Exp Hematol, 1989 Sep, 17(8), 865 - 71 A four-step procedure for the purification of thrombopoietin; McDonald TP et al.; The present work reports the preparation of a highly bioactive and stable thrombocytopoiesis-stimulating factor (TSF or thrombopoietin) by a four-step purification procedure, i.e., Sephadex column chromatography, ethanol precipitation, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and reverse phase-high performance liquid chromatography . The molecular weight (MW) of the purified product depended upon the method of purification, i.e., using denaturing buffers at 56 degrees C for 10 min, the MW was approximately 30,000 daltons; whereas, after preparing in denaturing buffers and heating to 100 degrees C for 10 min, the purified protein had an apparent MW of approximately 15 kd . Both moieties had significant biological activity . The data indicate that TSF may exist normally as a dimer (30 kd), but can disassociate to 15 kd without loss of bioactivity . The present work illustrates that the purified TSF has an isoelectric pH of 4.47 and exists in trace amounts in human embryonic kidney (HEK) cell culture media . The final product prepared in the presence of Tween-20 had a specific activity of approximately 21,000 U of TSF per mg of protein, representing a purification factor of approximately 164,000 . Using this four-step purification procedure, a homogeneous product was obtained as judged by SDS-PAGE and chromatofocusing . This purified material will be suitable for further studies, including amino acid sequencing. Zhongguo Yao Li Xue Bao, 1989 Sep, 10(5), 453 - 7 Effects of Tremella polysaccharides on immune function in mice; Xia D et al.; It was found in vitro that Tremella polysaccharides (TP) (50, 100, 150 and 200 micrograms/ml) augmented lymphocyte proliferation induced by Con A and did not antagonize the suppressive effect of hydrocortisone on lymphocyte proliferation . In vivo TP promoted the plaque-forming cell (PFC) response to SRBC in mice . TP 50 and 100 mg/kg ip for 5 d produced 77.6% and 81.8% increases in PFC response respectively . At the doses of 150 and 200 micrograms/ml, TP decreased the interleukin 2 (IL-2) activities in the supernatant of culture media of mouse spleen cells . TP (50 micrograms/ml) enhanced the lymphocyte proliferation induced by Con A and increased the PFC response to SRBC by 47.1% in 14-month-old mice. Am J Gastroenterol, 1989 Sep, 84(9), 1100 - 2 Eikenella corrodens isolated from a polymicrobial hepatic abscess; Massey BT; A 71-yr-old woman developed a hepatic abscess from which Eikenella corrodens was isolated as part of a polymicrobial flora . This facultatively anaerobic organism is now recognized as a true pathogen . Selective culture media may be required to isolate Eikenella corrodens, and it may be resistant to antibiotics commonly used for anaerobic infections . Special attempts to isolate this organism may be indicated when hepatic abscesses are likely to have resulted from spread of oral or abdominal infections. J Neurochem, 1989 Sep, 53(3), 912 - 6 Sulfation of rat apolipoprotein E; Gebicke-Haerter PJ et al.; The synthesis of a 37-kilodalton (kDa) protein which has been shown recently to be identical with apolipoprotein E (apo-E) was increased after sciatic nerve injury of the rat . When regeneration of the nerve was allowed, its synthesis returned to control levels at about 8 weeks post injury . In this report it is shown that similar time-course studies of the protein in the rat optic nerve revealed a delayed increase of the protein but a comparably high level of synthesis at 3 weeks post injury . This level was maintained up to at least 18 weeks after crush . Furthermore, two-dimensional electrophoresis revealed that the characteristic "trailing" of the protein is due to its sialylation, because it was reduced after neuraminidase treatment . This treatment, however, detected a neuraminidase-resistant heterogeneous form in CNS tissue and a homogeneous form in peripheral nervous tissue . The trailing persisted up to 18 days of culture of optic nerve explants, of CNS glial cells, and of peritoneal macrophages, but disappeared during the first culture days of sciatic nerve explants and was not observed in Schwann cell culture media . Incorporation studies with 35SO4 revealed that apo-E was the major sulfated protein in culture media conditioned by CNS glial cells, whereas sulfation of the protein was undetectable in Schwann cell cultures . Because macrophages are likely to be the major source of apo-E in both peripheral and central glial cell cultures as well as in injured optic and sciatic nerves, it is hypothesized that resident cells of sciatic nerves secrete potent sulfatases . As a result, sialic acid residues may be more susceptible to degradation.(ABSTRACT TRUNCATED AT 250 WORDS) Zhonghua Bing Li Xue Za Zhi, 1989 Sep, 18(3), 188 - 90 {The effect of prostaglandin E2 (PGE2) on growth, proliferation, morphology and protein synthesis of aortic smooth muscle cells (SMC) in vitro}; Lei ZZ et al.; SMC are the key element in the pathogenesis of atherosclerotic (AS) plaques . The effect of PGE2 on the growth, proliferation, morphology and protein synthesis of SMC were studied in vitro . SMC from tunica media of aorta of New Zealand white rabbits were used for cultivation . After 7 to 13 passages of subcultures, the cells were divided into control and experimental groups with a dosage of PGE2.5 micrograms,10 micrograms and 20 micrograms per milliliter of medium . After 3 days of successive culture, the cells were prepared for phase contrast microscopy, protein(P) and DNA(D) determination, and electron microscopy(TEM) . Additionally, similar groups of cells were grown on the cover glass for autoradiographic study of cell proliferation by adding 3H-thymidine in culture media . Under TEM, characteristic thin bundles of myofilaments and dense bodies were observed inside the cytoplasm . Mitochondria, Golgi complex,rER were also abundant in the control but not so in the experimental groups . Synchronously with the increase of PGE2 concentration, the P/D value which denotes protein synthesis was significantly decreased from 74.89 +/- 4.68 to 57.01 +/- 3.08, 45.81 +/- 4.61, 32.23 +/- 4.22 and the percentage of 3H-thymidine labelled cells from 37.60 +/- 5.30% to 15.60 +/- 4.20%, 10.18 +/- 3.00%, 3.75 +/- 0.80% respectively . Results showed that PGE2 may act as an inhibitor for growth, proliferation and protein synthesis of aortic SMC in vitro. Rev Esp Fisiol, 1989 Sep, 45(3), 227 - 33 {Proliferation and liberation kinetics of the tissue polypeptide antigen of MCF-7 cells . Hormonal influence}; Lopez-Gonzalez JD et al.; The presence or absence of tissular polypeptide antigen (TPA) in the culture medium of hormone dependent breast cancer tumoral cells, the relationship between TPA concentration and cellular proliferative activity, have been investigated as well as whether TPA levels change in response to steroid preparations or the action of different antiestrogens . Results show that in MCF-7 cell line culture media, proteins antigenically related to TPA can be detected in concentrations which parallel the number of cells in culture . Consequently, TPA can be considered a cell proliferation marker . Hydroxy-tamoxifen at a concentration of 10(-7) M inhibited both cell proliferation and TPA antigen release, while the relative proportion between number of cells (valued by DNA quantification) and TPA concentration remained unchanged. Mol Biol (Mosk), 1989 Sep-Oct, 23(5), 1364 - 72 {Properties and conditions of formation of the fibronectin-collagen complex in a culture of human embryo fibroblasts}; Belkin VM et al.; In our previous study the macromolecular complexes mostly consisting of fibronectin and procollagen were isolated from human fibroblast culture media using immobilized antibodies against fibronectin . At present an attempt was made to elucidate at what stage the formation of the fibronectin-collagen complex occurs--either in the course of incubation of the immobilized antibodies with labelled proteins secreted by fibroblasts, or in the extracellular space while labelling fibroblasts, or intracellularly . The results obtained show that the fibronectin-collagen complex: 1) pre-exists even before incubation with the immobilized antibodies and 2) it is of intracellular origin . Thus, the considerable amount of the fibroblast-secreted fibronectin (no less than 20%) is released from the cell not in the free form but in the complex with procollagen . It was suggested that the fibronectin-collagen complex presents a stage in the formation of the insoluble extracellular matrix. Nippon Hifuka Gakkai Zasshi, 1989 Sep, 99(10), 1059 - 65 {Production of monoclonal antibodies to human T-cell lymphotropic virus type 1 (HTLV-1) and studies of their specificity}; Iwatsuki K et al.; Mouse monoclonal antibodies to HTLV-1 core proteins, p19 and p24, were obtained by hybridoma technique . The specificity was studied by indirect immunofluorescence, enzyme-linked immunosorbent assay (ELISA), dot blot, and Western blot studies . Polyclonal antibodies reactive with HTLV-1 viral proteins were raised by rabbits and guinea pigs . A sandwich ELISA and a dot blot method using these antibodies detected the soluble viral antigens in the ATL cell lysate and ATL culture media . No reaction was observed when IL-2-activated human T-cell lysate and the culture media were used as controls . These data showed that our sandwich ELISA and dot blot systems are available for quantitative analysis of the soluble HTLV-1 viral antigens and contribute to understanding the ability of viral replication by ATL cells. Mol Cell Endocrinol, 1989 Sep, 66(1), 83 - 91 18-Hydroxylase activity in the Y1 adrenal cell line; Ramirez LC et al.; 18-Hydroxylase activity, reported here for the first time in the mouse adrenal tumor cell line (Y1), was expressed in the metabolism of 11-deoxycorticosterone (DOC) and corticosterone (B) . Detected after 24 h of incubation, it was more evident after 48 h and produced mostly 18-hydroxy-20 alpha-DHB from these exogenous substrates . However, 18-hydroxylation was quantitatively less significant than the metabolism of 20 alpha-reduction and 11 beta-hydroxylation (of DOC) . The latter is also the predominant metabolism of progesterone in this cell line, during the conversion of cholesterol from the serum-supplemented culture media . The cytochrome P-450 11 beta activity of the Y1 cells is similar to that of the mouse in vivo which catalyzes the production of an 11 beta 18-dihydroxylated metabolite as the principal 18-hydroxylated steroid . It is different from that of other species, such as the rat and the bovine, both in terms of the ratio of 11 beta- to 18-hydroxylated metabolites and of the structure of these metabolites. Vopr Med Khim, 1989 Sep-Oct, 35(5), 103 - 8 {Changes in the cAMP levels and acid phosphatase activity in a monolayer primary culture of hepatocytes from newborn rats during anoxia and substrate deprivation}; Nikulina SE et al.; Acid phosphatase activity and cAMP level were studied in primary hepatocyte culture of new born rats under conditions of anoxia and substrate deprivation (incubation of the cells in Hanks salt solution) . Incubation of hepatocytes in Hanks salt solution within one hour under conditions of anoxia caused a significant increase in free (cytosolic) enzyme activity . Substitution of Hanks salt solution by normal tissue culture medium and reoxygenation after 1 hr anoxia resulted in a decrease of free acid phosphatase activity, whereas activity of the enzyme in lysosomal fraction was increased . Content of cAMP was decreased distinctly after 15 min incubation of hepatocyte culture under conditions of anoxia and substrate deprivation and was increased above control values within 5 min of reoxygenation and substitution of Hanks salt solution by normal tissue culture media . Addition of cAMP-containing liposomes to hepatocyte culture under these experimental conditions led to a decrease in free acid phosphatase activity and to an increase of the enzyme activity in lysosomal fraction . cAMP appears to modulate the lability of hepatocyte lysosomal membrane . The mechanisms involved in these processes are discussed. Br J Cancer, 1989 Sep, 60(3), 343 - 50 Partial protection of oncogene, anti-sense oligodeoxynucleotides against serum nuclease degradation using terminal methylphosphonate groups; Tidd DM et al.; Under certain circumstances sequence-specific inhibition of gene expression may be achieved in intact cells using exogenous anti-sense oligodeoxynucleotides . The efficacy of this approach to investigating gene function is limited in part by the rapid serum nuclease mediated degradation of oligodeoxynucleotides in culture media . In order to determine the relative contributions of 3'-exonuclease, 5'-exonuclease and endonuclease activity in fetal calf serum to oligodeoxynucleotide destruction, we have tested chimeric N-ras anti-sense sequence molecules protected against exonuclease attack with terminal methylphosphonate diester linkages . An 18-mer with two methylphosphonate diester linkages at the 3'-terminus, a 20-mer with two methylphosphonate diester groups at both ends, and the 16-mer 3'-methylphosphonate monoester components of their respective piperidine hydrolysates were totally resistant to venom phosphodiesterase, whereas the 16-mer 3'-hydroxyl components of the hydrolysates were rapidly degraded . Both the chimeric oligodeoxynucleotides and 3'-methylphosphonate monoesters were considerably more stable than normal 3'-hydroxyl oligodeoxynucleotides at 37 degrees C in McCoy's 5A medium containing 15% heat inactivated fetal calf serum . Typically 20-30% of the former (initial concentration 10-100 microM) remained intact at 20 h as compared to the latter which were 88-100% degraded in 4 h and undetectable at 20 h . We conclude that a 3'-phosphodiesterase activity is a predominant nuclease responsible for oligodeoxynucleotide degradation by fetal calf serum, and that for cell culture studies, significant protection of oligodeoxynucleotides may be achieved by incorporating 3'-terminal methylphosphonate diester or even monoester end groups. J Clin Invest, 1989 Sep, 84(3), 863 - 75 Inhibition of tumor cell growth by interferon-gamma is mediated by two distinct mechanisms dependent upon oxygen tension: induction of tryptophan degradation and depletion of intracellular nicotinamide adenine dinucleotide; Aune TM et al.; Growth of a variety of human tumor cell lines is inhibited by interferon-gamma (IFN-gamma) in vitro . This mechanism is not well understood . The present experiments identify two separate mechanisms which account for the growth inhibitory activity of IFN-gamma . Cell lines most sensitive to IFN-gamma (inhibited by 10-30 U/ml IFN-gamma in 3 d) were stimulated by IFN-gamma to oxidize tryptophan in media to kynurenine and completely eliminated tryptophan from the culture media after 48-72 h . Addition of L-tryptophan, but not other aromatic amino acids, other essential amino acids, or D-tryptophan, prevented inhibition of cell growth by IFN-gamma . The amount of IFN-gamma required to yield 50% inhibition of cell growth was directly related to the concentration of L-tryptophan in culture media and increased from approximately 3 to 600 U/ml as the concentration of tryptophan in the media was increased from 25 to 1,000 microM . By contrast, inhibition of growth of the cell lines, BT20 and HT29, was not prevented by addition of tryptophan . Inhibition by IFN-gamma (100-300 U/ml after 5-6 d) was, however, completely prevented by addition of two inhibitors of adenosine diphosphate-ribosyl transferase (ADP-RT), 3-aminobenzamide or nicotinamide . Activity of ADP-RT was increased in these cell lines after addition of IFN-gamma . ADP-RT catalyzes the incorporation of the ADP moiety of nicotinamide adenine dinucleotide (NAD) into proteins and causes depletion of intracellular NAD . All tumor cell lines tested had reduced levels of intracellular NAD after treatment with IFN-gamma and loss of NAD preceded inhibition of cell growth by 12-24 h . Inhibitors of IFN-gamma-mediated inhibition of cell growth prevented loss of levels of intracellular NAD . Generation of reactive oxygen species lead to DNA strand breaks which result in activation of ADP-RT . Increased DNA strand breaks were induced in BT20 and HT29 cells but not ME180 and A549 cells after culture with IFN-gamma . The two enzymes known to catalyze the decyclization of tryptophan to kynurenine require superoxide anion for activity . Increased amounts of superoxide anion were released from ME180 and A549 cells after culture with IFN-gamma . Reduced oxygen concentration decreased the ability of IFN-gamma to inhibit tumor cell growth in vitro . Intracellular glutathione has been shown to protect cells against oxidative damage by various agents . Elevation or reduction of intracellular glutathione concentrations lowered or raised sensitivity of cell lines to IFN-gamma, respectively . These data indicate that at least two distinct mechanisms can account for IFN-gamma-madiated inhibition of tumor cell growth . Both mechanisms appear to be sensitive to oxygen tension and to changes in intracellular glutathione concentrations, and both mechanisms lead to loss of intracellular NAD. J Biol Chem, 1989 Aug 5, 264(22), 13150 - 6 The structure of avian type XII collagen . Alpha 1 (XII) chains contain 190-kDa non-triple helical amino-terminal domains and form homotrimeric molecules; Dublet B et al.; The monoclonal antibody 75d7, specific for type XII collagen (Sugrue, S.P., Gordon, M.K., Seyer, J., Dublet, B., van der Rest, M., and Olsen, B . R . (1989) J . Cell Biol., in press), was used to characterize the intact form of type XII collagen from chick embryo leg tendons . On an immunoblot of a 6% polyacrylamide gel of tendon extracts, one sharp band is recognized by the antibody at Mr = 220,000, while two fuzzy and poorly resolved bands are seen at Mr = 270,000 and Mr = 290,000 . By immunoprecipitation of radiolabeled tendon culture media and electrophoresis of the precipitated material, bands with the same mobilities are observed, indicating that type XII collagen is not proteolytically processed in the extracellular space . Type XII collagen was extracted from tendons with 1 M NaCl in a Tris-HCl buffer and partially purified by concanavalin A-Sepharose and gel permeation chromatographies, using dot immunoblots to monitor the purification . Fractions highly enriched in bacterial collagenase-sensitive proteins with the same electrophoretic properties as type XII collagen were obtained . These fractions did not stain with Alcian blue and neither they nor the immunostained type XII collagen were affected by chondroitinase ABC digestion, indicating that type XII collagen is not a proteoglycan . A disulfide-bonded trimeric CNBr peptide was isolated by affinity chromatography on an antibody column and further purified by gel electrophoresis . Its NH2-terminal amino acid sequence was shown to be unique, demonstrating that type XII collagen is a homotrimer {alpha 1 (XII)}3 . After bacterial collagenase digestion, both the immunopurified radiolabeled preparation and the purified tendon extract fraction showed by gel electrophoresis the presence of a large disulfide-bonded, 3 x 190-kDa, collagenase-resistant domain . Rotary shadowing and electron microscopy of the purified type XII fraction demonstrated that the molecule has the structure of a cross consisting of a 75 nm collagenase-sensitive tail, a central globule, and three 60 nm arms each ending in a small globule . After heat denaturation and renaturation, only a very large globule can be seen, attached to the triple helical tail . These results show that type XII collagen has a unique structure and is different from the other matrix constituents described so far. Biol Reprod, 1989 Aug, 41(2), 347 - 54 Immunological characterization and immunocytochemical localization of a progesterone-dependent cat endometrial secretory protein; Verhage HG et al.; The major objective of this study was to make a polyclonal antibody to a previously described group of progesterone (P)-dependent low molecular weight secretory proteins of the cat uterus . Proteins present in uterine flushings obtained from a P-treated cat were partially purified using Sephadex G-75, separated on two-dimensional polyacrylamide gels, and transferred to nitrocellulose membranes . The region containing the polypeptides were cut out, solubilized in dimethyl sulfoxide, mixed with Freund's adjuvant, and injected at 2-wk intervals into a male rabbit . The antiserum used in this study was obtained 8 wk after the initial injection and crossreacted with antigens on Western blots of uterine flushings and uterine culture medium obtained from ovariectomized estradiol (E2)-primed cats treated with P, and from pregnant preimplantation animals . Polypeptides in three molecular weight regions crossreacted with the antisera (Mr approximately equal to 28,000, pI 5.5 6.0; Mr approximately equal to 36,000, pI 6.0 6.5; Mr approximately equal to 41,000, pI 5.5 6.0), and each region consisted of several isoelectric variants . The Mr approximately equal to 28,000 proteins were the dominant form observed in culture media, and the Mr approximately equal to 36,000 proteins were the major form present in uterine flushings . The antigens were not detected in uterine flushings or culture medium obtained from ovariectomized, E2-treated, and estrous animals . The antigens were also absent in serum and other reproductive and nonreproductive tract tissues . Immunocytochemical analysis demonstrated that antigen staining was limited to the epithelial cells of the deeper uterine glands of the P-dominated animal . Immunoperoxidase staining was diffuse throughout the cytoplasm of these epithelial cells . Thus, the epithelial cells of the uterine glands in the P-dominated cat synthesize and secrete a complex group of P-dependent, uterine-specific proteins that may have potential functional significance during early blastocyst development and implantation. Liver, 1989 Aug, 9(4), 242 - 9 Human hepatic sinusoidal endothelial cells in culture produce von Willebrand factor and contain Weibel-Palade bodies; Harrison RL et al.; Human hepatic sinusoidal endothelial cells were derived from cadaveric human livers . Cells were grown in culture for several weeks to produce small patches of confluent endothelial cells . The ultrastructure of sinusoidal endothelial cells was examined, cell monolayers were stained immunocytochemically for von Willebrand factor antigen, and antigen in cell culture media was measured by enzyme-linked immunosorbent assay . Human hepatic sinusoidal endothelial cells contained von Willebrand factor antigen and Weibel-Palade bodies, were fenestrated, and released von Willebrand factor antigen into media in a time-dependent manner . Although in some respects human hepatic endothelial cells were different from vascular cells, there was no evidence that there were qualitative differences in their capacity to produce von Willebrand factor. Neurosurgery, 1989 Aug, 25(2), 196 - 201 Cyst fluids of malignant human brain tumors contain substances that stimulate the growth of cultured human gliomas of various histological type; Westphal M et al.; The contents of 14 cysts that were located within human intracranial tumors were obtained at surgery by needle aspiration . These tumor cyst fluids (TCFs) were mostly derived from glial tumors (10 cases) . TCFs from one metastasis from a mammary carcinoma, one cystic meningioma, one hemangioblastoma, and a cystic acoustic neurinoma were also included . These TCFs were added to primary cultures of human gliomas, established human glioma cell lines, and normal human arachnoid cells in culture . The presence of proliferation-promoting factors in all cyst fluids could be demonstrated . On the basis of the response patterns of the cultures, it was possible to distinguish different levels of growth autonomy and growth factor sensitivity among these cultures and to speculate about varying degrees of cellular autocrine activation . The TCFs appear to contain factors that are not normally present in fetal calf serum, which is a regular constituent of most cell culture media . Some primary cultured cells as well as cell lines react in an oversensitive manner to the addition of TCFs. J Clin Endocrinol Metab, 1989 Aug, 69(2), 259 - 66 Effects of human and salmon calcitonin on human articular chondrocytes cultivated in clusters; Franchimont P et al.; The effects of different pharmacological concentrations (0, 5, 10, 100, and 1000 ng/mL) of synthetic human calcitonin (hCT) and salmon calcitonin (sCT) on the incorporation of {3H}thymidine and production of proteoglycans (PG) and type II collagen (coll II) by human articular chondrocytes during a 20-day period were studied in a tridimensional chondrocyte culture model . {3H}Thymidine uptake was measured in chondrocyte clusters, and specific PG and coll II RIAs were performed every 4 days on the culture medium and cell aggregates; total PG and coll II production were also assessed at different culture durations by adding the amounts found in culture media and their corresponding clusters . Incubation with hCT or sCT did not affect {3H}thymidine uptake regardless of the dose . For each culture period, PG and coll II release into culture medium, cluster content, and total production increased significantly in a dose-dependent manner . Cumulative curves for these parameters showed a progressive significant increase with culture duration at hCT and sCT doses of 0, 5, and 10 ng/mL . Cumulative curves obtained with 10, 100, and 1000 ng/mL were seldom significantly different from one another . No differences emerged between the use of hCT or sCT . Thus, CT exerted no proliferative effect on human articular chondrocytes in tridimensional culture, but displayed a dose-dependent and prolonged stimulatory effect on PG and coll II production . CT may possess chondroprotective properties in addition to its other known effects. J In Vitro Fert Embryo Transf, 1989 Aug, 6(4), 213 - 7 Human amniotic fluid for fertilization and culture of human embryos: results of clinical trials in human in vitro fertilization (IVF) programs; Gianaroli L et al.; We report the outcome of clinical trials carried out in two IVF programs, comparing the use of human amniotic fluid (HAF) as a complete medium to Whittingham's T6 medium containing human serum (T6 + 10% HS) for egg incubation, insemination, embryo culture, and embryo transfer . There were no significant differences in the clinical trials between HAF used alone as a complete medium and T6 + 10% HS in fertilization rates of eggs, cleavage rates of embryos up to 48 hours in culture, pregnancy success rates after embryo replacement or the outcome of pregnancies . There was no advantage in using T6 + 10% HS for fertilization of eggs and HAF as a complete medium for embryo culture and transfer in any of the parameters examined . We conclude that HAF does not meet the complete requirements of human eggs and embryos in vitro and further developments of culture media are required to obtain embryo development equivalent to that in vivo. Mol Endocrinol, 1989 Aug, 3(8), 1197 - 206 Differential effects of estrogen on luteinizing hormone-releasing hormone gene expression in slice explant cultures prepared from specific rat forebrain regions; Wray S et al.; Five serially sectioned tissue slices (400 microns) from the preoptic area/hypothalamus of postnatal day 4 rats were cultured using a slice explant roller culture technique . After 18 days in culture, these slices thinned sufficiently to allow immunocytochemical and in situ hybridization histochemical assays for LHRH peptide and LHRH mRNA, respectively . Large numbers of neurons containing mRNA encoding LHRH were detected in these slices using in situ hybridization histochemistry (ISHH) . These 35S-labeled cells were distributed in the cultured slices in a pattern similar to that found with LHRH immunocytochemistry and ISHH in vivo, indicating that LHRH neurons were maintained in these cultures in an organotypic manner . Densitometric single cell analyses after ISHH of the culture slices were performed using a Loats image analysis system, so as to provide a density value per cell (density/cell) . Comparisons of these density values from the slice explants cultured in presence or absence of 10(-7) M estradiol found that: 1) under basal (control) culture conditions there were no consistent differences in the frequency distributions of the density/cell values between all the five slices derived from either male or female rats, 2) mean density/culture values under control conditions did not differ significantly between slices and sexes, 3) the presence of estradiol in the culture media resulted in an overall decrease in density/cell values, with the most significant decrease occurring in slice 3 which is comparable to the level of the organum vasculosum lamina terminalis/rostral preoptic area (OVLT/rPOA) in vivo, and 4) this decrease in density/cell values in slice 3 due to estradiol treatment, was greater in cultures derived from female vs . male tissues.(ABSTRACT TRUNCATED AT 250 WORDS) Jpn J Pharmacol, 1989 Aug, 50(4), 495 - 8 Effects of short chain fatty acids on the production of heat-labile enterotoxin from enterotoxigenic Escherichia coli; Takashi K et al.; Each addition of some short chain fatty acids (SCFAs) into casamino acids-yeast extract culture media at a concentration of 2 mg/ml reduced the production of heat-labile enterotoxin (LT) from enterotoxigenic Escherichia coli in proportion to the elongation of carbon chain from C-2 to C-7 . The LT-production was inversely recovered by the addition of longer chain fatty acids . The reduction of LT-production by SCFAs seems to depend on the disturbance of the biosynthesis of LT itself, since LT was not detected in the cells treated with n-heptyric acid at 2 mg/ml, which abolished the LT-production. Cutis, 1989 Aug, 44(2), 113 - 4 Recent developments in sexually transmitted diseases: chancroid--epidemiology, diagnosis, and treatment; Felman YM; The most important recent developments concerning chancroid have been in its epidemiology (a tremendous increase in the incidence of the disease in the United States over the past several years), diagnosis (better culture media), and treatment (several newer and older drugs that have been shown to be effective against chancroid in recent studies). Arch Biochem Biophys, 1989 Aug 1, 272(2), 386 - 92 Augmentation of inorganic pyrophosphate elaboration in cartilage by serum factors; Rosenthal AK et al.; The disordered production of inorganic pyrophosphate (PPi) by articular cartilage is thought to have an important role in the pathogenesis of calcium pyrophosphate dihydrate deposition disease and perhaps osteoarthritis . We have previously shown that fetal calf serum added to the culture media of porcine articular cartilage explants increases the elaboration of PPi into the ambient media . We have examined this PPi stimulatory activity by studying the effects of adult human serum (HS), serum derived from adult human plasma (HP), and an acid-alcohol extract of human platelets (PE) on PPi production in cartilage organ culture . Ten percent HS produces a 1.4-fold increase in PPi production after 48 h of culture, while cartilage incubated in media containing 10% HP produces no more PPi than that incubated in media alone . PE stimulates a mean 2-fold increase in PPi production at 48 h in the presence of low concentrations of HP, and has no effect alone . It does not appear to up-regulate the activity of the ectoenzyme nucleoside triphosphate pyrophosphohydrolase (NTPPPH), nor does it promote the release of enzyme substrate into the extracellular space . Cartilage exposed to 0.5% HP and PE has 1.51 +/- 0.36 units of NTPPPH activity whereas cartilage exposed to 0.5% HP alone has 1.52 +/- 0.41 units of enzyme activity . PE does not increase the release of {14C}adenine-labeled compounds into the media . Approximately 13% of soluble 14C counts was found in the media of chondrocytes treated with PE while 18% of counts was released in the presence of HP alone . We have demonstrated a factor or factors present in FCS, HS, and an acid-ethanol extract of human platelets which represent(s) the first known physiologic modulators of PPi production in articular cartilage and may increase PPi production without affecting NTPPPH activity. Shika Kiso Igakkai Zasshi, 1989 Aug, 31(4), 372 - 8 {In vitro cultivation of human pulpal fibroblast strains--permanent and deciduous teeth}; Tsukamoto Y et al.; We succeeded in separating and the cultivating stable monolayer cultures of dental pulp fibroblast strains derived from permanent and deciduous human teeth . Human permanent (n = 67) and deciduous teeth (n = 26) were extracted under acupuncture anaesthesia for the correction of malocclusion . After splitting the teeth, the pulp tissues were carefully removed, placed in tissue culture flasks, and grown in Dulbecco's modified Eagle medium supplemented with 10% fetal calf serum (FCS) . The human pulpal fibroblasts (HPF) of permanent teeth and deciduous teeth (DHPF) were subcultured . Both the HPF and DHPF appeared to migrate from adherent tissues within 24 to 48 hr after explanation . They proliferated in the pulp explants, and lined up in parallel rows of cells closest to the explant tissue within 7 to 10 days in all of the experimental cases . The outgrowing cells were subcultured at 1.3 x 10(4) cells/cm2 in tissue culture flasks every 4-11 days . They showed vigorous proliferation . The average number of cells in the 6-7 day cultures of HPF were 5.6 x 10(4) cells/cm2 from 3 to 16 passages . It was 4.7 x 10(4) cells/cm2 from 3 to 10 passages with DHPF . However, no difference was observed between HPF and DHPF in the amount of synthesized protein in culture flasks . Furthermore, the growth rate of DHPF was more sensitive than that of HPF to the FCS percentages of the culture media. Nippon Sanka Fujinka Gakkai Zasshi, 1989 Aug, 41(8), 971 - 80; discussion 1000-7 {Fundamental and clinical studies on biochemical properties of endometriosis in comparison with endometrium}; Taketani Y; 1 . Regulatory mechanism of cell growth of endometriosis in comparison with endometrium . Estradiol alone has no growth-promoting effect on both endometriotic and endometrial cells . Epidermal growth factor (EGF) stimulates cell growth of both cell types . Endometrial cells but not endometriotic cells produce and release EGF into culture media so that stimulatory effect of exogenous addition of EGF is blunted in endometrial cells . Estradiol exerts its mitogenic action by enhancing the mitogenic effect of EGF in endometrium . By contrast, the effect of estradiol is minimal in endometriotic cells, showing less dependency on estradiol for their proliferation . Progesterone inhibits cell growth of the both cell types in the same manner . 2 . A biological role of EGF in endometriosis . Endometriotic cells possess EGF receptors . The affinity of the receptor is the same as that of endometrial cells . However, the number of receptor per cell is about half of that for endometrium . Estradiol increases the number of EGF receptors in endometrial cells which may explain the mitogenic effect of estradiol in the face of EGF . However, stimulatory effect of estradiol for EGF receptors is less pronounced in endometriotic cells . Mitogenic action of EGF is suggested to be mediated by phosphorylation of tyrosine residues of 170 kd protein in the tissues . EGF increases the production of tissue plasminogen activator (t-PA) and activates the aromatase activity of the both cell types . However, the stimulatory action of EGF on progestin receptor is observed only in endometrial cells . 3 . Biochemical characterization of endometriotic cells in comparison with endometrial cells . Endometriotic tissues accumulate less amount of glycogen and XIII factor of blood coagulation as compared to endometrial tissues . The ability of endometriotic cells to release prostaglandin is also weaker, suggesting suppressed differentiated function of endometriotic cell . Endometriotic cells produce the same amount of CA125 as endometrial cells . Danazol and EGF inhibit the release of CA125 into culture media when standardized per cell . Therefore, normalization of CA125 levels during the treatment dose not always mean the reduction of the lesions but reflect the suppressed function of the endometriotic tissues . 4 . Altered microenvironment of endometriotic tissues . An analysis of peritoneal fluid . The amount of peritoneal fluid (PF) with endometriosis increased throughout the menstrual cycle . A number of macrophage is reported to increase in PF with endometriosis.(ABSTRACT TRUNCATED AT 400 WORDS) Biochem Biophys Res Commun, 1989 Jul 14, 162(1), 217 - 23 Concomitant secretion of big endothelin and its C-terminal fragment from human and bovine endothelial cells; Emori T et al.; A specific radioimmunoassay (RIA) for the carboxyl-terminal fragment (CTF) of big porcine endothelin (pET), an intermediate form of pET, was established to characterize big ET-like and its CTF-like immunoreactivity (LI) secreted from cultured bovine and human endothelial cells (EC) . The antibody used crossreacted equally with big pET(1-39) and its CTF(22-39), but not with pET(1-21) . Serial dilution curves of the culture media from bovine and human EC were parallel to that of standard CTF . Reverse-phase HPLC coupled with RIAs for big ET and ET of the culture media from bovine and human EC revealed essentially the same elution profiles: two major CTF-LI components, one corresponding to big pET(1-39) and the other to its CTF(22-39), in addition to one major ET-LI component corresponding to pET(1-21) . The amounts of CTF-LI were almost equal to that of ET-LI on a molar basis . These data suggest that big ET is processed by a putative ET converting enzyme to yield its CTF and the mature ET(1-21) in EC. Vet Clin North Am Food Anim Pract, 1989 Jul, 5(2), 291 - 300 Tremorgenic syndromes in livestock; Nicholson SS; Grasses that are essential components of livestock grazing programs sometimes are the source of tremorgenic toxicants to the animals consuming them . Morbidity can be high but mortality need not be if management closely observes the cattle daily and removes them at first sign of trouble . Specific treatment generally is not available nor needed . Survivors recover completely within a few days or weeks, except in chronic phalaris poisoning, where sheep and cattle may die after prolonged illness--or at least not make an economical recovery . Certain poisonous plants are responsible for tremorgenic signs in livestock and horses . White snakeroot and rayless goldenrod pose a public health risk to individuals who might drink milk from a goat or cow grazing toxic amounts of these weeds . Poisonous weeds and trees often are a local or regional problem, and often are seasonal . A veterinarian new to the area who has a food animal practice should seek out information relative to poisonous plants, nutritional deficiencies, and diseases endemic to the practice area . The ability of certain fungi to produce toxic metabolites in feed-stuffs creates the potential for tremorgenic or other types of toxicosis in most classes of livestock . Wet grain byproducts from ethanol production and other processes can provide the right culture media for fungi. Am J Respir Cell Mol Biol, 1989 Jul, 1(1), 13 - 20 Bronchial epithelial cells produce lung fibroblast chemotactic factor: fibronectin; Shoji S et al.; The interaction between the epithelial cells and the subjacent mesenchymal cells in the airway is thought to play a major role during tissue repair after airway injury and lung morphogenesis . To evaluate this interaction, we cultured human lung fibroblasts, and bovine and human bronchial epithelial cells, and determined that bronchial epithelial cell-conditioned medium has a chemotactic activity for lung fibroblasts . This activity had the characteristics of protein: it was nondialyzable, heat-labile, pepsin-labile, acid-stable, and lipid-inextractable . Molecular sieve chromatography on Sephadex G-150 and affinity chromatography on gelatin-Sepharose revealed that there was one peak of chemotactic activity in high molecular weight range, which bound to gelatin, thus suggesting that the chemotactic factor might be fibronectin . Production and secretion of fibronectin into the culture media were demonstrated by biosynthetic incorporation of radioactive amino acid into fibronectin followed by immunoprecipitation on SDS-PAGE and autoradiography . Release into the culture medium was confirmed by ELISA . The identity of fibronectin as the chemotactic activity was confirmed by the addition of antifibronectin antibody to the conditioned medium, which inhibited chemotaxis in dose-dependent manner . Thus, bronchial epithelial cells produce fibronectin which can function as a chemotactic factor for lung fibroblasts . This production of fibronectin by bronchial epithelial cells may play an important role in regulating interaction between the bronchial epithelial cells that line the lumenal surface of the bronchial epithelial wall and the mesenchymal fibroblasts that underlie the bronchial epithelial basement membrane. Diagn Microbiol Infect Dis, 1989 Jul-Aug, 12(4), 303 - 8 Evaluation of four mycobacterial blood culture media: BACTEC 13A, Isolator/BACTEC 12B, Isolator/Middlebrook agar, and a biphasic medium; Agy MB et al.; Four commercially available mycobacterial blood culture systems were compared for sensitivity and time to detection of growth . A 5-ml volume of SPS-anticoagulated blood was cultured in a BACTEC 13A vial and a modified M7H11/BHI biphasic medium . In addition, two aliquots of Isolator concentrates, each derived from 5 ml of blood, were inoculated into a BACTEC 12B vial and onto a pair of Middlebrook 7H11 agar plates (M7H11) . Mycobacteria were recovered from 32 of 180 cultured specimens (17.8%) . Growth was detected in 30 (93.7%) of the 13A vials, 27 (84.4%) of the M7H11 agar plates, 26 (81.2%) of the 12B vials, and 14 (43.8%) of the biphasic bottles . The mean times to growth detection in the 13A vial (14.2 days) and the 12B vial (13.7 days) were shorter than in either the M7H11 plates (20.8 days) or the biphasic medium (24.1 days) . When the Isolator/12B vial-and-M7H11 plates were evaluated as a single system, 29 cultures (90.6%) had a mean time to growth detection of 13.5 days . Colony-forming units per ml were inversely associated with time to growth detection . Delay in transport (greater than 24 h) appeared to reduce viability . The direct inoculation feature makes the 13A vial very suitable for mycobacterial blood cultures. J Neurochem, 1989 Jul, 53(1), 297 - 9 Direct evidence that excitotoxicity in cultured neurons is mediated via N-methyl-D-aspartate (NMDA) as well as non-NMDA receptors; Frandsen A et al.; Cultured GABAergic cerebral cortex neurons were exposed to the excitatory amino acid (EAA) L-glutamate, kainate (KA), N-methyl-D-aspartate (NMDA), or RS-alpha-amino-3-hydroxy-5-methyl-4-isoxazolopropionate (AMPA) . To ensure a constant glutamate concentration in the culture media during the exposure periods, the glutamate uptake inhibitor L-aspartic acid beta-hydroxamate was added at 500 microM to the cultures that were exposed to glutamate . Each of these EAAs was able to induce neurotoxicity . It was not possible to reduce or prevent glutamate-induced cytotoxicity by blocking only one of the glutamate receptor subtypes with either the NMDA receptor antagonist D-(-)-2-amino-5-phosphonopentanoate (APV) or with one of the specific non-NMDA antagonists 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and 6,7-dinitroquinoxaline-2,3-dione (DNQX) . However, if the cultures were exposed simultaneously to glutamate and the antagonists in combination, i.e., APV plus CNQX or APV plus DNQX, the toxicity was completely prevented . Furthermore, CNQX and DNQX were shown to be selective blockers of cytotoxic phenomena induced by non-NMDA glutamate agonists with no effect on NMDA-induced cell death . Likewise, APV prevented NMDA-induced cell death without affecting the KA- or AMPA-induced neurotoxicity . It is concluded that EAA-dependent neurotoxicity is induced by NMDA as well as non-NMDA receptors. J Endocrinol Invest, 1989 Jul-Aug, 12(7), 443 - 48 Effects of growth hormone-releasing hormone (GHRH) on densely granulated somatotroph adenomas and sparsely granulated somatotroph adenomas in vitro: a morphological and functional investigation; Kawakita S et al.; The effects of growth hormone-releasing hormone (GHRH) were studied on densely granulated somatotroph adenoma cells and sparsely granulated somatotroph adenoma cells in culture by measuring release of growth hormone (GH) as well as ultrastructural morphometrical parameters and comparing them with those of control adenoma cells . Both types of adenoma cells cultured with GHRH showed similar increases of GH release into culture media and exhibited similar increases in cytoplasmic volume densities (CVD) of endoplasmic reticulum and Golgi apparatus and decreases in CVD of secretory granules and secretory granule diameter . These results indicate that (1) both types of somatotroph adenoma cells react similarly to GHRH stimulation, despite their morphologic differences, and (2) GHRH stimulates GH synthesis as well as GH release by somatotroph adenoma cells. Rev Soc Bras Med Trop, 1989 Jul-Sep, 22(3), 119 - 24 Usefulness of serology for the evaluation of Trypanosoma cruzi transmission in endemic areas of Chagas' disease; Chuit R et al.; Thirteen communities from 7 Argentinian provinces were selected for the evaluation of serology as an indicator of transmission of Chagas disease . Of the communities appraised, 6 did not have a history of previous treatment with insecticides and 7 had received sporadic or continuous insecticide treatment . The inhabitants of 20% of the houses of each locality were studied by serology . The samples were obtained by finger pricking and 50 microliters of blood were mixed with 15 microliter of 50% glycerine solution in tissue culture media to be assayed by Indirect Hemagglutination and Indirect Immunofluorescence tests . In untreated areas, the prevalence of infection in infants 0-4 years old was 17.5%, reaching to over 22% for the 5-9 year old group, and to 33.3% in 10-14 year old individuals . The prevalence in treated and surveyed areas was 2.6% in 0-4 year old children, 5.4% in 5-9 year old and 6.2% in 10-14 year old youngsters . The differences between both areas were statistically significant (p less than 0.005) . This study favors serology as a valid indicator for the evaluation of transmission of Chagas disease in rural areas. J Invest Dermatol, 1989 Jul, 93(1), 10 - 7 Murine keratinocyte cultures grown at the air/medium interface synthesize stratum corneum lipids and "recycle" linoleate during differentiation; Madison KC et al.; In a recent investigation we showed that murine keratinocyte cultures grown at the air/medium interface in the presence of dermis exhibit morphologic differentiation comparable to that seen in vivo, including the formation of lamellar granules and stratum corneum intercellular lipid lamellae . In the present study, lifted cultures were found to more closely reproduce the lipid composition of the parent epidermal tissue than submerged cultures grown on plastic . In addition, the specific fatty acid profile of individual lipid classes in lifted cultures was, in general, remarkably well maintained in vitro . Acylceramides, which are highly enriched in linoleic acid in vivo, remained enriched in vitro; however, the linoleic acid content of the cultures was substantially lower than that in vivo, confirming previous reports of the relative essential fatty acid deficiency of standard culture media . As the lifted cultures differentiated over time, the lipid composition changed to reflect the formation of a stratum corneum with its different complement of lipids . Label from {U-14C}linoleic acid was specifically incorporated into linoleate-containing lipids during short pulses in both submerged and lifted cultures . Changes in label distribution over a long chase period in lifted cultures indicated that linoleate was transferred from phospholipids to ceramides, providing evidence for the "recycling" of essential fatty acids in epidermis. Brain Res, 1989 Jun 19, 490(1), 14 - 25 Endogenous opioid systems regulate growth of neural tumor cells in culture; Zagon IS et al.; Endogenous opioid systems (i.e., opioids and opioid receptors) play a role in neural cancer . Using a tissue culture system of S20Y murine neuroblastoma to assess the effects of opioids on growth, {Met5}-enkephalin was the most potent compound to influence cell replication . With a median effective concentration of 10(-10) M, this peptide inhibited cell proliferation in a stereospecific and naloxone-reversible manner . {Met5}-Enkephalin depressed both DNA synthesis and mitosis . {Met5}-Enkephalin was detected in neuroblastoma cells by radioimmunoassay, and was found to increase in concentration in culture media over time, suggesting that these cells produced the peptide . Immunocytochemistry showed {Met5}-enkephalin-like activity in the cortical cytoplasm, but not the cell nucleus, of neuroblastoma cells . Binding of {3H}-{Met5}-enkephalin specific and saturable, and Scatchard analysis yielded a Kd of 1.2 +/- 0.1 nM and a binding capacity of 50.2 +/- 4.3 fmol/mg protein . {Met5}-Enkephalin also depressed the growth of N115 murine neuroblastoma, SK-N-MC human neuroblastoma, and HT-1080 human fibrosarcoma . These results indicate that {Met5}-enkephalin, a naturally occurring pentapeptide that is derived from proenkephalin A, is a potent inhibitor of cell growth . Since cancer cells produce {Met5}-enkephalin, and contain a binding site to this ligand, endogenous opioid systems appear to control cell proliferation by an autocrine mechanism. FEBS Lett, 1989 Jun 19, 250(1), 85 - 90 A new family of growth factor-like peptides . 'Trefoil' disulphide loop structures as a common feature in breast cancer associated peptide (pS2), pancreatic spasmolytic polypeptide (PSP), and frog skin peptides (spasmolysins); Thim L; Four peptides present in completely different biological sources have been shown to exhibit a large degree of structural similarity . The peptides include: (i) a 60 amino acid residue breast cancer associated pS2 peptide isolated from human gastric juice and the culture media of the human breast cancer cell line MCF-7; (ii) a 106 amino acid residue pancreatic spasmolytic polypeptide (PSP) isolated from porcine pancreas and pancreatic juice; and (iii) a 49 and 50 amino acid residue peptide predicted from a cDNA isolated from the skin of the frog, Xenopus laevis . These peptides are characterized by having one (pS2 and the frog peptides) or two (PSP) domains of a highly conserved 38-39 amino acid residue consensus sequence not found in any other known peptides or proteins . The domain sequences contain 6 cysteine residues in nearly the same positions and it is suggested that these 6 residues are linked by 3 disulphide bonds to form a characteristic 'trefoil' disulphide loop structure common in all four peptides . From the sources of which the peptides have been isolated and from experiments showing that PSP has a growth factor stimulatory effect on MCF-7 cells, it is further suggested that these peptides may represent members of a new family of growth factors. Thromb Res, 1989 Jun 15, 54(6), 655 - 75 Biosynthesis of factor V by normal adult rat hepatocytes; Mazzorana M et al.; The synthesis of coagulation factor V was investigated in isolated rat hepatocytes maintained in long-term primary culture . Two culture conditions were compared . A clotting assay and an immunoprecipitation experiment with rabbit anti rat factor V IgG were used to demonstrate not only the presence of factor V in the cells but also active secretion into the culture medium . Both the inhibition of the clotting reaction in presence of the antibody and absence of thrombin in culture media confirmed the specificity of the clotting assays . Electron microscopic examination located factor V in the endoplasmic reticulum and Golgi apparatus of hepatocytes in common with other liver specific plasma proteins . Examination of liver tissue sections confirmed the production of factor V in hepatocytes but not in hepatic endothelial cells although it did not exclude a transit pathway of factor V through these cells . Addition of Russell viper venom factor V activating enzyme to the culture medium had no effect on the factor V activity . In contrast, treatment of cell extracts did increase the coagulant activity . This suggests that hepatocytes contained principally an unactivated form or procofactor, whereas factor V present into the culture medium was mainly in an activated form . These data provide evidence for synthesis and secretion of an hepatocytic factor V. J Immunol, 1989 Jun 15, 142(12), 4372 - 7 Expression of a binding structure for sialic acid-containing glycoconjugates on rat bone marrow-derived macrophages and its modulation by IFN, TNF-alpha, and dexamethasone; Gessl A et al.; Rat macrophages express a binding structure for sialic acid-containing glycoconjugates (sialic acid-binding receptor, SAR) which can be detected by a rosette assay utilizing SRBC coated with bovine brain gangliosides (E-G) . Freshly isolated rat bone marrow cells (BMC) contain about 5% SAR-positive cells . Rat BMC cultured for 1 wk with tissue culture media containing CSF-1 differentiate into a virtually pure population of bone marrow-derived macrophages (BMDM phi) . All BMDM phi bound E-G coated with an optimal concentration of gangliosides (100 micrograms/ml) . When BMC were cultured for 1 wk with murine recombinant granulocyte-macrophage CSF, irrespective of the dose of GM-CSF, approximately 90% of the cells were identified as rat macrophages, and practically all expressed SAR . Only about 50% of BMDM phi bound SRBC coated with a suboptimal concentration of gangliosides (20 micrograms/ml) . However, this percentage increased markedly after 8 to 72 h incubation with 1 to 10,000 U/ml purified murine IFN-alpha or IFN-beta, whereas murine or rat rIFN-gamma at doses above 10 U/ml led to a decrease of E-G binding . Human and murine rTNF-alpha enhanced rosette formation in a dose-dependent manner . These effects could be blocked by the respective anti-cytokine antibodies . Treatment of BMDM phi with dexamethasone also augmented E-G rosetting . The enhancement of E-G binding was abolished by pretreatment of BMDM phi with cycloheximide and actinomycin D but not with mitomycin C, suggesting that de novo synthesis of protein and RNA, but not DNA, is required . Our results demonstrate that all rat BMDM phi constitutively bear SAR, and that murine IFN-alpha, IFN-beta, and TNF-alpha, as well as dexamethasone, may augment SAR expression. Mol Biochem Parasitol, 1989 Jun 1, 35(1), 73 - 8 Trichomonas vaginalis and Tritrichomonas foetus secrete neuraminidase into the culture medium; Costa e Silva Filho F et al.; Supernatants taken from axenic cultures of Trichomonas vaginalis and Tritrichomonas foetus contain a neuraminidase activity, the detection of which is augmented when the trichomonad culture media are supplemented with 30% supernatant of confluent epithelial cultures . The enzyme was active against human erythrocytes, which became highly reactive to peanut agglutinin lectin . The specificity of the enzyme was checked by using a substrate specific to neuraminidase: 2'-(4-methylumbelliferyl)-alpha-D-N-acetylneuramic acid. Blood, 1989 Jun, 73(8), 2074 - 6 Multimeric composition of endothelial cell-derived von Willebrand factor; Tsai HM et al.; The multimeric composition of human endothelial cell (EC)-derived von Willebrand factor (vWF) was studied using SDS-agarose gel electrophoresis and autoradiography . Two multimers were found in lysates prepared from confluent cultures of human umbilical vein endothelial cells . The smaller multimer had a molecular weight (mol wt) of approximately 950 Kd, while the second was larger than those seen in plasma . When electrophoresis was performed using the discontinuous buffer system of Ruggeri and Zimmerman, the small multimer consisted of a single band migrating with the slowest-moving component of the corresponding plasma triplet . The large EC-vWF multimer was detected in culture media conditioned with EC monolayers for ten minutes . It remained the only multimer in media conditioned for up to three days . Calcium ionophore A23187 increased the amount of the large vWF multimer released into the culture media, but did not change its multimeric composition . The small multimer was never detected in the EC-conditioned media . These findings suggest that (1) a large, fully polymerized multimer of vWF is released from the ECs, while the small multimer probably represents a major intermediate component in the process of multimerization, and (2) plasma vWF multimers are probably generated from the large endothelial vWF after it is released into the circulation. J Endocrinol, 1989 Jun, 121(3), 513 - 9 Indomethacin inhibits the effects of oestrogen in the anterior pituitary gland of the rat; Rosental DG et al.; Two inhibitors of prostaglandin synthesis, indomethacin and aspirin, blocked the increase of oestrogen-binding sites in the nuclear subcellular fraction, an increase which occurs after the administration of oestradiol . Consequently the biological effects of oestrogens in the anterior pituitary gland of the rat (prolactin synthesis, concentration of progesterone-binding sites and cell proliferation) are diminished . The anterior pituitary gland synthesized prostaglandin F2 alpha (PGF2 alpha), PGE2 and PGD2 from arachidonic acid . This synthesis was blocked when indomethacin was added to the culture media . Oestrogen increased the concentration of PGE2: an increase that was partially prevented by indomethacin . Prostaglandins may have an important role on the effects of oestrogen in the anterior pituitary gland of the rat. Int J Dev Biol, 1989 Jun, 33(2), 267 - 75 Production of fibronectin and collagen types I and III by chick embryo dermal cells cultured on extracellular matrix substrates; Robert J et al.; Dermal cells isolated from the back skin of 7-day chick embryos were cultured on homogeneous two-dimensional substrates consisting of one or two extracellular matrix components (type I, III, or IV collagen, fibronectin and several glycosaminoglycans (GAGs): hyaluronate, chondroitin-4, chondroitin-6, dermatan and heparan sulfates) . The effect of these substrates on the production of fibronectin, of types I, III and IV collagen by cells was compared with that of culture dish polystyrene . Using immunofluorescent labeling of cultured cells, it was observed that, on all substrates, in 1-day and 7-day cultures, 85 to 95% of cells contain type I collagen in the perinuclear cytoplasm; label was absent from cell processes . Type I collagen was also detected in extracellular fibers extending between neighboring cells . By contrast, on all substrates, only 5 to 20% of cells produced type III collagen . Otherwise distribution of type III collagen was similar to that of type I collagen . With anti-type IV collagen antibody no staining of either cell content or extracellular spaces was detected . Staining with anti-fibronectin antibody revealed two types of distribution patterns . On polystyrene and on all but type I collagen substrates, labeling revealed clusters of short thick strands and patches of fibronectin-rich material in extracellular spaces . On type I collagen substrate, however, immunostaining revealed a delicate network of regularly spaced parallel fibrils of fibronectin extending between and along cells . Using quantitative radioimmunoassay of the culture media, it was shown that, after 7 days of culture, cells secreted more type I than type III collagen.(ABSTRACT TRUNCATED AT 250 WORDS) Cell Tissue Res, 1989 Jun, 256(3), 487 - 94 The effect of low- and high-density lipoprotein cholesterol on steroid hormone production and ACTH-induced differentiation of rat adrenocortical cells in primary culture; Heikkila P et al.; We studied the effects of lipoprotein-derived cholesterol on the ACTH-induced differentiation of cultured fetal rat adrenocortical cells . For this purpose human plasma high-density lipoprotein3 (HDL3) or low-density lipoprotein (LDL) was added to culture media devoid of cholesterol, and thereafter the morphological changes in cells were monitored and the amounts of steroids synthesized were measured . It could be demonstrated that, ultrastructurally, upon ACTH-stimulation the adrenocortical cells differentiated into fasciculata-like cells even in the absence of lipoproteins in the culture medium . The addition of either HDL3 or LDL caused an increase in the number and size of cytoplasmic lipid droplets suggesting uptake and deposition of lipoprotein-derived cholesterol into the differentiating cells . The amount of steroids secreted from cells differentiating in media devoid of cholesterol was only half that observed in cells differentiating in serum-supplemented medium . Addition of either HDL3 or LDL increased the ACTH-stimulated steroid synthesis to the levels observed in serum-supplemented medium . This study demonstrates that both HDL3 and LDL are able to provide cholesterol for steroid synthesis accompanying the ACTH-induced differentiation of fetal rat adrenocortical cells. Yan Ke Xue Bao, 1989 Jun, 5(1-2), 32 - 5 Study in cytotoxicity of gentamycin to corneal epithelium and endothelium in tissue culture; Lin N et al.; A study in cytotoxicity of gentamycin to tissue-cultured bovine corneal endothelial cells and rabbit corneal epithelial cells is reported . When the cultured cells reached confluence, they were exposed to tissue culture media containing gentamycin for 6 hours . We found that 0.5% gentamycin caused significant damage to corneal epithelial cells--diffuse plasmolysis, with scattered cell necrosis and 5% loss . While corneal endothelial cells were exposed to 1.6 mg/ml gentamycin, extensive cell loss (approximately 15%) was observed . The damaged cells recovered their normal morphology after 24 hours . When the concentration of gentamycin increased twice, serious damage to cells occurred . The area of cell loss reached 40%, and the recovery of cellular morphology was much slower . This study demonstrates that gentamycin potential cytotoxicity to corneal epithelium and endothelium, suggesting that gentamycin should be rationally used in the treatment of ocular diseases. Toxicology, 1989 May 31, 56(1), 107 - 17 Comparative toxicity of allylamine and acrolein in cultured myocytes and fibroblasts from neonatal rat heart; Toraason M et al.; Allylamine is toxic to the cardiovascular system causing aortic, valvular and myocardial lesions . Acute toxicity is believed to involve metabolism of allylamine to highly reactive acrolein . Comparative toxicity of allylamine and acrolein was evaluated in cardiac fibroblasts and myocytes, which were obtained from neonatal rat hearts by a differential plating technique . Allylamine and acrolein were added directly to serum supplemented culture media (M199) . Toxicity was assessed by measuring lactate dehydrogenase (LDH) release as an indicator of cell lysis . Spontaneous beating activity of myocytes, and adenosine 5' triphosphate (ATP) levels of myocytes and fibroblasts were also assessed . Cell lysis occurred 4 h after treatment of myocytes with 0.5 mM allylamine, whereas 20 mM allylamine was required to lyse fibroblasts . Acrolein, at a concentration of 0.05 mM, was equally toxic to fibroblasts and myocytes . Semicarbazide, a benzylamine oxidase inhibitor, protected myocytes from allylamine toxicity, but clorgyline, a monoamine oxidase inhibitor, was ineffective . Semicarbazide was ineffective against acrolein toxicity . Beating activity of myocytes was arrested by 0.05 mM acrolein and 0.5 mM allylamine, although 0.05-0.1 mM allylamine reduced beating activity . Myocyte ATP levels were reduced 4 h after exposure to 0.01 mM acrolein . Allylamine at 0.05 mM reduced ATP in myocytes, but 10 mM allylamine was required to reduce ATP in fibroblasts . ATP levels remained normal in myocytes exposed to 1 mM allylamine in the presence of 0.1 mM semicarbazide . The findings support the hypothesis that the toxicity of allylamine in cultured myocytes is dependent on its metabolism to acrolein, and that cytotoxicity may result from interference with energy production. Eur J Biochem, 1989 May 15, 181(3), 721 - 6 Thrombospondin is synthesized and secreted by human osteoblasts and osteosarcoma cells . A model to study the different effects of thrombospondin in cell adhesion; Clezardin P et al.; In this study we have shown by both immunofluorescence and immunoprecipitation techniques that human osteoblasts and osteosarcoma cells synthesize and secrete thrombospondin, a 450-kDa glycoprotein initially found in platelets . Immunofluorescence with a mouse monoclonal antibody to human platelet thrombospondin yielded specific granular staining within the cytoplasm of human osteoblasts . SDS/polyacrylamide gel electrophoresis analysis of immunoprecipitates obtained with polyclonal and monoclonal anti-thrombospondin antibodies allows the identification of thrombospondin in the cellular lysates and the culture media of biosynthetically labelled osteoblasts and osteosarcoma cells . Kinetic and dose/response studies of osteoblasts and of two osteosarcoma cell lines (MG-63, SaOs-2) were performed to assess the ability of these cells to adhere to thrombospondin and type-I collagen . Thrombospondin promoted the attachment of human osteoblasts whereas it inhibited the adhesion of MG-63 and SaOs-2 cells, both when it was directly adsorbed to plastic and when it was bound to type-I collagen . Therefore osteoblasts and osteosarcoma cells may be valuable tools to study the role of thrombospondin in cell adhesion. Int J Cancer, 1989 May 15, 43(5), 949 - 55 Presence of nerve growth factor-like immunoreactivity in carcinoid tumour cells and induction of a neuronal phenotype in long-term culture; Ahlman H et al.; Mid-gut carcinoid tumour cells expressed a neuronal phenotype, observed and characterized immunocytochemically in long-term culture . Initially the culture contained a main population of spherical tumour cells with granules immunopositive for serotonin (5-HT) and tachykinins (TK) . Production and secretion of these substances into media was verified biochemically . Cytoplasmic granules with 5-HT-like immunoreactivity (5-HT-LI) were markedly reduced during culture, while granules with TK-LI were unchanged in number, corresponding to the biochemical findings . After a few days in culture, tumour cells were flattened and fine neurite-like processes extended . After 2-3 weeks many endocrine tumour cells had converted to neuron-like cells with slender cell processes containing granules with TK-LI . Varicose enlargements and apparent growth cones were observed . When neurites were extended, 50-80% of the neuron-like cells were positive with antisera against the neurofilament triplet . Cells of both endocrine and neuronal phenotypes were positive with antisera against tetanustoxin, Thy 1-antigen, neuron-specific enolase, synapsin and a synaptic vesicle protein (p 38) supporting the concept of these tumour cells as para-neurons . Intermediate filaments, studied with monoclonal anti-vimentin, were found in all cells . Filaments were also observed ultrastructurally . Initially, nerve growth factor (NGF)-LI was found in granules of all spherical tumour cells . When neuritic processes were extended, the cells appeared to lose these granules . After 40 days in culture, NGF-LI was absent or very sparse . The studies indicate autocrine secretion of a growth factor, reacting with the NGF antiserum, by cultured mid-gut carcinoid tumour cells inducing a neuronal phenotype with enhanced NF and TK synthesis and suppressed 5-HT synthesis . In bioassay systems the culture media caused a delayed neurite reaction on PC12 cells, but no reaction on chick ciliary ganglion cells, indicating that the factor is not authentic NGF. Biochem Biophys Res Commun, 1989 May 15, 160(3), 1415 - 20 Effect of two different glucose concentrations on insulin receptor mRNA levels in human hepatoma HepG2 cells; Briata P et al.; Glucose is known to affect mRNA levels of several genes . In order to investigate possible effects of glucose on insulin receptor mRNA levels, we cultured human hepatoma cells (HepG2) in two different culture media: DMEM containing 100 mg/dl glucose and DMEM containing 450 mg/dl glucose . Insulin receptor mRNA levels and insulin binding activity were reduced in HepG2 cultured at lower glucose concentrations . These data suggest that glucose affects insulin receptor gene expression. Cancer Res, 1989 May 15, 49(10), 2584 - 7 Inhibition of carcinogen-inducible DNA amplification in a simian virus 40-transformed hamster cell line by ethacridine or ethanol; Burkle A; DNA amplification as a mechanism to increase gene expression has been established as a cause of cytotoxic drug resistance and appears to play a role in tumor cell progression . In order to investigate factors which control the process of DNA amplification we have been using a simian virus 40 (SV40)-transformed Chinese hamster cell line (CO60) as a model system . This cell line can be induced to amplify integrated viral DNA with a variety of agents . In this report the following is shown . (a) Addition of ethacridine, an intercalative compound, or ethanol to the culture media inhibits amplification induced by the alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine or by gamma-irradiation in a dose-dependent fashion . In the case of N-methyl-N'-nitro-N-nitrosoguanidine induction (50 microM), the highest concentrations of ethacridine (40 microM) or ethanol (2% v/v) tested reduced SV40 amplification from about 20-fold to less than 2-fold . (b) Neither substance induces significant amplification when applied alone over a wide range of concentrations (0.01-20 microM ethacridine; 0.001-2% ethanol) . (c) Significant inhibition of amplification is achieved with nearly nontoxic concentrations of both substances (10 microM; 1%), (d) Without direct interference with the inducer . It is concluded that ethacridine or ethanol treatment uncouples the toxic effects of an alkylating agent or ionizing radiation from their ability to induce amplification in CO60 cells. Anal Biochem, 1989 May 15, 179(1), 24 - 7 Bidimensional reversed-phase high-pressure liquid chromatography analysis of cultured cell neuropeptides: application to atrial natriuretic factor; Nguyen TT et al.; We report here a one-step procedure for extraction and analysis of neuropeptides in chromaffin cell culture media and acid extracts using reversed-phase HPLC . The bidimensional HPLC system consists of a precolumn connected to a six-port switching valve which is on-line with an analytical column . The direct injection of the biological samples onto the precolumn previously equilibrated with 15% acetonitrile allows the elimination of interfering substances . The samples purified on the precolumn can then be eluted onto the analytical column via the switching valve for neuropeptide separation . This trace-enrichment system allows a minimum of sample handling, both saving time and reducing possibilities of loss and contamination . This method has been applied to monitor the precursor and mature forms of atrial natriuretic factor from chromaffin cell secretion media and cell content extracts . The recovery of atrial natriuretic factor is in the range of 80-100% . This procedure could be applied to the study of the precursor-product relationship of any neuropeptide, e.g., from radiolabeled extracts of pulse-chase experiments performed on cultured chromaffin cells. J Immunol, 1989 May 15, 142(10), 3361 - 8 Regulation of antigen presentation . II . Anti-Ig and IL-2 induce IL-1 production by murine splenic B cells; Hawrylowicz CM et al.; Murine splenic B cells did not constitutively express IL-1 activity . After culture with anti-Ig and T cell-conditioned media and then fixation, B cells expressed membrane IL-1 and were able to stimulate growth of the IL-1-dependent T cell clone D10 . Expression of membrane IL-1 required stimulation of B cells for 2 days before fixation . Significant IL-1 activity was detectable in freeze-thaw lysates of identical B cell preparations by 12 h . B cells also released IL-1 into the culture media . In situ hybridization studies by using probes to murine IL-1 alpha and IL-1 beta genes supported these observations . Thus, messenger RNA for IL-1 alpha and IL-1 beta rose in parallel, were detected between 6 and 24 h of culture, and declined to low levels by 30 h . Despite the presence of mRNA for IL-1 alpha and IL-1 beta, only IL-1 alpha had functional activity as determined by the use of a mAb to IL-1 alpha . IL-2 was found to be an essential component of the T cell-derived supernatant . Although IL-4 or TNF did not induce significant B cell IL-1 expression, they both caused a modest, but reproducible enhancement when added in combination with IL-2 . IFN-gamma, by contrast, partially inhibited IL-1 induction. In Vitro Cell Dev Biol, 1989 May, 25(5), 424 - 34 Modulation of actin mRNAs in cultured vascular cells by matrix components and TGF-beta 1; Kocher O et al.; Alpha-smooth muscle actin is currently considered a marker of smooth muscle cell differentiation . However, during various physiologic and pathologic conditions, it can be expressed, sometimes only transiently, in a variety of other cell types, such as cardiac and skeletal muscle cells, as well as in nonmuscle cells . In this report, the expression of actin mRNAs in cultured rat capillary endothelial cells (RFCs) and aortic smooth muscle cells (SMCs) has been studied by Northern hybridization in two-dimensional cultures seeded on individual extracellular matrix proteins and in three-dimensional type I collagen gels . In two-dimensional cultures, in addition to cytoplasmic actin mRNAs which are normally found in endothelial cell populations, RFCs expressed alpha-smooth muscle (SM) actin mRNA at low levels . alpha-SM actin mRNA expression is dramatically enhanced by TGF-beta 1 . In addition, double immunofluorescence staining with anti-vWF and anti-alpha-SM-1 (a monoclonal antibody to alpha-SM actin) shows that RFCs co-express the two proteins . In three dimensional cultures, RFCs still expressed vWF, but lost staining for alpha-SM actin, whereas alpha-SM actin mRNA became barely detectable . In contrast to two-dimensional cultures, the addition of TGF-beta 1 to the culture media did not enhance alpha-SM actin mRNA in three-dimensional cultures, whereas it induced rapid capillary tube formation . Actin mRNA expression was modulated in SMCs by extracellular matrix components and TGF-beta 1 with a pattern very different from that of RFCs . Namely, the comparison of RFCs with other cell types such as bovine aortic endothelial cells shows that co-expression of endothelial and smooth muscle cell markers is very unique to RFCs and occurs only in particular culture conditions . This could be related to the capacity of these microvascular endothelial cells to modulate their phenotype in physiologic and pathologic conditions, particularly during angiogenesis, and could reflect different embryologic origins for endothelial cell populations. Endocrinology, 1989 May, 124(5), 2464 - 72 Immunological characterization and immunocytochemical localization of oviduct-specific glycoproteins in the baboon (Papio anubis); Verhage HG et al.; Oviducts obtained from estradiol-treated ovariectomized baboons synthesize and release a family of high mol wt (100,000-130,000) glycoproteins during short term explant culture . The objective of this study was to make a polyclonal antibody to these glycoproteins and then use the antibody to determine the presence of the glycoproteins in oviduct flushings, tissue culture media, and tissues obtained from cycling and steroid-treated baboons . Oviduct culture medium proteins from estradiol-treated baboons were separated on one-dimensional polyacrylamide gels and transferred to nitrocellulose membranes . The region containing the glycoproteins was cut out, solubilized in dimethylsulfoxide, mixed with Freund's adjuvant, and injected at 2-week intervals into a male rabbit . The anti-serum used in this study was obtained 6 weeks after the initial injection and cross-reacted with antigens on Western blots of oviduct flushings and oviduct culture media obtained from follicular stage and estradiol-treated baboons . The antigens were absent in oviduct flushings obtained from luteal stage, ovariectomized and estradiol-primed baboons treated with estradiol and progesterone or progesterone alone . The antigens were not detected on Western blots of other reproductive and nonreproductive tract culture media or in serum obtained from follicular stage or estradiol-treated baboons . Immunoperoxidase staining was limited to discrete granules in the apical cytoplasm of secretory cells in oviducts obtained from follicular stage and estradiol-treated baboons . Thus, the secretory cells of the baboon oviduct synthesize and secrete a family of estradiol-dependent oviduct-specific glycoproteins that may have potential functional significance during fertilization and embryo development. Kidney Int, 1989 May, 35(5), 1119 - 25 Micromolar aluminum levels reduce 3H-thymidine incorporation by cell line UMR 106-01; Blair HC et al.; Aluminum-induced osteomalacia is a frequent complication observed in patients on maintenance hemodialysis . However, it is not known whether there are direct effects of aluminum on osteoblasts, or alternatively, whether the observed changes are due to changes in PTH or other factors . We sought to determine the effect of micromolar levels of aluminum on osteoblasts using a well-defined cell line derived from a 32P induced osteosarcoma of rat, UMR 106-01, which is alkaline-phosphatase positive, responds to PTH, and synthesizes type I collagen . Aluminum exposure was controlled using tissue culture media with {Al } less than 1 microgram/liter (40 nM), produced by precipitation of aluminum salts at pH 8.5 . The effect of defined {Al }, from 20 to 800 micrograms/liter (0.7 to 30 microM), was then determined by adding back aluminum while measuring DNA and protein synthesis . We found that aluminum depressed DNA synthesis, as determined by 3H-thymidine incorporation, by 60%, with half maximal effect at 20 micrograms/liter (740 nM) in cells at a density of 20,000/cm2 . Alternatively, protein synthesis, as determined by 3H-leucine incorporation, did not decline, and in some cases increased . However, qualitative analysis of matrix proteins produced with and without 800 micrograms/liter (30 mM) {Al } showed no differences . Direct measurements of cell number and protein synthesis confirmed these findings . Al does not alter the PTH-induced cAMP response of these cells . Thus, aluminum has a direct effect on cell division, and probably on protein synthesis, in this osteoblast-like cell line . These effects occur at levels of aluminum below those commonly contaminating tissue culture media, and thus are seen reproducibly only in media of defined {Al }.(ABSTRACT TRUNCATED AT 250 WORDS) Appl Environ Microbiol, 1989 May, 55(5), 1209 - 13 Factors relevant in bacterial pyrroloquinoline quinone production; van Kleef MA et al.; Quinoprotein content and levels of external pyrroloquinoline quinone (PQQ) were determined for several bacteria under a variety of growth conditions . From these data and those from the literature, a number of factors can be indicated which are relevant for PQQ production . Synthesis of PQQ is only started if synthesis of a quinoprotein occurs, but quinoprotein synthesis does not depend on PQQ synthesis . The presence of quinoprotein substrates is not necessary for quinoprotein and PQQ syntheses . Although the extent of PQQ production was determined by the type of organism and quinoprotein produced, coordination between quinoprotein and PQQ syntheses is loose, since underproduction and overproduction of PQQ with respect to quinoprotein were observed . The results can be interpreted to indicate that quinoprotein synthesis depends on the growth rate whereas PQQ synthesis does not . In that view, the highest PQQ production can be achieved under limiting growth conditions, as was shown indeed by the much higher levels of PQQ produced in fed-batch cultures compared with those produced in batch experiments . The presence of nucleophiles, especially amino acids, in culture media may cause losses of PQQ due to transformation into biologically inactive compounds . Some organisms continued to synthesize PQQ de novo when this cofactor was administered exogenously . Most probably PQQ cannot be taken up by either passive diffusion or active transport mechanisms and is therefore not able to exert feedback regulation on its biosynthesis in these organisms. J Neurochem, 1989 May, 52(5), 1576 - 81 Role of intracellular pH in the axolemma- and myelin-induced proliferation of Schwann cells; Saunders RD et al.; In order to provide additional information on the biochemical events that interact to cause Schwann cells to proliferate, we have monitored the intracellular pH of Schwann cells that have been stimulated to divide with myelin-enriched fractions (MEF) or axolemma-enriched fractions (AEF) . The intracellular pH of Schwann cells was monitored using 2',7'-bis(carboxymethyl)-5(6)-carboxyfluorescein (BCECF), which displays an increase in fluorescence upon alkalinization . Both AEF and MEF caused dose-dependent increases in the intracellular fluorescence of the Schwann cell cultures . At their maximum doses, AEF and MEF stimulation resulted in a 260 and 300% increase in intracellular fluorescence, respectively . The increase in intracellular fluorescence was abolished when cells were stimulated in Na+-free media, suggesting a role for the Na+/H+ exchanger . Mitotic stimulation required integrity of the Na+/H+ exchanger, as inhibition of the Na+/H+ exchanger for periods up to 1 h after addition of mitogen caused a significant inhibition of subsequent mitosis . Phorbol esters, which can potentiate AEF- and MEF-induced Schwann cell proliferation, increased intracellular fluorescence fivefold, an effect which was also dependent upon the presence of Na+ in the culture media . The specificity of the increase in intracellular pH for AEF and MEF was tested by incubating Schwann cells with liver microsomes and a biologically inactive phorbol alcohol, neither of which is significantly mitogenic for Schwann cells . Neither liver microsomes nor phorbol alcohol had a significant effect on intracellular pH . The implications of the increase in intracellular pH in Schwann cells with respect to inositol phospholipid metabolism, protein kinase C activation, and cellular proliferation are discussed.(ABSTRACT TRUNCATED AT 250 WORDS) Am J Vet Res, 1989 May, 50(5), 747 - 50 Proadifen-induced production of prostacyclin by equine peritoneal macrophages; Morris DD et al.; A study was performed to determine the effect of proadifen hydrochloride on prostacyclin (prostaglandin I2 {PGI2}) and thromboxane A2 (TxA2) synthesis by equine peritoneal macrophages and the effect of proadifen on endotoxin-induced synthesis of PGI2 and TxA2 by equine macrophages . Peritoneal macrophages (2.5 x 10(6)/ml) were incubated for 6 hours in tissue culture media containing 1) nothing (nontreated control), 2) proadifen hydrochloride (20, 100, 250, and 500 mumol/L, 3) endotoxin (5 ng/ml), or 4) the calcium ionophore A23187 (0.95 mumol/L) . In a second series of experiments, peritoneal macrophages were incubated with endotoxin (5 ng/ml) and proadifen (250 umol/L), for 6 hours . Concentrations of 6-keto-prostaglandin F1 alpha (6-keto-PGF1 alpha) and thromboxane B2, the stable metabolites of PGI2 and TxA2, were determined in the incubation media by radioimmunoassay . Proadifen caused increased synthesis of PGI2 by equine macrophages, without affecting TxA2 production . The increased PGI2 production was similar to that induced by endotoxin and calcium ionophore; however, the latter 2 agents significantly stimulated TxA2 production as well (P less than 0.05) . There were no significant differences among mean concentrations of 6-keto-PGF1 alpha in media from macrophages treated with 100, 250, or 500 mumol/L proadifen, but there was a significant curvilinear regression between their concentrations . The ratio of thromboxane B2 to 6-keto-PGF1 alpha was significantly lower than baseline in incubation media from macrophages exposed to proadifen, endotoxin, and calcium ionophore . Proadifen hydrochloride did not significantly change equine peritoneal macrophage production of PGI2 or TxA2 in response to endotoxin. Endocrinology, 1989 May, 124(5), 2321 - 9 An insulin-like growth factor-binding protein in the baboon (Papio anubis) endometrium: synthesis, immunocytochemical localization, and hormonal regulation; Fazleabas AT et al.; The major secretory product of the baboon and human decidua during pregnancy is an insulin-like growth factor-binding protein (IGF-BP) . This study was designed to determine the site and regulation of synthesis of this protein in the nonpregnant baboon based on our previous findings that this molecule is biochemically and immunologically similar in the two species during pregnancy . Endometrial tissue was obtained from cycling baboons and steroid-treated ovariectomized animals . Portions of the tissue were fixed for immunocytochemical analysis, cultured in the presence or absence of cyclohexamide and actinomycin-D, or snap-frozen in liquid nitrogen for RNA isolation . Immunostaining using monoclonal antibodies to IGF-BP indicated that the protein was localized predominantly in the epithelium of the deep glands and was most intense during the late luteal stage of the cycle . Immunoreactive product was also present in tissue from estrogen-primed progesterone-treated ovariectomized animals . However, the staining pattern was more variable and less intense than that in intact animals . Western blots of explant culture media showed the presence of an immunoreactive product only in those tissues that also demonstrated immunocytochemical localization . The absence of an immunoreactive band in medium obtained from tissue incubated in the presence of cyclohexamide suggested that this protein was synthesized de novo . The mRNA coding for IGF-BP appeared to be stable, as synthesis in explant cultures continued in the presence of actinomycin-D . The cDNA probe hybridized to a single message transcript of 1.65 kilobases . The presence of mRNA in tissues coding for this protein correlates with the immunochemical data relating to the site and hormonal regulation of its synthesis . The presence of this protein in the glandular epithelium of the baboon endometrium may have implications in the autocrine and/or paracrine regulation of trophoblast growth and penetration during implantation. Endocrinology, 1989 Apr, 124(4), 1595 - 601 Glucocorticoids stimulate plasminogen activator production by rat granulosa cells; Wang C et al.; We studied the direct effects of glucocorticoids on plasminogen activator (PA) production by rat granulosa cells . PA production was assayed by culturing rat granulosa cells on {125I}fibrin plates and determining the extent of fibrinolysis after addition of the specific substrate plasminogen . In granulosa cells from preantral follicles of immature rats, treatment with FSH caused dose-dependent increases in PA production whereas glucocorticoids by itself was without effect . Increasing concentrations (10(-10) to 10(-6) M) of both natural and synthetic glucocorticoids potentiated the stimulating effect of FSH on PA production by 120 to 170% . The stimulatory potencies of the natural corticosteroids correlated with the glucocorticoid potencies (cortisol/corticosterone greater than aldosterone/11-deoxycorticosterone) . In granulosa cells from Graafian follicles of mature rats, glucocorticoids on its own had direct stimulating effect on PA production . The stimulatory action of glucocorticoids on FSH-dependent PA production was completely blocked by simultaneous treatment with antiglucocorticoid RU 486 . Antiserum directed against tissue-type PA (tPA) neutralized the increased fibrinolytic actions of glucocorticoids . Using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fibrin autography techniques, we showed that the increase in fibrinolytic activity in response to glucocorticoids resulted from increased production of tPA rather than urokinase-like PA . The effect of glucocorticoids on the production of PA inhibitors (PAI) was examined by 1) neutralization of urokinase activity by increasing amounts of culture media from granulosa cells treated with glucocorticoids, 2) reverse fibrin autography, and 3) Western blot analysis with a specific PAI antiserum . All these methods failed to detect a stimulatory action of glucocorticoids, with or without FSH, on PAI production by rat granulosa cells in the culture media . Our data showed that glucocorticoids have a direct stimulating effect on tPA production, but unlike its action on other in vitro systems, they have no significant effect on PAI production by rat granulosa cells in vitro. Prenat Diagn, 1989 Apr, 9(4), 227 - 42 Differentiation in human chorionic villus cultures: hCG and HLA expression; Phillips CN et al.; The present report describes methods to separate, culture, and study syncytio-cytotrophoblast and mesenchymal core of the first-trimester human chorionic villus . The cultured outer layer cells (syncytio-cytotrophoblast) are multinucleated, pleomorphic, and active in the formation of human chorionic gonadotrophin (hCG) . The mesenchymal core cells are more fibroblast-like in appearance, do not show multinucleation, and have less hCG in their culture media . Both cultured cell types express HLA (ABC) Class I histocompatibility antigens but not HLA (DR) Class II antigens . These and previous studies from this laboratory postulate different embryonic origins: (1) Syncytio-cytotrophoblast cultures of chorionic villus derive from differentiated trophoblast and preserve multinucleation as well as hCG hormone function . (2) Cells cultured from the chorionic villus core originate from extraembryonic mesenchyme . (3) Amniocytes (AF cells) cultured from amniotic fluid resemble the multipotential and early-stage trophoblast, retaining pleomorphism, multinucleation, and lacunae formation as well as production of hCG, progesterone, oestrogen, basement membrane glycoprotein, and Type IV collagen . These cell types cultured from the chorionic villus and amniotic fluid provide a means for in vitro study of specific embryonic cell lineages. Hum Reprod, 1989 Apr, 4(3), 327 - 30 Platelet aggregating activity in human embryo culture media free of PAF-acether; Amiel ML et al.; A factor activating human platelets and liberated by the embryo was sought in the culture media of human embryos using two bioassays . The first bioassay demonstrated the existence of a thrombocytopaenic factor after the i.p . injection of culture media into splenectomized mice . This factor was found more frequently in media which contained an embryo compared to those which contained a non-fertilized oocyte . PAF-acether (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) was searched for with a specific bioassay, using washed rabbit platelets . This remained negative for all the media studied, including those which had contained an embryo giving rise to a pregnancy . In these experiments it was not possible to relate embryo-derived platelet activating factor (EDPAF) to PAF-acether . Neither were we able to use the detection of EDPAF to test embryonic viability, or attempt to identify those embryos which were susceptible to lead to a pregnancy after intrauterine transfer from among all the embryos transferred. In Vitro Cell Dev Biol, 1989 Apr, 25(4), 358 - 64 Influence of branched-chain amino acid composition of culture media on the synthesis of plasma proteins by serum-free cultured rat hepatocytes; Montoya A et al.; Supplementation of Ham's F12 culture medium with essential amino acids (EAA) up to the rat plasma levels increased the rates of synthesis of albumin and transferrin by cultured rat hepatocytes by 1.3 and 1.7, respectively . Fifty percent of this increase could be attributed to three of the EAA: the branched-chain amino acids (BCAA: Leu Ile and Val) . Non-branched-chain essential amino acids (non-BC-EAA) stimulated only 25% of the increase produced by the whole EAA mixture . When each EAA was tested individually, none of them caused an appreciable increase in albumin and transferrin in culture medium . When the concentrations of all EAA were raised to rat postprandial portal levels, albumin and transferrin synthesis rates reached a maximum, increasing by 3.2 and 3.5, respectively . Supplementation with BCAA at postprandial portal concentrations increased albumin and transferrin synthesis rates by 2.2 and 2.0, respectively, and had no noteworthy effect on the synthesis of cellular proteins . Non-BC-EAA at their postprandial portal concentrations increased albumin and transferrin synthesis rates by 1.7 and 1.9, respectively . Supplementation with alanine to reach a nitrogen content equal to that of the modified EAA-enriched medium had no stimulatory effect . Our results show that EAA have a specific effect on the synthesis of plasma proteins by cultured hepatocytes, and that BCAA at physiologic concentrations account for the major part of this stimulatory effect . Consequently, EAA and particularly BCAA concentration should be elevated in serum-free nutrient media to sustain maximum plasma protein synthesis. Ann Vasc Surg, 1989 Apr, 3(2), 167 - 9 Pericardial cells for graft seeding: isolation, culture and identification; Sugimoto JT et al.; The purpose of this study was to determine the feasibility of using the pericardium as a source of endothelial cells . Nineteen pieces of fresh pericardium were obtained from nine mongrel dogs . Cells were prepared by collagenase digestion of the pericardium for 24 minutes followed by centrifugation . The cells were divided into three groups: The supernatant subjected to no further steps, Group I (N = 6); filtration through a 15 micron porous mesh, Group II (N = 6); and Percoll gradient separation with medium 199, Group III (N = 7) . The cells obtained were cultured for seven days in tissue culture media . Yield (cells x 10(5)/gram fresh tissue) was determined with Methods I, II, and III, producing 32.4 +/- 25.9 (SD), 0.96 +/- 0.6 and 0.57 +/- 0.5, respectively (I vs II or III, p less than 0.01) . Fibroblast contamination determined by phase contrast light microscopy was demonstrated in 6/6 cultures with Method I, 3/6 with II and 1/7 for III (I vs III, p less than 0.01) . An assay for endothelial cells (Factor VIII) was positive in 2/6 cultures with Method I, 5/6 with II and 7/7 for III (I vs III, p less than 0.01) . The pericardium is a suitable organ for procurement of endothelial cells . Though reducing yield, filtration and Percoll gradient separation allows for isolation of a relatively pure culture of endothelial cells. J Anim Sci, 1989 Apr, 67(4), 928 - 33 Acute and long-term lipogenic response to insulin and clenbuterol in bovine intramuscular and subcutaneous adipose tissues; Miller MF et al.; The present study was conducted to determine whether insulin and clenbuterol affected either short-term (2-h) incubations or long-term (48-h) tissue cultures of i.m . and s.c . adipose tissue explants . Samples were taken from control steers and steers fed 7 mg.head-1.d-1 clenbuterol for 50 d, after which time the drug was withdrawn from the diet for 90 d prior to slaughter . Neither short-term incubations nor long-term explant cultures contained bovine serum albumin (BSA) . Insulin (6.67 x 10(-9) M) had no effect (P greater than .05) on lipogenesis in s.c . and i.m . adipose tissue in 2-h tissue incubations of fresh adipose tissue . There was a substantial decrease in activity during the culture period, which was ameliorated somewhat in s.c . adipose tissue by the presence of insulin in the culture media . Clenbuterol exposure for 48 h in vitro decreased the production of lipids from acetate in both adipose tissue depots but had no effect in short-term adipose tissue incubations . Results from the present study confirm that omitting BSA from incubation media does not enhance the responsiveness of bovine s.c . adipose tissue or the less mature i.m . adipose tissue to insulin . Insulin may maintain greater cell viability in 48-h explant cultures. Nippon Hifuka Gakkai Zasshi, 1989 Apr, 99(5), 529 - 36 {Effects of dihydrotestosterone on cultured hair papilla cells and localization of its receptors}; Katsuoka K et al.; We have reported that cultured papilla cells (PC) grown by isolation and cultivation of human hair papillae show some biological characteristics . In the present study, localization of androgen binding proteins in PC and effects of dihydrotestosterone (DHT) on PC in vitro were examined . Cytochemical staining of PC using DHT-peroxidase conjugate gave positive reactions in the nucleo of PC originating from scalp- and axilla-hair papillae . The cultivation of PC with added DHT in media for two weeks showed increases 3H-thymidine uptake and 14C-proline uptake over that of dermal fibroblasts . These results suggest that androgen receptors exist in PC and that DHT in culture media induces an accelerating effect upon the DNA synthesis and protein synthesis. Cardiovasc Res, 1989 Apr, 23(4), 279 - 85 An in vitro system for study of effects of angiotensin I on cultured endothelial cells; Bagby SP et al.; Since certain non-vascular angiotensin II (AII) receptors may be activated by angiotensin I (AI), and since sustained increase in AI levels accompanies chronic treatment with converting enzyme inhibitors (CEI) which block conversion of AI to AII, the question of whether AI has significant biological effects is of clinical relevance . We therefore sought to develop an in vitro culture system in which effects of angiotensin I, independently of its conversion to AII, could be studied in cloned aortic vascular endothelial cells (VEC) . This was complicated by peptide degradation during the period of observation, both by angiotensin converting enzyme (ACE) on the surface of VEC and by angiotensinases in either the serum component of culture media or associated with the cell monolayer . Accordingly, we examined the half life of AI under relevant cell culture conditions, with and without confluent fetal bovine aortic endothelial cells (FBAEC) . Factors assessed included (1) fetal calf serum: commercial source, concentration in culture media, effects of converting enzyme inhibitor (CEI: MK422) and/or heat inactivation (superimposed on the commercially performed process); and (2) effect of FBAEC in monolayer culture, with and without CEI . Results showed that (1) in the absence of cells, loss of AI in culture media, when present, was solely due to the presence of fetal calf serum (FCS) and showed a dose dependent response; (2) FCS from differing sources may vary dramatically in capacity for AI breakdown; and (3) serum related AI disappearance included a heat resistant ACE like component (inhibitable by CEI) and a heat sensitive/CEI resistant component dominant at concentrations of FCS exceeding 5%.(ABSTRACT TRUNCATED AT 250 WORDS) J Virol Methods, 1989 Apr-May, 24(1-2), 91 - 101 Concentration and purification of feline leukaemia virus (FeLV) and its outer envelope protein gp70 by aqueous two-phase systems; Hammar L et al.; The major protective antigens of retroviruses are considered to be their glycosylated envelope proteins . However, the methods commonly employed to enrich and purify virus from culture media such as pelleting and density-gradient centrifugation result in a low recovery of the viral external glycoproteins . This is an obvious drawback when the virus is intended for use in a vaccine . In search for alternative methods to concentrate and purify FeLV, we have attempted extraction in two-phase systems based on water-soluble polymers (Albertsson PA., Biochem Biophys Acta 1958; 27: 378-395) . A variety of polymer systems was tested . Some of them seem attractive for a large-scale concentration of the virus and/or its glycoprotein . The distribution between the phases of two FeLV proteins, the outer envelope protein, gp70, and the gag protein, p27, was determined . With a system composed of dextran sulfate and polyvinyl alcohol both the glycoprotein and the gag protein were almost completely recovered in the lower phase which constitutes about 3% of the total system in weight . The two proteins were more than 40-fold purified as calculated on protein basis . The proteins can be extracted readily. Acta Med Okayama, 1989 Apr, 43(2), 97 - 103 Composition of culture media for steroid hormone secretion by murine adrenal tumor cells, Y-1 clone; Ichikawa Y; Murine adrenal tumor cells (Y-1 clone) were stimulated by adrenocorticotropic hormone (ACTH) and cyclic adenosine 3',5'-monophosphate (cyclic AMP) to produce steroid hormone (delta 4, 3-keto steroids) . The steroids were secreted into the medium immediately after synthesis . The optimum concentrations of ACTH and cyclic AMP for stimulation of steroid production were 10(-2) U/ml and 1.0 mM, respectively . In serum-free medium, ACTH and cyclic AMP stimulated steroidogenesis in Y-1 cells, but the amount of steroid hormone in the culture medium was low . However, a high level of steroid production was maintained with medium containing 10 mg/ml bovine serum albumin (BSA) . In culture medium containing a higher concentration of BSA, Y-1 cells did not become spherical as is usually the case when steroid production is stimulated by ACTH or cyclic AMP . The morphological changes did not always correlate with steroid secretion by Y-1 cells. Arch Invest Med (Mex), 1989 Apr-Jun, 20(2), 113 - 22 Steroid conjugate formed by human endometrium; Garzon P et al.; Progesterone -6,7-3H (P*) was incubated in minces of human secretory and proliferative endometrium in absence as well as in presence of 10 and 100 micrograms/ml of unlabelled progesterone (P), in Eagle's Culture Medium throughout 72h . The following metabolites of P* were found in culture media: 1 . C-21 derivatives of P* reduced at C-5, and C-20 positions . Also, a 3 beta-hydroxy-5 alpha pregnane-20-One conjugated to a glucuronic acid moiety was identified . 2 . Concentrations of water-soluble derivative accounted for 21% and 29% of the recovered radioactivity in proliferative and secretory endometria, respectively . 3 . After beta-glucuronidase cleavage, 80% to 95% of the water soluble derivatives were released as free steroids . 4 . Approximately 60% corresponded to 3 beta -hydroxy- 5 alpha pregnane-20-one of the pooled water extracts . 5 . Alson, 17% and 13% as well as 9% and 8% recoveries under 10 and 100 micrograms/P were observed in proliferative and secretory endometria, respectively . Glucuronidation seems to be a compensatory route to metabolize P and P* in human endometrium as might also occur in other species. Inflammation, 1989 Apr, 13(2), 233 - 44 Stimulus-specific production of cyclooxygenase and lipoxygenase metabolites of arachidonic acid by bovine alveolar macrophages; Laegreid WW et al.; Alveolar macrophages (AMs) are capable of producing a variety of inflammatory mediators including those derived from arachidonic acid, the prostaglandins (PGs), leukotrienes (LTs) and hydroxyeicosatetraenoic acids (HETEs) . Inflammation associated with release of arachidonate-derived mediators is a result of the combined actions of all of these mediators . Thus, it is critical to determine the entire spectrum of arachidonate-derived metabolites that AMs are capable of producing . In this study bovine AMs were prelabeled with {3H}arachidonic acid prior to stimulation with serum-treated zymosan, phorbol myristate acetate (PMA), or the calcium ionophore A23187 . The total release of arachidonate metabolites into the culture media was measured by reverse-phase HPLC with on-line radiometric detection . All stimuli used induced production of metabolites of the cyclooxygenase pathway with thromboxane B2 and HHT being the major metabolites . Lesser amounts of PGF2 alpha, PGE2, and PGD2 were produced . Only stimulation with A23187 resulted in production of LTB4 and 5-HETE, products of the 5-lipoxygenase pathway . This latter result indicates that the two major pathways of arachidonate metabolism in AMs may be selectively stimulated . Such an effect could have important consequences in the development of pulmonary inflammation . Furthermore, the spectrum of arachidonic acid metabolites produced by bovine AMs closely resembles that of human AMs, in contrast to rodent AMs. Thromb Res, 1989 Apr 1, 54(1), 41 - 52 Interleukin-1, endotoxin or tumor necrosis factor/cachectin enhance the level of plasminogen activator inhibitor messenger RNA in bovine aortic endothelial cells; Medina R et al.; It is known that either endotoxin (LPS) or interleukin-1 (IL-1) increase the activity of plasminogen activator inhibitor (PAI) in the culture media of human and bovine endothelial cells . We have confirmed these results in bovine aortic endothelial cells (BAEC) . To determine if this effect was mediated by increases in the level of PAI messenger RNA (mRNA) we examined the effects of these cytokines on PAI mRNA levels in BAEC, using RNA blot analyses . Treatment of BAEC with either IL-1, LPS, or human recombinant tumor necrosis factor/cachectin (TNF) dramatically increased the level of PAI mRNA . Since elevated levels of PAI will decrease fibrinolytic potential, this mechanism is in concert with the known increase in in vivo procoagulant potential induced by these agents and could contribute to thromboembolic phenomena. Biol Reprod, 1989 Apr, 40(4), 873 - 85 Characterization of an insulin-like growth factor binding protein, analogous to human pregnancy-associated secreted endometrial alpha 1-globulin, in decidua of the baboon (Papio anubis) placenta; Fazleabas AT et al.; The major secreted protein of the human decidua (pregnancy-associated endometrial alpha 1-globulin {alpha 1-PEG}), is an insulin-like growth factor-binding protein (IGF-BP) that is immunologically and biochemically similar to placental protein 12 (PP12) extracted from term human placenta . Since previous studies have demonstrated that the baboon and human endometrium synthesize and release a number of biochemically and immunologically related polypeptides in culture, this study was undertaken to further characterize a related IGF-BP in baboon placental tissues . Decidua, chorio-amniotic membranes with adhering decidua (CAM-D), and placental villi were obtained from pregnant baboons between Days 134 and 160 of gestation by Cesarean sections . Portions of tissue were either cultured in the presence of 35S-methionine, fixed for immunocytochemistry, or frozen in liquid nitrogen for cytosol extraction . Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) of tissue culture media (TCM) revealed that the major secretory product of the decidua and CAM-D was an acidic polypeptide (Mr 33,000) . Western blot analysis and immunoprecipitation of TCM with murine monoclonal antibody (B2H10) against human alpha 1-PEG demonstrated that this molecule, secreted by the baboon decidua and CAM-D, but not the placental villi, was immunologically identical to the human IGF-BP . Immunocytochemical localization of IGF-BP was intense in the cytoplasm of stromal cells in decidua and CAM-D and absent in the placenta . Gel filtration of TCM and cytosol followed by screening of eluates for 125I-IGF-I binding resolved two peaks (Mr greater than 100,000 and 35,000) of specific IGF-BP in decidua and CAM-D . The 35,000 peak had 100-200 times the binding capacity of the Mr greater than 100,000 peak and a Kd of 1.14-1.83 nM . The eluates contained in the Mr 35,000 peak were also immunoreactive to alpha 1-PEG, as accessed by a polyclonal radioimmunoassay . Affinity cross-linking with 125I-IGF-I followed by sodium dodecyl sulfate-PAGE revealed an immunoreactive complex of Mr 36,000, confirming that the baboon protein represents a high affinity IGF-BP . These studies indicate that the hypertrophied stromal cells of the baboon decidua and CAM-D synthesize and release an IGF-BP as their major secretory product, analogous to the situation in humans . The results of this study suggest that this protein may play a role in the regulation of IGF action during pregnancy. FEBS Lett, 1989 Mar 27, 246(1-2), 25 - 9 Characterization of vesicles, containing an acylated oligopeptide, released by human colon adenocarcinoma cells . NMR and biochemical studies; Masella R et al.; RNA-containing vesicles, recovered from the supernatant of high-density cell samples of human colon carcinoma, produce a high-resolution 1H NMR spectrum of lipids characterized by isotropic tumbling; these vesicles contain large amounts of triglycerides and cholesterol esters . Both findings have strict analogies to what is displayed by the proteolipid complexes isolated from the sera of tumor-bearing patients {(1985) Proc . Natl . Acad . Sci . USA 82, 3455-3459; (1986) FEBS Lett . 203, 164-168} . Lipid analysis and enzymatic tests indicate that these vesicles are selected micromaps of plasma membranes, analogous to those that can be recovered from culture media in which tumor cells are grown {(1985) Dev . Biol . 3, 33-57} . Peculiar lipids, an acylated oligopeptide and a modified phospholipid, are also present in the vesicles. Int J Cancer, 1989 Mar 15, 43(3), 478 - 86 Immunodetection of cathepsins B and L present in and secreted from human pre-malignant and malignant colorectal tumour cell lines; Maciewicz RA et al.; Pre-malignant and malignant human colorectal tumour epithelial cell lines both secreted precursor forms of the 2 cysteine proteinases, cathepsins B and L . The amount of proteinases secreted by these cell lines varied according to the cell density . Comparison at similar cell densities showed that the pre-malignant, adenoma-derived cell line (PC/AA) secreted as much, or more, of both cathepsin B and L precursors as did the malignant, carcinoma-derived cell line (PC/JW/FI) . However, mature forms of cathepsins B and L were detected in the culture media of only the carcinoma-derived cell line, thus indicating that the invasive potential of a tumour may be related to its ability to process extracellularly the secreted precursor enzyme to a mature and consequently active enzyme, rather than to the amount of proteinase synthesized and/or secreted . Similar results were obtained using 2 other epithelium-derived tumour cell lines, HT/29 (carcinoma) and SP/AN (adenoma) . Immunolocation studies showed that cathepsin B was lysosomal while cathepsin L appeared to have a distribution more consistent with a plasma membrane association . Purified human cathepsins B and L (mature form) were capable of solubilizing an isolated basement membrane matrix (bovine anterior lens capsule) in vitro, thus indicating that the secreted mature enzymes and the membrane-associated cathepsin L could potentially degrade basal laminae or sub-endothelial basement membranes in vivo. J Rheumatol, 1989 Mar, 16(3), 355 - 62 Proteoglycan metabolism in tissue cultured human articular cartilage . Influence of piroxicam; Verbruggen G et al.; Proteoglycan metabolism was investigated in longterm tissue cultured human cartilage . Visually intact cartilage from adult donors showed improving accumulation rates for 35sulfate labelled proteoglycans over a 6-week period . The loss of newly synthesized molecules in the nutrient culture media was low and constant . Fibrillated cartilage from a 17-year-old male showed higher basal 35S incorporation rates and the proportions of 35S proteoglycan aggregates were higher than in normal tissue . These observations may reflect the immature status of the tissue or an attempt at repair . However samples lost increasing amounts of 35S proteoglycans in