suggest that the reverse reaction of choline phosphotransferase may be responsible for the conversion of alkylacylGPC to alkylacylGPE.

Biochem Biophys Res Commun, 1992 Jan 15, 182(1), 70 - 7
A heterozygous mutation (the codon for Ser447----a stop codon) in lipoprotein lipase contributes to a defect in lipid interface recognition in a case with type I hyperlipidemia; Kobayashi J et al.; Previously, we reported a case with type I hyperlipidemia due to a lipid interface recognition deficiency in lipoprotein lipase (LPL) (1) . The LPL from postheparin plasma of this patient did not hydrolyze TritonX-100-triolein or very low density lipoprotein-triolein but did hydrolyze tributyrin and LysoPC-triolein substrates . Sequence analysis of the probands DNA revealed a heterozygous nucleotide change: a C----G transversion at position of 1595, resulting in changing the codon for Ser447 to a stop codon . Expression studies of this mutant LPLcDNA in Cos-1 cells produced and secreted considerable amounts of LPL mass in the culture media . The mutated LPL hydrolyzed much less TritonX-100-triolein than wild type LPL, whereas hydrolysis of tributyrin and LysoPC--triolein was the same with both the mutant and wild type LPL . These results suggest that this mutation might be responsible for the property of the LPL with a defect in lipid interface recognition in the type I patient we reported.

Microsc Res Tech, 1992 Jan 15, 20(2), 136 - 51
Utilization of electron microscopic techniques in the in vitro study of adenohypophysial function and regulation; Asa SL et al.; Morphologic studies of human adenohypophysial cells using immunocytochemistry and electron microscopy have characterized the hormone-producing cell types of the normal gland and pituitary adenomas . The classifications which have emerged allow more accurate clinicopathologic correlations than ever before, but have also raised new questions concerning cytogenesis, pathogenesis, and structure-function correlations . We report the results of studies which marry the conventional morphologic techniques of light microscopy, immunohistochemistry, electron microscopy, and ultrastructural immunocytology with functional analyses using tissue culture and radioimmunoassay of hormones released into culture media . The hormone secretory activity of nontumorous and adenomatous pituitary cells is correlated with their structural features; their secretory responses to several adenohypophysiotropic factors are compared with morphologic alterations which are characterized at the light and electron microscopic levels by morphometric analysis . These studies have shown that hypothalamic stimulating hormones increase hormone release by their target cells and alter the ultrastructural appearance of the affected cells by increasing organelles involved in hormone synthesis . Inhibitory drugs and adrenal and gonadal steroids are capable of suppressing hormone release by some tumors and also give rise to morphologic changes which correlate with the functional inhibition . Hormone release by clinically nonfunctioning adenomas has been characterized and the behavior of these tumor cells in vitro sheds some light on the reasons for lack of clinical symptomatology . The plurihormonal nature of several nontumorous and adenomatous pituitary cell types has been characterized in vitro . The results of these studies provide the basis for more accurate structure-function correlations which can be used to study the hormonal milieu in vivo, to predict the role of pathogenetic factors in pituitary tumorigenesis, and to assess the therapeutic value of stimulating or inhibiting hormones and drugs.

J Invest Dermatol, 1992 Jan, 98(1), 45 - 9
Culture of basal cell carcinoma; Brysk MM et al.; Studies of basal cell carcinoma have been hindered by a lack of a suitable and reproducible tissue-culture model system . We have succeeded in growing this tumor in primary culture from eight different patients . We can separate and grow the tumor cells and the stromal cell component . We also culture normal keratinocytes and fibroblasts from the same patient for comparative studies . All the cell types have been subcultured four to five times and cryopreserved . The normal keratinocytes were indistinguishable from the tumor cells in ploidy, in rate of growth, and in the failure to express ICAM-1 . Both cell types also fail to synthesize the matrix proteins: types I and IV collagens . Differences were noted in the expression of fibronectin and the bullous pemphigoid antigen, with the normal cells expressing the antigens although the tumor cells did not . Interferons exogenously added to the culture media preferentially killed the basal cell carcinoma cells, as compared to normal keratinocytes from the same patients . We believe that our culture system opens possibilities for biochemical and molecular studies of this disease, and for in vitro testing of antitumor agents for clinical therapy.

DNA Cell Biol, 1992 Jan-Feb, 11(1), 21 - 30
Primary structure and characterization of the precursor to human pituitary adenylate cyclase activating polypeptide; Ohkubo S et al.; Human cDNAs encoding the precursor to pituitary adenylate cyclase activating polypeptide (PACAP) were cloned from human testis and cerebral cortex cDNA libraries . Nucleotide sequencing revealed that cDNA from the testis library encoded the entire precursor for PACAP, while cDNA from the brain library represented only the carboxy-terminal half of the precursor . The predicted human PACAP precursor consisted of 176 amino acid residues and was very similar to the ovine one (82%) . Both human and ovine precursors contained both PACAP and another peptide, PACAP-related peptide (PRP), having 29 amino acids . PACAP and PRP were preceded and followed by paired basic amino acids, recognized as important for post-translational processing . The PACAP precursor resembles the vasoactive intestinal peptide (VIP) precursor, which contains VIP and peptide histidine methionine/isoleucine amide (PHM/PHI) . Structurally, PRP had some similarity to PHM/PHI, growth hormone-releasing hormone (GRH) and PACAP . Northern blot analysis indicated that a 3.0-kb transcript was expressed in the ovine hypothalamus . Tissue distribution of PACAP mRNA was also clarified in the rat . Southern blot analysis of human genomic DNA gives single bands with six restriction enzymes, indicating that a single copy of the PACAP gene is contained in a haploid genome . The cDNA for human PACAP precursor was expressed using COS-7 and Chinese hamster ovary (CHO) cells . Immunoreactive PACAP was secreted into the culture media of both transfected cell lines.

J Clin Invest, 1992 Jan, 89(1), 46 - 52
Interleukin 6 . A potential autocrine/paracrine factor in Paget's disease of bone; Roodman GD et al.; Pagetic osteoclasts are greatly increased in number and size and have increased numbers of nuclei per cell compared to normal osteoclasts . The mechanisms responsible for enhanced osteoclast formation in Paget's disease are unknown . We have used our recently described model system for pagetic osteoclast formation to evaluate culture media conditioned by these atypical multinucleated cells (MNC) to determine if pagetic osteoclasts produce an autocrine or paracrine factor that enhances osteoclast formation . Conditioned media from long-term bone marrow cultures from patients with Paget's disease stimulated osteoclast-like MNC formation in normal marrow cultures . At least part of this activity could be ascribed to interleukin 6 (IL-6) . In contrast, conditioned media from normal marrow cultures contained lower levels of IL-6 and did not stimulate formation of osteoclast-like MNC . 7 of 8 bone marrow plasma samples taken from involved bones and 18 of 27 peripheral blood serum samples from Paget's patients had high levels of IL-6 . Normal marrow plasma and peripheral blood serum had no or very low levels of IL-6 . These results suggest that IL-6 produced by marrow and/or bone cells in patients with Paget's disease may be an autocrine/paracrine factor for pagetic osteoclasts.

Nihon Kyobu Shikkan Gakkai Zasshi, 1992 Jan, 30(1), 66 - 75
{Eosinophil colony stimulating factor production by lymphocytes from patients with bronchial asthma}; Kamei T et al.; To investigate the eosinophilia in patients with bronchial asthma (BA), we examined the release of eosinophil colony stimulating factor (Eo-CSF) from blood mononuclear cells (MNC) and lymphocytes from BA . We also investigated the effect of specific antigens on the release of Eo-CSF to determine its relation to other known Eo-CSFs . 1) Phytohaemagglutinin (PHA) and interleukin (IL)-2 at optimal concentrations stimulated mononuclear cells from BA, and induced Eo-CSF release . In contrast, MNC from normal volunteers released Eo-CSF with only PHA, but did not release Eo-CSF with IL-2 . 2) Lymphocytes from BA who were sensitive to house dust mite and Dermatophagoides farinae (D . farinae) antigens responded to specific antigens with Eo-CSF production, but those from normal volunteers did not . 3) The anti-IL-3, anti-IL-5, and anti-granulocyte-macrophage (GM)-CSF antibody inhibited Eo-CSF activity in culture media of lymphocytes from BA . These results indicate that the increase in responsiveness of lymphocytes from BA to specific antigens and cytokines produced by T cells play an important role in the induction of eosinophilia in BA.

Blood Cells, 1992, 18(1), 75 - 88; discussion 88-9
Inactivation of viruses with photoactive compounds; Matthews JL et al.; The transmission of human immunodeficiency virus (HIV-1) and other enveloped virus by blood transfusion is a major concern . Photosensitive dyes such as hematoporphyrin derivative (HPD), dihematoporphyrin ether (DHE), benzoporphyrin derivatives (BPD), extended ring porphyrins, sapphyrins and texaphyrins, and various cyanines were used with viral cultures to test the feasibility of using those light-excitable dyes to kill virus . A photodynamic flow cell was used to irradiate viral suspensions or viral infected cells in culture media or in whole blood . Herpes virus (HSV-1) was used to screen compounds . Effective compounds were subsequently tested for their ability to kill HIV-1, CMV, and SIV in culture medium and in blood and proved to effectively kill free virus and infected cells at significant viremias . Irradiation was achieved with a filtered xenon light source and/or tunable dye laser . Concentrations of dyes at 10 times viral kill dose were irradiated in blood which was tested for damage to erythrocytes (RBC), platelets, and blood proteins . No damage to RBC, complement factors, and immunoglobulins was evident immediately after photodynamic treatment . Platelet condition is minimally modified with time . Photodynamic treatment of blood appears to be a feasible means of eradicating virus and some protozoans from blood.

Gynecol Obstet Invest, 1992, 33(3), 129 - 33
Effects of prolactin on secretion and synthesis of human chorionic gonadotropin in human term placentas in vitro: short-term increase in secretion, followed by medium-term suppression of synthesis and secretion; Wurfel W et al.; We studied the influence of human prolactin on the secretion and de novo synthesis of human chorionic gonadotropin (hCG) in the human term placenta in culture . Placental tissue from 14 patients with uncomplicated pregnancies and deliveries was prepared mechanically, with addition of a Percoll gradient step . hCG levels were determined in the culture media and in the cytosolic fraction of cells by means of an enzyme immunoassay with coated beads . The amount of newly synthesized hCG was measured by the extent of incorporation of 35S-methionine into the hCG molecule . Our results showed that human prolactin had two different effects in vitro: between 1/2 and 1 h, prolactin slightly increased secretion of hCG into the culture medium without affecting de novo synthesis; after 2 h, prolactin began to cause a significant decrease in both secretion and de novo synthesis of hCG over several hours . It appears that both effects are receptor mediated, for ovine prolactin failed to produce any response . We conclude that prolactin is one of the main factors regulating the synthesis and secretion of hCG in the human trophoblast at term.

Thymus, 1992, 19 Suppl 1, S71 - 8
Modulation by ST 789 of in vitro lymphocyte activation and cytokine production; Famularo G et al.; Our results indicate that ST 789 exhibits complex immunomodulant properties . In fact, we found that ST 789 inhibits the expression of activation antigens, such as interleukin-2 and transferrin receptors by peripheral blood mononuclear cells (PBMCs) from healthy subjects following mitogen stimulation, but we were not able to detect under the same experimental conditions any effect on the in vitro production of soluble CD8 antigen and of interleukin-4 as well as on the proliferative response of antigen-specific and autoreactive human T cell lines . Finally, we showed that ST 789 is able to strongly enhance the in vitro production of interleukin-6 by PHA-stimulated PBMCs . The reduced expression of activation antigens in the presence of ST 789 does not seem to be mediated by CD8+ suppressor T lymphocytes, as indicated by the normal soluble CD8 levels in culture media, but rather reflects a direct inhibitory action on T helper proliferation and likely on interleukin-2 secretion . The strong enhancement by ST 789 of the in vitro interleukin-6 production seems to indicate the most relevant possibility of clinical applications in human diseases.

Toxicology, 1992, 72(3), 315 - 27
Importance of hepatocyte culture conditions in dimethylnitrosamine-induced suppression of antibody response in the mixed cultures of murine hepatocytes and splenocytes; Jeong TC et al.; The role of the hepatocyte culture media in dimethylnitrosamine (DMN)-induced suppression of antibody responses by splenocytes against sheep erythrocytes (SRBCs) was investigated using the mixed culture system of murine hepatocytes and murine splenocytes . It was observed that hormone-supplemented complete media was required for hepatocyte cultures to optimally activate DMN to its immunosuppressive form(s) . In the absence of the hormone supplement, the concentration of DMN required to produce a 50% suppression (IC50) was increased by over 10-fold (i.e., compare the IC50 in complete media of less than 0.5 mM to the IC50 in basal media of almost 10.0 mM) . In contrast, the activation of cyclophosphamide (cytoxan, CTX), which was used in these studies as a comparative control, was not affected by the absence of the hormone supplement . These results indicate that the observed effect on the activation of DMN was not due to a generalized loss of metabolic capability of hepatocytes cultured without hormones . To examine the role of drug metabolizing capabilities of hepatocytes in the differential activation of DMN, we compared phase I and phase II enzyme activities of hepatocytes cultured for 24 h in either basal media or hormone-supplemented complete media . Our results indicated that there was a significant decrease of phase I monooxygenase activities of cultured hepatocytes when compared to freshly isolated hepatocytes . However, our results failed to show any difference in the activities of hepatocytes cultured in the two media . Most notably, there was no difference in the activity of either high- or low-affinity DMN demethylase, as measured by the generation of formaldehyde . We observed a similar profile with phase II conjugative capabilities, specifically sulfotransferase and glucuronyltransferase . These results indicate that the activation of DMN to its immunosuppressive form(s) can be modulated in the co-culture system by culturing hepatocytes under different conditions . Because we failed to show any differences in the metabolic capabilities of hepatocytes cultured under the two media conditions, the results suggest that the modulation of immunosuppressive activity may not be related to a change in the generation of the immunosuppressive metabolite(s).

Mol Reprod Dev, 1992 Jan, 31(1), 72 - 7
Effect of oocyte maturation medium on in vitro development of in vitro fertilized bovine embryos; Rose TA et al.; The objective of this study was to examine the effects of different culture media used for maturation of bovine oocytes on in vitro embryo development following in vitro fertilization . Oocytes were aspirated from 2-5 mm follicles of ovaries collected at a local abattoir . The oocyte-cumulus complexes (OCCs) were cultured for 23-25 h in one of seven commercially available media supplemented with 6 mg/ml bovine serum albumin (BSA), 0.25 mM pyruvate, 10 micrograms/ml luteinizing hormone (LH), 0.5 microgram/ml follicle-stimulating hormone (FSH), and 1 microgram/ml estradiol . After maturation for 23-25 h, all eggs were subjected to the same in vitro fertilization protocol using modified TALP medium and subsequently cultured in the same serum-free embryo culture medium (HECM-1/BSA) for 8 days, after which embryo development was assessed . Five media (SFRE, MEM alpha, TCM199, MEM alpha/+, RPMI:MEM alpha) better supported normal oocyte maturation as determined by embryo development to the two-cell (76-82%), morula/blastocyst (25-32%), and blastocyst (12-19%) stages . Oocytes that were matured in Waymouth's medium MB 752/l or Ham's F-12 had a significantly reduced incidence of cleavage to the two-cell stage (52% and 37%, respectively), which was not attributed to failure of fertilization . Of the eggs that did cleave to the two-cell stage in these two media, 27% and 9% developed to morulae/blastocysts but only 6% and 3%, respectively, developed into blastocysts.(ABSTRACT TRUNCATED AT 250 WORDS)

J Reprod Fertil, 1992 Jan, 94(1), 33 - 43
Development and viability of bovine embryos derived from oocytes matured and fertilized in vitro and co-cultured with bovine oviducal epithelial cells; Xu KP et al.; A co-culture system using a suspension of detached bovine oviducal epithelial cells (BOEC) has been developed as an effective culture method for supporting the development of bovine embryos derived from oocytes matured and fertilized in vitro . Four commercially available culture media (Waymouth's, Ham's F-10, TCM 199 and Menezo's B2) supplemented with 10% oestrous cow serum, and a modified Tyrode's medium (TALP) supplemented with 0.6% bovine serum albumin were used . Menezo's B2 resulted in the highest percentages of total uncleaved presumptive zygotes, and of the cleaved zygotes that reached at least the morula stage (31-46% and 66-74%, respectively) . The embryos produced in vitro in B2 with BOEC resembled embryos produced in vivo with regard to numbers of cells (averaging 45.4 in morulae, 101.5 in blastocysts, 174.7 in hatching blastocysts and 195.9 in hatched blastocysts), rate of development (hatching on Day 8-9 of culture in vitro), rate of hatching (66% of cleaved zygotes) and pregnancy rates (63%) resulting from the transcervical transfer of selected embryos.

J Reprod Fertil, 1992 Jan, 94(1), 189 - 201
Purification of ovine transferrin and study of the hormonal control of its secretion in enriched cultures of ovine Sertoli cells; Monet-Kuntz C et al.; Ovine transferrin (o-transferrin) was purified from sheep serum by fractionated precipitation with ammonium sulphate, ion-exchange chromatography on DEAE trisacryl and finally by affinity chromatography on Affigel blue to remove albumin . Ovine transferrin was identified by its apparent molecular weight in sodium dodecyl sulphate polyacrylamide gel electrophoresis and by its N-terminal amino-acid sequence . The procedure presented in this report permits the preparation of highly purified o-transferrin with a good recovery (52% of initial total immunoactivity) . An antiserum against o-transferrin was then raised in rabbits, using this highly purified preparation . A specific radioimmunoassay was set up using 125I-labelled o-transferrin . Its detection threshold (4 ng/ml) was low enough to measure o-transferrin in spent culture media of ovine Sertoli cells, which ranged between 15 and 600 ng/ml . Sheep seminiferous tubule cells, containing approximately 80% Sertoli cells, were cultured at a high density (1.5 x 10(6) cells/cm2) on a thin layer of reconstituted basement membrane . Kinetic studies showed that basal daily secretion of o-transferrin was reduced by half (-49%) between Day 1 and Day 2 of culture, and progressively decreased thereafter . Under FIRT (500 ng ovine follicle-stimulating hormone (FSH)/ml + 10 micrograms insulin/ml + 500 ng retinol/ml + 5 x 10(-7) mol/l testosterone) stimulation, the ratio of stimulated to basal secretions increased 11-fold between Day 1 (1.1) and Day 6 (12) . When 10% fetal calf serum was added, mean o-transferrin secretion was a third of that in serum-free medium, suggesting that fetal calf serum contains factors that inhibit secretion of ovine Sertoli cell transferrin . In the presence of serum, the ratio of FIRT-stimulated to basal secretions doubled between Day 1 (1.0) and Day 4-6 (2.0) . Between Days 2 and 4 of culture, insulin had a slight stimulatory effect on o-transferrin secretion (128% of control at 10 micrograms insulin/ml), as well as epidermal growth factor (124% of control at 50 ng/ml) . Testosterone at up to 5 x 10(-7) mol/l had no effect; 500 ng retinol/ml doubled o-transferrin secretion (218% of control) as did 500 ng FSH/ml (220% of control) . A combination of retinol and FSH increased the secretion 4-fold, indicating that maximal stimulation of o-transferrin secretion by ovine Sertoli cells requires the combined actions of mechanisms dependent and independent of cAMP.

Arch Androl, 1992 Jan-Feb, 28(1), 53 - 9
Immunosuppressive activity in culture media containing human oocytes fertilized in vitro; Pinkas H et al.; A possible cause for implantation failure following embryo transfer is the rejection of pre-embryos by the maternal immune system . To elucidate the mechanisms underlying the rescue of the pre-embryo from maternal rejection, immunosuppressive activity was investigated in culture media containing human oocytes fertilized in vitro by employing the graft-versus-host reaction and the active rosette test . Normal donor lymphocytes were incubated with culture medium containing either fertilized or nonfertilized oocytes . Control experiments were carried out using lymphocytes incubated in culture medium only, as well as in culture medium containing human sperm . In 67% of the tests (12 out of 18), graft-versus-host reaction was inhibited, as compared to 29% of those performed with medium in which oocytes failed to fertilize (p less than .03), and 33% with medium in which only sperm was present . The frequency of an inhibitory active rosette test was similar in both groups . It is possible that immunosuppressive properties are acquired by the human pre-embryo as it grows in culture . Detection of such substances, which could contribute to the establishment of pregnancy, may improve monitoring of embryo quality prior to transfer.

Antisense Res Dev, 1992 Fall, 2(3), 211 - 22
Cellular uptake and subcellular distribution of phosphorothioate oligonucleotides into cultured cells; Iversen PL et al.; A phosphorothioate oligonucleotide that has been employed to inhibit HIV-1 viral expression in chronically infected H9 cells was examined for cellular uptake and subcellular distribution . The relationship between extracellular oligonucleotide concentration and the distribution and accumulation into subcellular organelles is important to the design, potential side effects, and understanding of a therapeutically useful antisense oligonucleotide . These studies employed uptake of both 35S- and fluorescence-labeled phosphorothioate oligonucleotides . Experiments with V79, HeLa, H9, and fresh human peripheral blood monocytes indicate that accumulations of oligonucleotide inside cells exceeds the concentration of oligonucleotide in culture media by over 100 times following 1 h of exposure at 37 degrees C . Uptake is more efficient at low concentrations, suggesting a saturable process . The total oligonucleotide that remains in cells begins to reach a plateau after 45-60 min, indicating either that efflux pathways exist or that uptake is saturable . Subcellular fractionation studies with 35S-labeled phosphorothioate demonstrate the oligonucleotide is sequestered into both the nuclei and the mitochondria of cultured HeLa cells in a time-dependent manner . The subcellular fractionation was examined with fluorescence-labeled phosphorothioate by both confocal and fluorescence microscopy, which confirmed the rate and localization of oligonucleotide into cultured cells . Finally, cellular uptake is not uniform for all cells in a nonsynchronous culture.

Dev Biol Stand, 1992, 76, 69 - 82
Integration and stability of CHO amplicons containing plasmid sequences; Wurm FM et al.; Fluorescence in situ hybridization (FISH, 15) and high density, non-selective long term perfusion culture were used to study aspects of genetic stability and productivity in recombinant CHO cells . We analysed the distribution and structure of recombinant amplicons in chromosomes of CHO cells used for the production of proteins . In the presence, but not in the absence, of methotrexate (MTX) we found a high proportion of cells (40-60%) with multiple and/or unusually structured and extended chromosome regions containing amplified sequences . Removal of MTX from culture media resulted in the rapid decline in the frequency of cells containing amplified sequences exhibiting multiple and heterogeneous integrations . In cloned lines cultivated in the absence of MTX, a well defined signal motif on a specific chromosome, interpreted as the "master integration" unit, became increasingly abundant over time until almost all cells contained that signal motif . We used fluorescence in situ hybridization (FISH) with chemically modified DNA probes complementary to the integrated sequences to verify the stability of master integrations in cells cultured for extended periods in medium lacking MTX . In a second approach, we studied long-term stability of recombinant sequences during non-selective perfusion culture of CHO populations with non-cloned, MTX resistant heterogeneous populations of cells producing CD4IgG chimeric molecules . Due to the mode of transfection and primary selection the resulting cell lines consisted of subpopulations of cells derived from various independent integration events . The amplification and MTX selection procedure used consecutively generated multiple, structurally different amplicon types in the cell population, thus increasing the degree of heterogeneity . High density perfusion culture of these cells in the absence of MTX was used to maximize growth rates and was thought to select against cells with reduced growth rates, due to the highly amplified state of their introduced DNA, with concomitantly high productivity . However we found no evidence for such a selection; cells showed no reduction in copy number or total loss of amplified sequences at the end of the culture . More significantly, specific productivity of these cell lines grown under non-selective conditions did not decrease over the 100 days observation period.

Dev Biol Stand, 1992, 76, 319 - 24
Insect cells: adventitious agents; Vaughn JL; The varied associations between insects and micro-organisms provides ample opportunity for the contamination of insect cell cultures . As with cell cultures from vertebrates, the addition of serum or serum products into cell culture media risks the introduction of mycoplasma and bovine viruses into cell cultures . Rarely do these organisms persist or multiply in insect cultures; however, there have been reports of the growth of Acholeplasmas introduced with fetal bovine serum . The contamination of cultures with plant or mammalian pathogens that are transmitted by insects is more common . The micro-organisms that are associated with insects, either as pathogens or commensals, frequently contaminate primary cell cultures and may also contaminate cell lines.

Ger J Ophthalmol, 1992, 1(1), 58 - 61
The effects of basic fibroblast growth factor on bovine retinal pigment epithelium in vitro; Esser P et al.; Basic fibroblast growth factor (bFGF) has been reported to initiate DNA synthesis in a variety of cells involved in the pathogenesis of proliferative vitreoretinal disorders, e.g., glial cells and fibroblasts . We analyzed the mitogenic effects of bFGF on cultured bovine retinal pigment epithelial cells in relation to time and dose response regulations and culture conditions . Maximum stimulatory effect of bFGF (+70% compared to control group) was found on day 3 following treatment of cultures with 80 ng/ml bFGF . The action of bFGF seems to depend on the serum concentration in the culture media, which means that cofactors may be present in the serum and potentiate the effects of bFGF on cell proliferation.

Acta Vet Hung, 1992, 40(1-2), 39 - 45
Comparative studies on in vitro mitogen-induced proliferation of peripheral blood lymphocytes in dog and breeding fox; Kostro K et al.; In vitro blastogenesis of dog and fox lymphocytes was compared by a microculture technique . The highest 3H-thymidine incorporation in cultures of dog lymphocytes was observed at day 3, while in those of fox at day 2, incubated either at 37 degrees C or at 39 degrees C . Lymphocytes cultured at 39 degrees C incorporated more tritiated thymidine than did cells cultured at 37 degrees C . The stimulation index (SI) of dog peripheral blood lymphocytes to both mitogens concanavalin A (Con A) and leucoagglutinin (LA) was in a similar range, while pokeweed mitogen (PWM) showed a weaker but significant stimulatory action . The blastogenesis of fox lymphocytes was the greatest in Con A stimulated cultures . The mitogenic potency of LA and PWM was about half of that of Con A, with no essential difference between them . Maximum lymphocyte proliferation of dog and fox was observed when culture media were supplemented with 10% fetal calf serum (FCS).

Breast Cancer Res Treat, 1992, 24(1), 35 - 41
A bioassay for antiestrogenic activity--potential utility in drug development and monitoring effective in vivo dosing; DeGregorio M et al.; Monitoring effective antiestrogenic activity of the triphenylethylenes in patients with breast cancer is usually determined by the duration of response . The pharmacokinetics of toremifene and tamoxifen have been shown to be highly variable but patient specific . In the present study, we developed a method to accurately assess the antiestrogenic activity of these agents using plasma specimens, cell culture, and cell cycle measurements . Plasma specimens (4-5mls) obtained from patients receiving toremifene (360mg/day for 5 days in a phase I trial) or tamoxifen (20mg/day) were extracted and reconstituted in tissue culture media (4-5mls), and growth inhibition was determined in estrogen responsive MCF-7 cells . Additionally, plasma specimens were quantified for toremifene or tamoxifen concentrations using HPLC . Growth inhibition of plasma specimens containing either toremifene or tamoxifen and their metabolites was also examined . Cell cycle measurements were determined following in vitro exposure with flow cytometric techniques . Our results show that a dose-response relationship exists between cell growth inhibition and cell cycle measurements for human plasma with added toremifene or tamoxifen, and also for human plasma specimens containing drug and its metabolites after treatment . Our antiestrogenic bioassay can address clinical research problems such as patient-specific pharmacokinetics, dosing compliance, and acquired antiestrogen resistance.

Reprod Fertil Dev, 1992, 4(4), 387 - 98
Metabolic and developmental responses of preimplantation embryos to platelet activating factor (PAF); Ryan JP et al.; Platelet activating factor (PAF) is an ether phospholipid produced by preimplantation embryos of a number of species . Production of PAF by embryos has been measured by detecting thrombocytopenia in a splenectomized mouse bioassay, platelet aggregation bioassays in vitro and a specific radioimmunoassay . Production is highly variable and is adversely affected by culture in vitro . It has, however, been correlated with morphology, development rates in vitro and the pregnancy potential of embryos following transfer . Investigations using PAF-antagonists have established an essential role for PAF in early pregnancy . Together with studies that have shown PAF to have direct effects on embryonic metabolism during culture in vitro, these observations suggest that PAF acts as an embryonic autocoid . Hence, a major site of action for embryo-derived PAF in vivo is the embryo itself . Supplementation of embryo culture media with PAF had no effect on the rate of development in vitro of 2-cell mouse embryos through to the blastocyst stage . However, PAF increased cell numbers of blastocysts cultured from the 2-cell stage and the mitotic index of embryos at both the 8-cell and blastocyst stages . Supplementation of culture media with PAF has also been shown to increase the implantation potential of both mouse and human embryos cultured in vitro . In the mouse, the effect of PAF in enhancing implantation rates was most evident when the developmental potential of untreated embryos was suboptimal . These observations suggest that the production of embryo-derived PAF is one limiting factor in maintaining the viability of embryos cultured in vitro.

Chin J Biotechnol, 1992, 8(1), 41 - 9
Production of monoclonal antibody in hollow fiber culture system with serum-free medium; Zhou W et al.; Hybridoma cells 7E8, secreting mouse monoclonal antibody (McAb) IgG against potato virus X, were cultured in hollow fiber cell culture system (module VF-2) with serum-free medium 1640SFM . The maximum viable cell density was found to be 2.34 x 10(6) cells/ml that was 3.7 and 2.2 times as high as those achieved in spinner and static flask culture respectively . About 10,000ml of cell culture media had been collected in the cultural period of 42 days . The ELISA titer of the McAb in the media was about 1:20,000 that was 25 times as high as those attained from the cultures in either spinner or static flask . From 1,000ml of cell culture media, an amount of 51.2mg of 7E8 McAb IgG had been purified by 50% saturated ammonium sulfate precipitation and Sephadex G-200 chromatography . The average yield of McAb in the hollow fiber cell culture system was 12.3mg/day . The results of the study suggest that it is hopeful for hybridoma cell culture in the hollow fiber cell culture system with serum-free medium for McAb production on large-scale.

Surg Today, 1992, 22(4), 333 - 8
The hemostatic effect of deacetylated chitin membrane on peritoneal injury in rabbit model; Fukasawa M et al.; In this study, we determined the effect of 80% deacetylated chitin (DAC-80) membrane on postsurgical bleeding after visceral and parietal peritoneal abrasion . Japanese white rabbits underwent a midline laparotomy followed either by a bilateral peritoneal sidewall abrasion (4 x 4 cm) or an abrasion of liver surface (3 x 2 cm) . The injured surface was then covered with a 0.2 mm thick DAC-80 membrane . On postsurgical day 2, the rabbits were sacrificed and the amounts of postsurgical bleeding was determined by quantitating the number of red blood cells recovered in 50 ml peritoneal lavage fluid . The DAC-80 membrane was found to reduce postsurgical bleeding after the abrasion of liver surface (treated with DAC-80 membrane: 2.9 +/- 0.8; control: 24.6 +/- 5.9 x 10(8) cells/peritoneal cavity, P less than 0.005) . This same hemostatic activity was not observed after application in the peritoneal sidewall abrasion model . We also measured plasminogen activator activity (PA) and urokinase inhibitory (PAI) activity in the spent culture media of macrophages recovered from the postsurgical peritoneal exudate . The DAC-80 membrane reduced the PA secretion from postsurgical macrophages after liver surface abrasion (treated with DAC-80: 2.8 +/- 0.7; control: 3.9 +/- 0.9 mPU/ml) . The DAC-80 membrane also showed similar effects on PA secretion after peritoneal sidewall abrasion . No significant effects were found in the secretion of PAI by postsurgical macrophages in both surgical models . These findings suggest that the DAC-80 membrane may have hemostatic activity through the modulation of fibrinolytic activity of peritoneal exudative macrophages.

J Cell Physiol, 1992 Jan, 150(1), 76 - 83
Monkey retinal pigment epithelial cells in vitro synthesize, secrete, and degrade insulin-like growth factor binding proteins; Waldbillig RJ et al.; Cultured monkey retinal pigment epithelial (RPE) cells rapidly secrete large amounts of insulin-like growth factor binding proteins (IGF-BPs) . IGF-II tracer binding activity in conditioned media is two to three times greater than that of IGF-I . Under reducing SDS-PAGE conditions, 125I-IGF affinity-crosslinked binding protein (BP) is visualized as a broad band between 36 +/- 2.9 and 49 +/- 3.3 kDa . Because the electrophoretic mobility of the crosslinked BP is increased under non-reducing conditions (33-45 kDa), intramolecular sulfhydryl bonding may be present . Frequently, the radiographic band representing affinity-crosslinked binding protein exhibits a complex pattern of non-uniform densities that suggests structural or functional IGF-BP micro-heterogeneity . IGF-BPs synthesized by RPE also exhibit heterogeneity with respect to the absence or presence of oligosaccharide side chains . In particular, the larger, but not the mid-sized or smaller IGF-BPs exhibit side chains linked to the core protein with N-glycosidic linkage . None of the crosslinked IGF-BPs exhibit O-linked side chains . Long-term (12, 24, 48 hr) conditioning studies revealed that IGF-BP fails to accumulate in culture media beyond 12 hr, but that replacement of conditioned media with fresh media allows a second period of binding protein accumulation . Other short-term (12 hr) experiments indicate that, in fresh medium, the levels of IGF-BP increase during the first 6-8 hr and then remain stable . To examine the processes contributing to these steady state levels of IGF-BP, aliquots of 8-hr conditioned medium were removed from the cells and either frozen on dry ice or incubated at 37 degrees C for 16 hr . Importantly, it was found that incubation at 37 degrees C resulted in a near total loss of binding activity . This is the first report of IGF-BP degrading activity in a cell culture system . These findings indicate that 1) primate RPE cells rapidly secrete a complex mixture of N-glycosylated and non-glycosylated IGF-BPs, and 2) the steady state levels of secreted IGF-BP are tightly regulated at least in part through a concomitant IGF-BP inactivating activity . Cultured RPE cells may be of utility in examining the mechanisms of IGF-BP synthesis, secretion, and degradation at the cellular level.

Dev Neurosci, 1992, 14(5-6), 377 - 85
Release of neurotransmitters and neurosecretory substances during in vitro maturation of mouse hypothalamic cultures; Miranda-Contreras L et al.; The maturation of the neurosecretory activity of a hypothalamic nerve cell population grown in vitro, prepared from 10-day-old mice and cultured for 6 days, has been demonstrated in the present report . A low-molecular weight polypeptide of 30-kD was found to be released into the culture media during the 6-day period of incubation, as analyzed by SDS-polyacrylamide gel electrophoresis . Comparative electrophoresis of the in situ hypothalamic, neurohypophysis and cerebral cortex homogenates revealed the presence of a 30-kD protein component in both the hypothalamus and neurohypophysis but not in the cerebral cortex . The release of the 30-kD polypeptide into the incubation media indicates an expression of the neurosecretory activity of the peptidergic neurons of the hypothalamus during in vitro maturation . On the other hand, high pressure liquid chromatography with electrochemical detection showed appreciable quantities of released dopamine (DA), epinephrine and serotonin (5-HT) in the incubation media in which the neurons were allowed to differentiate . There was a steady release of DA during the 6-day incubation period, varying from 0.21 +/- 0.02 to 0.49 +/- 0.05 ng/mg protein . The epinephrine level increased progressively from day 1 to 6 of culture, from 3.73 +/- 0.57 to 12.08 +/- 1.81 ng/mg protein, respectively . The measured 5-HT level was 0.07 +/- 0.001 on day 2 and increased to 0.38 +/- 0.05 ng/mg protein on day 6 of culture . These data demonstrate the functional maturation of catecholaminergic, serotoninergic and peptidergic neurons in these rotary histotypic cultures of the mouse hypothalamus.

Res Commun Chem Pathol Pharmacol, 1992 Jan, 75(1), 39 - 48
Inhibition of fibroblast proliferation in L-valine reduced selective media; Lazzaro VA et al.; A selective cell culture medium, D-valine minimal essential medium (92 mg/l), has been developed to inhibit the proliferation of fibroblasts in cell culture (Gilbert & Migeon 1975) . Substitution of D-valine for L-valine prevents fibroblast growth due to the absence of D-amino acid oxidase in these cells . Most cell cultures require foetal bovine serum as an essential component of the culture media, however foetal bovine serum contains L-valine, negating the value of D-valine selective media . To overcome this difficulty, we have produced a modified selective media for cell culture, by the dialysis of foetal bovine serum and confirmed its ability to inhibit fibroblast growth whilst still allowing the proliferation of epithelial cells in culture.

J Egypt Public Health Assoc, 1992, 67(1-2), 163 - 9
Estimation of the infectivity of herpes simplex virus type I passage III on two different tissue culture; Arafa RM; This technique has been developed for the estimation of the infectivity of herpes simplex virus type I passage III on a tissue culture of Vero and HBK21 . The plaque assay method was performed and the number of plaques on the two culture media was counted per each dilution from (10(-5) to 10(-8)) . It was found that the number and the size of plaques in 10(-5) dilution are the best in the two types of tissue cultures . The number indicates the number of virus particle (per each dilution point), which can be used for infection of experimental animals to demonstrate its infectivity character.

Am Rev Respir Dis, 1992 Jan, 145(1), 85 - 91
Neutrophil chemotactic factors in the respiratory tract of patients with chronic airway diseases or idiopathic pulmonary fibrosis; Ozaki T et al.; This study was designed to clarify the contributions of specific neutrophil chemotactic factors (NCF) in neutrophil accumulation in the human respiratory tract associated with various diseases . The activity and characteristics of the NCF in the bronchoalveolar lavage (BAL) fluid and culture media of alveolar macrophages obtained from normal volunteers, control patients, patients with chronic airway diseases (CAD) and patients with idiopathic pulmonary fibrosis (IPF) were examined . The BAL fluid from normal volunteers contained NCF comparable with the chemotactic factors interleukin-8 (IL-8) and leukotriene B4 (LTB4) . Analysis of the biochemical characteristics of NCF released from alveolar macrophages suggests that they are derived from alveolar macrophages . The NCF activities in BAL fluids from patients with CAD and IPF were higher than those in BAL fluids from normal volunteers and control patients . Biochemical analysis demonstrated that several kinds of NCF, including those derived from the complement component C5 and alveolar macrophages, were present in the BAL fluid from patients with CAD and respiratory infections . The especially marked increase of C5-derived NCF indicate their importance in neutrophil accumulation in the respiratory tract of patients with CAD . Alveolar macrophages released different types of NCF after different lengths of culture periods (4 h and 24 h) . Alveolar macrophages from patients with IPF released larger amounts of NCF than alveolar macrophages from normal volunteers, indicating the importance of alveolar-macrophage-derived NCF as well as C5-derived NCF in neutrophil accumulation in the respiratory tract of patients with IPF . These results suggest that various types of NCF increase in response to different disease states of the respiratory tract and serve to regulate the accumulation of neutrophils.

Arch Virol, 1992, 122(1-2), 119 - 31
Cell fusion caused by herpes simplex virus-1 (HSV-1) strains tsB5 and MP is inhibited at pH 6.7 and pH 7.0; Baghian A et al.; We investigated the effect of different pH conditions on Vero cell cultures infected with herpes simplex virus-1 (HSV-1) wild-type strain KOS, and syncytial mutants HSV-1 HFEM (tsB5) and HSV-1 mp (MP) . Cell fusion was inhibited when infected cells were continuously incubated with culture media adjusted to pH 6.7 or pH 7.0 . Inhibition of cell fusion was rapidly reversible when infected cell cultures were returned to pH 7.5 . The rate of synthesis and cell-surface expression of virus-specified glycoproteins gB, gC, gD, and gH were not affected during continuous incubation at pH 7.0, but they were reduced at pH 6.7 in comparison to pH 7.5 . At later hours p.i . however, these glycoproteins continued to accumulate at all tested pH levels . Accumulation of infectious virions was substantially reduced for MP, KOS, and tsB5 at pH 6.7 . At pH 7.0, KOS and tsB5 titers were greatly reduced but MP titers were not affected.

Mem Inst Oswaldo Cruz, 1992 Jan-Mar, 87(1), 53 - 8
A medium for inducing conversion of Histoplasma capsulatum var . capsulatum into its yeast-like form; Fressatti R et al.; Histoplasma capsulatum var . capsulatum is a dimorphic fungus that, under special conditions, converts from its more common mycelial form to a yeast-like form . Achieving this conversion, however, has been problematical for researchers . The present study tested conversion rates in ten Histoplasma capsulatum var . capsulatum strains using seven culture media, four of which were conventional and three novel . One of our novel media, MLGema, induced complete conversion of two strains within five days of incubation at 35 degrees C, and of all strains that eventually converted by the time of the second subculturing transfer, under defined experimental conditions . MLGema is also inexpensive and easy to produce.

Connect Tissue Res, 1992, 28(4), 289 - 305
Isolation and characterization of an abundant elastase inhibitor from NaCl extracts of bovine nasal septa and articular cartilage; Homandberg GA et al.; Extracts of cartilage have been reported to inhibit many serine proteinases and metalloenzymes . Such inhibition may be important in protecting cartilage against degradation by chondrocytic proteinases such as collagenase, stromelysin and by leukocytic proteases, such as elastase . We report here isolation and partial characterization of a 17-kD elastase inhibitor from 0.5 M NaCl extracts of both nasal septum cartilage and articular cartilage, which inhibits elastase and represents 0.08% of the weight of nasal cartilage and 0.002% of the weight of articular cartilage . The protein was highly specific for elastase and did not inhibit cartilage metalloproteinases, suggesting that it may be mainly directed toward protecting cartilage against leukocytic proteases . The inhibitor had a blocked amino-terminus, was high in serine and glycine and lacked carbohydrate . The ease with which the inhibitor was extracted from cartilage suggests that it may function in vivo as a highly abundant elastase inhibitor which is secreted into synovial fluid from cartilage . The inhibitor was shown to be synthesized by bovine articular cartilage in explant culture and nearly all of the metabolically labeled material was secreted into the culture media . The inhibitor cross-reacted with polyclonal antibodies to bovine neck ligament alpha-elastin and antibodies to the inhibitor reacted with bovine neck ligament elastin . The properties of this inhibitor are different than those of any other reported cartilage derived inhibitor.

Reprod Fertil Dev, 1992, 4(5), 573 - 83
Evaluating an in vitro culture system of bovine uterine and oviduct epithelial cells for subsequent embryo co-culture; Thibodeaux JK et al.; Three experiments were conducted to evaluate the effects of culture medium and incubation temperature on bovine uterine and oviduct epithelial cell growth, so that the most efficient combination could then be used to develop a co-culture system for bovine embryos . In the first experiment, uterine and oviduct epithelial cells at either the second or third subpassage were incubated for 8 days at 37 degrees C with 5% CO2 in Tissue Culture Medium-199, CMRL-1066, Minimal Essential Medium, Menezo's B2 or Ham's F-12 medium . In addition to plotting growth curves of cell populations, the cell cycle was monitored for 8 days by flow cytometry . Uterine and oviduct epithelial cells incubated in CMRL-1066 exhibited the highest growth rates during the 8-day culture period . However, there were no differences in cell cycle analysis among treatment groups during the incubation period . In the second experiment, CMRL-1066 medium was used to evaluate growth and proliferation of uterine and oviduct epithelial cells incubated at 37 degrees C or 39 degrees C; temperature had no significant effect on growth rates or proliferation rates for either uterine or oviduct cells during the 8-day incubation . In the third experiment, the more promising culture media for epithelial cell culture studies were chosen for in vitro maturation and subsequent in vitro fertilization (IVF) of bovine oocytes . Early cleavage-stage embryos produced by IVF procedures were subsequently cultured in vitro for 7 days in medium alone or with oviduct epithelial cells . In this study, the culture medium did not influence fertilization or cleavage rates . However, more embryos co-cultured with oviduct epithelial cells were considered viable after 7 days of incubation compared with embryos incubated in medium alone . These results indicate that various incubation conditions can influence the growth of bovine uterine and oviduct epithelial cells in vitro . However, in spite of changes in cell growth patterns, there does not appear to be a change in their embryotropic capabilities in vitro.

J Immunol Methods, 1991 Dec 15, 145(1-2), 175 - 83
Commercial serum-free media: hybridoma growth and monoclonal antibody production; Mariani E et al.; Various growth factors, hormones and proteins present in serum are presumed to be responsible for its growth-stimulating activity in culture media . However, synthetic or serum substitute media supporting cell growth are advantageous when it is necessary to standardize culture conditions, particularly when cell products are used . In this study we have evaluated and compared the effects of some commercially available serum substitute media (10% NU Serum, 10% BMS, 2% Ultroser HY, 1% ITS + Premix, 1% Nutridoma, Ultradoma, FEB100, DCCM1 and DCCM2) on growth and immunoglobulin production in different hybridoma cell lines . Six different hybrids were studied: OKT3, OKT4 and OKT8 (producing monoclonal antibodies against CD3, CD4 and CD8 molecules), HB43 and HB57 (producing monoclonal antibodies against human IgG and IgM), and CRL 8019 (producing monoclonal antibodies against high-molecular weight CEA) . Several parameters were evaluated, such as viability, doubling time, DNA synthesis, monoclonal antibody production and cell cycle under different culture conditions . Since not all of the hybridomas grew equally well in the same serum substitute media, one synthetic medium cannot be used for all the lines . We found that the serum-free media that best supported hybridoma growth were Nutridoma, DCCM1, DCCM2, NU Serum and FEB100.

Prostaglandins, 1991 Dec, 42(6), 533 - 40
Effects of prostaglandin F2 alpha prostaglandin E2 and arachidonic acid on the induction of oviposition in vivo and in vitro in oviparous lizards; Guillette LJ Jr et al.; Gravid females of four different species of oviparous lizard were treated in vivo with varying doses of prostaglandin F2 alpha (PGF2 alpha), prostaglandin E2 or arachidonic acid (AA) . In contrast to previous studies examining birds and viviparous lizards, no dosage induced oviposition in any of the treated females . All females, however, did exhibit behaviors associated with oviposition . Intact oviducts removed from gravid females and placed in organ culture did oviposit when treated with 30 or 100 ng PGF2 alpha/ml of culture media . Arachidonic acid at similar concentrations also was effective in stimulating birth . These data suggest that prostaglandins can stimulate oviposition in oviparous lizards but further suggest that their action may be inhibited by oviducal innervation until just prior to natural birth.

FEBS Lett, 1991 Dec 2, 294(1-2), 23 - 6
Immunological identification and sequence characterization of a peptide derived from the processing of neuroendocrine protein 7B2; Paquet L et al.; A newly raised antiserum against the C-terminal region of neuroendocrine protein 7B2 was used to purify a novel peptide from the culture media of the mouse corticotroph cell line AtT-20 . Based on partial sequencing, this peptide, which we call Cter-7B2, begins at Ser156 and appears to result from the cleavage of pro7B2 after a five-basic-residue sequence . Thus, 7B2 processing may contribute to the diversity of peptides found in neuronal and endocrine cells.

Endocrinology, 1991 Dec, 129(6), 3321 - 30
pH-dependent cytotoxicity of contaminants of phenol red for MCF-7 breast cancer cells; Grady LH et al.; The pH indicator phenol red (phenolsulfonphthalein) is present in most tissue culture media . Contaminants of this indicator have shown substantial estrogenic activity for estrogen-dependent cells in culture, including the human breast cancer-derived MCF-7 cell line . In the course of other studies, we observed that brief (1- to 4-h) incubations of these cells at 37 C in serum-free medium (Hanks' or Earle's Balanced Salts Solution) could be toxic to MCF-7 cells when the pH was increased above 7.4, but only if phenol red (10 micrograms/ml) was present in the medium . Because damaged/killed cells detached from the substratum (greater than 98% of detached cells stained with trypan blue), we used DNA assay of the cells remaining after treatment and wash (98% of the remaining cells were dye excluding) to further assess cytotoxicity . The MCF-7 cells were more susceptible to the cytotoxicity at lower cell densities, so further characterization of phenol red cytotoxicity was performed at cell densities of 1-10 micrograms DNA/2-cm2 well, or approximately 40,000-400,000 cells/ml medium . In the pH range of 7.0-8.2, 50% cell death was observed in the presence of phenol red at pH as low as 7.6-7.7, with nearly 100% of the cells killed by pH 8.0 . Little effect was seen in phenol red-free medium at any part of the tested pH range or in medium that contained phenol red at pH less than or equal to 7.4 . In time-course studies of cytotoxicity at pH 8.0 (phenol red, 10 micrograms/ml), greater than 50% cell damage could be observed after less than 1 h, and little cell recovery was observed if the pH was restored to 7.4 . For phenol red samples from two major commercial sources, the concentration for half-maximal cytotoxicity (TD50) in dose-responses after 4 h at pH 8.0 showed TD50 values of 2 and 6 micrograms/ml, while the estrogenic activities, as half-maximal stimulation of estrogen-dependent proliferation, were identical at 2 micrograms/ml . Both the cytotoxic and estrogenic activities could be removed from the phenol red by extraction with diethyl ether . A number of contaminants of the commercial phenol red were detected by reverse phase C18 HPLC . Cytotoxicity and estrogen bioassays of each of the HPLC fractions indicated that the pH-dependent cytotoxicity was separate from the estrogenic activity and confirmed that neither activity was associated with the phenol red itself.(ABSTRACT TRUNCATED AT 250 WORDS)

Blood, 1991 Dec 1, 78(11), 2823 - 33
Production of erythropoietic bursts by progenitor cells from adult human peripheral blood in an improved serum-free medium: role of insulinlike growth factor 1; Correa PN et al.; Several culture media for the growth of human circulating erythroid burst-forming units (BFU-E) that have been claimed to be "serum-free" ("SF") have actually included albumin preparations known to be contaminated with an undefined burst-promoting activity (BPA); a BPA has also been found in the preparations of other "SF" medium components . This has precluded reliable investigation of the growth factor (GF) requirements of these progenitors . Using a defatted, BPA-free bovine serum albumin (BSA) and the recombinant human growth factors (GFs) erythropoietin (rHu Epo), insulinlike growth factor 1 (rHu IGF-1), and interleukin-3 (rHu IL-3), we have developed an improved serum-free (SF) medium for the production of erythroid bursts from normal adult human peripheral blood mononuclear cells (PBMNC), which requires both hemin and retinyl acetate for its optimal performance . In the presence of BSA without IL-3 or Epo, no burst or colony formation was observed . With IL-3 and Epo alone, only a small number of day 14 erythroid colonies was obtained (12 +/- 1/10(5) PBMNC) . Addition of hemin (0.1 mmol/L) allowed the direct scoring of day 14 hemoglobinized colonies and increased their number sevenfold (86 +/- 5) . Inclusion of retinyl acetate at physiologic concentrations further augmented the number of colonies threefold to fourfold . Under these apparently optimal conditions, we found that IGF-I could entirely replace Epo . However, IGF-I required a 100-fold higher molar concentration than that of Epo to reach maximal stimulation . The combined effect of Epo and IGF-I was found to be less than the sum of their individual effects, suggesting an overlap in the sensitivities of erythroid progenitors to these GFs . The colony-forming efficiencies of erythroid progenitors in the improved SF medium was very high: 700 single, day 14 erythroid colonies/10(5) PB MNC (at 0.25 mmol/L hemin) distributed as 126 clusters (bursts), with a mean of 5.6 component colonies per burst . These findings show that IGF-I has an Epo-like activity that targets circulating early erythroid progenitors or their progeny, providing strong evidence for the existence of an Epo-independent pathway for normal human adult erythropoiesis, possibly operative when Epo levels are low.

Yan Ke Xue Bao, 1991 Dec, 7(4), 176 - 80
{Retinoblastoma-culture in vitro and establishment of cell line}; Zheng J et al.; 60 cases of retinoblastoma cells have been cultured in vitro in recent 6 years . 10 of them grew more than 3 months, and one continuous retinoblastoma cell line (SO-Rb50) has been established for more than 3 years . In addition to the degree of malignancy of the tumor cells, selection of tumor tissues, components of culture media, optimal selection of seeding, passage and densities of cells play important roles in the establishment of immortal retinoblastoma cell line.

Steroids, 1991 Dec, 56(12), 578 - 80
19-Nordeoxycorticosterone synthesis by rat kidney inner medullary collecting duct cells; Azar ST et al.; 19-Nordeoxycorticosterone (19-nor-Doc), a potent mineralocorticoid, was found to be synthesized by the isolated rat kidney perfused by an adrenal precursor (19-oxo-Doc) . To determine if this bioconversion is a function of renal tubular cells, various adrenal precursors of 19-nor-Doc were added separately to rat kidney inner medullary collecting duct cells culture media at a concentration of 10 nM . While 4.6% +/- 1.0% of 19-oxo-Doc (n = 3) and 14.4% +/- 1.4% of 19-oic-Doc (n = 3) were converted to 19-nor-Doc after 24 hours of incubation, Doc, and 19-OH-Doc were not converted . This represents further evidence that Doc has to be metabolized to 19-oxo-Doc or 19-oic-Doc (19-carboxy-Doc) before it can be converted by the kidney inner medullary collecting duct cells to 19-nor-Doc.

Ann Trop Med Parasitol, 1991 Dec, 85(6), 591 - 7
Giardia lamblia: phospholipid analysis of human isolates; Mohareb EW et al.; Thin layer chromatograms for phospholipids obtained from 11 human Giardia lamblia isolates and their culture media have shown that phosphatidylcholine and sphingomyelin are the predominant phospholipid classes in all samples . A decrease in the relative percentage of the different classes, especially of phosphatidylcholine, was noticed in the medium after Giardia growth . Fatty acid analysis of the parasite phosphatidylcholine demonstrated that while oleate and palmitate were the major fatty acids in most isolates, arachidonate predominated in two of those studied . Some isolates contained small amounts of myristate, which was not present in the phosphatidylcholine of the culture medium . Moreover, stearate and linoleate predominated in phosphatidylcholine obtained from both media types . The saturated/unsaturated fatty acid ratio also varied for the different isolates . These results appear to suggest heterogeneity in the metabolic activity and utilization of lipid molecules between Giardia isolates.

Clin Lab Med, 1991 Dec, 11(4), 909 - 22
Laboratory diagnosis of Leishmania; Palma G et al.; Leishmaniasis should be considered in the differential diagnosis of individuals living in or with a history of having traveled to known endemic areas and who present with signs and symptoms of visceral infection or with cutaneous or mucosal lesions . Because leishmaniae are capable of producing a wide spectrum of disease in humans, the clinical manifestations overlap with many other diseases . Definitive diagnosis of Leishmania infection in the laboratory requires demonstration of the parasite in smears, in biopsies, or by isolation of the organism in culture media or in experimental animals . Many other methods for demonstration of parasites (histochemical and immunohistochemical) or for detecting the presence of antibodies against leishmaniae (serologic) have been described . Many advances have been made in these areas, but the methodology and the technology involved in immunohistochemistry and serology remain outside the reach of the standard clinical diagnostic laboratory, which both in developed and less developed countries still relies on demonstration of the parasites in smears stained with Giemsa stain and on biopsy specimens processed and stained with hematoxylin and eosin stain . The newer serologic techniques, such as ELISA with several variations, IFAT, and others, are largely research tools with the greatest use in seroepidemiologic surveys.

Atherosclerosis, 1991 Dec, 91(3), 229 - 40
Proteoglycans produced by cholesterol-enriched macrophages bind plasma low density lipoprotein; Owens RT et al.; Proteoglycans (PG) produced by {35S}sulfate and {3H}serine labeled cultures of cholesterol-enriched macrophages obtained from atherosclerosis-susceptible White Carneau (WC) and -resistant Show Racer (SR) pigeons were characterized and assessed for their capacity to bind low density lipoprotein (LDL) . The majority of 35S-labeled PG was released into the culture media in both WC and SR macrophage cultures and consisted of large and small size PG as determined by Sepharose CL-4B chromatography . Large PG were identified as chondroitin sulfate-PG comprised of 4-sulfated disaccharides whereas small PG consisted of primarily 4-sulfated chondroitin sulfate-PG and lesser amounts of heparin sulfate-PG . Experiments demonstrated that 32-34% of 35S-labeled large PG and 86-93% of small PG bound to LDL-substituted Sepharose . Interactions between PG and LDL-substituted Sepharose were inhibited in the presence of heparin or soluble LDL . Glycosaminoglycans derived from macrophage PG had a decreased binding affinity demonstrating the importance of an intact PG . The results suggest that macrophage PG may facilitate trapping of LDL in the intimal intima and promote foam cell formation through a mechanism involving the uptake of PG-LDL complexes.

J In Vitro Fert Embryo Transf, 1991 Dec, 8(6), 304 - 7
Synergy between tumor necrosis factor and endotoxin decreases early embryo development in vitro; Randall GW et al.; The purpose of the present study was to investigate the individual and combined effects of low levels of endotoxin and physiological levels of tumor necrosis factor on in vitro fertilization and preimplantation embryo development . B6D2F1 mice were superovulated by utilizing pregnant mare serum gonadotropin and human chorionic gonadotropin . Oocyte-cumulus complexes (3221 oocytes) were collected, pooled, and randomized into control and treatment groups . Sperm were collected from the caudae epididymides of mature male mice and allowed to capacitate . Treatments included culture media supplemented with increasing amounts of endotoxin (0.5, 1.0, and 2.0 ng/ml; sp act, 3000 IU/ng) and/or tumor necrosis factor (1, 10, and 50 pg/ml; sp act, 0.01 IU/pg) throughout the fertilization and preimplantation development process . Percentage cleavage and percentage expanded blastocyst formation were evaluated . No significant effects were observed for percentage cleavage or percentage expanded blastocyst formation in either the endotoxin (E) or the tumor necrosis factor (TNF) groups . The combination of endotoxin and tumor necrosis factor at any of the levels tested did not significantly decrease cleavage; however, percentage blastocyst formation was decreased with any combination of TNF and E (P less than 0.05-P less than 0.001) . We conclude that TNF and E exert significant synergistic effects which are detrimental to in vitro preimplantation embryonic development.

Am J Physiol, 1991 Dec, 261(6 Pt 1), L415 - 23
Effect of protein kinase C activating agents on respiratory glycoconjugate release from feline airways; Rieves RD et al.; Abnormal regulation of airway glycoprotein secretion may underlie many respiratory diseases . Experimental activation of the protein kinase C (PKC) family of cytosolic enzymes has been shown to induce a secretory response in many tissues . To estimate the effect of PKC activation on airway secretion, alteration in the amount of radiolabeled respiratory glycoconjugate (RGC) released into culture media was determined following feline airway explant exposure to PKC activating agents . Exposure to two known activators of PKC, phorbol 12-myristate 13-acetate (PMA) and mezerein (MEZ), resulted in profound increases in respiratory glycoconjugate release over a seven day experimental period . The response evolved over several hours and was dose dependent . Maximal RGC release, 90% above control, occurred 2 days after exposure to either PMA or MEZ . Pharmacological inhibition of the PKC effect using two PKC inhibitors, 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine and sphingosine, resulted in dose-dependent antagonism of the maximal PMA (10(-7) M)-stimulated RGC release, suggesting altered PKC activity was responsible for augmenting RGC release . Since altered arachidonic acid metabolism has been implicated in mediating some PKC effects, eicosanoids were assayed in airway explant supernatants following PMA exposure . Enhanced release of both cyclooxygenase and lipoxygenase pathway products was detected by radioimmunoassay . Cotreatment of explants with PMA and an inhibitor of oxidative arachidonic acid metabolism, nordihydroguaiaretic acid, blocked RGC release . These data demonstrate prolonged augmentation of respiratory glycoconjugate release from airway explants following exposure to PKC-activating agents.

J Steroid Biochem Mol Biol, 1991 Dec, 39(6), 975 - 86
In vivo and in vitro studies on sex steroid binding protein (SBP) regulation in rainbow trout (Oncorhynchus mykiss): influence of sex steroid hormones and of factors linked to growth and metabolism; Foucher JL et al.; The respective roles of sex steroids and hormones related to growth and metabolism, on SBP regulation have been studied in rainbow trout . In vivo, oestradiol (E2) supplementation induces a slow but significant increase of plasma SBP concentration . Testosterone or cortisol injections have no effect . In vitro, the steroid binding protein that accumulates in incubation medium of hepatic cell primary cultures has been characterized and found to be similar to blood SBP . Its production is increased by addition of E2 (maximum: +300%) . This effect develops slowly over several days of culture and is dose dependent; as little as 1-10 nM E2 is effective . Recombinant rainbow trout GH (rtGH)--0.01 to 1 microgram/ml--also increases SBP accumulation as compared to control cells and seems to maintain SBP production over culture duration . In preliminary experiments, (1) insulin-like growth factor (IGF) and SBP concentrations were found to change inversely after a 4 days stimulation with increasing concentrations of GH; (2) recombinant human IGF1 (250 ng/ml) tended to be inhibitory when SBP production was expressed per mg of total cellular protein, and a micromolar concentration of bovine insulin was clearly inhibitory . Other hormones tested in vitro: triiodothyronine (10-1000 nM), thyroxine (100 nM), 17 alpha, 20 beta-dihydroprogesterone (10-2000 nM), and testosterone (1-1000 nM) did not influence SBP concentration in hepatic cells culture media.

J Cell Physiol, 1991 Dec, 149(3), 536 - 43
Germ cell mitogenic activity is associated with nerve growth factor-like protein(s); Onoda M et al.; The mitogenicity of germ cell proteins released from round spermatids (RS) and pachytene spermatocytes (PS) was investigated . Germ cells were isolated by centrifugal elutriation from 90-day-old rat testes and incubated in a supplement enriched culture media that lacked exogenous proteins . The conditioned culture media of RS and PS were dialysed/concentrated and lyophilized to prepare RS protein (RSP) and PS protein (PSP) . Mitogenic activity of RSP and PSP was determined by 3H-thymidine incorporation into Swiss 3T3 fibroblasts . RSP and PSP stimulated 3H-thymidine incorporation by fibroblasts in a dose-dependent manner . At a higher concentration of RSP (300 micrograms/ml), fibroblast proliferation was stimulated from 6- to 20-fold of control cultures, whereas PSP (300 micrograms/ml) stimulated fibroblast proliferation 2.5-fold of control cultures . Since RSP exhibited substantially greater mitogenic activity than PSP we further investigated the RSP mitogenic substance(s) by immunoneutralization with antibodies against several growth factors . The mitogenic activity of RSP was significantly reduced by treatment with nerve growth factor (NGF) antibody, while neither the treatment of RSP with acidic fibroblast growth factor (aFGF) antibody, nor basic fibroblast growth factor (bFGF) antibody significantly modified the mitogenic activity of RSP . Interestingly, murine NGF-beta, recombinant human NGF-beta, and bovine serum albumin (BSA) did not exhibit mitogenic activity on 3T3 fibroblasts . Nevertheless, the presence of a NGF-like protein in RS and PS was confirmed by indirect immunofluorescence staining with a murine NGF antibody . Subsequently, a Western blot analysis with the NGF antibody identified two immunoreactive bands of 41 +/- 2 kDa and 51 +/- 1 kDa in both RSP and PSP under reduced conditions . These germ cell NGF-like proteins were apparently different from similarly prepared murine and human NGFs (13 kDa) in their molecular weight . Furthermore, neurite outgrowth from pheochromocytoma cells (PC-12), a functional bioassay for NGF-like activity, was stimulated by addition of RSP and PSP to the culture media of the PC-12 cells . These results demonstrate mitogenic activity in germ cell proteins (RSP and PSP) and identify a NGF-like protein(s) which is associated with most of this activity.

Proc Soc Exp Biol Med, 1991 Dec, 198(3), 818 - 25
Zinc depletion modifies CD5 expression by 70Z/3 murine pre-B leukemia cell line; Jyonouchi H et al.; CD5 expression on B cells is regulated by certain humoral factors . In a pre-B leukemia cell line 70Z/3, we found that interleukin 4 down-regulates it . Herein, we report that zinc influences spontaneous CD5 expression by this cell line as well as actions of these factors on CD5 expression considerably . In zinc-depleted culture media, spontaneous CD5 expression by 70Z/3 cells was enhanced . In contrast, the down-regulatory action of interleukin 4 was significantly reduced under culture conditions of zinc depletion . The supplementation of zinc to physiologic concentrations (1 to 2 microM) abolished such effects of zinc-depleted medium . The reduction of the suppressive action of interleukin 4 was observed at the level of gene expression . However, CD5 mRNA expression enhanced by lipopolysaccharide or NZB-SF was not further enhanced under conditions of zinc deficiency . These observations may suggest that CD5 expression by malignant or even normal B cells may be influenced by cellular/serum zinc levels.

J Neurosci Methods, 1991 Dec, 40(2-3), 193 - 202
Membrane depolarization by isotonic or hypertonic KCl: differential effects on mRNA levels of tyrosine hydroxylase and dopamine beta-hydroxylase mRNA in PC12 cells; Kilbourne EJ et al.; Membrane depolarization is an important and common manipulation used to study the result of enhanced neuronal activity on adaptive changes, including alterations in gene expression . In this study, the effect of elevated KCl, under isotonic and hypertonic conditions, on the changes in mRNA levels of the catecholamine biosynthetic enzymes, tyrosine hydroxylase (TH) and dopamine beta-hydroxylase (DBH) was compared . Treatment of PC12 pheochromocytoma cells for several hours with 50 mM KCl, under conditions where osmolarity was maintained, induced TH mRNA levels several fold, without changing DBH mRNA levels (Kilbourne and Sabban, 1990) . In contrast, 50 mM KCl added to culture media without adjusting the osmolarity did not alter TH mRNA levels for up to 24 h . Longer continuous exposure to this hypertonic depolarization condition reduced TH mRNA levels to about 10% of control levels . DBH mRNA levels also declined when PC12 cells were treated from 12 h to 5 days with hypertonic 50 mM KCl . The effect appeared to be specific, since actin mRNA levels were elevated about 2-fold with these same hypertonic treatments . As a control for osmotic changes, 50 mM NaCl was used and did not alter TH or DBH mRNA levels . Viability of the cells was maintained and total protein synthesis was reduced somewhat after 12 h of exposure to hypertonic 50 mM KCl, and remained relatively constant for as long as 4 days . Thus, there appears to be an interaction between osmolarity and elevated KCl since very different results of the effects of membrane depolarization on the mRNA levels for the catecholamine biosynthetic enzymes were obtained depending on the osmolarity of the cultures . The extent of elevation of TH mRNA with isotonic KCl was also dependent on cell density . At high cell densities, membrane depolarization no longer induced TH mRNA levels . The results of this study indicate the experimental parameters which can be crucial in studies of membrane depolarization.

Int J Dev Biol, 1991 Dec, 35(4), 473 - 9
Changes in surface glycoconjugates in adhesion-defective variants of P19 embryonal carcinoma cells; Sakalian M et al.; Embryonal carcinoma cells defective in their ability to adhere to tissue culture dishes were isolated from mutagenized P19X1 and P19S1801A1 cells . Three independently isolated variants were analyzed for their morphology, surface properties and ability to differentiate in vitro . Two of the mutant cell lines expressed similar amounts of stage-specific embryonic antigens TEC-1, TEC-4 and Thy-1 as parental cells, whereas all three showed significant reduction in the expression of uvomorulin as determined by a direct radioantibody binding assay . Variant cells exhibited a decrease in their ability to aggregate in media with or without CA2+ and were unable to form compact aggregates when cultured for two days in complete culture media . In the presence of retinoic acid variant cells formed aggregates which exhibited significantly lower frequency neuron formation after transfer to tissue culture dishes . The combined data indicate that the adhesion-defective phenotype of P19-derived cells is in part the result of a reduced surface expression of uvomorulin.

Anal Biochem, 1991 Nov 15, 199(1), 86 - 92
An ion-exchange chromatography procedure for the isolation and concentration of basic amino acids and polyamines from complex biological samples prior to high-performance liquid chromatography; Gilbert RS et al.; The original objective of this study was to develop a selective and sensitive method for the analysis and quantification of basic amino acids from biological samples via reversed-phase high-performance liquid chromatography . Using various previously described techniques for the separation of amino acids, we were unsuccessful in measuring levels of histidine, arginine, ornithine, and lysine in biological samples due to the presence of interfering compounds . A "cleanup" procedure for the isolation of the basic amino acids using a weakly acidic cation exchange resin, Biorex-70 (Bio-Rad), is described in detail . Upon separation from the bulk of the neutral and acidic amino acids, the basic amino acids were subjected to precolumn fluorescence derivatization using 9-fluorenylmethyl chloroformate (FMOC) and the fluorescent derivatives were separated by RP-HPLC . The advantages of this method over previously described amino acid analysis techniques are (i) isolation and stable recovery (greater than 95%) of the desired basic amino acids, (ii) sensitivity of detection (low pmol range), (iii) complete resolution of derivatized amino acids via HPLC, (iv) limited amount of sample required for analysis, and (v) samples readily concentrated by lyophilization or rotoevaporating . This ion-exchange cleanup procedure was also adapted for the analysis of polyamines in concentrated culture media samples and proved additionally advantageous by eliminating the use of costly C-18 extraction columns required by previously described techniques.

Biol Psychiatry, 1991 Nov 15, 30(10), 966 - 72
Changes in serine metabolism by a serum factor present in a group of episodic psychotic patients; Fekkes D et al.; Addition of serum, obtained from patients suffering from an acute psychosis characterized by dysperceptions, to the culture media of fibroblasts altered the amino acid metabolism in these cells . After subculturing of fibroblasts in the presence of serum obtained from these patients, the concentrations of both serine and methionine were decreased in the medium as well as in the fibroblasts . Moreover, the concentration of taurine in the fibroblasts was increased . The specific activities of serine hydroxymethyltransferase and cystathionine beta-synthase were also measured in the fibroblasts . It was found that both enzyme activities were significantly higher after subculturing with patients' serum as compared with serum obtained from healthy controls . It is concluded that a factor, present in the serum of these acute psychotic patients, is responsible for the observed changes in serine, taurine, and methionine concentrations in the fibroblasts as well as for the increased enzyme activities mentioned.

Mol Cell Biochem, 1991 Nov 13, 108(1), 39 - 48
Effects of cholesterol oxidase on cultured vascular smooth muscle cells; Liu KZ et al.; Cholesterol oxidase (3 beta-hydroxy-steroid oxidase) catalyzes the oxidation of cholesterol to 4-cholesten-3 one and other oxidized cholesterol derivatives . The purpose of the present study was to investigate its effects on cultured vascular smooth muscle cells . Cultured rabbit aortic smooth muscle cells were morphologically altered after exposure to cholesterol oxidase in the presence of culture medium containing 10% fetal calf serum . If fetal calf serum was absent, cells were unaffected by the treatment . The extent of morphological change of the smooth muscle cells was dependent upon the time of exposure to the enzyme and the concentration of cholesterol oxidase employed . After moderate treatment with cholesterol oxidase, cells excluded trypan blue . Further, a specific mitochondrial marker DASPMI (dimethyl aminostyryl-methyl-pyridiniumiodine) which was used as a fluorescent index of cell viability, revealed that cell viability was unchanged after moderate cholesterol oxidase treatment . Nile red, a hydrophobic probe which selectively stains intracellular lipid droplets, was applied to detect the cellular lipid content after treatment with cholesterol oxidase . Cellular nile red fluorescence intensity increased linearly with the time and concentration of cholesterol oxidase treatment . These results demonstrate that cholesterol oxidase alters lipid deposition in the cell and changes cell morphology . The primary site of action of cholesterol oxidase appears to be independent of the cell membrane itself and instead is dependent upon the lipid content in the surrounding culture media . These changes occur prior to the cytotoxic effects of extensive oxidation . Because oxidized cholesterol may play an important role in the pathogenesis of atherosclerosis, our results have implications for intracellular accumulation of lipids in smooth muscle cells during the atherosclerotic lesion.

APMIS, 1991 Nov, 99(11), 1038 - 48
Effects of alloxan and reducing agents on macrophages in culture; Zhang H et al.; The diabetogenic effect of the quinonoid compound alloxan is not understood in detail although it supposedly involves reactions mediated by alloxan and oxygen radicals . These reactive species may form extra- or intracellularly and cause cell damage through a variety of complex interactions with several macromolecules . The purpose of this study was to elucidate early (less than or equal to 60 min) effects of alloxan and reducing agents (cysteine and ascorbic acid) on cultured macrophages, as assayed by the trypan blue dye exclusion test and the sensitive fluorescein diacetate and propidium iodide (FDA/PI) double staining technique . During the reactions between alloxan and reducing agents, oxygen was consumed as a sign of superoxide anion radical formation . When alloxan alone was added to two different culture media without serum, oxygen was still consumed, indicating formation of oxygen radicals due to the occurrence of reducing substances in cell culture media . This finding demonstrated the necessity of performing further studies in solutions without reducing capacity, e.g . in phosphate-buffered saline . The experiments showed that exposure of normal and malignant macrophages to alloxan and reducing substances resulted in rapidly occurring plasma membrane damage and ensuing cell death . Separate addition of catalase, desferrioxamine or superoxide dismutase resulted in evident, slight and no protection, respectively . The combinations of (i) catalase and desferrioxamine, and (ii) catalase, desferrioxamine and superoxide dismutase, however, inhibited cell damage in a pronounced and complete way, respectively . The results are interpreted as indicating cell damage due to the extracellular formation of hydrogen peroxide and hydroxyl radicals . The latter in close proximity to the cells and acting on the plasma membrane, while the former, after diffusing into the cell, may have several intracellular targets . The FDA/PI technique proved its value as a quantifiable method for the evaluation not only of cell death but also of cell damage with computer-based fluorometry.

Nippon Sanka Fujinka Gakkai Zasshi, 1991 Nov, 43(11), 1527 - 32
{Effects of various growth factors on the proliferation and the differentiation of trophoblastic cells in vitro}; Aoki Y et al.; EGF, PDGF, IL-6, TGF-beta 1 and supernatants from decidual cell cultures were added in various concentrations to cultures of normal trophoblastic cells in vitro . Effects on cell proliferation and differentiation were assessed by using 3H-thymidine uptake test and measuring hPL, hCG production in culture media, respectively . EGF stimulated only hPL, hCG production and in contrast, PDGF stimulated 3H-thymidine uptake only . IL-6, TGF-beta 1 and decidual supernatants stimulated both hPL, hCG production and 3H-thymidine uptake, especially TGF-beta 1 and decidual supernatants . These results suggest that EGF is related to the differentiation, PDGF is to the proliferation, and IL-6, TGF-beta 1 and decidual supernatants are to both the differentiation and proliferation of trophoblastic cells . Further understanding of the effects of these products on trophoblastic growth may provide important insights into mechanisms of early pregnancy development.

Arterioscler Thromb, 1991 Nov-Dec, 11(6), 1667 - 77
Connective tissue proteinases and inhibitors in abdominal aortic aneurysms . Involvement of the vasa vasorum in the pathogenesis of aortic aneurysms; Herron GS et al.; Recent studies have shown that increases in proteolytic activity are associated with abdominal aortic aneurysms (AAAs) . We have studied samples of the dilated aortic wall, taken during corrective surgery for AAAs, in terms of the number, type, and tissue location of connective tissue proteinases and their inhibitors . Five distinct caseinolytic serine proteinases and six gelatinolytic metalloproteinases were resolved by molecular weight by use of sodium dodecyl sulfate-substrate gel electrophoresis . Isoforms of the Mr 92,000 neutrophil gelatinase were identified by immunoprecipitation of biosynthetically labeled organ culture media . About 50% of the total radiolabeled protein secreted by AAA organ cultures was identified as the Mr 30,000 glycoprotein, tissue inhibitor of metalloproteinase (TIMP), by immunoprecipitation . Both TIMP and gelatinase were localized to the vasa vasorum by immunoperoxidase staining . However, interstitial collagenase could not be detected by any method . These results suggest the involvement of the vasa vasorum in the maintenance and possibly the genesis of AAAs.

Arch Biochem Biophys, 1991 Nov 1, 290(2), 497 - 502
Extracellular poly(3-hydroxybutyrate) depolymerase from Penicillium funiculosum: general characteristics and active site studies; Brucato CL et al.; An extracellular poly(3-hydroxybutyrate) (PHB) depolymerase has been isolated from Penicillium funiculosum cultural medium by a single hydrophobic column chromatography . The enzyme is a glycoprotein composed of a single polypeptide chain with a molecular mass of about 37,000 Da as analyzed by denatured sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by native gel filtration on Sephadex G-100 . Its optimum activity occurs at pH 6.0 . It has an isoelectric point of 5.8 and has a Km for PHB (average molecular weight = 45,000 Da) of 0.17 mg/ml . Various nonionic detergents competitively inhibit the enzyme with Ki values of 0.56 and 0.014% for Tween 80 and Triton X-100, respectively . The enzyme is extremely sensitive to diisopropyl fluorophosphate, mercuric ion, and dithiothreitol (DTT) . However, sulfhydryl reagents have little or no effect on its activity . The inactivation by mercuric ion and DTT is reversible by mercaptoethanol and hydrogen peroxide, respectively . These data suggest that the enzyme may be a serine esterase and may contain an important disulfide bond . The enzyme is also inactivated by diazoacetyl and epoxide compounds at low pH, which can be prevented by PHB, indicating the presence of a critical carboxyl group at the active site . These characteristics of the enzyme are compared to other extracellular polymerases isolated from bacterial culture media.

Zhonghua Yan Ke Za Zhi, 1991 Nov, 27(6), 351 - 3
{Selection of culture media for human and rabbit corneal epithelia}; Yang H; Five (5) culture media of MEM, DMEM, Ham's F12, DMEM+F12, and RPMI 1640 were used to culture the human and rabbit corneal epithelia, in order to find the best suited media for the purposes . It was found that rabbit corneal epithelium grew best in DMEM+F12, with good adhesion to the wall, cell multiplication, and sheet formation, while RPMI 1640 was suitable for human corneal epithelium . The authors discussed the ingredients of suitable media and their effects on the growth of cells.

Andrologia, 1991 Nov-Dec, 23(6), 439 - 42
Sperm motility analysis using multi-exposure photography (MEP) before and after in vitro-treatment of the semen; Oliva A et al.; Different methods and culture media were used on the same semen samples . Ejaculated and treated samples were evaluated by means of multiple exposure photography (MEP) in order to study changes in sperm motility . A statistical analysis of the normal sperm population distribution curve was done with two objectives: (1) evaluating semen improvement after treatment; and (2) finding out whether the post-treatment sample differed from the original one . When variables were analysed in the individual subject, a significant percentage improvement of mean velocity was observed . Only two methods improved the percentage of motile sperm . The influence of treatment and of culture media on sperm responses was also tested and a significant influence of the media was found (Ham F10 showed the best responses) . No significant differences between the ejaculated and treated samples were found when mean velocity, percentage of motile sperm, variance population, and kurtosis were analysed, as grouped treatment variables.

J Endocrinol Invest, 1991 Nov, 14(10), 861 - 5
Patterns of gastrin secretion in patients with nonfunctioning pituitary adenomas; Persani L et al.; The presence of gastrin in pituitary tissue as well as gastrin hypersecretion by some pituitary adenomas have been documented using different methodological approaches . In the present study, serum gastrin levels were measured in 93 patients with nonfunctioning pituitary adenoma, i.e . a condition lacking a reliable marker of the disease . Elevated gastrin levels (85-2, 180 ng/l; normal range: 15-80 ng/l) were found in 14/93 patients (15%), the highest values being observed in one patient with MEN I syndrome . In all but MEN I hypergastrinemic patient, a severe gastric hypochlorhydria (Basal Acid Output: 0.04 +/- 0.1 mmol H+/h) unresponsive to pentagastrin (Maximum Acid Output: 0.1 +/- 0.2 mmol H+/h) was seen . Secretin injection caused gastrin to increase in the patient with MEN I and in another hypergastrinemic patient . Antiparietal cells autoantibodies were positive in 3/11 patients . No changes in gastrin concentrations were found after administration of several agents usually employed in the evaluation of pituitary function, except a significant gastrin reduction after octreotide injection . In two hypergastrinemic patients who underwent pituitary adenomectomy, the high gastrin levels did not change after surgery . Finally, gastrin was undetectable in the culture media of 15 pituitary adenomas surgically removed from both normo- and hypergastrinemic patients and immunocytological studies of tumor cells did not show any gastrin staining . In conclusion, although in patients with pituitary adenomas serum gastrin evaluation is indicated in order to document the presence of a MEN I syndrome, the present data show that high gastrin levels cannot be taken as a specific marker of nonfunctioning pituitary adenomas unless the peripheral origin of hypergastrinemia is excluded.

Mol Reprod Dev, 1991 Nov, 30(3), 226 - 31
Reduction of embryotoxicity by protein in embryo culture media; Flood LP et al.; Experiments tested the hypothesis that one role of protein in embryo culture media is protection of embryos against potentially embryotoxic substances in the media . Mouse embryos were cultured in modified Krebs-Ringer bicarbonate medium and in modified Tyrode's medium, aliquots of which were supplemented with 4 mg/ml of the protein bovine serum albumin (BSA), while other aliquots were left protein free . The media were prepared using water samples that differed in purity, as reflected by differences in conductivity, with tap water being least pure (and considered to have the greatest potential for being embryotoxic) and water that had been purified by reverse osmosis, Milli-Q filtration, and triple distillation being most pure . Embryos were placed in the media while in the two-cell stage of development and their development was assessed after 24, 48, and 72 hr of culture . Rate of embryo development in BSA-supplemented media was greater than that in protein-free media only when the media were prepared with the least purified water samples . Because these water samples would have contained substances not contained in media prepared with purer water, or would have contained the substances in higher concentration, the data supported the hypothesis that protein can protect embryos during culture by negating effects of embryotoxic substances in the media.

Hum Reprod, 1991 Nov, 6(10), 1343 - 8
Medium-associated luteinization expressed as progesterone release in granulosa-luteal cells isolated from patients undergoing in-vitro fertilization; Holst N et al.; The present study describes the effect of culture medium components on progesterone release from human granulosa-luteal cells isolated from patients undergoing in-vitro fertilization (IVF) . Progesterone release was selectively measured as a central parameter of in-vitro luteinization, a process believed to decrease the success rate of IVF treatments . Ten different media of relevance to embryo culture were investigated for their effect on progesterone release in unstimulated granulosa cell cultures and in cultures stimulated with human chorionic gonadotrophin (HCG) (1 IU/ml) during 4 days in vitro . Culture media supplemented with human serum yielded the greatest secretion of progesterone . Supplementation with fetal calf serum caused an intermediate pattern of progesterone release . Substitution of serum with a synthetic replacement (Medi-CultR SSR 1 and 2), lacking hormones, cholesterol and growth factors, led to a minimal output of progesterone from granulosa-luteal cells . Complex media (RPMI 1640 and Ham's F10) generally caused a greater progesterone release than simple salt solution (EBSS) . No effect of insulin was detected when added to serum-free media.

Mol Cell Endocrinol, 1991 Nov, 82(1), R13 - 6
Sertoli cells are the site of interleukin-1 alpha synthesis in rat testis; Gerard N et al.; To elucidate the cellular origin of interleukin-1 (IL-1) in the mammalian testis, we assayed IL-1 activity in culture media of Sertoli cells collected from rats at 20, 35 and 45 days of age as well as in culture media of interstitial, peritubular and germ cells from adult rats . IL-1 was detected in Sertoli cell culture media isolated from 35- and 45-day-old rats . At 45 days approximately twice as much IL-1 was produced than at 35 days . In contrast, IL-1 activity was not detected in 20-day-old rat Sertoli cell culture media nor in interstitial, peritubular and germ cell culture media . The Sertoli cell IL-1 activity was specifically neutralized by an IL-1 alpha antiserum . It is concluded that Sertoli cells produce IL-1 alpha and that this production increases during sexual maturation.

Biol Reprod, 1991 Nov, 45(5), 736 - 42
In vitro-matured/in vitro-fertilized bovine oocytes can develop into morulae/blastocysts in chemically defined, protein-free culture media; Pinyopummintr T et al.; Development of bovine embryos derived from in vitro-matured/in vitro-fertilized (IVM/IVF) oocytes was examined in two culture media: hamster embryo culture medium (HECM), a relatively simple, chemically defined, protein-free medium containing 20 amino acids; and tissue culture medium (TCM)-199, a more complex medium designed for culture of somatic cells . The first experiment studied (1) effects of glucose and/or phosphate (Pi) using HECM and (2) the development of bovine IVM/IVF embryos in four different conditions: HECM, TCM-199, TCM-199 + 10% unheated bovine calf serum (BCS), and oviduct cell-conditioned TCM-199 + 10% BCS . After IVF, 45% of the inseminated oocytes developed to the morula/blastocyst stages (M&B) when cultured in HECM; blastocyst development was depressed in the presence of glucose and Pi when compared to Pi alone . When the four culture conditions were compared, there was no significant difference in M&B development (42-51% of inseminated oocytes) . However, blastocyst development in TCM-199 supplemented with 10% BCS (29.7%) or with BCS + oviduct cell-conditioned medium (21.6%) was significantly greater than in nonsupplemented HECM (9.7%) or TCM-199 (13.8%) . In the second experiment, the effects of serum supplementation and/or oviduct cell conditioning on HECM and TCM-199 were examined in a 2 x 2 x 2 factorial experiment . Oviduct cell conditioning of either HECM or TCM-199 without serum supplementation did not enhance bovine embryo development . Serum supplementation exhibited a biphasic effect, with inhibition at the first cleavage and stimulation of morula compaction and blastocyst formation.(ABSTRACT TRUNCATED AT 250 WORDS)

Jpn J Cancer Res, 1991 Nov, 82(11), 1292 - 8
Antitumor effector mechanism of interleukin-1 beta at a distant site in the double grafted tumor system; Ebina T et al.; Recombinant human interleukin-1 beta (IL-1 beta) inhibited the growth of not only the right, but also the left non-treated tumor in a double grafted tumor system . Since the antitumor activity of IL-1 beta against the right and left tumors was not seen in nude mice, lymphocytes have a key role in the antitumor effect of intratumoral administration of IL-1 beta . TIL (tumor-infiltrating leukocytes) obtained from left and right side tumors treated with IL-1 beta were examined by Winn assay for their antitumor activity against Meth-A sarcoma in BALB/c mice . TIL from the right side clearly inhibited the growth of admixed Meth-A cells, but control TIL did not . Spleen cells and right and left regional lymph node cells prepared from IL-1-treated mice were examined for Lyt-1, Lyt-2 and L3T4 phenotypes . The number of Lyt-1-positive lymphocytes increased in the spleen and in the right regional lymph nodes after intratumoral administration of IL-1 . Isolated tumor cells obtained from the right tumor treated with IL-1 beta and the left side tumor on day 6 were cultured in RPMI 1640 with 10% fetal calf serum for 24 h . The culture supernatants were harvested and tested for the presence of chemotactic activity for neutrophils or macrophages . Significant neutrophil chemotactic factor and macrophage chemotactic factor activities were detected in the culture media from IL-1-treated tumor tissues cultured for 24 h . Neither significant neutrophil nor macrophage chemotactic activity was detected in the media from untreated tumor tissues . These results suggest that intratumoral administration of IL-1 first induces neutrophils and macrophages in the right tumor, then Lyt-1-positive cells in the right regional lymph nodes and in the spleen, and subsequently induces macrophages in the left, non-treated tumor.

J Cell Physiol, 1991 Nov, 149(2), 339 - 46
Adipogenic activity produced by hepatocyte-derived cell lines and by normal hepatocytes in primary culture; Zaitsu H et al.; Culture media conditioned by several hepatocyte derived cell lines were analyzed for their ability to stimulate adipose differentiation of the adipogenic cell line 1246 . The results presented here show that culture media from HepG2 and Hep3B cell lines contain a high level of the activity, whereas media from hepatoma and hepato adenocarcinoma cell lines Huh-7, PLC/PRF/5, and SK-Hep-1 do not contain adipogenic activity . Conditioned medium from HepG2 cells also stimulated differentiation of 3T3-L1 cells and of rat epididymal adipocyte precursors in primary culture . Partial biochemical characterization of the adipogenic activity carried out using HepG2 conditioned medium indicates that the hepatocyte derived adipogenic factor has an apparent molecular weight between 445 and 232 kDa, is destroyed by treatment at 100 degrees C, with protease, with 2-mercaptoethanol and in acidic conditions . The activity is stable at alkaline pH . Culture media conditioned by normal rat hepatocytes in primary culture also contained adipogenic activity . In contrast, medium conditioned by primary culture of nonhepatocyte cell also isolated from liver was deprived of this activity . The data presented in this paper suggest that hepatocytes could be a physiological site of production of adipogenic activity.

Pancreas, 1991 Nov, 6(6), 625 - 30
Survival and B-cell function of neonatal pig pancreatic islet-like cell clusters in an extracellular matrix; Ohgawara H et al.; Islet-like cell clusters (ICCs), formed from single cells of pig pancreas in suspension culture, were embedded in an extracellular matrix . It was recently reported that nicotinamide prevented dissolution of the extracellular matrix by the ICCs . In this experiment, various conditions for embedded culture of ICCs in an extracellular matrix were studied, in an attempt to maintain the function as well as the extracted insulin content of culture specimens . The ICCs in the matrix were refed with RPMI 1640, containing 10 mM nicotinamide, 10% fetal bovine serum (FBS), 11 mM D-glucose and with or without 0.1 mM 3-isobutyl-1-methylxanthine (IBMX) or 1.0 micrograms/ml caerulein . A comparison between the different culture media showed that embedded ICCs, maintained in RPMI 1640 with caerulein, in the presence of nicotinamide, had higher insulin content accumulation than when maintained in medium containing nicotinamide alone, but had impaired glucose-stimulated insulin secretion . In the medium containing IBMX and nicotinamide, embedded ICCs showed higher insulin accumulation but lower insulin content, compared to ICCs maintained in the presence of caerulein, and also showed impaired glucose-stimulated insulin release . Thus, the effect of nicotinamide on the survival and function of B-cells is amplified by the presence of caerulein or IBMX.

Agents Actions, 1991 Nov, 34(3-4), 410 - 5
Formation of a clastogenic factor by asbestos-treated rat pleural mesothelial cells; Emerit I et al.; The data of the present study indicate that chrysotile induces the formation of a clastogenic factor (CF) when mesothelial cells are exposed to these fibers in vitro . Ultrafiltrates of culture media induce chromosome damage in human lymphocytes used as a test system for the detection of clastogenic activity in conditioned media . According to the cut off of the filters (10,000 dalton), CF is a small molecule . Its exact nature is unknown . The intermediacy of active oxygen species in CF formation is suggested by the anticlastogenic effect of antioxidant enzymes such as superoxide dismutase and catalase . The data are similar to those obtained with other membrane-active agents, in particular the tumor promoter tetra-decanoylphorbol acetate (TPA) . The model of membrane-mediated chromosome damage with CF formation is proposed for asbestos-induced cell injury.

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