Prostate, 1987, 10(2), 95 - 114 Heterotransplantation of human prostatic tissue; Hymer WC et al.; Pieces of human prostatic tissue (approximately 1 mm3) were encapsulated in XM-50 Amicon hollow fibers and either implanted into the testis of an adult male rat or placed in culture . The protein synthetic capacity of such tissue pieces removed 1-42 days later was monitored by TCA precipitation, SDS-PAGE, and autoradiography . The results showed that tissue pieces retained their functional protein synthetic elements after this procedure . A newly developed solid-phase enzyme immunoassay for human prostatic antigen (PA), described in this report, was used to monitor PA levels in the serum of the recipient host or culture media . In some instances PA was detected 1-2 weeks postimplantation, a result that implies maintenance of functional secretory elements in vivo . Finally, morphology of tissue pieces 1-2 weeks postimplantation sometimes showed the presence of ductal epithelia and stromal elements in distribution patterns typical of those seen in fresh biopsy samples . We conclude that the function and structure of prostatic tissue implanted intrastesticularly compares favorably with that maintained in conventional explant culture . As such, the hollow fiber method offers promise for a new way of monitoring hormonal and/or chemotherapy testing of human prostatic tissue. Biochem Biophys Res Commun, 1986 Dec 30, 141(3), 885 - 91 Estrogenic activity of phenol red in rat anterior pituitary cells in culture; Hubert JF et al.; The estrogenic activity of phenol red, a pH indicator widely used in cell culture media, was studied in rat anterior pituitary cells . After 72 hours of incubation with 40 microM phenol red, a 40-50% increase in prolactin cell content and a 100% stimulation of luteinizing hormone-releasing hormone induced luteinizing hormone release was observed . Both effects could be completely reversed by simultaneous incubation with the antiestrogen LY156758 . In the rat uterine {3H} estradiol binding assay, phenol red showed a significant displacement at concentrations above 10 microM while its concentration in the commonly used culture media is about 40 microM . From the present results, we conclude that phenol red acts as a weak estrogen in normal tissues and that its estrogenic activity should be taken into account in studies using estrogen-sensitive cell or tissue cultures. Thromb Res, 1986 Dec 15, 44(6), 729 - 37 Release of both urokinase and tissue plasminogen activator from veins in vitro; Kjaeldgaard A et al.; Specimens of human veins were incubated in culture media, in which the concentrations of urokinase (UK) and tissue plasminogen activator (t-PA) were determined by radioimmunoassay and immunoradiometric assay . Both UK and t-PA were released to the media, in which the concentration of plasminogen activators was correlated to the weight of the specimens . The release of t-PA was maximal on the first day, after which it rapidly decreased, and t-PA was not demonstrable after the third day of incubation . By contrast, UK was continuously liberated to the media during the study period of 5 days . The present results indicate that both UK and t-PA are present in the vein wall and probably released by different cells. Biochim Biophys Acta, 1986 Dec 10, 884(3), 402 - 8 Comparative kinetic analysis of the activation of human plasminogen by natural and recombinant single-chain urokinase-type plasminogen activator; Lijnen HR et al.; Single-chain urokinase-type plasminogen activator (scu-PA) may be obtained from conditioned cell culture media (natural scu-PA) or by expression of the cDNA encoding human scu-PA in Escherichia coli (recombinant scu-PA) . The activation of Glu-plasminogen by natural and recombinant scu-PA can be described by a sequence of three reactions, each of which obeys Michaelis-Menten kinetics . Initial activation of plasminogen to plasmin by scu-PA (reaction I) occurs with a high affinity (Km below 0.8 microM) for both scu-PAs, while the catalytic rate constant (k2) is 0.017 s-1 for recombinant scu-PA but only 0.0009 s-1 for natural scu-PA . Subsequent conversion of scu-PA to urokinase (two-chain urokinase-type plasminogen activator, tcu-PA) by generated plasmin (reaction II) occurs with a comparable affinity (Km about 5 microM) for natural and recombinant scu-PA and with a k2 of 0.23 s-1 for natural and 1.2 s-1 for recombinant scu-PA . Finally, activation of plasminogen by tcu-PA (reaction III) occurs with low affinity (Km 30-50 microM) but with a high catalytic rate constant (k2 about 5 s-1) for both natural and recombinant tcu-PA . The differences in the kinetic parameters of the activation of plasminogen by natural or recombinant scu-PA are thus mainly due to differences in turnover rate in the first reaction . Indeed, the catalytic rate constant of the first reaction is about 20-times higher for recombinant scu-PA than for natural scu-PA . Thus, surprisingly, the artificial, unglycosylated recombinant scu-PA molecule has a better catalytic efficiency than its natural glycosylated counterpart. Mol Biochem Parasitol, 1986 Dec, 21(3), 269 - 76 Discovery of a specific double-stranded RNA virus in Giardia lamblia; Wang AL et al.; Nucleic acid samples purified from trophozoites of Giardia lamblia Portland I strain contain an ethidium-stainable band that comigrates with 7.0 kilobase DNA in agarose gel electrophoresis . The band was degradable by alkali, ribonuclease A and ribonuclease T1, but the susceptibility toward the ribonucleases decreased with increasing ionic strength, suggestive of double-stranded RNA (dsRNA) . This identification was confirmed by electron micrographs of the purified samples, which showed linear double-stranded structures with an estimated average length of 1.5 micron . In crude homogenates of G . lamblia, this dsRNA was protected against added ribonuclease A but disappeared upon adding sodium dodecyl sulfate or proteinase K . Differential centrifugations suggested an association of the dsRNA with the nuclear fraction, but it was freed to the 109,000 X g pelletable fraction with increasing homogenization . The dsRNA was purified by CsCl buoyant density gradient centrifugations in a distinct band with a rho value of 1.368 g ml-1 . Electron microscopy revealed spherical virus-like particles (VLP) with a diameter of 33 nm . VLP of similar shape and size were also identified in the nuclei of sectioned G . lamblia trophozoites . VLP yield a major protein with an estimated molecular weight of 66,000 in sodium dodecyl sulfate polyacrylamide gel electrophoresis . VLP are abundant in the culture media of stationary-phase G . lamblia Portland I strain and are able to infect the G . lamblia WB strain, which is free of the virus . There is no sequence homology between the dsRNA and the nuclear DNA of G . lamblia and thus no apparent integration of viral genome into host DNA. Am J Physiol, 1986 Dec, 251(6 Pt 1), E707 - 14 Expression of a cholecystokinin precursor-related peptide in vertebrate and invertebrate tissues; Schneider BS et al.; We have developed a radioimmunoassay for the nonapeptide predicted by cDNA sequence analysis to reside at the extreme C-terminus of the mouse cholecystokinin (CCK) precursor . Sensitivity of the assay is 1 pg synthetic CCK precursor-related peptide (CCK-PRP)/ml . The antibody has no cross-reactivity with cholecystokinin, gastrin, or a variety of other known neuropeptides . We have employed this assay to demonstrate the presence, in rodent brain, gut, and peripheral plasma, of peptides with immunological properties that are identical to, and gel filtration characteristics that are very similar to, those of the synthetic CCK-PRP . We have also detected a similar peptide in the culture media of a human CCK-producing tumor . The molar ratios of immunoreactive CCK-PRP/CCK vary widely among tissues of origin and during ontogeny, suggesting regional and developmental differences in the turnover rates or in posttranslational modification of the two peptides . Our studies suggest that peptides very similar to intact CCK-PRP are posttranslationally liberated from the cholecystokinin precursor in a variety of tissues and may have neurotransmitter and/or hormonal functions distinct from those of CCK . Relatively high quantities of material immunologically indistinguishable from CCK-PRP were also found in several coelenterate species, indicating that this epitope arose as early in evolution as did CCK. Fertil Steril, 1986 Dec, 46(6), 1150 - 2 Inhibin concentration in the culture media of human oocyte-cumulus-corona cell complexes is not related to subsequent embryo cleavage; Demoulin A et al.; We have demonstrated an in vitro secretion of inhibin by human OCCC, not related to subsequent egg cleavage . A putative signal, released by OCCC, that could modulate inhibin secretion by granulosa cells has not been discovered . Inhibin secretion by OCCC has no predictive value concerning the further fertilization and cleavage of the oocytes. Exp Cell Res, 1986 Dec, 167(2), 440 - 52 Rabbit chondrocytes maintained in serum-free medium . I . Synthesis and secretion of hydrodynamically-small proteoglycans; Malemud CJ et al.; The biosynthesis of sulfated proteoglycan in vitro by rabbit articular chondrocytes in first passage monolayer culture maintained in fetal bovine serum (FBS) or in serum-free conditions was compared . Neosynthesized proteoglycan in the culture medium in the most dense fraction of an associative CsCl density gradient (fraction dAl) declined with increasing time under serum-free conditions, but not when cells were maintained in the presence of serum . After one day, the major peak of incorporated 35SO4 in medium fraction dAl eluted as a retarded peak (Kav 0.28) on Sepharose CL-2B, whether cells were maintained under serum-free or serum-containing conditions . The hydrodynamic size of proteoglycan monomer fraction dAlDl obtained after one day of exposure to serum-free culture media was smaller than dAlDl from serum-containing cultures . The hydrodynamic size of dAlDl obtained from serum-free culture media became even progressively smaller after 2 and 3 days' exposure to these conditions . Hydrodynamically small sulfated proteoglycans were identified in the cell-associated dAlDl fraction as early as one day after switching chondrocytes from serum-containing to serum-free medium . Culture medium fraction dAlDl from serum-free culture medium aggregated poorly when incubated with human hyaluronic acid (HA) in the presence of bovine link protein or when dialysed against bovine nasal cartilage proteoglycan aggregate . Proteoglycan monomer from serum-containing medium reaggregated more efficiently under both conditions . No change in the size of glycosaminoglycan chains was seen in the smaller proteoglycan subpopulations, nor was there any indication of marked changes in the glycosaminoglycan types. Metabolism, 1986 Dec, 35(12), 1128 - 36 Insulin effects on apolipoprotein B lipoprotein synthesis and secretion by primary cultures of rat hepatocytes; Sparks CE et al.; Lipoprotein synthesis and secretion were examined in primary cultures of rat hepatocytes cultured on collagen-coated plates and incubated with pharmacologic and physiologic concentrations of insulin . Media insulin concentration declined rapidly over the course of incubation indicating that hepatocytes rapidly degrade insulin . When insulin was present in the media, cellular triglyceride accumulated while lipid secretion declined . Insulin inhibited the incorporation of labeled amino acids into total secretory lipoprotein apoproteins and apolipoprotein B (apo B) as well as apo B mass as measured by monoclonal radioimmunoassay . The effect of insulin on apo B secretion occurred as early as three hours after the addition of insulin to the culture media and both apo B of higher molecular weight (apo BH) and apo B of lower molecular weight (apo BL) were affected . Cellular apo B did not accumulate within cells . The majority of secretory lipid radioactivity synthesized from acetate was in VLDL density lipoproteins . The composition of newly synthesized lipids as assessed by thin layer chromatography was not significantly altered with insulin . These studies support the finding that insulin inhibits VLDL secretion by hepatocytes while at the same time stimulating overall triglyceride synthesis . A suggested mechanism is that insulin uncouples triglyceride and apo B synthesis, which influences subsequent lipoprotein assembly and secretory pathways . These results are consistent with the concept that postprandial insulin release inhibits hepatic lipoprotein secretion while intestinal lipoprotein metabolic pathways are most active. Hum Genet, 1986 Dec, 74(4), 378 - 81 Assignment of human transcobalamin II (TC2) to chromosome 22 using somatic cell hybrids and monosomic meningioma cells; Arwert F et al.; Human transcobalamin II (TC2), a vitamin B12 binding serum protein, is synthesized and secreted into the medium by cells growing in vitro . Mouse-man somatic cell hybrids were analyzed in order to map the locus of TC2 . The presence of human TC2 in the culture media was correlated with the results of genetic marker and chromosome analysis of the hybrid cells . Chromosome 22 showed 100% concordancy . However, chromosome 6 (90% concordancy) and chromosome 7 (96% concordancy) were not completely excluded . Meningioma cells obtained from patients heterozygous for TC2 showed a concomitant loss of one chromosome 22 and one of the TC2 alleles, strongly supporting the assignment to chromosome 22. Cancer Res, 1986 Dec, 46(12 Pt 1), 6095 - 100 Release of a sodium transport inhibitor (inhibitin) from cultured human cancer cells; Morgan K et al.; In this study we investigated whether the sodium transport inhibitor, inhibitin, originally isolated from leukemic promyelocytes, was also elaborated by some other neoplastic cells in culture . Like culture medium from the leukemic promyelocytes (HL60), the media from two other leukemic cell lines (erythroblasts K562 and monoblasts U937) also showed significant inhibitory activity on ouabain-insensitive sodium efflux rate constant in normal erythrocytes . Similarly, culture media from three neoplastic cell lines (H.Ep2, MRC5, and HX99) also showed significant inhibitin-like inhibitory activity . Using high-performance liquid chromatography to isolate inhibitin, culture media from HL60 and H.Ep2 cells were identically treated, and inhibitin isolated from H.Ep2 cells had the same retention time as that shown by promyelocyte inhibitin . H.Ep2 inhibitin reduced ouabain- and bumetanide-insensitive sodium efflux rate constant from 0.1510 +/- 0.0275 (SD) to 0.0988 +/- 0.0110 (P less than 0.005) . Like promyelocyte inhibitin, H.Ep2 inhibitin reduced sodium efflux and influx by equivalent amounts suggesting thereby that it is a sodium/sodium exchange inhibitor . These studies show that a factor exhibiting inhibitory activity on sodium/sodium exchange is secreted by a variety of leukemic and neoplastic cells in culture. Endocrinol Exp, 1986 Dec, 20(4), 393 - 400 Effect of exogenous hormones on estrogen and progesterone release from cultured rat granulosa cells from various stages of estrous cycle; Madej E; Progesterone and estrogen levels in the culture media of granulosa cells isolated from rat ovarian follicles in all stages of estrous cycle were estimated using RIA, with special regard to hormone levels in proestrus . In control cultures the peak of progesterone release occurred at 20.00 h in proestrus . The addition of LH and FSH to the culture medium resulted in a remarkable stimulation of progesterone release by cells isolated in estrus . The highest concentration of estrogens in control cultures was observed in granulosa cells isolated at 18.00 h in proestrus . Exogenous LH and FSH stimulated the release of estrogen from the cells isolated on the days of estrus, metestrus and diestrus . Cultured granulosa cells maintained similar pattern of progesterone and estrogen release as the whole follicles did in vitro and in vivo. Kidney Int, 1986 Dec, 30(6), 852 - 61 Demonstration and characterization of C3 receptors on rat glomerular epithelial cells; Kasinath BS et al.; Receptors for C3 have been demonstrated on the glomerular podocyte in humans . There is conflicting evidence regarding the presence of C3 receptors on rat glomerular cells . Even when shown to be present, the ligand specificity of the receptor has not been determined . Decapsulated rat glomeruli obtained from male Sprague-Dawley rats weighing 50 to 100 g were placed in enriched culture media . On days four to eight, cells of epithelial morphology were observed growing out of glomeruli . Receptors for C3 were detected by rosette formation of sheep erythrocytes (E) coated with antibody (A) and complement (EAC) around the glomerular epithelial cells in culture . The EACs were prepared by incubating antibody-coated sheep erythrocytes with C5-deficient mouse serum or with individual components of complement . Results indicate the presence of two types of C3 receptors on glomerular epithelial cells--CR1 for C3b and CR2 for C3d . The functional roles of these receptors remain to be elucidated. Mech Ageing Dev, 1986 Nov 28, 37(1), 55 - 67 Culture media variation as related to in vitro aging of human fibroblasts: I . Effects on population doubling, nuclear volume and nuclear morphology; Mukherjee AB et al.; The relative effect of five commonly used culture media (MEM, BME, McCoy's 5A, M199 and HMEM) on the population doubling level (PDL), nuclear volume and nuclear morphology was examined during in vitro senescence of WI-38 human fetal fibroblasts . Statistical analyses showed that cells grown in M199 had a significantly lower PDL than that of cells cultured in any other medium . The PDL in McCoy's 5A was significantly lower compared to that in BME, MEM and HMEM . Cells grown in BME, MEM and HMEM showed similar PDL . It was found that the nuclei of aged cells grown in M199 were significantly larger in volume than cells aged in any other medium . The average increases in nuclear volume of cells during aging in BME, MEM and McCoy's 5A were statistically equivalent . The increase in nuclear volume in HMEM was significantly smaller than that of cells aging in M199 and was longer than that of cells aging in BME or MEM . Linear regression analysis showed that there was a linear increase in nuclear volume as a function of PDL for cells aged in all five media . However, the rate of increase in nuclear volume with increasing PDL varied from medium to medium . There was no significant difference between media on induction of abnormal nuclear morphology as related to PDL . The relative effects of all five media were not uniform on the three cellular parameters investigated during in vitro aging of WI-38 cells . It is, therefore, suggested that one should keep this medium differential in mind to allow meaningful comparison of possible changes in various morphological parameters during in vitro senescence of diploid human fibroblasts such as WI-38. FEBS Lett, 1986 Nov 24, 208(2), 439 - 44 Identification of an insulin-like growth factor-binding protein in human cerebrospinal fluid with a selective affinity for IGF-II; Hossenlopp P et al.; Human cerebrospinal fluid (CSF) has been found to contain several different molecular forms of IGF-specific binding proteins (BPs) . Qualitatively, they are similar to those present in serum, although their relative proportions are very different, as well as to those present in the culture media of brain tissue from which these BPs presumably arise . One particular form of BP is predominant in CSF . It has an Mr of 34,000, as estimated by SDS-polyacrylamide gel electrophoresis followed by transfer onto nitrocellulose, and an isoelectric point around 5.0 based on chromatofocusing . It has a selective affinity for IGF-II (approximately 4 X 10(10) M-1) as shown by competitive binding experiments in which biosynthetic IGF-I was about 40-times less potent than native IGF-II in displacing 125I-labelled IGF-II . These findings are in agreement with the preponderance of IGF-II in nervous tissue and in CSF and suggest that this BP plays an important role in the interaction of IGF-II with its target cells. J Immunol Methods, 1986 Nov 20, 94(1-2), 197 - 200 Stimulation of resting normal human peripheral blood mononuclear cells by fetal calf sera . Activation to an interleukin-2 responsive state; Lakhanpal S et al.; Normal adult human peripheral blood mononuclear cells which are negative for interleukin-2 (IL-2) receptors as assessed by flow cytofluorometry, acquire IL-2 receptors and IL-2 responsiveness after culture in media supplemented with fetal calf sera . Thus, in the absence of any known external stimuli, fetal calf sera used to supplement culture media can induce the transformation of resting (G0) peripheral blood mononuclear cells to an activated (G1) state . The activated (G1) cells are able to progress through the rest of the cell cycle (S, G2, M) in the presence of IL-2 . As a result, studies of human peripheral blood mononuclear cells in fetal calf serum-supplemented culture media should be interpreted with appropriate caution. J Immunol, 1986 Nov 15, 137(10), 3183 - 8 Down-regulation of interleukin 1 (IL 1) receptor expression by IL 1 and fate of internalized 125I-labeled IL 1 beta in a human large granular lymphocyte cell line; Matsushima K et al.; The regulation of interleukin 1 (IL 1) receptor expression on a human large granular lymphocyte cell line, YT, and fate of internalized 125I-labeled IL 1 beta (125I-IL 1 beta) were studied . YT cells were selected for this study, because this cell line expresses a large number of specific high-affinity receptor for IL 1, responds biologically to exogenously added IL 1 by expressing high-affinity IL 2 receptors, and does not produce IL 1 . YT cells constitutively express approximately 7 X 10(3) IL 1 receptors/cell with a Kd approximately 10(-10) M . Neither IL 2, phorbol myristic acid, nor lipopolysaccharide affected the total binding of 125I-IL 1 beta by YT cells . In contrast, the capacity of YT cells to bind 125I-IL 1 beta when incubated at 37 degrees C for 3 to 16 hr with a low dose of purified IL 1 beta (approximately 6 U/ml) was reduced by greater than 80% . The loss of binding capability gradually recovered by 16 hr after removal of IL 1 beta from cultured YT cells . The apparent loss of IL 1 receptor expression was accompanied by the internalization of 125I-IL 1 beta into cells . Acid treatment of YT cells to remove bound 125I-IL 1 beta at 4 degrees C showed that 50% of the 125I-IL 1 beta bound to cells could no longer be recovered after 30 min at 37 degrees C, and this increased to 80% after 3 hr at 37 degrees C . Fractionation of cell extracts on Percoll gradient additionally showed 125I-IL 1 beta to appear intracellularly after receptor binding on plasma membranes, and to be successively transferred to some membranous organelles (d approximately equal to 1.037) through an intermediate density organelle (d approximately equal to 1.050), and to finally end up in lysosomal cell fractions (d approximately equal to 1.05 to 1.08) after approximately 3 hr at 37 degrees C . Only approximately 5% of internalized 125I-IL 1 beta was released into culture media by 6 hr of incubation at 37 degrees C . However, the radioactivity in the TCA soluble fraction of the culture media increased gradually by 6 hr and a lysosomotropic enzyme, ethylamine, significantly inhibited both the transfer of internalized 125I-IL 1 beta to the lysosomal fraction and the degradation of 125I-IL 1 beta . This study represents the first evidence of autoregulation of IL 1 receptors by IL 1 and internalization of IL 1 molecules after binding to receptors.(ABSTRACT TRUNCATED AT 400 WORDS) Life Sci, 1986 Nov 10, 39(19), 1751 - 3 Peptides bound to albumin; Menezo Y et al.; Preparations of albumin (placenta or serum) do bind peptides . Dissociation of these peptides from the albumin core can occur partially in water but is much more efficient in the presence of amino acids and salts, in complex culture media for example . Contrary to common belief, we observed that arginine vasopressin (AVP) can bind to serum albumin, at least in commercial powder form. Anal Biochem, 1986 Nov 1, 158(2), 262 - 7 Application of a nitrocellulose immunoassay for quantitation of proteins secreted in culture media; LaDuca FM et al.; A macro-dot immunoassay was developed to quantitate proteins (antigens) secreted in the culture media of primary rat hepatocytes . Dilutions of protein standards and undiluted spent culture media were applied to numbered sheets of nitrocellulose (NC) paper by vacuum filtration (in volumes up to 1 ml) through a specially designed macrofiltration apparatus constructed of plexiglass . Sequential incubation of the NC with bovine serum albumin blocking buffer, monospecific antibody, and 125I Protein A enabled quantitation of protein concentration by determination of NC bound radioactivity . Linear and reproducible standard curves were obtained with fibrinogen, albumin, transferrin, and haptoglobin . A high degree of coefficient of correlation between radioactivity (cpm) and protein concentration was found . Intra- and interest reproducibility was excellent (C.V.'s less than 7%) . By using monospecific antibodies, single proteins (i.e., fibrinogen), as low as 32 ng/ml, could be quantified in heterogeneous protein mixtures and in spent culture media . The assay was sensitive to the difference of fibrinogen secretion under nonstimulatory (serum-free hormonally defined medium, SFHD) and stimulatory (SFHD plus hydrocortisone) culture conditions . The procedure and techniques described are applicable to the quantitation of any protein in a suitable buffer. J Reprod Fertil, 1986 Nov, 78(2), 711 - 7 Melatonin directly stimulates the secretion of progesterone by human and bovine granulosa cells in vitro; Webley GE et al.; Melatonin, at concentrations and periods of exposure reflecting those present during the circadian cycle, was investigated for its influence on steroid production by granulosa cells cultured in serum-supplemented medium . At high (200 pg/ml) but not low (20 pg/ml) physiological concentrations, melatonin significantly stimulated progesterone production by human granulosa cells . This response was independent of the overall level of cell activity and was seen under the different culture conditions associated with different culture media . Exposure to melatonin for 8 h significantly stimulated progesterone secretion to a level similar to that achieved under continuous exposure, and the effect was reduced to control levels during subsequent periods in which no melatonin was added . Melatonin had no consistent effect on aromatase activity in the conversion of stored or serum-available androgen to oestradiol . Melatonin significantly stimulated progesterone production by bovine granulosa cells in vitro, at concentrations similar to those present during the endogenous nocturnal rise (100-400 pg/ml) . This response to physiological conditions by human and bovine cells suggests a role for melatonin in the regulation of progesterone production by the ovary. J Endocrinol, 1986 Nov, 111(2), 343 - 8 Effect of androgen on androgen receptors in cultured human epididymis; Vazquez MH et al.; Optimal assay conditions were developed to determine androgen receptor concentrations in samples of human epididymis . Pretreatment of cytosol with mersalyl, as well as the inclusion of molybdate in the homogenization buffers, resulted in a substantial increase in the number of soluble sites detected . A high yield of nuclear and microsomal binding sites was obtained by prolonged incubation (12 h) in 0.6 mol NaCl/l . Organ culture for 6 days resulted in a major loss of androgen-binding sites . In the absence of androgen in the culture media, only 20% of the original sites were found in cultured tissue . Inclusion of dihydrotestosterone (0.1 mumol/l) in the media resulted in samples containing twice as many sites as controls . It is concluded that androgens influence the number of androgen-binding sites in cultured human epididymis in a manner analogous to that described for rat epididymis in vivo. Gut, 1986 Nov, 27(11), 1298 - 305 Establishment of an animal model of ovalbumin sensitised mouse to study protein induced enteropathy; Malo C et al.; The protein induced modifications of the small bowel mucosa from ovalbumin-sensitised mouse have been studied in organ culture . A decrease in gamma-glutamyl transpeptidase, alkaline phosphatase, lactase, sucrase, and glucoamylase activities was observed in the explants cultured in the presence of ovalbumin . In contrast, a large increase of those enzymatic activities was noted in the culture media, the overall effect observed being a net stimulation of the total enzymatic activities of the culture system . The enzymes accumulated in the particulate fraction of the medium (brush border membrane fraction) suggesting an increased turnover of membrane components by a process of shedding or microvesiculation . This model serves as a useful tool in evaluating the local response of the small bowel mucosa induced by a specific protein. Mol Biochem Parasitol, 1986 Nov, 21(2), 151 - 9 Schistosoma mansoni: phospholipid methylation and the escape of schistosomula from in vitro cytotoxic reaction; Parra JF et al.; The ability of Schistosoma mansoni schistosomula to evade in vitro cytotoxic activity of antibodies plus complement is shown to be increased by incubation with Concanavalin A (Con A) or with non-immune inactivated human serum . This effect was not observed if S-adenosyl-homocysteine (SAH) a methyltransferase inhibitor was added to the incubation medium . Methyl group incorporation occurs in schistosomulum phospholipids if parasites are incubated in Earle's balanced salt solution . This incorporation is increased by Con A addition and this increase is inhibited by SAH . Supernatants of schistosomula incubated in culture media containing Con A were able to promote phospholipid methylation, showing that methyltransferases were liberated into the culture media . The possible roles played by these phenomena in host-parasite interactions are discussed. Epilepsia, 1986 Nov-Dec, 27(6), 685 - 96 Identification of a teratogenic drug-protein complex in sera of phenytoin-treated monkeys; Clapper ML et al.; Whole rat embryos were cultured for 48 h on sera drawn from monkeys before and 10 h after phenytoin gavage (275 mg/kg body weight) . Sera from treated monkeys caused exencephaly, anophthalmia, microcephaly, and incomplete ventral curvature when used as culture media, whereas sera drawn from the same monkeys before treatment supported normal embryonic development . To identify the cause of serum teratogenicity, isolated constituents of teratogenic sera were added to nonteratogenic sera for testing by embryo culture . Serum extracts containing free phenytoin and its free metabolites were not teratogenic . Teratogenicity was found associated with serum proteins . Using polyacrylamide gel electrophoresis (PAGE) and an antibody that recognized phenytoin and its metabolites, we were able to demonstrate that phenytoin was bound to a protein of 80,000 daltons . Addition of this same antibody to teratogenic sera from dosed monkeys improved the development of cultured embryos and provided additional support for this complex as the proximal teratogen . Use of the antibody to follow the uptake and distribution of phenytoin in cultured embryos suggested that only the phenytoin-protein complex (and not phenytoin itself) was able to pass through the yolk sac and reach the tissues of the embryo proper . These results suggested that a drug-protein complex may serve to transport drugs from their site of activation in the maternal liver to the developing embryo. J Exp Zool, 1986 Nov, 240(2), 265 - 73 Calcium effects on progesterone accumulation and oocyte maturation in cultured follicles of Rana pipiens; Francisco SK et al.; Pituitary homogenates (FPH) provoke a cascade of responses in the amphibian ovarian follicle, culminating in progesterone biosynthesis and oocyte maturation (GVBD) . Calcium may play an important role as an intracellular second messenger in regulating these physiological responses . Experiments were carried out on cultured, isolated follicles of Rana pipiens to assess the effects of varying extracellular calcium on follicular progesterone accumulation and oocyte maturation . In hormonally unstimulated follicles, an increase in extracellular Ca2+ alone produced a significant increase in progesterone in methanol extracts of follicles after 4 hours of culture, and in some cases also provoked oocyte maturation assessed after 24 hours of culture . In no case did elevated Ca2+ alone stimulate maximal progesterone accumulation as compared with FPH-stimulated follicles, although the time-course of accumulation was similar . The calcium ionophore, A-23187, similarly increased progesterone accumulation in a dose-dependent manner when introduced in amphibian Ringer's (1.35 mM Ca2+), but inhibited progesterone elevation caused by increasing calcium concentrations in the culture media and FPH stimulation . Depleting free calcium from the culture medium with graded doses of the chelator EGTA decreased FPH-induced progesterone accumulation and inhibited FPH- and progesterone-induced GVBD . The calcium channel blocker, verapamil, also inhibited FPH-induced progesterone accumulation and GVDB in a dose-dependent manner, while having no effect on progesterone-induced meiotic resumption . These data strongly implicate intracellular calcium levels regulating progesterone production by ovarian follicle cells and subsequent oocyte maturation. J Cell Biol, 1986 Nov, 103(5), 1799 - 805 Regulation of myogenic differentiation by type beta transforming growth factor; Olson EN et al.; Type beta transforming growth factor (TGF beta) has been shown to be both a positive and negative regulator of cellular proliferation and differentiation . The effects of TGF beta also are cell-type specific and appear to be modulated by other growth factors . In the present study, we examined the potential of TGF beta for control of myogenic differentiation . In mouse C-2 myoblasts, TGF beta inhibited fusion and prevented expression of the muscle-specific gene products, creatine kinase and acetylcholine receptor . Differentiation of the nonfusing muscle cell line, BC2Hl, was also inhibited by TGF beta in a dose-dependent manner (ID50 approximately 0.5 ng/ml) . TGF beta was not mitogenic for either muscle cell line, indicating that its inhibitory effects do not require cell proliferation . Inhibition of differentiation required the continual presence of TGF beta in the culture media . Removal of TGF beta led to rapid appearance of muscle proteins, which indicates that intracellular signals generated by TGF beta are highly transient and require continuous occupancy of the TGF beta receptor . Northern blot hybridization analysis using a muscle creatine kinase cDNA probe indicated that TGF beta inhibited differentiation at the level of muscle-specific mRNA accumulation . These results provide the first demonstration that TGF beta is a potent regulator of myogenic differentiation and suggest that TGF beta may play an important role in the control of tissue-specific gene expression during development. Ann Inst Pasteur Immunol, 1986 Nov-Dec, 137D(3), 369 - 82 Isolation of follicular dendritic cells from human tonsils and adenoids . VI . Analysis of prostaglandin secretion; Heinen E et al.; Follicular dendritic cells (FDC) are able to fix high amounts of immune complexes by C3b or Fc receptors without endocytosis and for long periods of time . In order to determine the function of this retention, we analysed the secretion of prostaglandin E2 (PGE2) by FDC in vitro; indeed, it is well-known that immune complex fixation on cells may induce PGE2 production . FDC were isolated by enzymic digestion of lymph follicles dissected under the biomicroscope from human tonsils or adenoids . Isolated FDC appeared as spherical clusters where they enveloped lymphoid cells with their cytoplasmic extensions . Tests were performed in synthetic culture media or in media supplemented with foetal calf serum . PGE2 production in FDC suspensions was compared to that of lymphocyte or macrophage-enriched populations prepared from the same human tonsils . In all experimental conditions, FDC and macrophage-enriched cell populations produced high levels of PGE2, inversely to lymphoid cell populations . This secretion was inhibited by indomethacin . At the ultrastructural level, we also showed that 3H-arachidonic acid was metabolized in cell membranes of all three cell types . The PGE2 produced in the culture media, according to our experimental conditions, do not influence cell proliferation, as assessed by 3H-thymidine incorporation tests on phytohaemagglutinin-stimulated lymphocytes. Calcif Tissue Int, 1986 Nov, 39(5), 334 - 41 In vitro bone resorption activity produced by a hypercalcemic adenocarcinoma tumor line (CAC-8) in nude mice; Rosol TJ et al.; Tumor extracts and conditioned tissue culture media from a canine adenocarcinoma tumor line (CAC-8) propagated in nude mice significantly increased in vitro bone resorption in neonatal mouse calvaria as measured by release of previously incorporated 45Ca . In vitro bone resorption activity was induced in a dose-dependent manner, was not suppressible by indomethacin, and was heat- and acid-stable . Gel exclusion chromatography demonstrated peak bone resorbing activity at a relative molecular mass of approximately 28,000 . The parathyroid hormone (PTH) antagonist (8,18norleucine, 34tyrosine) bovine PTH (3-34) amide did not inhibit CAC 8-stimulated or (1-34) bPTH-induced bone resorption . There was an increased number of tartrate-resistant, acid phosphatase-positive cells in calvariae exposed to CAC-8 extract . Ultrastructural evaluation of calvaria revealed hypertrophy and maturation of osteoclasts in calvaria exposed to CAC-8 extract . The maturation effects included close contact to bone surfaces and the presence of clear zones and ruffled borders in osteoclasts . Similar structures were observed infrequently in osteoclasts of control calvaria . These data demonstrate that the tumor line (CAC-8) contained activity capable of stimulating in vitro bone resorption by increasing osteoclast numbers and the activity of existing osteoclasts. J Endocrinol, 1986 Nov, 111(2), 255 - 61 Sexual dimorphism in immunoneutralization of bioactivity of rat and ovine inhibin; van Dijk S et al.; Inhibin was partially purified from bovine follicular fluid using chromatography on immobilized Procion Red 3B and anion-exchange chromatography . Ovariectomized Texel ewes were immunized against the inhibin-containing fraction from the Procion Red 3B column and the immune response was subsequently boosted with similar fractions or with the preparation obtained from the anion-exchange column . The potencies of the resulting antisera were evaluated in an in-vitro bioassay system for estimating inhibin activity, using dispersed rat pituitary cells . The antisera were found to inhibit the bioactivity of inhibin preparations from ovarian follicular fluid of bovine, porcine, ovine or human origin, as well as inhibin activity in ovine testicular lymph and rete testis fluid, in culture media from rat granulosa and rat Sertoli cells and in homogenates of rat ovaries and testes . These results indicate that the inhibin molecules from several species contain a common bioactive moiety . The results also showed that the antiserum was more effective in neutralizing inhibin activity from ovarian than from testicular sources in both sheep and rat, indicating a sex-related difference in the inhibin molecules within a species. Acta Endocrinol (Copenh), 1986 Nov, 113(3), 340 - 5 Stimulation of thyroid adenylate cyclase activity by sera from patients with non-thyroidal illness; Kakezono F et al.; We have previously shown that sera from many hypothyroid patients stimulated adenylate cyclase activity as measured by serum bioactive TSH concentrations produced by FRTL-5 cell line . This TSH-stimulating activity did not correlate with serum immunoreactive TSH . IgG fractions of these sera did not stimulate FRTL-5 cells . The present study was, therefore, undertaken to investigate the thyroid stimulating activity of sera from patients with non-thyroidal illness . Studies were performed in 36 patients with various non-thyroidal illness . In these patients, serum concentrations of T4, free thyroxine, T3, and TSH were determined . In addition, sera were incubated with FRTL-5 cells or porcine thyroid cells in primary culture in the presence of 0.4 mM MIBX, and medium cAMP concentrations were determined by radioimmunoassay . Sera obtained from some patients with various non-thyroidal illness increased cAMP concentrations in culture media of FRTL-5 cells as well as that of porcine thyroid cells . The thyroid stimulating effects of sera were not disease specific and significantly correlated inversely with serum T3 and T4 concentrations . Serum TSH concentrations in these patients were within the normal range even by the newly developed ultrasensitive assay . Although the nature of substance(s) present in sera of patients with low T3 syndrome which stimulates thyroid adenylate cyclase is not entirely known, it is conceivable that there exist mechanisms independent of TSH to compensate the decreased serum T3 levels in low T3 syndrome. Exp Cell Res, 1986 Nov, 167(1), 218 - 26 Chondrocyte activation in response to factor(s) produced by a continuous line of lapine synovial fibroblasts; Watanabe S et al.; We have established a permanent line of lapine synovial fibroblasts called HIG-82 . Upon appropriate stimulation, these cells mimicked primary cultures of lapine synovial cells in producing substances which activated primary cultures of lapine articular chondrocytes . Activated chondrocytes secreted prostaglandin E2 (PGE2) and latent neutral collagenase, gelatinase, and caseinase, but not acid hydrolases, into their culture media . PGE2 itself did not activate the chondrocytes . Heating the crude, synovial-conditioned media at 70 degrees C for 30 min reduced their activating activity by 49.3 +/- 20.5% (n = 7) . Production of PGE2 by chondrocytes was maximal during the first day of exposure to synovial conditioned media, whereas the production of neutral proteinases peaked during the second day . All the chondrocyte-stimulating activity was present in a fraction of Mr 10,000-25,000 . Unlike the crude conditioned medium, this partially-purified material retained full activity following heating to 70 degrees C for 30 min . These data indicate that synovial fibroblasts (type B synoviocytes) are a source of chondrocyte activator(s) and that neutral, but not acid, proteinases may be involved in extracellular proteolysis which leads to the resorption of the cartilaginous matrix seen in bioassays of catabolin. Biochim Biophys Acta, 1986 Oct 31, 889(1), 23 - 34 Abnormal procollagen synthesis in fibroblasts from three patients of the same family with a severe form of osteogenesis imperfecta (type III); Bonaventure J et al.; Dermal fibroblast cultures from three siblings with a severe form of osteogenesis imperfecta were established in order to analyze their procollagen and collagen synthesis . Cell strains from clinically normal consanguineous parents (first cousins), were also obtained for comparison . Total collagen production in culture media was diminished by 55% in the patients fibroblasts and to a lesser extent in the parents . This decrease was specific for collagenous proteins . From polyacrylamide gel electrophoresis, it appeared that the three children had not only the same defective secretion of pro alpha 1(I) molecules but that their pro alpha 1(I) migrated slightly faster than the parental and control counterparts . Analysis of secretion confirmed a reduced rate in procollagen synthesis and the absence of intracellular storage . Upon pepsin treatment, extracellular alpha 1(I) and alpha 2(I) chains were found in the expected ratio of 2:1 and migrated normally, suggesting that the altered mobility of pro alpha 1(I) chains was related to COOH or NH2 terminal propeptides . In agreement with the reduced type I collagen production, an increase in the alpha 1(III)/alpha 1(I) ratio was also detected . Furthermore, after a 2.5-h labelling followed by alkylation with iodoacetamide, free intracellular pro alpha 2(I) and alpha 1(I) chains were detected in the absence of reduction, consistent with an abnormal intracellular ratio of pro alpha 1(I)/pro alpha 2(I) that was measured after dithiothreitol reduction . Analysis of intracellular collagen chains from parental strains following a 4-h incubation demonstrated that pro alpha 1(I) appeared as a doublet, one band with normal mobility and a less intense band migrating faster and corresponding to the defective chain found in the patients . Absence of the abnormal molecules in culture media was related to the demonstration of a defective collagen secretion by parental fibroblasts . Correlation between these biochemical findings and clinical data strongly support a recessive inheritance of the disease that could be classified as a type III form of osteogenesis imperfecta . Patients would be homozygous for the same defective allele and the asymptomatic parents would most likely be heterozygous carriers of the mutation . Although the exact location of the alteration is not yet elucidated, a splicing mutation is suggested. Biochem J, 1986 Oct 15, 239(2), 325 - 31 Identification of a new tissue-kallikrein-binding protein; Chao J et al.; We have identified a tissue-kallikrein-binding protein in human serum and in the serum-free culture media from human lung fibroblasts (WI-38) and rodent neuroblastoma X glioma hybrid cells (NG108-15) . Purified and 125I-labelled tissue kallikrein and human serum form an approximately 92,000-Mr SDS-stable complex . The relative quantity of this complex-formation is measured by densitometric scanning of autoradiograms . Complex-formation between tissue kallikrein and the serum binding protein was time-dependent and detectable after 5 min incubation at 37 degrees C, with half-maximal binding at 28 min . Binding of 125I-kallikrein to kallikrein-binding protein is temperature-dependent and can be inhibited by heparin or excess unlabelled tissue kallikrein but not by plasma kallikrein, collagenase, thrombin, urokinase, alpha 1-antitrypsin or kininogens . The kallikrein-binding protein is acid- and heat-labile, as pretreatment of sera at pH 3.0 or at 60 degrees C for 30 min diminishes complex-formation . However, the formed complexes are stable to acid or 1 M-hydroxylamine treatment and can only be partially dissociated with 10 mM-NaOH . When kallikrein was inhibited by the active-site-labelling reagents phenylmethanesulphonyl fluoride or D-Phe-D-Phe-L-Arg-CH2Cl no complex-formation was observed . An endogenous approximately 92,000-Mr kallikrein-kallikrein-binding protein complex was isolated from normal human serum by using a human tissue kallikrein-agarose affinity column . These complexes were recognized by anti-(human tissue kallikrein) antibodies, but not by anti-alpha 1-antitrypsin serum, in Western-blot analyses . The results show that the kallikrein-binding protein is distinct from alpha 1-antitrypsin and is not identifiable with any of the well-characterized plasma proteinase inhibitors such as alpha 2-macroglobulin, inter-alpha-trypsin inhibitor, C1-inactivator or antithrombin III . The functional role of this kallikrein-binding protein and its impact on kallikrein activity or metabolism in vivo remain to be investigated. Exp Eye Res, 1986 Oct, 43(4), 617 - 29 Growth of non-pigmented ciliary epithelial cells in serum-free hormone-supplemented media; Coca-Prados M et al.; Bovine non-pigmented ciliary epithelial (NPE) cells are cultured following a dissecting procedure that preserves the original cellular polarity . Cell growth of primary cultures of NPE cells is supported by hormonally defined serum-free culture media and an artificial substrate with high binding affinity to attach cells in culture . Cell proliferation of primary cultures of bovine NPE cells was limited to 3-5 weeks . Nerve growth factor (NGF) was found to have a profound effect on the cellular junctions of growing NPE cells when added to the culture media . Nerve growth factor induced the formation abundant microfilament bundles associated with junctional complexes of undetermined nature between adjacent cells . Electron microscopy of NPE in culture shows a high intracellular transport activity with abundant coated pits and clathrin-coated vesicles, a complex network of microfilaments, microtubules and intermediate filaments associated with desmosomes. Eur Heart J, 1986 Oct, 7(10), 904 - 9 Spontaneous coronary artery dissection: case report and evidence for a defect in collagen metabolism; Bonnet J et al.; Spontaneous coronary artery dissection is a rare disease with a higher prevalence in women, especially in the post-partum state . In one case, we attempted to relate this pathology to a disorder in collagen metabolism . A 32-year-old woman presented two episodes of myocardial infarction, 2 and 4 months after delivery which were shown to be due to two consecutive coronary artery dissections on coronary angiogram . Collagen metabolism was investigated in skin fibroblast cultures derived from the patient, and in control fibroblast cultures . After protein labelling in culture, total protein and collagen synthesis were determined . Quantification of procollagen synthesized in cell cultures and their rate of conversion into collagen were determined both in the culture media and a cell layer extract by DEAE cellulose chromatography . The results showed a reduced total collagen synthesis in the cultures of the patient in comparison with control cultures . The ratio between type I and III procollagen was not altered . The rate of conversion of procollagen into collagen was higher in the pathological cultures than in control cultures . Impaired collagen synthesis due perhaps to a change in hormonal equilibrium in the post-partum state might therefore have been responsible for coronary artery dissection. Drug Alcohol Depend, 1986 Oct, 18(2), 115 - 26 Lipids and fatty acids in membranes from astroglial cells cultured in ethanol-containing media; Alling C et al.; A study was undertaken to evaluate the usefulness of a primary brain cell culture for the assessment of general membrane phenomena caused by ethanol . The major aim was to study the inertness versus vulnerability of membrane lipids for 8 days of ethanol exposure . Since brain cells in cultures could be more easily influenced by nutrition than in vivo, effects of varying levels of essential fatty acids in the medium were also studied . Astroglial cells from cerebral hemispheres of newborn rats had a fatty acid composition of major phospholipids resembling that of whole brain . Addition of essential fatty acids to the medium profoundly altered the composition of cell membranes, contrary to what is found in whole animal experiments . Ethanol, in graded levels up to 75 mmol/l and added daily up to 8 days, did not significantly change the fatty acid composition of phosphatidylcholine and phosphatidylethanolamine . The ratio between neutral and acidic phospholipids diminished, which was more pronounced after 8 days of ethanol exposure than after 3 days . This study on ethanol exposure on glial cells focuses on the importance of nutritional composition of culture media and on the role of dynamics among phospholipid classes. Mol Cell Endocrinol, 1986 Oct, 47(3), 257 - 67 Metabolism of {3H}ecdysone by isolated tissues of the female ixodid tick Amblyomma hebraeum (Ixodoidea; Ixodidae); Connat JL et al.; Malpighian tubules, gut, ovaries and carcasses of the adult female tick Amblyomma hebraeum were incubated in vitro in the presence of 2 microM {3H}ecdysone . Organs and media were separately extracted after 6, 24 and 48 h incubations and the patterns of ecdysone metabolites were analyzed by HPLC . Esterase-susceptible apolar metabolites similar to the AP2 already described in the soft tick Ornithodoros moubata and thus presumably corresponding to the same conjugates (C-22 esters with fatty acids) were rapidly produced in all tissues investigated . They were mainly found within the organs but they were also released into the medium to some extent . By contrast, less apolar metabolites corresponding to the AP1 esters were mainly found in the media . Malpighian tubules and gut were the most active organs regarding the conversion of ecdysone into 20-hydroxyecdysone (20E) . However, only low quantities of 20E were formed, reaching respectively 12.5% and 11.6% of the total metabolites after 48 h incubations . In the carcass and in the ovary the formation of 20E was only a minor pathway (1.7% and 3.1% of the total metabolites after 48 h) . In ovaries we observed a massive conversion of ecdysone into 3-epiecdysone, which (as in insects) presumably proceeded through the intermediate formation of 3-dehydroecdysone . These two compounds were identified among the metabolites by CI/D mass spectrometry . The 3-epimer was released into the media, in contrast with the AP2 which were essentially stored within ovaries . Epimerization was also realized to some extent by carcasses, and again the epimer was released into the culture media . The different pathways are compared with those found in other tick species and in insects, and the significance of the various metabolites is discussed. Gen Comp Endocrinol, 1986 Oct, 64(1), 99 - 106 Preparation and culture of dispersed avian pituitary cells, and age-related changes in donor pituitary weight and growth hormone content; Vasilatos-Younken R; Techniques were perfected for the enzymatic dissociation of chicken pituitary glands and a number of factors evaluated for their effects upon growth hormone (GH) production by dispersed chicken pituitary cells in culture . Age-related changes in donor pituitary weight and GH content were also determined . A procedure involving digestion of minced glands with a solution of 0.1% trypsin in S-MEM tissue culture medium (0.1% BSA) for 1 hr at 37 degrees under an atmosphere of 5% CO2-95% air yielded greater than 2.0 X 10(6) cells per gland with 80-90% viability . Five tissue culture media (D-MEM, alpha-MEM, RPMI 1640, Med-199, Earle's salts), two serum sources (calf serum (CS), horse serum (HS), and two levels of serum (5, 20%) were tested for their ability to support GH synthesis over 4 days in culture . Additionally, two culture regimes (continuous culture vs daily media changes) were evaluated for their effects on GH production . alpha-MEM resulted in the numerically highest net GH synthesis (over starting cell content), although not statistically different from RPMI 1640 or Earle's salts . Neither serum type nor percentage was significant; therefore the lower serum percentage (5) was adopted for future studies . Culture regime significantly altered the proportion of secreted vs stored hormone harvested at the end of the culture period . Changing media daily resulted in a 40% reduction in final cell GH content compared to continuous culture, whereas total cumulative media GH was approximately 39% greater (P less than 0.01) . Pituitary weight increased with age until approximately 9 weeks, whereas GH content plateaued earlier, at 5 weeks of age.(ABSTRACT TRUNCATED AT 250 WORDS) J In Vitro Fert Embryo Transf, 1986 Oct, 3(5), 319 - 25 Immunofluorescent localization of immunoglobulins on the cell surface of mouse oocytes and preimplantation embryos; Wiley LM et al.; Living mouse oocytes and preimplantation embryos were assayed by indirect immunofluorescence for their ability to adsorb heterologous serum proteins from culture media to their cell surfaces . Bovine and human immunoglobulins of the IgG class were adsorbed by the oocytes and all stages of preimplantation embryos, while IgG of mouse or goat origin was not . In contrast, none of the serum albumins was adsorbed to the cell surfaces of mouse oocytes and preimplantation embryos . From the differential binding of IgG of some, but not all, of the species that were tested, we concluded that these cell surface IgG "receptors" on mouse oocytes and preimplantation embryos are likely to be heterophilic in nature . Similar observations were made irrespective of the strain of mice used to provide the oocytes and embryos . These observations raise the question of whether human oocytes and preimplantation embryos should also be assayed for their ability to adsorb animal serum proteins that are possible candidates as a substitute protein supplement for human serum in culture medium used in human in vitro fertilization/embryo transfer programs. Mol Biochem Parasitol, 1986 Oct, 21(1), 75 - 82 Partial inhibition of trypomastigote entry into cultured mammalian cells by monoclonal antibodies against a surface glycoprotein of Trypanosoma cruzi; Alves MJ et al.; Monoclonal antibodies were raised against the surface of trypomastigote forms of Trypanosoma cruzi . Although some of these antibodies reacted against antigens shared by trypomastigote and epimastigote or amastigote forms, the majority were trypomastigote-specific . Trypomastigote-specific monoclonal antibodies recognized all infective stages, including trypomastigotes from the bloodstream of infected mice, insect feces, tissue culture and those resulting from differentiation of epimastigotes in axenic culture media . The monoclonal antibodies H1A10 and 6A2, as well as Fab fragments from H1A10, partially prevented T . cruzi invasion of LLC-MK2 cell monolayers (inhibition of 50-70%) when present throughout the entire experiment . Both antibodies recognized an 85 kDa glycoprotein (Tc-85) of the trypomastigote surface which contains N-acetyl-D-glucosamine and/or sialic acid. Acta Pathol Microbiol Immunol Scand {C}, 1986 Oct, 94(5), 181 - 6 Expression of MHC class II antigen, interleukin-2 receptor, transferrin receptor and gp 40/80 glycoprotein during different phases of a normal PHA-driven lymphocyte activation in vitro; Konttinen YT et al.; This study characterizes the temporal profile of MHC class II antigen, interleukin-2 receptor, transferrin receptor and gp 40/80 glycoprotein lymphocyte activation markers in relation to each other during different phases of PHA-dependent cellular activation in vitro . Binding of these monoclonal lymphocyte activation-probes was visualized by the avidin-biotin-peroxidase complex method . Maximum PHA-dependent MHC class II antigen expression of 27 +/- 3% was observed on culture day 1, but later no significant differences were observed in MHC class II antigen expression between PHA-driven or culture media-containing control cultures . On the contrary, interleukin-2 receptor (78 +/- 6%) and transferrin receptor (75 +/- 5%) expression reached a maximum on culture day 3, coinciding with a maximum proliferative response . On culture day 5, when 3H-thymidine incorporation was already on the decline, gp 40/80 glycoprotein reached a maximum PHA-dependent expression of 78 +/- 2%, which differed significantly from interleukin-2 receptor (60 +/- 8%, p less than 0.05) and transferrin receptor (51 +/- 8%, p less than 0.01) expression . This study suggests that MHC class II antigen, interleukin-2 receptor, transferrin receptor and gp 40/80 glycoprotein, although all of them are lymphocyte-activation markers, differ as to the chronological sequence of their appearance and disappearance . Their combined use in lymphocyte-activation marker profile assay therefore gives valuable information about the lymphocyte activation state. Fundam Appl Toxicol, 1986 Oct, 7(3), 494 - 501 Mechanisms for the release and redistribution of 2,4,5,2',4',5'-hexachlorobiphenyl (6-CB) from hepatic tissues in the rat; Rau LA et al.; The translocation of 6-{14C}CB from rat hepatic tissues to various media was studied employing in situ hepatic perfusion and primary hepatocyte culture techniques . 6-{14C}CB release from the hepatic tissues of female rats pretreated with 2 microCI 6-CB was dependent on the relative proportion of perfusate buffer components . Approximately 10% of hepatic 6-CB was released into buffer containing either 4% BSA or 4% BSA and 100 mg/dl exogenous human very low density lipoproteins (VLDL) . 6-CB release was significantly increased under simulated hyperlipidemic conditions (400 mg VLDL/dl) . Release declined when BSA was eliminated or replaced with Dextran . The distribution of 6-CB between the triacylglycerol (TG)-rich VLDL and the protein buffer components was found to be dependent on the ratio of TG:protein . Under hyperlipidemic perfusate conditions, approximately 83% of the 6-CB associated with the BSA fraction . Under normolipidemic conditions, 99% of the 6-CB associated with BSA . The concentration of 6-CB in TG was greatly increased under hyperlipidemic conditions . Thus, 6-CB distribution under simulated normolipidemic conditions could not be explained by saturation of the VLDL fraction . Approximately 15% of 6-CB was released from hepatocytes prepared from late pregnant or age-matched control rats . Eighty percent of 6-CB was associated with VLDL secreted from hepatocytes . The TG:protein ratio in culture media was approximately 1: 6 while ratios of 1:20 or 1:600 occurred in the perfusion studies . These data suggest that 6-CB may be released from hepatic tissues in association with newly synthesized TG, but that once in the circulation, its distribution is dependent on the ratio of TG to protein present. Appl Environ Microbiol, 1986 Oct, 52(4), 723 - 6 Selective partitioning of conidia of some Penicillium and Aspergillus species in aqueous two-phase systems; Strom GB et al.; Conidia of Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger, Penicillium brevi-compactum, Penicillium frequentans, Penicillium spinulosum, and Penicillium verrucosum var . cyclopium were subjected to partition at varying pH values in an aqueous two-phase system containing charged polyethylene glycol . In the system, the partition behavior of the conidia of the Penicillium species varied when the pH was raised, while the conidia of the Aspergillus species seemed unaffected . P . brevi-compactum was separated from P . verrucosum var . cyclopium after only 10 transfers when subjected to stepwise partitioning . In the same way, 10 transfers were needed to separate P . verrucosum var . cyclopium from a mixture of conidia of three Aspergillus species . The partition behavior was influenced by the culture media used. Eur J Immunol, 1986 Oct, 16(10), 1289 - 96 T90/44 (9.3 antigen) . A cell surface molecule with a function in human T cell activation; Lesslauer W et al.; T90/44 is a cell surface antigen which is present on human T cells of the helper and cytotoxic subsets and which binds the 9.3 monoclonal antibody (9.3 mAb) . It is expressed in the form of 90-kDa disulfide-bonded dimers of a 44-kDa polypeptide and of free 44-kDa subunits . The function of T90/44 was investigated in a series of T cell function assays . 9.3 mAb was found to inhibit the activation of class II-restricted cloned T helper cells derived from leprosy patients and reactive with M . leprae antigens . The inhibition was first found at 1-10 ng/ml 9.3 mAb and regularly increased with the antibody concentration . The extent of the inhibition varied among different T cell clones in proportion to the respective different levels of T90/44 expression at their cell surface . The proliferative responses of peripheral blood lymphocytes (PBL) to purified protein derivative of M . tuberculosis (PPD) and tetanus toxoid were enhanced by the 9.3 mAb resulting in up to 20-30-fold increase of {3H}-thymidine incorporation . After phytohemagglutinin-induced activation of PBL, the number of T90/44 molecules per cell expressed at the cell surface rose from day 0 to day 7 by a factor of about 10 . High concentrations of 9.3 mAb (5-10 micrograms/ml) at low cell densities and in the presence of monocytes in culture media supplemented by fetal calf serum were directly mitogenic for resting lymphocytes . The cytolytic effector functions of class I-restricted cytotoxic T lymphocytes (CTL) were not modulated by 9.3 mAb . The mixed lymphocyte reactions of three class I-restricted CTL to their specific target cells were found not to be significantly influenced by 9.3 mAb . In conclusion it is proposed that an antigen-independent T cell activation pathway can be entered at T90/44. Life Sci, 1986 Sep 29, 39(13), 1201 - 6 Estrogen receptors and growth response in cultured human periodontal ligament cells; Lewko WM et al.; The periodontal ligament (PDL) is a connective tissue involved in the remodeling process associated with tooth development and positioning . PDL cells grown in culture were analyzed for the capacity to specifically bind steroid hormones and for growth response to estradiol-17 beta . Using {3H}estradiol-17 beta as the ligand, PDL cells in first passage cultures exhibited a specific estrogen binding capacity of 881 fmol/mg cell protein . With {3H}dexamethasone as a ligand, the binding capacity of the glucocorticoid receptor was 143 fmol/mg protein . With {3H}R5020 as a ligand, the progestin receptor exhibited a binding capacity of 5 pmol/mg protein . Scatchard analysis of estradiol binding at 37 degrees revealed a dissociation constant of 2.7 X 10(-9) M, representative of the estrogen receptor . The addition of estradiol-17 beta at concentrations of 10(-9) and 10(-8) M to culture media induced a dose-dependent decrease in growth (DNA content) to 62% and 38% control values, respectively . The addition of the antiestrogens tamoxifen and 4-hydroxytamoxifen at concentrations of 10(-7) and 10(-6) M similarly depressed cell growth . These results show that PDL cells contain high affinity receptors for several steroid hormones and further that these cells are targets for the action of estrogens. Biochim Biophys Acta, 1986 Sep 12, 878(2), 168 - 76 Production and use of an inhibitory monoclonal antibody to human lipoprotein lipase; Goldberg IJ et al.; Studies were performed to produce a monoclonal antibody to human lipoprotein lipase, verify the specificity of the antibody for lipoprotein lipase, and use this antibody for detection of lipoprotein lipase protein in human post-heparin plasma . Partially purified lipoprotein lipase from human milk was used as an antigen for the production of anti-lipoprotein lipase antibodies in mice . The spleen was removed from the animal having the highest titer of inhibitory antibodies to lipoprotein lipase and the cells were fused mouse myeloma cells . Culture media from the resulting hybridomas were screened for their ability to inhibit lipoprotein lipase catalytic activity . This screening procedure thus identified only those hybridomas which produced antibodies directed against lipoprotein lipase . One monoclonal antibody, from one clone, was selected for detailed study . The specificity of this antibody for lipoprotein lipase protein was established by three methods . First, post-heparin plasma lipoprotein lipase activity and immunoreactivity detected by an enzyme-linked immunosorbent assay (ELISA) co-eluted during heparin-agarose and phenyl-Sepharose chromatography . Second, the antibody detected a protein which was released into the circulation after intravenous injection of heparin into humans . Third, both immunoreactive lipoprotein lipase protein and lipoprotein lipase enzymatic activity were lost by heat-inactivation of lipoprotein lipase . The use of active enzyme as an antigen and the procedure used to screen the monoclonal antibody-producing hybridomas allowed the production of an inhibitory anti-human lipoprotein lipase monoclonal antibody . This antibody is useful for detection of lipoprotein lipase protein in plasma and should allow for immunohistochemical staining of active lipoprotein lipase enzyme in tissues . Moreover, the methods described for screening hybridomas may be modified and used to produce specific antibodies against other partially purified enzymes. J Biochem Biophys Methods, 1986 Sep, 13(2), 97 - 102 Oligodeoxynucleotide stability in subcellular extracts and culture media; Wickstrom E; Oligodeoxynucleotide degradation was studied in four systems in order to assess the importance of degradation in hybridization arrest experiments dependent on oligodeoxynucleotides complementary to mRNA sequences . Oligodeoxynucleotides were not detectably degraded over 2 h at 37 degrees C in rabbit reticulocyte lysate or Dulbecco's modified essential medium with 5% fetal calf serum, but were degraded over 2 h in HeLa cell postmitochondrial cytoplasmic extract, and were degraded within 15 min in bovine calf serum. Biol Reprod, 1986 Sep, 35(2), 455 - 62 The detection of oviduct-specific proteins in the baboon (Papio anubis); Fazleabas AT et al.; Oviducts obtained from cycling baboons were first flushed with saline, then minced and cultured in the presence of L-{3H} leucine . The flushings, tissue culture media, and solubilized tissues were analyzed by electrophoresis for secretory products that were oviduct-specific . The fluorographs of one-dimensional 7.5% slab gels demonstrated that a macromolecule of Mr 130,000 was present in the tissue culture medium of oviducts obtained during the mid-to late follicular stages, and absent during the mid-to late luteal stages . Two-dimensional analysis revealed that the Mr 130,000 band consisted of one basic protein and two acidic proteins . The basic protein formed the major component of this band and was the protein either absent or greatly reduced in intensity during the luteal stage . A macromolecule with similar electrophoretic properties was present in silver-stained, two-dimensional gels of follicular stage flushings, and absent in both luteal stage flushings and follicular stage serum . Two other macromolecules (Mr approximately equal to 160,000 and 88,000) appeared to be secretory products that were oviduct-specific . While both were present at all stages of the menstrual cycle, the Mr 160,000 protein showed a midcycle intensification, and the Mr 88,000 protein showed a midluteal stage intensification . Thus, the synthetic ability of baboon oviduct tissue and macromolecular content of the oviduct lumen changed with the stage of the menstrual cycle . The major alteration was in the synthesis and secretion of the Mr 130,000 basic macro-molecule, which was the major protein present during the follicular stage of the cycle.(ABSTRACT TRUNCATED AT 250 WORDS) In Vitro Cell Dev Biol, 1986 Sep, 22(9), 508 - 14 Confrontation of an invasive (MO4) and a noninvasive (MDCK) cell line with embryonic chick heart fragments in serum-free culture media; Bracke ME et al.; Confronting cultures of precultured embryonic chick heart fragments (PHF) with aggregates of malignant cells in vitro have been shown to be relevant for a number of aspects of tumor invasion in vivo . Preculture of the heart fragments, formation of cell aggregates, and subsequent culture of confronting pairs have so far been done only in serum-containing culture media . We describe here confronting cultures of PHF with invasive MO4 mouse cell aggregates or noninvasive MDCK dog kidney cell aggregates in serum-free media . Heart fragments precultured in the absence of serum seemed to be necrotic after confronting culture in serum-free media . However, preculturing in media supplemented with 10% fetal bovine serum allowed us to do subsequent confronting cultures in absence of serum . Cell aggregates were also prepared in serum-containing medium . MO4 cells occupied and replaced the heart tissue within 4 d, whereas MDCK cells remained at the periphery of the PHF . This indicates that serum-free confronting cultures can discriminate between invasive and noninvasive cells . The viability of individual PHF and cell aggregates cultured in the same way as in confrontations was ascertained by histology and by explantation and postculturing on a solid tissue culture substrate . Growth of the cultures was smaller in serum-free media than in media supplemented with 10% fetal bovine serum . The main advantage of serum-free culture conditions in vitro is the elimination of the influence of serum components on invasion, and the ability to examine the effect on invasion of drugs that are susceptible to inactivation by serum. Thromb Res, 1986 Sep 1, 43(5), 569 - 77 Quantitation of urokinase antigen in plasma and culture media by use of an ELISA; Binnema DJ et al.; An ELISA was set up using polyvinylchloride microtiter plates coated with rabbit anti-UK IgG's and affino-purified goat anti-UK IgG's as second antibody . Detection occurred with rabbit anti-goat IgG antibodies conjugated with alkaline phosphatase . The assay is specific for urokinase (UK) with a detection limit of 100 pg/ml sample . Tissue-type plasminogen activator, up to concentrations of 100 ng/ml, does not interfere . The assay measures the antigen of the inactive zymogen pro-UK, the active enzyme UK and the UK-inhibitor complex with equal efficiency and gives the total UK antigen present, irrespective of its molecular form . Culture media of fibroblasts, endothelial- and kidney cells showed, despite the absence of active UK, antigen levels of 1.2, 23 and 65 ng/ml, respectively . In human plasma the UK concentration was found to be 3.5 +/- 1.4 ng/ml (mean +/- SD, n = 54) . The inter- and intra-assay variations were 20% and 6%, respectively. J Natl Cancer Inst, 1986 Sep, 77(3), 809 - 14 Protective effect of pregnancy against transplantation of lymphoma in rats; Ioachim HL et al.; Pregnancy was considered to aggravate concomitant neoplasia; however, recent observations in humans and experimental animals suggest that on the contrary tumor growth may be unfavorably affected by pregnancy . For this relationship to be investigated, a serially transplantable virus-induced lymphoma was inoculated into female rats of two different strains (WF and SD) in early or in late pregnancy . While all nonpregnant control rats died with lymphoma in 8-10 weeks, 36-75% of rats inoculated with lymphoma cells in the 1st week of pregnancy remained free of tumors, and 20-54% had tumors half the size of those in controls . When lymphoma cells were inoculated in the last week of pregnancy, the protective effect of pregnancy, although less clearly manifested, still resulted in 11% rats free of tumors and 78-91% rats with tumors markedly smaller than those of controls . Nonpregnant rats receiving pregnant rat serum in twice weekly injections grew tumors only 25-60% the size of those in rats receiving sera of nonpregnant rats . In vitro, pregnant rat sera added to the tissue culture media of rat lymphoma (AS line) or human leukemia (MOLT-4) cells resulted in reductions of viable target cells of 67-71%, respectively . The present experiments show that transplanted lymphoma cells that are 100% lethal in nonpregnant rats were totally or partially inhibited in their growth when the recipients were pregnant . Similar inhibiting effects were observed when pregnant rat serum was administered to lymphoma cells in vivo and in vitro . The protective effect against tumor growth was greater during early pregnancy, less accentuated during late pregnancy, and ceased entirely post partum and particularly during lactation, when tumor growth resumed on an accelerated course. Prenat Diagn, 1986 Sep-Oct, 6(5), 351 - 61 Cultured amniotic fluid cells for prenatal diagnosis of lysosomal storage disorders: a methodological study; Arnon J et al.; The influence of culture conditions on the ultrastructure and enzyme activities of amniotic fluid cells are reported . Morphological changes were determined as a function of the number of lysosomal-like inclusion bodies per cell, and these results correlated to the activity of beta-hexosaminidase, alpha-mannosidase, beta-glucuronidase, arylsulphatase C and 5' nucleotidase . The parameters examined were pH of the culture media, type of media, increasing cell passage and day of harvest . Our results indicate that enzyme activities are less sensitive to changes in culture conditions as compared to ultrastructural changes . We therefore recommend that in order to obtain reliable ultrastructural results for the diagnosis of storage disorders, cultures should be grown in MEM as the culture medium, the pH of the medium carefully monitored to remain below pH 7.4, examining the cultures no later than the eighth cell passage and no later than the 10th day after subculture. J Biol Chem, 1986 Aug 25, 261(24), 11295 - 301 Domain-specific distribution of carbohydrates in human fibronectins and the transformation-dependent translocation of branched type 2 chain defined by monoclonal antibody C6; Nichols EJ et al.; Fibronectins purified from human plasma (termed pFN), spent culture media of human fibroblasts WI38 (termed cFN), and SV40 virus-transformed WI38/VA13 cells (termed tFN) and their cleavage fragments were compared with respect to their binding activities to lectins and anti-carbohydrate antibodies reacting with chemically well-defined structures . The following findings were of particular interest . About 25-35% of cFN and tFN carried a binary sialosyl type 2 chain (NeuAc alpha 2----3/or 6Gal beta 1----4GlcNAc) linked beta 1----3/beta 1----6 to the galactose residue and defined by monoclonal antibody C6 . This structure was not detected in pFN . In cFN, the C6-defined structure was localized within the gelatin-binding domain, whereas in tFN the same structure was absent from this domain but was located at the NH2-terminal region of the central domain . Other carbohydrate determinants, defined by Ricinus communis lectin and concanavalin A before and after sialidase treatment, showed essentially identical domain distribution patterns among cFN, tFN, and pFN and were all located at the gelatin-binding domain (44 kDa), its precursor (60 kDa), and the Cell/Hep-2 domain (155/145 kDa) . Although both cFN and tFN were reactive with lentil lectin, pFN was not . Fibronectin from transformed cells (tFN) showed much greater reactivity than cFN and pFN with wheat germ lectin before sialidase treatment and showed enhanced reactivity with R . communis lectin and peanut lectin after sialidase treatment, indicating that tFN is more highly sialylated than cFN and pFN . All fibronectins examined were strongly reactive with monoclonal antibody AH8-28, which binds to Gal beta 1----3GalNAc residues, and this reactivity was localized to both the NH2-terminal half and COOH-terminal half of the S-cyanylation-cleaved fibronectin molecule. Int J Cancer, 1986 Aug 15, 38(2), 237 - 44 Characterization of the platelet-aggregating activity of cancer cells with different metastatic potential; Grignani G et al.; We studied the mechanisms of platelet activation by sublines exhibiting different metastatic potential of two murine experimental tumors: sublines M4 and M9 of the benzopyrene-induced mFS6 sarcoma and sublines B77-AA6 and B77-3T3 of RSV-transformed BALB/c 3T3 fibroblasts . The neoplastic cells of both models induced platelet aggregation, secretion and prostaglandin biosynthesis . In the first model but not in the second, all these processes correlated with the in vivo malignancy of cells . Pretreatment of B77-AA6 and B77-3T3 cells with apyrase significantly decreased platelet aggregation, while pretreatment of M4 cells was ineffective . However, pretreatment with trypsin or neuraminidase was effective in reducing platelet aggregation induced by M4 cells, but not that induced by any of the others; furthermore, phospholipase A2 reduced the platelet response by all sublines . Finally, platelet-activating activity was also found in the pellets obtained following centrifugation of culture media . These results suggest that platelets are stimulated by cancer cells through different mechanisms; platelet activation by a sialo-lipo-protein complex of the cellular membrane was found to be characteristic of the model in which the platelet-aggregating activity of neoplastic cells correlated with their in vivo metastatic behavior. Pharmacol Res Commun, 1986 Aug, 18(8), 759 - 67 Prevention by thymidine against toxicity and glucose uptake inhibition of methotrexate on cultured Ehrlich ascites tumour cells; Fung KP et al.; Methotrexate (MTX) arrested cell growth and inhibited 2-deoxy-D-glucose uptake of Ehrlich ascites tumour cells in vitro . The changes of glucose concentrations in the culture media did not affect the degree of suppression of cell growth by MTX . Thymidine (dThd) protected against the toxic effect of MTX on the cells . It also prevented the inhibition effect on glucose uptake of the cells by MTX . MTX significantly suppressed the maximal uptake rate of glucose (Vmax) while addition of dThd alleviated the suppression . The half-saturation constant of the uptake (Km) remained constant. J In Vitro Fert Embryo Transf, 1986 Aug, 3(4), 215 - 7 Successful fertilization, embryo development, and pregnancy in human in vitro fertilization (IVF) using a chemically defined culture medium containing no protein; Caro CM et al.; A randomized control trial involving the fertilization and culture of human embryos in culture medium (T6) containing either 10% maternal serum or no protein or amino acid supplement was carried out to assess the effect of deletion from culture of all fixed nitrogen on fertilization, embryo development, and embryo viability . There was no difference in fertilization rates (68 vs 69%), development of apparently normal embryos (96 vs 97%), pregnancy rate (18 vs 14%), or birth rate (13 vs 11%) between protein-containing and protein-free media . Deletion of protein from the culture medium may enable the constitution of more appropriate and defined culture media for human in vitro fertilization (IVF). J Dairy Res, 1986 Aug, 53(3), 419 - 29 Radioimmunoassay for measurement of bovine alpha-lactalbumin in serum, milk and tissue culture media; Akers RM et al.; A sensitive, specific radioimmunoassay for bovine alpha-lactalbumin (alpha-la) suitable for measurement of serum, tissue culture media and milk was developed . alpha-La was not detected in serum from prepubertal male or female cattle, but was detected as early as 60 d of gestation in nulliparous Holstein heifers, the level being greatly increased during the last one third of gestation . Serum from cross bred beef heifers contained less alpha-la and it was not detected until late gestation . Concentrations of alpha-la in serum samples from pregnant multiparous Holstein cows decreased at drying-off and subsequently increased just before parturition . Secretion of alpha-la by mammary tissue explants from steroid-primed prepubertal Holstein heifers was induced by the addition of bovine prolactin, ovine prolactin or human growth hormone to tissue culture media. Biochim Biophys Acta, 1986 Aug 1, 887(3), 304 - 14 Fluorescence studies of macrophage recognition and endocytosis of native and acetylated low-density lipoprotein; Berg KA et al.; Macrophage recognition and endocytosis of 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (diI)-labeled low-density lipoprotein (LDL) and acetyl LDL (Ac-LDL) was studied using fluorescence flow cytometry and fluorescence video intensification microscopy . RAW264 macrophages and U937 monocytes were grown in the tissue culture media in the presence and absence of LDL and Ac-LDL . Several lines of evidence indicate that receptor-mediated endocytosis of diI-labeled LDL or Ac-LDL was taking place . Binding can be distinguished from binding plus endocytosis by incubation at 4 and 37 degrees C, respectively . Binding was saturable at 4 degrees C and uptake at 37 degrees C was time- and ligand dose-dependent . Also, unlabeled LDL and Ac-LDL compete for their receptors . Macrophages grown in the presence or absence of LDL demonstrated distinct labeling patterns . LDL receptors were significantly increased by culture in defined medium without serum lipoproteins, while Ac-LDL receptors remained unaffected . Flow cytometry can provide an important tool to examine receptor levels, modulation of these levels and receptor-mediated endocytosis . Video intensification microscopy of similarly labeled cells has been performed . Receptors appear as punctate fluorescence, usually distributed randomly across the cell surface. J In Vitro Fert Embryo Transf, 1986 Aug, 3(4), 218 - 23 Oocyte maturation inhibitor activity in human follicular fluid: quantitative determination in unstimulated and clomiphene citrate- and human menopausal gonadotropin-stimulated ovarian cycles; Winer-Sorgen S et al.; Since removal of the oocyte from the intrafollicular milieu allows meiotic resumption and germinal vesical breakdown to proceed, the concept of an intrafollicular oocyte maturation inhibitor (OMI) has evolved . Accordingly, we asked the following questions: Is there OMI activity in human follicular fluid? Does OMI activity change with ovarian hyperstimulation? and Does OMI activity correlate with oocyte fertilization or the concentration of steroids in the corresponding follicular fluid? Fresh cumulus enclosed porcine oocytes from small follicles were incubated with human follicular fluid aspirates from normally menstruating patients with or without treatment: unstimulated follicles (N = 10), clomiphene citrate (150 mg/day) (N = 10)-treated cycles, and human menopausal gonadotropin (hMG) (N = 12)-treated cycles . A lyophylized porcine follicular fluid standard and serum-free culture media were used as positive and negative controls, respectively . After a 40-hr incubation with test materials, the oocytes were fixed, stained, and evaluated for oocyte maturation as determined by germinal vesical breakdown . Human follicular fluid, estradiol, progesterone, androstenedione, and testosterone levels were determined by radioimmunoassay . The 50% inhibitory dose (ID50) for OMI activity in follicular fluid from untreated, spontaneously menstruating women was less than that for follicular fluid from clomiphene-stimulated patients, which was less than that for follicular fluid from hMG-stimulated patients . The difference between OMI values from untreated and hMG-stimulated follicular fluids was statistically significant . Human oocytes removed from follicular fluid with higher OMI activity tended not to fertilize in vitro compared to the relatively lower OMI activity present in follicular fluid yielding oocytes which did fertilize.(ABSTRACT TRUNCATED AT 250 WORDS) J Lipid Res, 1986 Aug, 27(8), 875 - 83 Parameters of cholesterol metabolism in the human hepatoma cell line, Hep-G2; Erickson SK et al.; The human hepatoma cell line Hep-G2 has been shown to express the major enzymes of intra- and extracellular cholesterol metabolism . These include lecithin:cholesterol acyltransferase, acyl coenzyme A:cholesterol acyltransferase, 3-hydroxy-3-methylglutaryl coenzyme A reductase, and cholesterol-7 alpha-hydroxylase . Regulatory mechanisms that have been described in other hepatic systems also appear to be active in Hep-G2 cells: perturbations of cholesterol and triglyceride metabolism affected the enzyme activities and the accumulation of specific apolipoproteins in the culture media . The results indicate that studies of Hep-G2 cells may provide useful information for the elucidation of mechanisms of regulation of human hepatocyte cholesterol, lipoprotein, and biliary metabolism. Clin Immunol Immunopathol, 1986 Aug, 40(2), 326 - 34 Previous immunization of mice with herpes simplex virus type-1 strain MP protects against secondary corneal infection; Sandstrom IK et al.; Herpes simplex virus (HSV)-induced ocular disease is occurring in epidemic proportions throughout the world, and is the number one cause of unilateral corneal blindness in all developed countries . We have found, in a mouse model of herpes simplex keratitis (HSK), that products encoded by the Igh-1 locus on chromosome 12 exert a profound influence on the immune/inflammatory response in the cornea after HSV inoculation in the cornea . Thus, mice with Igh-1c or Igh-1d phenotype routinely develop extreme keratopathy and loss of corneal clarity after HSV encounter in the eye, while congenic strains expressing other Igh-1 phenotypes develop substantially less keratopathy . We examined the effect of previous subcutaneous immunization with the mutant, less virulent, MP strain of HSV on the development of keratitis and encephalitis after secondary corneal inoculation with strains MP, mP, F, and KOS . A/J mice (Igh-1c), 5-6 weeks old, were injected sc with live HSV-1 strain MP . Controls were injected with culture media without virus . Three weeks later both immunized and control nonimmunized animals were challenged in the cornea with HSV-1, strains MP, mP, F, and KOS . The animals were clinically scored for keratitis and encephalitis at regular intervals for 21 days following corneal challenge . None of the immunized animals challenged in the cornea with strain MP, 5 X 10(4) plaque-forming units (PFU), developed clinical signs of encephalitis compared to 86% of unimmunized controls . Of the immunized animals challenged in the cornea with strain MP, 5 X 10(4) PFU, only 18% developed a mild keratitis, while 96% of unimmunized controls developed severe keratitis . Mice immunized subcutaneously with MP and subsequently challenged corneally with other HSV-1 strains (mP, F, or KOS) were also protected from development of severe keratopathy. Blood, 1986 Aug, 68(2), 586 - 91 Folate requirements of methotrexate-resistant human acute lymphoblastic leukemia cell lines; Kano Y et al.; We studied the folate requirements of a human acute lymphoblastic leukemia cell line, MOLT-3, and methotrexate (MTX)-resistant sublines established in vitro . The requirement of pteroylglutamate (PGA) for optimal cell growth was different for each cell line . With increasing MTX resistance, there was progressive increase in PGA requirements, moving the PGA concentration-cell growth curve (dose-response curve) 1 log order of magnitude to the right . The increases in the requirement of 5-methyltetrahydrofolate (5-methyl-THF) by the resistant sublines were more pronounced than PGA requirement, moving the dose-response curve nearly 3 log orders in magnitude to the right . The concentrations in vitro of 5-methyl-THF required for optimal growth of the MTX-resistant sublines far exceeded the normal serum 5-methyl-THF concentrations known in humans . These observations show that MTX-resistant cell established in vitro in culture media containing PGA instead of 5-methyl-THF, a physiological folate, cannot be expected to grow in vivo . The collateral sensitivity of transport-impaired MTX-resistant sublines to 2,4-diamino-5-methyl-6-{(3',4',5'- trimethoxyanilino) methyl} quinazoline (trimetrexate, TMQ) was negated in the absence of PGA . With the addition of 5-methyl-THF, the parent cells became more resistant than the transport-impaired sublines to TMQ These data indicate that the collateral sensitivity of MTX resistant cells to the substituted 2,4-diaminoquinazoline is due to functional folate deficiency by virtue of the impaired transport of folate. Biochem Biophys Res Commun, 1986 Jul 31, 138(2), 974 - 80 Stimulation of normal human fibroblast collagen production and processing by transforming growth factor-beta; Varga J et al.; Transforming growth factor-beta (TGF beta) a growth factor with diverse effects on cellular growth and metabolism, caused dramatic stimulation of total protein and collagen synthesis by confluent normal human dermal fibroblasts in culture in a dose-dependent manner . Gel electrophoresis of the newly synthesized macromolecules from the culture media of TGF beta treated cultures demonstrated accelerated procollagen processing . These results indicate that TGF beta is capable of qualitatively and quantitatively influencing the biosynthesis of matrix molecules by fibroblasts, and raise the possibility that TGF may play a role in the development of normal and pathologic fibrogenesis. J Biol Chem, 1986 Jul 25, 261(21), 9622 - 8 Expression, purification, and characterization of recombinant gamma-carboxylated factor IX synthesized in Chinese hamster ovary cells; Kaufman RJ et al.; Factor IX has been expressed to high levels within a recombinant host cell and the biologically active fraction subsequently purified to homogeneity for characterization . The coding sequence for Factor IX was inserted into a mammalian cell expression vector and transfected into dihydrofolate reductase-deficient Chinese hamster ovary cells . The integrated DNA was amplified to a high copy number by selection for increasingly higher expression levels of the marker gene, dihydrofolate reductase, contained within a co-transfected plasmid . Cloned cell lines secreting over 100 micrograms/ml Factor IX antigen and up to 1.5 microgram/ml native Factor IX antigen have been obtained . Expression of biologically active Factor IX was dependent on the presence of vitamin K in the culture media . The gamma-carboxylated Factor IX was isolated from cell culture fluid by immunoaffinity chromatography using antibodies conformation-specific for the metal-stabilized conformer of Factor IX . This conformation is dependent upon metal ions and gamma-carboxyglutamic acid . Purified recombinant Factor IX migrated as a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with an electrophoretic mobility equivalent to plasma-derived Factor IX . The purified recombinant Factor IX demonstrated Factor IX coagulant activity, measured in Factor IX-deficient plasma, of 35-75 units/mg . Amino acid analysis of the alkaline hydrolysate of recombinant Factor IX demonstrated an average of 6-7 mol of gamma-carboxyglutamic acid per mol of Factor IX . NH2-terminal sequence analysis of the first 17 residues revealed equivalent amino acid sequences for both purified recombinant and plasma-derived Factor IX . The results represent the first purification and characterization of a biologically active, gamma-carboxylated vitamin K-dependent protein expressed in a recombinant DNA system. Biochim Biophys Acta, 1986 Jul 11, 887(2), 204 - 13 Dexamethasone-induced stimulation of arachidonic acid release by U937 cells grown in defined medium; Duval D et al.; Since the presence of serum in culture media has been shown to alter prostaglandin production, as well as to interfere with the action of anti-inflammatory drugs, we have studied the effect of dexamethasone, a potent steroidal anti-inflammatory drug, on the metabolism of arachidonic acid by human monocyte-like cells (U937) grown in a fully defined medium . Under these culture conditions, dexamethasone (10(-6) M, 24 h) induced a marked stimulation of the release of unmetabolized arachidonic acid into the culture medium . The steroid also induced an inhibition of cell proliferation which became significant only after 48 h of treatment . The accumulation of arachidonic acid in the medium after steroid treatment was associated with a significant inhibition of cell acyltransferase activity, suggesting that steroids may also act upon arachidonic acid metabolism at sites other than those of phospholipase activity. Science, 1986 Jul 11, 233(4760), 209 - 12 Neutralization of the AIDS retrovirus by antibodies to a recombinant envelope glycoprotein; Lasky LA et al.; Mammalian cell lines have been engineered to produce a secreted form of the AIDS retrovirus envelope glycoprotein . The recombinant protein has been isolated from growth-conditioned culture media and used to immunize animals . Antibodies directed against the recombinant molecule were found to react with the envelope glycoprotein produced in virus-infected cells . Furthermore, these antibodies were able to directly inactivate the AIDS retrovirus in a neutralization assay in vitro . The expression system reported here should provide sufficient quantities of the AIDS retrovirus envelope protein for biological and vaccination studies. Nippon Sanka Fujinka Gakkai Zasshi, 1986 Jul, 38(7), 1071 - 8 {Establishment and characterization of TA-4 producing cell line (OMC-1) originating from a human squamous cell carcinoma of the uterine cervix}; Ueda M et al.; A new human cell line designated OMC-1 was established from a metastatic lesion of Virchow lymph node of a large cell non-keratinizing squamous cell carcinoma of the uterine cervix . OMC-1 was successively subcultured 30 times in about 15 months . The monolayer cultured cells appeared to be epithelial with a pavement-like arrangement and tendency to pile up without contact inhibition . Electronmicroscopy showed desmosomes and well-developed tonofilaments . The population doubling time was 43-70 hours, the saturation density was 1.3-1.7 X 10(5) cells/cm2, the plating efficiency was 23-25% and the mitotic index was 3.3-4.8% . Chromosome studies showed aneuploidy and a modal number of 45 . After subcutaneous transplantation into nude mice, the cells grew into solid large cell non-keratinizing squamous cell carcinomas . By the flow cytometric analysis, the phase fractions of the cells was G1 + G0 = 40.1%, S = 24.9% and G2 + M = 35.0% . The OMC-1 cells produced TA-4 in culture media . TA-4 was also demonstrated immunohistochemically in the original tumor tissue, cultured cells and tumors in nude mice. Fertil Steril, 1986 Jul, 46(1), 66 - 72 Correlation between the follicular gonadotropin inhibitor and the maturity of the ovum-corona-cumulus complex; Saito H et al.; We assessed the correlation between the maturity of the ovum-corona-cumulus complex, the gonadotropin inhibitor in follicular fluids (FFs), and culture media derived from the granulosa cells of these follicles . FFs were obtained from patients (n = 20) stimulated with clomiphene citrate and human chorionic gonadotropin . The percentages of the inhibition of the ovarian weight augmentation (71.5% +/- 4.4%) and the number of ova (91.3% +/- 3.7%) in the mice given injections of FF from follicles containing mature ova and pregnant mare serum gonadotropin (PMS) were markedly greater when compared with those given FF from follicles containing immature ova and PMS (weight, 33.7% +/- 6.4%; ova, 60.8% +/- 5.8%) . The immature granulosa cell culture media of days 4 to 6 and the mature cell media of days 1 to 3 greatly inhibited ovarian responses to gonadotropin (percent inhibition, immature, weight, 27.2% +/- 6.7%, ova, 48.1% +/- 13.2%; mature, weight, 42.9% +/- 9.2%, ova, 37.4% +/- 7.3%) . It appears that mature follicles contain more gonadotropin inhibitor than immature follicles, that mature granulosa cells secrete the inhibitor more during days 1 to 3 of the culture than during days 4 to 6, and that immature cells secrete more during days 4 to 6 than during days 1 to 3. J Urol, 1986 Jul, 136(1 Pt 2), 219 - 24 Sertoli cell production of müllerian inhibiting substance in vitro; LaQuaglia M et al.; Sertoli cell cultures were monitored for mullerian inhibiting substance with a solid phase radioimmunoassay . Sertoli cells from newborn calf testes were placed in defined media free of serum in monolayer culture after treatment with trypsin-collagenase followed by gravity separation . Immunoreactive mullerian inhibiting substance was detected in the culture media of Sertoli cells but not National Institutes of Health 3T3 cells . To verify that the Sertoli cells were intact, cyclic adenosine monophosphate levels were determined after follicle-stimulating hormone stimulation . Cyclic adenosine monophosphate levels were elevated in Sertoli cells but not National Institutes of Health 3T3 cells . The newborn calf Sertoli cell culture provides a useful system in which to study factors affecting mullerian inhibiting substance production and release, and documents this substance as another reliable marker for the Sertoli cell . Mullerian inhibiting substance levels also could be measured in media beneath testicular fragments in organ culture, and were increased and prolonged in comparison to mullerian inhibiting substance release from Sertoli cell monolayer cultures . Modulation of mullerian inhibiting substance release from monolayer and organ cultures then was attempted . Neither gonadotropin nor steroid additions affected the release of mullerian inhibiting substance during 24 to 72 hours in organ or tissue culture . We are using the mullerian inhibiting substance radioimmunoassay to monitor attempts to immortalize a Sertoli cell line capable of continuous mullerian inhibiting substance production using viral deoxyribonucleic acid transfection techniques. Transfusion, 1986 Jul-Aug, 26(4), 331 - 4 Liquid storage of unseparated human bone marrow . Evaluation of hematopoietic progenitors by clonal assay; Lasky LC et al.; Marrow from five donors was aspirated into a small volume of heparinized culture media, filtered, and stored in 10-ml nominal plastic bags at 4, 22, and 37 degrees C, respectively, for 7 days with and without agitation . Samples were taken at 0, 1, 2, 3, and 7 days, respectively, for determination of cell numbers, trypan blue viability, and committed colony-forming unit-granulocyte-monocyte (CFU-GM) and erythroid burst-forming unit (BFU-E) and colony-forming unit (CFU-E), as well as multipotent (CFU-MIX), hematopoietic progenitors . Glucose and pH decreased with storage, most markedly at higher storage temperatures . Recovery of recovered cells exceeded 90 percent under all conditions at 7 days . Viability of committed and multipotent progenitors was maintained fairly well for 3 days at 4 and 22 degrees C, but fell off rapidly after 1 day at 37 degrees C . Recovery of progenitor cells stored 7 days was relatively poor . Best maintenance was achieved for 3 days using 4 degrees C storage without agitation. Carcinogenesis, 1986 Jul, 7(7), 1161 - 4 Studies on the role of oxygen radicals in asbestos-induced cytopathology of cultured human lung mesothelial cells; Gabrielson EW et al.; The possible role of oxygen radicals in mediating the cytopathologic effects of asbestos was studied using human mesothelial cells in culture . Electron paramagnetic resonance measurements of intact cells using the spin trap 5,5-dimethyl-1-pyrroline-1-oxide failed to detect any increase in oxygen radicals in mesothelial cells after exposure to amosite asbestos, although oxygen radicals were readily detected in cells exposed to menadione, an uncoupler of oxidation-reduction reactions . Cellular thiol levels were reduced after exposure to menadione, but were not affected by exposure to asbestos . Addition to the culture media of the free radical scavengers superoxide dismutase, reduced glutathione, N-acetylcysteine, or D-alpha-tocopherol had no affect on the dose-dependent cytotoxicity of amosite fibers . Furthermore, exposure of the mesothelial cells to amosite fibers resulted in no significant increase in the level of DNA single-strand breaks . These results all suggest that for cultured human mesothelial cells, oxygen free radicals are not important mediators of the cytopathic effect of asbestos. J Biol Chem, 1986 Jun 5, 261(16), 7485 - 90 Vitamin D-dependent calcium-binding protein of mouse yolk sac . Biochemical and immunochemical properties and responses to 1,25-dihydroxycholecalciferol; Bruns ME et al.; The present studies were performed to further characterize a mouse yolk sac protein which is similar or identical to the vitamin D-dependent intestinal calcium-binding protein (CaBP) . Yolk sac protein and purified rat intestinal CaBP displayed full identity upon immunodiffusion (Ouchterlony) using antiserum to the rat intestinal CaBP . Immunoreactive CaBP in yolk sac homogenates eluted from gel permeation columns with the low molecular weight peak of 45Ca2+ binding (Chelex assay), and the electrophoretic mobility of the protein was markedly increased by EDTA . On days 11-13 of gestation, the concentrations of immunoreactive CaBP in yolk sac were 4-5-fold higher than in placenta; by days 16-17, the concentrations in yolk sac and placenta were similar . Incubation of yolk sac with {3H}leucine demonstrated synthesis of immunoprecipitable {3H}CaBP . A single band of 3H-labeled protein was seen on sodium dodecyl sulfate gel electrophoresis of the immunoprecipitate . This protein co-migrated with radioactive placental CaBP with an apparent Mr of 10,050 . Addition of 1,25-dihydroxycholecalciferol (calcitriol) to organ culture media with or without serum increased the amount and concentration of CaBP in yolk sac (p less than 0.001) at 48 h . CaBP synthesis in yolk sac appeared to be independent of calcitriol concentrations in the maternal circulation since injection of the hormone into the maternal compartment produced no change in yolk sac CaBP despite increases of maternal intestinal and renal CaBP . These studies demonstrate that yolk sac immunoreactive CaBP is synthesized in yolk sac and has an apparent molecular size and calcium-binding properties characteristic of mammalian vitamin D-dependent calcium-binding proteins . The in vitro response of yolk sac CaBP to calcitriol is the first evidence of a vitamin D effect on the fetal membranes and suggests one function for calcitriol receptors in these tissues. Kidney Int, 1986 Jun, 29(6), 1172 - 9 Nephron segment and calcium as determinants of anoxic cell death in renal cultures; Wilson PD et al.; Proximal tubules of the S1, S2 and S3 segments, medullary thick ascending limbs of Henle's loop (MAL) and cortical collecting tubules (CCT) were individually microdissected from rabbit kidneys and cultured for seven days in hormonally defined media . Anoxia was induced by incubation of cultures in normal medium for 45 min at 25 degrees C in an atmosphere of nitrogen (N2), and cell death was measured by nigrosine dye uptake . Immediately after anoxia, cell death was highest in S3 and MAL segments greater than S2 greater than S1 = CCT . The combined effects of anoxia and substrate (glucose, vitamins, amino acid) omission determined after incubation of cultures in phosphate buffered saline containing Ca2+ and Mg2+ (PBS) for 45 min in N2 also showed differential killing dependent on segment of origin: MAL greater than S3 greater than S2 CCT greater than S1 . The effects of in vitro "reflow" were tested by returning cells to their normal oxygenated culture media at 37 degrees C . After the 45 min of anoxia and four to six hr of reflow in normal calcium-containing media, all cells from each segment were dead . Reflow in media lacking calcium for two hr immediately after anoxia then followed by return to normal calcium-containing media was associated with the survival of a significant percentage of cells for 48 hr: S1 (35.3 +/- 2.0%), S2 (30.0 +/- 2.0%), S3 (46.2 +/- 3.0%), MAL (38.7 +/- 3.0%), CCT (28.2 +/- 2.0%).(ABSTRACT TRUNCATED AT 250 WORDS) Nippon Seikeigeka Gakkai Zasshi, 1986 Jun, 60(6), 663 - 70 Possible involvement of prostaglandin endoperoxides in cartilage-synovial interactions; Yamamoto K; The depletion of proteoglycans in cartilage matrix is the beginning of cartilage breakdown . Prostaglandins and related compounds might play an important role in inhibition of cartilage metabolism under arthritic conditions . Prostaglandin endoperoxides, intermediate metabolites of arachidonic acid, are more potent chemical mediators than prostaglandins, but their action can only be demonstrated in cartilage co-incubated with synovial tissue, because they are short-lived and active only within a small restricted space . Human rheumatoid synovialis highly inhibited sulfation of cartilage matrix co-incubated . The inhibition of cartilage metabolism was released by indomethacin added to the co-incubating system, showing its responsiveness to indomethacin . The magnitude of inhibition was time-dependent and substantially greater than that by prostaglandin in cell-free rheumatoid synovial culture media . The results suggest a possible involvement of prostaglandin endoperoxides in potent inhibition of cartilage metabolism under arthritic conditions. Neurochem Res, 1986 Jun, 11(6), 913 - 25 Protein metabolism and electrophoretic profiles in astroglial primary cultures from different regions of newborn rat brain; Hansson E et al.; Primary astroglial cultures (14 days of age) from cerebral cortex, striatum, and hippocampus of newborn rat brain contained similar amounts of soluble proteins . Uptake and incorporation of {3H}valine into soluble protein measured after 30 and 60 min of incubation, respectively, was on a similar level in the various cultures . {3H}valine labeling of protein bands from the cell soluble fractions and incubation media of hemisphere cultures, and which were separated by isoelectric focusing (IEF) or sodium-dodecyl-sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), indicated that proteins are released to the extracellular medium after being synthesized within cultivated cells . Acidic and high molecular weight proteins were more heavily labelled in the incubation media than in the cell soluble fractions . Two-dimensional electrophoresis (IEF X SDS-PAGE) of soluble proteins from the different cultures showed similar patterns, which were quite different from the serum-free extracellular protein patterns . Both fractions were different from the pattern obtained from fetal calf-serum . In striatum and hippocampus culture media a "spot" was seen with Ip 6.0-6.8 and m.w . 105,000, and in the media from cerebellar cultures another "spot" was observed with Ip 5.2-5.6 and m.w . 55,000 . The results show that the different cultures are similar in their protein synthetic capacity and protein composition . The specific differences observed in proteins obtained from the serum-free incubation media might indicate specific properties among astroglial cells from various brain regions. Neurochem Res, 1986 Jun, 11(6), 813 - 24 Growth and lipid composition of rat brain glial cells cultured in lipoprotein deficient serum; Shah SN et al.; The purpose of this study was to examine the effects of substituting lipoprotein deficient serum (LPDS) for complete fetal calf serum (FCS) in culture media on the growth and lipid composition of cells dissociated from 1 to 2-day-old rat brain . The results show that in FCS cultures DNA, protein and all lipids increase with an increase in the number of days in culture . Substitution of LPDS for FCS in the culture media caused a slower increase in each of these constituents . Esterified cholesterol remained unaltered with time in LPDS cultures but increased continuously in FCS cultures . Substitution of LPDS for FCS reduced the DNA:protein ratio, and unesterified cholesterol:phospholipid ratio but the protein:phospholipid ratio and the proportion of individual phospholipids were not affected . The data indicate that removal of low density lipoprotein (LDL) from serum used in culture media reduces cell proliferation and causes alterations in cellular lipid composition specifically ratio of cholesterol:phospholipids. Clin Orthop, 1986 Jun, (207), 227 - 38 Differentiation of cartilage on three substrata under the influence of an aggregate of morphogenetic protein and other bone tissue noncollagenous proteins (BMP/iNCP); Takahashi S et al.; A cellulose acetate membrane was fashioned into a cone to serve as a substratum for an outgrowth of connective tissue from normal neonatal muscle, and a container for diffusion of bone morphogenetic protein (BMP) of an aggregate of BMP and cold water insoluble noncollagenous protein (BMP/iNCP) . The BMP/iNCP was prepared by dissociative extraction and differential precipitation with other bone matrix proteins that slowly become soluble and diffusible in culture media at 37 degrees . The BMP/iNCP was applied either on, within, or beneath the surface of the explants or suspended in the culture medium . Under the influence of BMP in tissue cultures, without any bone matrix or bone collagen in the system, connective tissue outgrowths of muscle differentiate into cartilage on three substrata: (1) cellulose acetate membranes with pore size of 0.45-5.0 micron; (2) remnants of undissolved BMP/iNCP; and (3) degenerating myofibers . The cartilage developed in the interior of muscle, possibly by phenotypic cell transformation, when the pore size