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