J Rheumatol, 1990 Dec, 17(12), 1600 - 7 Antibody to interleukin 1 inhibits the cartilage degradative and thymocyte proliferative actions of rheumatoid synovial culture medium; Yodlowski ML et al.; Cartilage breakdown in rheumatoid arthritis results from (a) lytic action by synovial enzymes, and (b) release of synovial catabolin, now believed to be a form of interleukin 1 (IL-1), causing chondrocytes to degrade their matrix . Rheumatoid synovial culture media were tested for their ability to stimulate cartilage degradation (proteoglycan release from bovine nasal cartilage discs) and thymocyte proliferation (3H-thymidine incorporation) in the absence or presence of anti-IL-1 . Degradation of living cartilage, stimulated 2-fold by synovial culture media, was inhibited up to 80% by anti-IL-1 . Residual breakdown in living cartilage and synovial culture media induced breakdown in dead cultures were of similar magnitude, and both were unaffected by antibody treatment . Proteoglycan products released from synovial culture media treated cartilage were of smaller average molecular weight (Sepharose CL-2B), and such size reduction was inhibited by anti-IL-1 treatment . Synovial culture media that stimulated cartilage degradation also stimulated thymocyte proliferation; the latter was fully suppressible by anti-IL-1 . One of 8 synovial culture media contained an inhibitor(s) of thymocyte proliferation, removable by dialysis . We conclude (1) rheumatoid synovial catabolin activity is due to a form of IL-1 . (2) A minor nonsuppressible component of synovial culture media stimulated breakdown, identical in living and killed cartilage, is due to passive transfer of enzymic activity . (3) Cultured rheumatoid synovium releases both IL-1 and an inhibitor(s) of IL-1 action. J In Vitro Fert Embryo Transf, 1990 Dec, 7(6), 347 - 50 Embryotoxicity of micropore filters used in liquid sterilization; Harrison KL et al.; Microfiltration is the usual method of sterilization of tissue culture media . The filtrates of eight commercial filters were tested for embryotoxicity using mouse embryo growth . Four of five disk filters tested showed embryo-toxicity in the first 5 ml of filtrate and the three larger-capacity filters showed embryotoxicity for up to the first 10 ml of filtrate . The ethylene oxide-sterilized filters appeared to give poorer results than the gamma-irradiated filters . The importance of discarding the initial filtrate or preflushing such filters before use is emphasized. J Appl Physiol, 1990 Dec, 69(6), 1953 - 60 Cholinergic reactivity of tracheal smooth muscle after infection with feline herpesvirus I; Killingsworth CR et al.; Airway responsiveness was studied in cats 3 or 6 days after exposure to feline herpesvirus I . Control cats were sham inoculated with tissue culture media . Intrathoracic airway caliber was evaluated by pulmonary resistance (RL) and dynamic compliance (Cdyn) . Trachealis shortening was quantitated with microfoil strain gauges, which measured the external diameter of tracheal ring 4 . Airway smooth muscle contraction was produced using vagal stimulation and local infusion of acetylcholine . The diameter of tracheal ring 4 decreased with increasing frequency of vagal stimulation, and there was more constriction at 3 (PID3) than at 6 days postinfection (PID6) or in control cats . RL increased and Cdyn tended to decrease with increasing frequency of stimulation, but there was no difference between control and infected cats . Infected and control cats did not differ in their response to locally infused acetylcholine . Virus was consistently cultured from conjunctival, nasal, and oral mucous membranes, trachea, and main stem bronchi at PID3 but not from the trachea and main stem bronchi at PID6 . Virus was never isolated distal to the main stem bronchi . Tracheal hyperresponsiveness to vagal stimulation correlates with the presence of virus at PID3 and is apparently presynaptic in origin. J Protein Chem, 1990 Dec, 9(6), 663 - 72 Recombinant human prorenin from CHO cells: expression and purification; Holzman TF et al.; The gene for human preprorenin was obtained from total RNA prepared from primary human chorion cells . An expression vector was constructed containing an SV40 early promoter, a human preprorenin cDNA, bovine growth hormone poly-A addition signal, and a dihydrofolate reductase (dhfr) expression cassette . This vector was inserted into the DXB-11 Chinese hamster ovary (CHO) cell line . The recombinant protein was exported by CHO cells into the tissue culture media . At harvest the prorenin levels ranged from approximately 1-5 mg/L . For prorenin isolation the cell culture supernatants were processed by filtration, concentration, dialysis, and batch extraction . Preparative-scale isolation of prorenin was accomplished using blue-dye chromatography and size-exclusion chromatography . The isolated prorenin yielded a single SDS-gel band with Mr approximately 40,000 . The proprotein was characterized with respect to N-terminal sequence and N-linked sugar composition . Trypsin-activated renin prepared from the proprotein was characterized with respect to N-terminal sequence and pH-activity profile . Enzyme activity was measured with a newly developed fluorogenic peptide substrate containing the P6-P'3 sequence of human angiotensinogen. Acta Orthop Scand, 1990 Dec, 61(6), 539 - 45 Long-term storage effects on canine osteochondral allografts; Wayne JS et al.; We have studied long-term (to 60 days) effects of 4 degrees C storage in culture media on the histologic, mechanical, and chemical properties of the cartilage from osteochondral shell allografts from the dog . The structural integrity of the cartilage matrix was intact up to 60 days of storage, for the mechanical properties represented by the aggregate modulus and apparent permeability remained normal . These data are supported by normal safranin-O staining as well as normal glycosaminoglycan content and total collagen concentration . However, chondrocyte viability, as assessed by 35SO4 uptake and hematoxylin and eosin preparations, decreased dramatically with time . We believe that the longer storage to 60 days is not indicated, unless conditions can be modified to maintain cell viability. Jpn J Cancer Res, 1990 Dec, 81(12), 1281 - 5 Inhibitory effects of acyclic retinoid (polyprenoic acid) and its hydroxy derivative on cell growth and on secretion of alpha-fetoprotein in human hepatoma-derived cell line (PLC/PRF/5); Fukutomi Y et al.; Acyclic retinoid (polyprenoic acid) has a slightly different structure from retinoic acid . However, acyclic retinoid acts similarly to retinoic acid, because both bind to cellular retinoic acid-binding protein and cellular retinoid-binding protein . F-type, with the same strong binding affinity . We studied the effects of acyclic retinoid, the 7-hydroxy derivative of acyclic retinoid (7OH-acyclic retinoid) and retinoic acid on a human hepatoma-derived cell line PLC/PRF/5 (Alexander cells) . Acyclic retinoid inhibited cell growth with an ID50 value of 14 microM, and reduced cell viability with an LD50 value of 86 microM . The ratios of LD50 value to ID50 value were 6.1 for acyclic retinoid, 2.4 for 7OH-acyclic retinoid and 1.4 for all-trans-retinoic acid . Taking this ratio as a parameter of relative cytotoxicity, we concluded that acyclic retinoid is the least toxic compound . Growth inhibition of cells by acyclic retinoid was associated with the incorporation of 3H-thymidine in the logarithmic phase . Acyclic retinoid reduced secretion of alpha-fetoprotein (AFP) and reciprocally increased secretion of albumin in the culture media, suggesting that acyclic retinoid influences gene expression of these proteins . Thus, acyclic retinoid, one of the less toxic retinoids, inhibits cell growth of human cancer cell line PLC/PRF/5 and appears to alter gene expression of AFP and albumin toward a "normal" direction. J Virol, 1990 Dec, 64(12), 5874 - 82 Increased human immunodeficiency virus (HIV) expression in chronically infected U937 cells upon in vitro differentiation by hydroxyvitamin D3: roles of interferon and tumor necrosis factor in regulation of HIV production; Locardi C et al.; We have investigated the roles of cytokines in the modulation of human immunodeficiency virus (HIV) production in chronically infected U937 cells upon in vitro differentiation by hydroxyvitamin D3 . HIV-infected U937 cells exhibited markedly lower levels of CD4 and HLA-DR antigens than uninfected cells did . Vitamin D3 induced a time-dependent macrophagelike differentiation, as determined by monitoring the expression of some surface antigens by means of the monoclonal antibodies OKM1, OKM5, OKM13, OKM14, OKT4, anti-HLA-DR, TecMG2, TecMG3, LeuM3, LeuM1, anti-HLA-DP, and anti-HLA-DQ . Treatment with hydroxyvitamin D3 resulted in a marked increase in HIV production compared with control cultures . Interleukin 1 beta (IL-1 beta) and tumor necrosis factor alpha (TNF-alpha) were detected in the culture media, whereas interferon (IFN) was not generally found . Using the polymerase chain reaction technique, we found HIV-infected U937 cells to express detectable levels of mRNAs for alpha interferon (IFN-alpha), IFN-beta, TNF-alpha, and IL-1 beta . The addition of TNF resulted in a marked increase of HIV production, whereas IL-1 beta was ineffective . In contrast, both IFN-alpha and IFN-beta exerted some inhibitory effect on HIV production, which was more marked in vitamin D3-treated cultures than in untreated cultures . HIV production was significantly increased by antibodies to IFN-alpha in both untreated and vitamin D3-treated cultures . Anti-IFN-beta antibody increased HIV production only in vitamin D3-treated cells . In contrast, anti-TNF-alpha antibodies markedly decreased HIV production in both control and differentiating U937 cells . Vitamin D3 treatment resulted in a higher expression of TNF receptors in differentiating cells than in control HIV-infected cells . These data demonstrate a strong correlation between HIV production and macrophagelike differentiation in chronically infected U937 cells and suggest that endogenous IFN and TNF exert opposite effects in the regulation of virus production in both undifferentiated and vitamin D3-treated cell cultures. Brain Res, 1990 Nov 19, 533(2), 239 - 47 Culture of mature hippocampus slices for 4 days in a newly developed medium: preservation of transmitter release and leucine incorporation into protein; Kleinberger-Doron N et al.; A procedure and a medium for sustaining mature hippocampus slices in vitro for 4 days are described . The ionic composition of the medium, in which the slices were incubated for 1 h of recovery following preparation, strongly affected their ability, 4 days later, to take up and to release D-{3H}aspartate and {14C}GABA . A medium deficient in Na+ and Ca2+ proved best for recovery of the fresh slices prior to transfer to culture medium . The newly developed CSF-like culture medium was the best among several media tested in maintaining the potential of the slices for uptake and for induced release of D-{3H}aspartate and {14C}GABA . Glutamine, present in most culture media, appeared to be particularly toxic . Relative to fresh slices, the slices after 4 days in culture maintained 118% and 97% of the uptake of D-{3H}aspartate and of {14C}GABA respectively . K(+)-induced release of D-{3H}aspartate and of {14C}GABA was 104% and 82% of the respective values in fresh slices . Under the optimal culture conditions worked out, the slices also regained a considerable capacity for incorporation of labelled leucine into protein, which was low in fresh slices. Biochem J, 1990 Nov 15, 272(1), 39 - 44 Inhibitor studies indicate that active cathepsin L is probably essential to its own processing in cultured fibroblasts; Salminen A et al.; The lysosomal cysteine proteinase cathepsin L is synthesized in cultured mouse NIH 3T3 cells as a 39 kDa precursor and processed intracellularly into active 29 kDa and 20 kDa + 5 kDa lysosomal forms . Addition to culture media of the peptidyl aldehyde leupeptin, a non-covalent inhibitor of cathepsin L, results in the accumulation of the 20 kDa mature form of the enzyme, resulting in increased activity of cathepsin L as measured in an in vitro assay system in the absence of leupeptin . The more potent irreversible cathepsin L inhibitors benzyloxycarbonyl-Phe-Ala-diazomethane and L-transepoxysuccinyl-L-leucylamino-(4-guanidino)butane, when added to living cells at low concentrations, result in accumulation of all partially processed forms of cathepsin L, especially the 29 kDa form, suggesting that cathepsin L is responsible for its own processing . Exogenous procathepsin L introduced into CHO cells by endocytosis via the mannose 6-phosphate receptor is processed in a manner similar to endogenous procathepsin L . We conclude that the major intracellular pathway for processing of procathepsin L, either endogenous or exogenous, probably requires active cathepsin L. Arerugi, 1990 Nov, 39(11), 1492 - 8 {Allergenicity of the osmophilic fungus Aspergillus restrictus evaluated by skin prick test and radioallergosorbent test}; Sakamoto T et al.; Recently large amounts of Aspergillus restrictus, a species of osmophilic fungi, have been detected in house dust using culture media with low water activity . But little attention has been paid to this fungus as an allergen . In the present study, the authors examined the allergenic activity of A . restrictus by skin prick tests and radioallergosorbent tests (RAST) on 94 asthmatic patients (mean age 12.0, range 3-18) . Aspergillus fumigatus, Alternaria alternata and house dust were used for comparison . In the skin prick tests, A . restrictus, A . fumigatus, A . alternata and house dust elicited positive reactions in 8 (8.5%), 8 (8.5%), 15 (16.0%) and 69 (73.4%) patients, respectively . RAST showed positive reactions in 27 (28.7%) subjects for A . restrictus, 22 (23.4%) for A . fumigatus, 35 (37.2%) for A . alternata, and 75 (79.8%) for house dust . These results indicated that some asthmatic individuals showed immediate-type hypersensitivity to A . restrictus, and the prevalence of hypersensitivity of A . restrictus determined by skin prick tests and RAST was comparable with that of A . fumigatus but lower than that of A . alternata or house dust . This indicates that this fungal species may be of importance as a causative agent in atopic diseases. J Burn Care Rehabil, 1990 Nov-Dec, 11(6), 504 - 9 Persistence of fetal bovine serum proteins in human keratinocytes; Johnson MC et al.; Cultured human keratinocytes are used for skin grafts, but their success is limited by late graft loss . Development of antibody to fetal bovine serum (FBS) protein used in culture media for in vitro keratinocyte growth has been identified . The persistence of FBS antigen in skin grafts is important in the induction of the immune response and the susceptibility of the keratinocytes to immune-mediated injury . The magnitude and longevity of FBS protein persistence on human keratinocytes was studied . Secondary passage human keratinocytes were grown in media supplemented with 5% FBS . The media was changed to one supplemented with pooled human AB serum, and the amount of FBS protein incorporated in the tissue was measured over the following 8 days by an ELISA reaction directed against FBS antigen . Incorporated FBS antigen decreased for the first 3 days to 31% of maximum . There was no further significant decrease for 5 days . Keratinocytes grown in alternative serum supplements (NuSerum {Collaborative Research Inc., Bedford, Mass.} and Serum Plus {Hazelton Research Products Inc., Lenexa, Kan.}), which contain reduced amounts of FBS, offered no significant reduction in FBS protein incorporation . This duration of antigen persistence would make human keratinocytes susceptible to cell destruction by immune response to FBS and may contribute to delayed loss of human keratinocyte grafts. J Exp Zool, 1990 Nov, 256(2), 189 - 99 Rat renal papillary tissue explants survive and produce epithelial monolayers in culture media made hyperosmotic with sodium chloride and urea; Woolverton WS et al.; The capacity of papillary cells to adapt to elevated osmotic concentrations is unusual among mammalian cells . This capacity was evaluated by using primary tissue culture . Viability and growth of cells in rat renal papillary tissue explants were assessed after culture in media adjusted with urea and sodium chloride to various osmotic concentrations between 300 and 1,500 mOsm/kg water . The survival of cells, including cells resembling those of the collecting ducts and the loop of Henle, was greatest in medium adjusted to 1,000 mOsm with equiosmolar amounts of the two solutes . At 1,500 mOsm only cuboidal tubular epithelium resembling collecting duct epithelial cells survived . In contrast, cells of cortical tissue survived and grew at 300 and 640 mOsm, but not at 1,000 mOsm or above . Epithelial monolayers appeared to proliferate from collecting ducts and spread over the surface of the explants as well as onto the glass surface in the culture dish . Epithelial growth of medullary tissue was most rapid at 300 mOsm and was slower at 700 and 1,000 mOsm . Monolayers did not form at 1,500 mOsm; however, epithelial overgrowth of explants did occur . Hydropenia in the donor animal did not significantly affect the viability or growth of cultured papillary tissue . Explants cultured for 5 days at 300 mOsm followed by a stepwise increase in medium osmolality to 1,100 or 1,500 mOsm and cultured for 3 more days showed low or no survival whereas explants cultured at 700 mOsm survived such increases . Explants cultured for 5 days at 1,500 mOsm survived and grew monolayers when lowered to 300 mOsm . Poor viability and no epithelial proliferation were observed in explants cultured in medium adjusted to 900 mOsm with either urea or sodium chloride alone, suggesting that a mixture of the two solutes in the extracellular space, as found in vivo, may be essential in achieving elevated osmolalities. Toxicol Appl Pharmacol, 1990 Nov, 106(2), 209 - 21 Studies on the mechanisms of Ni2(+)-induced cell injury: I . Effects of Ni2+ on microtubules; Lin KC et al.; Cytoskeletal perturbations have been associated with exposures to a variety of toxic agents as well as a number of human pathological conditions . We have observed dramatic alterations in the organization of microtubules (MT), a major component of the cytoskeleton, in 3T3 cells exposed to Ni2+ . Severe perinuclear bundling and aggregation of MT occurred in both a time- and dose-dependent fashion, and this MT damage was reversible upon removal of Ni2+ from the culture media . To understand the mechanism of the Ni2(+)-induced MT change, we investigated the effect of Ni2+ (0.01 to 3.0 mM) on in vitro tubulin polymerization . Ni2+ at lower concentrations (0.01 to 1.0 mM) had little or no significant effect on the kinetics of MT polymerization . In contrast, in the presence of 1.5 to 2.0 mM Ni2+, a significant promoting effect on both the rate and the final extent of polymerization was observed . However, at Ni2+ concentrations higher than 2.0 mM, such stimulatory effect on the rate and the final extent of tubulin polymerization declined . Furthermore, the promoting effects of Ni2+ on MT polymerization were accompanied by a significant decrease in the lag period . Electron microscopic examination of samples of the polymerization product showed that MT, polymerized in the presence of 2.0 mM Ni2+, appeared more numerous and shorter (1.10 +/- 1.02 microns) than those of control (3.81 +/- 2.29 microns; p less than 0.005) . This was probably a direct result of an increase in the number of initiation centers in the presence of Ni2+ as a consequence of the decreased critical concentration (7%, p less than 0.05) necessary for polymerization to occur . Our results suggest that Ni2+ may exert its toxic effect on MT in cultured cells by altering the normal kinetics of MT polymerization. Vet Clin North Am Small Anim Pract, 1990 Nov, 20(6), 1457 - 74 Management of dermatophyte infections in catteries and multiple-cat households; Moriello KA; Successful elimination of dermatophytosis from cats requires clipping of the haircoat, weekly or twice-weekly antifungal dips, and systemic antifungal therapy . In addition, the environment should be repeatedly decontaminated with an appropriate chemical, for example, household bleach . Successful treatment may take weeks to months depending on the circumstances . Monitoring response to therapy is best done via toothbrush-culturing techniques and inoculation of the bristles onto fungal culture media . Currently, there is no successful fungal vaccine, and prevention of reinfection can be difficult and requires careful quarantine measures. Proc Natl Acad Sci U S A, 1990 Nov, 87(22), 8869 - 73 Cultured normal human hepatocytes do not synthesize lipoprotein-associated coagulation inhibitor: evidence that endothelium is the principal site of its synthesis; Bajaj MS et al.; Human plasma contains a factor Xa-dependent inhibitor of tissue factor/factor VIIa complex termed lipoprotein-associated coagulation inhibitor (LACI) . The present study examines the site(s) of LACI synthesis . In this study, cultured hepatocytes isolated from normal human liver were found to be essentially negative in LACI mRNA as revealed by Northern blot analysis using a full-length LACI cDNA as probe . The conditioned media from these cultures were also essentially negative for LACI activity . Similarly, poly(A)+ RNA obtained from normal human liver did not contain detectable LACI mRNA . In contrast, cultured human umbilical vein endothelial cells and human lung tissue (rich in endothelium) both contained abundant amounts of LACI mRNA . Moreover, erythrocyte lysates and culture media from normal monocytes, lymphocytes, or neutrophils did not contain measurable LACI activity; these cells were also negative for LACI mRNA . Platelets, however, contained LACI activity . The likely source of platelet LACI is the megakaryocyte cell since a megakaryocyte cell line (MEG-01) was found to contain LACI mRNA and to secrete small amounts of LACI activity . Additionally, human vascular smooth muscle cells and lung fibroblasts were also found to synthesize only small amounts of LACI . From these observations, we conclude that normal liver does not synthesize LACI and that endothelium is the principal source of plasma LACI . The undegraded LACI synthesized by endothelial cells had a molecular weight of approximately 41,000. Lab Invest, 1990 Nov, 63(5), 676 - 82 Increased production of tumor necrosis factor by normal immune cells in a model of the humoral hypercalcemia of malignancy; Sabatini M et al.; The rat Leydig cell tumor is a well characterized model of the humoral hypercalcemia of malignancy . The studies reported here were provoked by the observation that tumor-bearing rats become extremely cachectic and develop hypertriglyceridemia as they become hypercalcemic . Since the bone resorbing cytokine tumor necrosis factor (TNF)/cachectin is associated with cachexia and hypertriglyceridemia, we examined hypercalcemic tumor-bearing rats for evidence of increased TNF production using a TNF radioimmunoassay . We found that immunoreactive TNF was increased in the plasma of tumor-bearing rats . The increase in plasma TNF was comparable to that previously shown in hypercalcemic nude mice bearing Chinese hamster ovarian cell tumors transfected with the human TNF gene . There was no detectable TNF activity in tumor culture media which suggested that the tumor itself was not the source of excess TNF production . However, we found that tumor cell conditioned media enhanced the production of TNF activity by normal macrophages in vitro, indicating that increased TNF production in vivo may result from a tumor factor(s) which stimulates TNF production by normal immune cells . When TNF was added together with tumor products to organ cultures of fetal rat long bones, osteoclastic bone resorption was potentiated . These data are consistent with the concept that in this model of the humoral hypercalcemia of malignancy, increased TNF production by normal immune cells is increased, has systemic effects as suggested by cachexia and hypertriglyceridemia, and may work in concert with factors produced directly by tumor cells to overwhelm normal calcium homeostasis. Proc Natl Acad Sci U S A, 1990 Nov, 87(21), 8296 - 300 A 170-kDa membrane-bound protease is associated with the expression of invasiveness by human malignant melanoma cells; Aoyama A et al.; Malignant spreading of cancer cells requires cell surface proteases that cleave the crosslinked collagenous matrix of connective tissues . From correlating the morphologically defined invasiveness of tumor cells with the presence of specific membrane-associated proteases, we have identified a malignant human melanoma cell line, LOX, that invades crosslinked gelatin films in vitro and contains uniquely a neutral 170-kDa gelatinase in the cell membrane . A similar gelatinase was found in membranes recovered from culture media conditioned with LOX . The 170-kDa gelatinase is a wheat germ agglutinin-binding protein . The proteolytic activity is maximal at neutral pH, enhanced by EDTA and dithiothreitol, inhibited by the cysteine protease inhibitors N-ethylmaleimide, HgCl2, and phenylmethylsulfonyl fluoride, and can bind to an organomercurial adsorbent, suggesting that it is a neutral sulfhydryl-sensitive protease . This 170-kDa gelatinase of LOX cells was not found in a control melanoma cell line, SK-MEL28, or in 32 other tumor cell lines that did not show extracellular gelatin degradation . Thus, we have identified a large membrane-bound protease that may be a specific marker molecule for melanoma cell invasiveness. Cell Regul, 1990 Nov, 1(12), 907 - 19 The transmembrane anchor of the T-cell antigen receptor beta chain contains a structural determinant of pre-Golgi proteolysis; Wileman T et al.; Studies with the T-cell antigen receptor (TCR) have shown that the endoplasmic reticulum, or an organelle closely associated with it, can retain and degrade membrane proteins selectively . The observation that only three (alpha, beta, and delta) of the six (alpha beta gamma delta epsilon zeta) subunits of the TCR are susceptible to proteolysis implies that structural features within the labile proteins mark them for degradation . The TCR beta chain is degraded in the endoplasmic reticulum, and, in this study, we have started to define the domains of the protein that make it susceptible to proteolysis . The experiments show that the transmembrane anchor and short five-amino-acid cytoplasmic tail of the protein contain a dominant determinant of proteolysis . When these residues were removed from the beta chain, the protein became resistant to proteolysis . Even though the resulting ectodomain of the beta chain lacked a transmembrane anchor, it was not secreted by cells and was retained in the endoplasmic reticulum . We conclude that retention in the endoplasmic reticulum alone does not lead to degradation . The results suggest that structural features within the membrane anchor of the protein predispose the beta chain to proteolysis . This was confirmed by replacing the membrane anchor of the interleukin 2 (IL2) receptor, a protein that was stable within the secretory pathway, with that of the TCR beta chain . The unmodified IL2 receptor was transported efficiently to the surface of cells, and an "anchor minus" construct was secreted quantitatively into the culture media . When the membrane anchor of the IL2 receptor was replaced with that of the TCR beta chain, the chimera was unable to reach the Golgi apparatus and was degraded rapidly. Biol Reprod, 1990 Nov, 43(5), 828 - 34 Follicle-stimulating hormone regulation of cytochrome P-450 side-chain cleavage messenger ribonucleic acid accumulation by porcine granulosa cells isolated from small and medium follicles; Mulheron GW et al.; The experiments described here were conducted to examine regulation of cytochrome P-450 side-chain cleavage (SCC) mRNA accumulation in porcine granulosa cells isolated from small (1-4-mm) and medium (5-6-mm) follicles . Granulosa cells were cultured under the following conditions: 1) for 48 h or 96 h with 0, 50, or 200 ng/ml porcine FSH; 2) for 96 h with 200 ng/ml FSH and aminoglutethimide (100 microM); and 3) for 96 h with forskolin (100 microM) . Total RNA was extracted and examined by Northern and dot-blot hybridization analysis, and culture media were assayed for progesterone concentration . Northern blot analysis revealed a single band approximately 2.1 kb in size . Accumulation of SCC mRNA by granulosa cells was both FSH dose- and culture time-dependent (p less than 0.05) with maximal increases approximately 4.5 times control levels . Aminoglutethimide reduced progesterone production by about 80% while having no effect on granulosa cell accumulation of SCC mRNA compared to cells stimulated with 200 ng/ml of FSH . Forskolin-treated cells produced significantly more progesterone than did cells treated with FSH, but accumulation of SCC mRNA was similar . In response to FSH, concentration of SCC mRNA did not vary with follicle size, but granulosa cells from small follicles produced significantly more progesterone than did those from medium follicles . These results demonstrate that concentration of SCC mRNA in cultured porcine granulosa cells is FSH dose-dependent, does not vary significantly in cells from small- and medium-sized follicles, and is correlated with progesterone production, but may not parallel progesterone secretion . This last observation indicates that control at sites other than SCC mRNA can affect progesterone production. J Clin Invest, 1990 Nov, 86(5), 1678 - 83 Mechanism of Pneumocystis carinii attachment to cultured rat alveolar macrophages; Pottratz ST et al.; Pneumocystis carinii (PC) pneumonia begins as an intra-alveolar process resulting in injury to the alveolar epithelium with subsequent invasion of the lung interstitium . The clearance of PC organisms from the alveolar space is a critical function of alveolar macrophages (AM), the resident alveolar phagocytic cells . In this study the mechanism of PC attachment to AM was determined using 51Cr-labeled organisms, with PC attachment reaching a maximum of 18.9 +/- 2.5% after 4 h . Attachment was significantly decreased by preincubation of the AM with a monoclonal anti-fibronectin antibody directed against the cell attachment site of fibronectin (from 17.8 +/- 2.2% to 8.3 +/- 1.0%, P less than 0.01), or by addition of the fibronectin cell binding site analogue Arg-Gly-Asp-Ser (RGDS) (from 18.1 +/- 2.3% to 2.9 +/- 0.8%, P less than 0.01) . An anti-fibronectin monoclonal antibody directed against the heparin binding domain of fibronectin had no effect on PC attachment . Addition of the specific calcium ion chelating agent EGTA to the culture media similarly decreased attachment from 16.9 +/- 2.0% to 5.1 +/- 1.1% (P less than 0.01) . Fibronectin-mediated attachment of PC to AM did not result in phagocytosis of the organisms by the AM as determined by chemiluminescence measurements . Therefore, the data indicate that PC attachment to AM is a calcium-dependent process mediated by the cell binding domain of fibronectin which does not trigger a phagocytic response by the AM. Invest Ophthalmol Vis Sci, 1990 Nov, 31(11), 2412 - 9 Scleral fibroblasts . Human leukocyte antigen expression and complement production; Harrison SA et al.; The authors investigated the ability of recombinant human gamma-interferon (rhIFN-gamma) to influence production of complement and expression of human leukocyte antigens (HLA) by human scleral fibroblasts in culture . Cell cultures were established by explanting sclera from normal human donor eyes . To study complement production, fibroblasts were treated with 500 units/ml rhIFN-gamma in cell culture, and media were tested for complement components by hemolytic assay after 0, 1, 3, 6, 9, and 11 days . To induce Class II HLAs, fibroblasts were exposed to rhIFN-gamma at concentrations ranging from 10-500 units/ml and incubated for 1, 3, and 6 days . The HLAs were detected by immunofluorescence in conjunction with flow cytometry . Class I antigen was detected using a monoclonal antibody directed against beta 2-microglobulin . Class II histocompatibility antigens were identified using monoclonal antibodies specific for HLA-DR, -DP, and -DQ . Although complement component C1 was produced constitutively in cell culture, the addition of rhIFN-gamma resulted in an increase in production . Complement components C2 and C4 were detected only after treatment with rhIFN-gamma . Complement production was completely inhibited by cycloheximide, and C3, C5, C6, and C7 were not present in cell culture media with or without rhIFN-gamma . Class I antigen was present on all cells before induction, and an increase in expression was noted after exposure to rhIFN-gamma . Class II antigens were absent before induction with rhIFN-gamma . After treatment with rhIFN-gamma, scleral fibroblasts expressed HLA-DR, -DP, and -DQ in a dose-dependent, time-related fashion . These findings suggest that rhIFN-gamma has multiple effects on scleral fibroblasts: (1) increased production of C1, (2) production of C2 and C4, (3) up-regulation of Class I antigen expression, and (4) expression of Class II antigens . They also suggest that scleral fibroblasts have the potential to participate in immunologic diseases of the eye. J Clin Endocrinol Metab, 1990 Nov, 71(5), 1235 - 8 Highly vectorial secretion of inhibin by primate Sertoli cells in vitro; Handelsman DJ et al.; Having recently demonstrated highly vectorial and FSH-stimulated inhibin secretion by immature rat Sertoli cells in vitro, we wished to determine if vectorial secretion of inhibin was also characteristic of primate Sertoli cells . By adapting techniques for isolation of Sertoli cells from testes of the immature rat and cynomolgus monkey . Sertoli cells were isolated from immature baboon testes . Sertoli cells were then plated onto matrix-impregnated porous filters and cultured in twin chamber assemblies in fully defined, supplemented HEPES-buffered Eagles medium . Inhibin was measured in conditioned culture media by an heterologous RIA validated for primate inhibins . Throughout 28 days of culture immunoreactive inhibin was readily detectable in the upper chambers whereas inhibin was undetectable or just detectable in the lower chambers . The median ratio of upper to lower chamber inhibin content was 15.3 under basal conditions rising to 41 under FSH stimulation . Inhibin secretion into the upper chamber was increased 2.5 +/- 0.4 times by stimulation with ovine FSH (100 ng/ml) . We conclude that immature Sertoli cells from a nonhuman primate demonstrate FSH-responsive and highly vectorial secretion of inhibin almost exclusively into the upper chamber . These data suggest that during maturation mammalian Sertoli cells secrete inhibin vectorially mainly from the apical surface of the cell towards the seminiferous tubular lumen . The predominance of inhibin secretion into the seminiferous tubule during testicular maturation suggests that inhibin may have an important paracrine or autocrine role in the developmental biology of spermiogenesis. FEMS Microbiol Lett, 1990 Nov, 60(3), 253 - 7 Genome analysis of Legionella ssp . by orthogonal field alternation gel electrophoresis (OFAGE); Bender L et al.; Various Legionella isolates from different sources and origins were analysed by orthogonal field alternation gel electrophoresis of NotI cleaved genomic DNA . The genome of L . pneumophila Philadelphia I, the original isolate of the epidemics in 1976, exhibits only five NotI fragments . Two virulent derivatives, derived from L . pneumophila Philadelphia I, which were obtained by prolonged passage on artificial culture media, did not differ from their isogenic virulent strain according the NotI fragment pattern . By summing the lengths of the NotI fragments, the genome size of L . pneumophila Philadelphia I was calculated as approximately 3.9 Mb . Environmental L . pneumophila strains exhibited different NotI patterns, as did Legionella strains not belonging to the species pneumophila . The usefulness of DNA long range mapping of Legionella ssp . with NotI for epidemiology and evaluation of their evolutionary relationships is discussed. Mol Reprod Dev, 1990 Nov, 27(3), 216 - 23 Development of preimplantation rabbit embryos in uterine flushing-supplemented culture media; Fischer B et al.; The development of cultured rabbit preimplantation embryos grown in standard media (Ham's F-10 or BSM II supplemented with bovine serum albumin (BSA) or homologous serum) or in Ham's medium supplemented with uterine flushings was compared . The uterine flushings derived from donors of 0.5-6 years of age . Uterine flushing supplemented media were used natively or after treatments like sterilization by filtration, lyophilization, three times freezing/thawing, heat denaturation, dialysis, or ultrafiltration . Compared with in vivo controls, embryonic growth was substantially reduced during in vitro culture, demonstrably by smaller diameters and impaired cell proliferation (measured by thymidine incorporation) . The growth retardation was more pronounced in blastocysts (recovered at day 4 post coitum {p.c.}) than in morulae (recovered at day 3 p.c.) . Development in uterine flushing media was notably better than in standard media but did not comply with in vivo development . Highest thymidine incorporation was observed in media with increased concentrations of uterine secretions and after sequential supplementation of flushings from subsequent progestational stages . Advanced donor ages, heating up to 80 degrees C, freezing, and lyophilizing did not affect incorporation data statistically significantly, whereas sterilization by filtration, ultrafiltration, and dialysis led to a significantly reduced thymidine incorporation in the cultured embryos . The positive effects of uterine flushing supplementation are attributed to the supply of components more adjusted to the needs of the cultured embryos and/or to a reduction of pathological effects in vitro like washing out of nutritive and regulatory components from the embryo into the surrounding culture medium. J Cell Physiol, 1990 Nov, 145(2), 222 - 37 Polarized membrane expression of brush-border hydrolases in primary cultures of kidney proximal tubular cells depends on cell differentiation and is induced by dexamethasone; Ronco P et al.; To analyze the influence of cell differentiation and the effects of hormones on the subcellular distribution of apical antigens in polarized epithelial cells, we have compared the localization of three brush border (BB) hydrolases {neutral endopeptidase (ENDO), aminopeptidase N (APN), and dipeptidylpeptidase IV (DPPIV)} in primary cultures of renal proximal tubule cells grown in various culture media . The degree of cell differentiation modulated by medium composition was estimated by measuring proximal functions, including glucose transport, specific enzymatic activities, and PTH responsiveness . In the dedifferentiated state observed in cells grown in 1% fetal calf serum (FCS)-supplemented medium, the three hydrolases are abnormally concentrated in a cytoplasmic vesicle compartment with weak expression on both membrane domains . By contrast, in serum-free hormonally defined medium (DM: insulin, 5 microgram/ml; dexamethasone, 5 x 10(-8) M), which markedly enhances morphological and functional cell differentiation, the distribution of hydrolases parallels that observed in the normal tubule . When added to the DM devoid of hormones, insulin has little polarizing effect, whereas dexamethasone dramatically increases the apical expression of the hydrolases, which then almost disappear from the basolateral membrane and cytoplasmic vesicular compartments . This glucocorticoid hormone augments the amount of immunoreactive antigen detectable on the apical domain in paraformaldehyde-fixed cells but does not change the total enzymatic activity . This suggests the presence in tubular cells of a dexamethasone-dependent polarizing machinery that requires de novo RNA and protein synthesis, and probably acts mainly by targeting a storage cytoplasmic pool of enzyme to the apical domain. Cell Regul, 1990 Nov, 1(12), 895 - 905 Plasminogen activation by t-PA on the surface of human melanoma cells in the presence of alpha 2-macroglobulin secretion; Bizik J et al.; Several human melanoma cell lines produced tissue-type plasminogen activator (t-PA), as detected by zymography and immunocapture assay of culture media and cell lysates . Urokinase (u-PA) was found at only less than or equal to 1% the level of t-PA . Acid eluates of the cell surface indicated that the melanoma cells had t-PA bound on their surface, but no u-PA, and also had a very low capacity to bind exogenous u-PA . After incubation of the melanoma cells with 10% plasminogen-depleted fetal calf serum and human plasminogen, bound plasmin activity could be eluted from the cell surface with tranexamic acid, an analogue of lysine . This indicated that plasminogen was activated on the cell surface . The cell-surface plasmin formation was inhibited by an anti-catalytic monoclonal antibody to human t-PA, and not by an anti-catalytic antibody to u-PA . The melanoma cells also synthesized and secreted alpha 2-macroglobulin (alpha 2M), as shown by alpha 2M-specific mRNA in Northern blotting and detection of alpha 2M protein in conditioned cell culture media . The media were found to inhibit u-PA but not t-PA . This inhibition was related to their alpha 2M content, and immunoabsorption of alpha 2M removed the inhibitory activity . These studies suggest that t-PA can bind to the surface of melanoma cells and generate surface-bound plasmin . Because t-PA and cell-bound plasmin are unaffected by alpha 2M, t-PA may, in the case of melanoma cells, serve an analogous function to u-PA in supporting tumor cell invasion. J Neuroimmunol, 1990 Nov, 30(1), 71 - 9 Lymphokine production by encephalitogenic and non-encephalitogenic T-cell clones reactive to the same antigenic determinant; Tokuchi F et al.; Among the myelin basic protein (MBP)-specific T-cell clones mediating experimental allergic encephalomyelitis (EAE), which were established from SJL/J mice, one clone was found to have lost its encephalitogenicity during long-term passages in vitro, although the clone keeps its specific reactivity to the encephalitogenic determinant lying in the sequence of guinea pig MBP 89-101 . To clarify the difference between the encephalitogenic T-cell clone (4b.14a) and non-encephalitogenic T-cell clone (4b.14a/n), we examined various lymphokines secreted into the culture media of 4b.14a and 4b.14a/n . The results show that the activities of lymphotoxin, interferon-gamma or interleukin-2 were not different between encephalitogenic clones and 4b.14a/n, whereas the activity of tumor necrosis factor-alpha, possibly secreted from antigen-presenting cells, was higher in culture media of 4b.14a/n . Moreover, the culture fluid of both 4b.14a/n and 4b.14a revealed suppressive effect on the proliferation of 4b.14a stimulated by MBP 89-101, but the effect was not different between the two clones . Thus, it is suggested that neither production of lymphokines examined so far nor soluble suppressive substance is related to the loss of encephalitogenicity of the T-cell clone. Biochem Biophys Res Commun, 1990 Oct 30, 172(2), 661 - 8 Insulin-like growth factor-I stimulates endothelin-3 secretion from rat anterior pituitary cells in primary culture; Matsumoto H et al.; Since we found relatively high concentrations of immunoreactive (ir-) ET-3 in the rat pituitary gland (190 pg/g tissue), we have investigated the possible ET-3 secretion from the primary culture of anterior pituitary cells and the effects of various growth factors on the ET-3 secretion . The ir-ET-3 was detected in the incubation medium within 2 h, and 24 h of culture attained the concentrations of 1.15 +/- 0.26 pg/well/6 x 10(5) cells . The ir-ET-3 secretion was stimulated by insulin, insulin like growth factor-II (IGF-II), and most effectively by insulin like growth factor-I (IGF-I) in a dose- and time-dependent manner, whereas the production of ir-ET-1 and ir-big ET-1 was slightly inhibited by IGF-I and IGF-II . In reverse-phase HPLC, the ir-ET-3 released into the culture media showed identical retention time with authentic ET-3 . Although ir-ET-1 and ir-big ET-1 secretion was stimulated by transforming growth factor-beta (TGF-beta), ir-ET-3 secretion was inhibited . These results indicate that the anterior pituitary cells secrete ET-3 and the secretion is stimulated by IGF-I. Mol Cell Endocrinol, 1990 Oct 22, 73(2-3), 217 - 24 Inhibition of N-glycan processing affects iodide organification in porcine thyroid cells; Long Y et al.; The N-glycan-processing inhibitors swainsonine (Sw) and deoxymannojirimycin (dMM) were used to study the influence of N-glycans on iodide organification in cultured porcine thyroid cells . Incubations with {125I}NaI were followed by determination of labeled trichloroacetic acid-insoluble material in culture media, follicular contents and cells . In controls, most of this material was in the follicular contents . With Sw and dMM, total acid-insoluble material was less than 10% of control . Iodide uptake was slightly inhibited and hydrogen peroxide release was not affected by inhibitors . Cell-surface thyroid peroxidase (TPO) activity, assayed by its ability to iodinate bovine serum albumin, was strongly inhibited . Pronase glycopeptide analysis indicated that with drugs the content in complex-type N-glycans was strongly decreased while that in hybrid or oligomannosidic type was increased . In conclusion, inhibition of N-glycan processing prevents iodide organification in cultured porcine thyroid cells by decreasing the recovery of cell-surface TPO activity. Thromb Res, 1990 Oct 15, 60(2), 141 - 56 Dipyridamole inhibits O2- release and expression of tissue factor activity by peripheral blood monocytes stimulated with lipopolysaccharide; Brozna JP et al.; Monocytes can be induced to synthesize and express tissue factor procoagulant activity . They can also be stimulated to release a broad spectrum of inflammatory agents including superoxide anion (O2-) that are thought to contribute to the pathogenesis of inflammatory diseases . Dipyridamole, an inhibitor of platelet aggregation blocks the lipopolysaccharide (LPS)-induced increase in monocyte-associated tissue factor activity and phorbol myristate acetate (PMA) stimulated O2- release from monocytes and polymorphonuclear leukocytes (PMN) . Dipyridamole inhibition of O2- release can be reversed by increased glucose in the culture media, whereas dipyridamole inhibition of tissue factor can not be reversed by increased glucose in the culture media . These results reveal that dipyridamole influences monocytes by at least two distinct mechanisms . Further, it may serve as an anti-thrombotic agent by virtue of its effect on both platelet aggregation and monocyte tissue factor activity. Toxicol Ind Health, 1990 Oct, 6(5), 133 - 45 Presence of viable mould propagules in the indoor air of houses; Verhoeff AP et al.; The aim of the first part of this study was to select the optimal technique for the enumeration and identification of viable mould propagules in the indoor air of houses . A comparison was made between the results obtained with six commercially available air sampling devices in combination with four culture media . The optimal technique was defined as the technique with the best precision and the highest yield . The coefficients of variation were high (generally greater than 20%) for all combinations . Statistical analysis showed that the Slit sampler and the N6-Andersen sampler in combination with DG18 and MEA gave the best precision and the highest yield in terms of CFU/m3 and number of species isolated . In the second part of this study the presence of viable mould propagules in the indoor air of 46 houses in relation to the dampness of these houses was investigated, using the N6-Andersen sampler in combination with DG18 . To assess the variability in time, the measurements were repeated after five weeks . Overall, between the two periods no difference was found between the average number of CFU/m3 in the investigated homes . However, the variation between homes was much smaller than the variation within homes . The mean number of CFU/m3 was somewhat higher in "damp" houses than in "dry" houses . However, this difference was not significant . Furthermore, there were no demonstrable differences in the presence of specific mould species in "damp" and "dry" houses. J In Vitro Fert Embryo Transf, 1990 Oct, 7(5), 254 - 6 The prevalence of human immunodeficiency virus in patients and their spouses entering a large in vitro fertilization program; Edelstein MC et al.; Between September 1987 and August 1989, all patients and their spouses entering our in vitro fertilization (IVF) program were screened for the human immunodeficiency virus (HIV) using the enzyme-linked immunosorbent assay (ELISA) . Of 848 patients and 848 spouses tested, all but 4 patients and 1 husband tested negative . Of those who tested positive on repeat testing with ELISA, only one was positive on Western blotting (HIV prevalence, 0.59 per 1000) . During this same time period 1187 samples of human cord blood were used to make tissue culture medium for the IVF embryology laboratory . One sample was discarded because of positive HIV on ELISA and Western blotting; two other samples were discarded because of positivity to the hepatitis B surface antigen . While we believe that routine HIV screening of IVF patients and their spouses is indicated, this population is of low risk for HIV positivity . Furthermore, continued screening of human sera used to make tissue culture media for IVF is mandatory. J Cell Biochem, 1990 Oct, 44(2), 69 - 79 Monoclonal antibody 425 inhibits growth stimulation of carcinoma cells by exogenous EGF and tumor-derived EGF/TGF-alpha; Rodeck U et al.; Carcinoma cells frequently coexpress transforming growth factor (TGF)-alpha and its receptor, the epidermal growth factor (EGF) receptor, implicating an autocrine function of carcinoma-derived TGF-alpha . Using a monoclonal antibody (425) to the EGF-receptor, we investigated the role of exogenous and tumor cell-derived EGF/TGF-alpha mitogenic activities in proliferation of cell lines derived from solid tumors . Monoclonal antibody 425 was chosen for these studies because it inhibits binding of EGF/TGF-alpha to the EGF-receptor and effectively blocks activation of the EGF-receptor by EGF/TGF-alpha . Seven malignant cell lines originating from carcinomas of colon, pancreas, breast, squamous epithelia, and bladder expressed surface EGF-receptor and secreted EGF/TGF-alpha-like mitogenic activities into their tissue culture media . All cell lines were maintained in a defined medium free of exogenous EGF/TGF-alpha . EGF and TGF-alpha added to the culture medium stimulated proliferation of five cell lines to comparable levels . EGF/TGF-alpha-dependent proliferation was significantly reduced by addition of MAb 425 to culture media . In addition, monoclonal antibody 425 reduced proliferation of the five EGF/TGF-alpha responsive cell lines in the absence of exogenous EGF/TGF-alpha . Antiproliferative effects induced by monoclonal antibody 425 were reversible and could be overcome by addition of EGF to culture media . Our results indicate that tumor-derived EGF-receptor-reactive mitogens can promote proliferation of carcinoma cells in an autocrine fashion. APMIS, 1990 Oct, 98(10), 901 - 8 Effect of polyoxyethylene sorbate compounds (Tweens) on colonial morphology, growth, and ultrastructure of Mycobacterium paratuberculosis; van Boxtel RM et al.; Polyoxyethylene sorbate (Tween) compounds were tested to compare their growth stimulation effects on Mycobacterium paratuberculosis . Three low passage and three high passage clinical isolates and ATCC strain 19698 were used . Tween 20, 40, 60, and 80 were tested at concentrations of 0, 0.001, 0.01, 1.0, and 3.0% (w/v) in radiometric broth culture media and in Middlebrook 7H9 agar plates . Scanning and transmission electron microscopy were used to examine cell wall appearance and ultrastructure, respectively . In broth culture, 0.1% (w/v) Tween 60 most dramatically enhanced growth of M . paratuberculosis ATCC strain 19698 . The effects of Tween 40 and 80 on growth took a bimodal form, enhancing growth at concentration ranges of 0-0.01% and 0.1-1.0% (w/v) but suppressing growth at concentrations of 0.01-0.1% (w/v) . Two of three high passage clinical isolates grew optimally in the presence of 1.0% (w/v) Tween 80, while the remaining high passage isolate and all three low passage isolates grew best in media containing 0.1% (w/v) Tween 80 . Colonial morphology of all strains grown on Middlebrook 7H9 agar without Tween 80 was irregular and granular whereas colonies on plate media containing greater than 0.01% (w/v) Tween 80 were entire, smooth, and domed . Scanning electron microscopy also revealed a transition from rough to smooth cell walls with increasing Tween 80 concentration . Transmission electron microscopy showed the presence of low electron dense intracellular vacuoles in Tween 80 grown M . paratuberculosis cells . Thus, Tweens altered colonial morphology, the cell wall surface, and ultrastructure of M . paratuberculosis and stimulated its growth in vitro in a concentration-dependent, and often bimodal, fashion.(ABSTRACT TRUNCATED AT 250 WORDS) Invest Ophthalmol Vis Sci, 1990 Oct, 31(10), 1945 - 9 Generation of complement-derived anaphylatoxins in normal human donor corneas; Mondino BJ et al.; Complement-derived anaphylatoxins (C3a, C4a, and C5a) are potent, stable mediators of acute inflammation . Because human corneas contain functional complement, the authors subjected normal human donor corneas to various forms of immunologic or chemical injury to determine if the complement system could be activated and anaphylatoxins generated . The experimental cornea of each donor pair was injected with lipopolysaccharide (LPS) or immune complexes or injured by application of acid or alkali . The remaining cornea of each donor pair served as a control . After incubation of corneas in tissue culture media for 6 hours and elution in phosphate-buffered saline for 24 hours, C3a, C4a, and C5a were measured in corneal eluates by radioimmunoassay . Compared with control corneas, C3a levels were significantly increased in corneas injected with LPS or immune complexes and in corneas injured with acid or alkali . C4a levels were significantly elevated in corneas injected with immune complexes and in corneas injured with acid or alkali but not in corneas injected with LPS . C5a levels were detectable only in corneas injured with acid or alkali . These results suggest that immunologic reactions in the human cornea may activate the classic or alternative complement pathways and generate anaphylatoxins . Additionally, chemical injuries with acid or alkali generate anaphylatoxins in the cornea . Anaphylatoxins may participate in the acute inflammatory response of the human cornea to chemical or immunologic injury. Endocrinology, 1990 Oct, 127(4), 1682 - 8 Demonstration of neuropeptide Y and its precursor in plasma and follicular fluid; Jorgensen JC et al.; The present investigation provides three lines of evidence for the presence of a pro-form of neuropeptide Y (NPY) in plasma and follicular fluid . First, by the demonstration of NPY-immunoreactive material of a size corresponding to the estimated mol wt of pro-NPY . Second, an antiserum specific for the C-terminal tyrosine amide of NPY and peptide YY does not react with this material . Third, it was possible to convert the pro-NPY extracted from plasma and follicular fluid using the protease, Endoproteinase-Lys C, to a NPY-immunoreactive form eluting slightly before NPY on a G-50 column . The size of the digested product was consistent with a cleavage of pro-NPY resulting in an immunoreactive species, NPY-Gly-Lys . Pro-NPY was also found in tissue culture media from the human neuroendocrine cell line SH-SY5Y . As in the case of plasma and follicular fluid, another NPY immunoreactive species eluted from a G-50 gel filtration column slightly before synthetic human NPY . Analysis of this material with an antibody directed against the tyrosine amide of NPY in combination with isoelectric focusing revealed that this peak consisted of at least two immunoreactive forms of NPY . In conclusion, at least three different forms of NPY immunoreactivity are likely to be present in plasma, follicular fluid, and cell tissue culture media; pro-NPY, a degradation form of pro-NPY, or a biosynthetic intermediate and NPY. Nippon Sanka Fujinka Gakkai Zasshi, 1990 Oct, 42(10), 1309 - 16 {Possible involvement of leukotriene B4 on human luteal function: special reference to determination of leukotriene B4 produced by cultured luteal cells}; Ichikawa F et al.; The present study was undertaken to determine the production of leukotriene B4 (LTB4) by cultured human luteal cells in the mid-luteal phase using a reverse phase column (C8) . The luteal cells were cultured with or without hCG at 100 ng/ml for 8 days . In the preliminary experiment, methods for extracting culture media samples were assessed prior to radioimmunoassay . Reverse phase column C8, but not C18, made possible the determination of LTB4 produced by human luteal cells . Progesterone (P) production by cultured luteal cells reached its maximum on day 4 following exposure to hCG, and then declined gradually . The concentrations of LTB4 produced by luteal cells varied from 100 to 500 pg/10(5) cells/2 days . However, exposure to hCG did not affect LTB4 production by cultured luteal cells . The level of LTB4 in culture medium (115.0 +/- 37.8 pg/10(5) cells/2 days) was reduced on day 4, but increased thereafter . LTB4 production appeared to decrease concomitantly with increased P production of cultured luteal cells . In conclusion, cultured luteal cells produced considerable amounts of LTB4 throughout the entire culture period . These results suggest that lipoxygenase activity of luteal cells may be closely related to steroidogenic potential. Diabetologia, 1990 Oct, 33(10), 597 - 602 Effects of hyperglycaemia on sorbitol and myo-inositol contents of cultured embryos: treatment with aldose reductase inhibitor and myo-inositol supplementation; Hashimoto M et al.; To demonstrate the myo-inositol depletion hypothesis in hyperglycaemia-induced embryopathy, rat conceptuses of 9.5 days of gestation in the early head-fold stage were grown in vitro during neural tube formation for 48 h with increasing amounts of glucose . The effects of an aldose reductase inhibitor and the myo-inositol supplementation were also investigated . Sorbitol and myo-inositol contents were measured in separated embryos and extra-embryonic membranes including yolk sac and amnion at the end of culture . After addition of 33.3 mmol/l and 66.7 mmol/l glucose to the culture media, the myo-inositol content of the embryos was significantly decreased by 43.1% (p less than 0.05) and 64.6% (p less than 0.01) of the control group, while a marked accumulation of sorbitol was observed (25 and 41 times that of the control) . Although the addition of an aldose reductase inhibitor (0.7 mmol/l) to the hyperglycaemic culture media containing an additional 66.7 mmol/l glucose significantly reduced the sorbitol content of embryos to approximately one-eighth, the myo-inositol content of embryos remained decreased and the frequency of neural lesions was unchanged (23.1% vs 23.9%, NS) . Supplementation of the myo-inositol (0.28 mmol/l) completely restored the myo-inositol content of the embryos and resulted in a significant decrease in the frequency of neural lesions (7.1% vs 23.9%, p less than 0.01) and a significant increase in crown-rump length and somite numbers . Much less significantly, sorbitol accumulation was also observed in the extra-embryonic membrane in response to hyperglycaemia, neither hyperglycaemia nor the myo-inositol supplementation modified the myo-inositol contents of the extra-embryonic membrane.(ABSTRACT TRUNCATED AT 250 WORDS) Arch Mal Coeur Vaiss, 1990 Oct, 83(11), 1661 - 7 {New valvular homografts . Prospects and limits of their viability . Report of 42 implantations}; Bloch G et al.; The regain of interest in aortic homograft bioprostheses is related to the prospects of improved viability resulting from explanation from organ donors, preservation in rich tissue culture media, together with the progress made in techniques of cryopreservation . Viability studies examining morphology of electron microscopy and tests of tissue culture confirm this notion of longer viability . These properties raise hopes of satisfactory long-term results while acknowledging outstanding antigenic problems which require strict A-B-O system compatibility . The results of a preliminary series of 42 valve homografts implanted at Henri Mondor Hospital over the last 5 years are reported . Twenty-one bioprostheses were implanted on the right side in congenital heart disease with good results in every case . Twenty-one were implanted in the aortic position in children and show no signs of degeneration as yet . One poor result was related to a technical error in calibration . The rebirth of this technique raises certain hopes, especially in aortic valve replacement. Hepatogastroenterology, 1990 Oct, 37(5), 457 - 60 Integration of hepatitis B virus DNA into cells of six established human hepatocellular carcinoma cell lines; Fujise K et al.; The authors successfully established 5 hepatocellular carcinoma cell lines, JHH-1, 2, 4, 5 and 6, derived from hepatitis B surface antigen seronegative patients and one line . JHH-7, from a patient considered to be a hepatitis B virus carrier . In the culture media of any of the JHH cell lines, including JHH-7, hepatitis B surface antigen was not detected by radioimmunoassay . However, in JHH-7, integration of hepatitis B virus DNA was confirmed at two sites on the chromosomes of this line by Southern blot hybridization . In contrast, in other JHH cell lines derived from hepatitis B surface antigen seronegative patients, integration of hepatitis B virus DNA into the chromosomes of cells was not detected . These cell lines will be useful in the investigation of hepatocellular carcinoma development, especially for research into non-A/non-B hepatitis viruses. Toxicol Ind Health, 1990 Oct, 6(5), 133 - 45 Presence of viable mould propagules in the indoor air of houses; Verhoeff AP et al.; The aim of the first part of this study was to select the optimal technique for the enumeration and identification of viable mould propagules in the indoor air of houses . A comparison was made between the results obtained with six commercially available air sampling devices in combination with four culture media . The optimal technique was defined as the technique with the best precision and the highest yield . The coefficients of variation were high (generally > 20%) for all combinations . Statistical analysis showed that the Slit sampler and the N6-Andersen sampler in combination with DG18 and MEA gave the best precision and the highest yield in terms of CFU/m3 and number of species isolated . In the second part of this study the presence of viable mould propagules in the indoor air of 46 houses in relation to the dampness of these houses was investigated, using the N6-Andersen sampler in combination with DG18 . To assess the variability in time, the measurements were repeated after five weeks . Overall, between the two periods no difference was found between the average number of CFU/m3 in the investigated homes . However, the variation between homes was much smaller than the variation within homes . The mean number of CFU/m3 was somewhat higher in "damp" houses than in "dry" houses . However, this difference was not significant . Furthermore, there were no demonstrable differences in the presence of specific mould species in "damp" and "dry" houses. J Biol Chem, 1990 Sep 25, 265(27), 16096 - 101 Immunoaffinity purification and characterization of lipoprotein-associated coagulation inhibitors from Hep G2 hepatoma, Chang liver, and SK hepatoma cells . A comparative study; Wun TC et al.; A polyclonal antibody against a synthetic peptide corresponding to amino acids 3-25 of mature lipoprotein-associated coagulation inhibitor (LACI) was raised in rabbits . The antibody was used to study the production of LACI by Hep G2 hepatoma, Chang liver, and SK hepatoma cells, and to purify LACI from the culture media . By using an amidolytic assay for factor Xa, it was found that the culture media from these liver-derived cell lines contain inhibitors of factor Xa . In Hep G2 hepatoma culture medium, approximately 50% of Xa inhibitory activity was due to LACI . In the Chang liver and SK hepatoma culture media over 95% of the Xa inhibitory activity was due to LACI . The LACIs were purified from these media by immunoaffinity chromatography on an anti-LACI-lg-Sepharose 4B column and preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis . The purified LA-CIs varied in molecular weight depending on whether the media were concentrated before chromatography . An Mr approximately 38,000 LACI was obtained by chromatography of unconcentrated media . Chromatography of concentrated media yielded a LACI of Mr approximately 35,000 with the same amino-terminal sequence, suggesting partial proteolysis in the carboxyl-terminal region . In addition, an Mr approximately 25,000 form of LACI was also present . The purified Mr approximately 38,000 and approximately 35,000 LACI species from the above cells possess similar specific activities when measured by an anti-Xa/amidolysis assay . To study the role of LACI in the control of coagulation, pooled human plasma was depleted of LACI antigen by immunoaffinity absorption and reconstituted with varying amounts of purified LACI to examine the effect on tissue factor (TF)-induced coagulation . LACI depletion shortens the time of TF-induced clotting of plasma and the clotting time is linearly related to the LACI concentration after reconstitution . These results suggest that LACI plays an important role in limiting TF-induced coagulation in human plasma . Comparison of the potencies of various purified LACIs in the prolongation of TF-induced coagulation revealed that LA-CIs from different sources are not equivalent . The plasma LACI, SK hepatoma LACI, and Chang liver LACI are approximately 7-, 6-7, and 1.3-fold higher in specific activity than Hep G2 hepatoma LACI in the TF-induced clotting assay when compared on an anti-Xa/amidolysis unit basis, suggesting possible differences in post-translational modification of these LA-CIs. Mol Pharmacol, 1990 Sep, 38(3), 401 - 9 Hemoregulatory peptide pGlu-Glu-Asp-Cys-Lys: a new synthetic derivative for avoiding dimerization and loss of inhibitory activity; Paukovits WR et al.; The hemoregulatory peptide (pGlu-Glu-Asp-Cys-Lys, pEEDCK) is a potent inhibitor of stem cell recruitment, which is a major source of hematological complications after cytostatic tumor therapy . By preventing recruitment, pEEDCK can keep hemopoietic stem cells in their normal nonproliferative state and in this way prevent damage by certain cell cycle-specific cytostatic drugs . pEEDCK could play a role as hemoprotector in tumor chemotherapy . As a thiol-containing peptide, pEEDCK is highly sensitive to oxidation, resulting in the formation of a dimer . Although monomeric pEEDCK is a strong inhibitor of colony-forming units-granulocyte/macrophage (CFU-GM) clonal growth, the dimer was previously found to enhance colony-stimulating factor-triggered CFU-GM colony formation . It seemed, thus, necessary to find methods that avoid undesired dimerization reactions . A solid phase strategy for pEEDCK synthesis is presented . The primary synthetic product, S-tert-butyl-sulfenyl-pEEDCK, was purified and stored with the thiol-protecting group remaining attached . Conversion to active monomeric pEEDCK was achieved by reductive treatment in situ before application and removal of tert-butyl-mercaptane in vacuo . The activation reagent (dithioerythritol) prevented reoxidation also in culture media, where unprotected peptide was oxidized rapidly (t1/2 less than 13 min) . The purified synthetic peptide was found to be a potent inhibitor of CFU-GM colony formation (IC50 = 1.1 x 10(-12) M) in vitro . It was also found to inhibit colony formation of some leukemic cell lines (HL-60, RAJI) although at much higher concentrations (10(-8) to 10(-9) M) . Friend leukemia cells were not inhibited in the dose range where CFU-GM were sensitive. J Surg Res, 1990 Sep, 49(3), 248 - 51 Competitive inhibition of LDL binding and uptake by HDL in aortic endothelial cells; Alexander JJ et al.; High-density lipoprotein (HDL) may inhibit the binding and cellular uptake of low-density lipoprotein (LDL) as one means of regulating the delivery of exogenous cholesterol to nonhepatic tissues . This may play an important role in atherogenesis, by altering lipid metabolism in cells of the arterial wall . To verify and better characterize this effect, endothelial cells were harvested from bovine aorta and maintained in tissue culture . Following initial preincubation in lipid-deficient culture media, these cells were incubated for 2 hr at 4 degrees C in media containing 125I-LDL (10 micrograms protein/ml) and varying concentrations of either HDL (0-400 micrograms protein/ml) or comparable amounts of Apoprotein A (Apo A), the major protein component of HDL . Intracellular and trypsin-released counts were assayed separately, as a measurement of cellular uptake and membrane bound LDL, respectively . Results of this study indicated an inhibition of LDL binding and uptake by HDL (P less than 0.005, ANOVA) . A similar inhibition was found with Apo A alone (P less than 0.005) . When identical studies were performed using 125I-Apoprotein B, the protein component of LDL, and Apo A, the latter was found to inhibit the binding of Apo B to the same extent (P less than 0.0006) . These results indicate that HDL does inhibit LDL binding and uptake by bovine aortic endothelial cells and that, because this effect is seen equally with only the protein component of these lipoprotein particles, it is most likely due to competitive binding at the receptor level rather than to stearic hindrance or an alteration of the cell membrane. J Clin Invest, 1990 Sep, 86(3), 845 - 50 Effects of interleukin 2 on cardiac function in the isolated rat heart; Sobotka PA et al.; Adoptive immunotherapy with IL 2 is associated with severe cardiovascular toxicities including peripheral and pulmonary edema, hypotension decreased systemic vascular resistance, increased heart rate, and an increased cardiac index . The purpose of this investigation was to determine whether IL 2 alone or in combination with lymphokine-activated killer cells (LAK) cells depress cardiac function using the isolated, perfused, working rat heart preparation . Male Sprague-Dawley rats (250-350 g) were anesthetized and the hearts were removed and placed on the perfusion apparatus . Hearts were perfused with oxygenated Krebs-Henseleit buffer (KHB), or oxygenated KHB containing IL 2 alone, IL 2-Media (cell culture media supplemented with 1,500 U IL 2/ml), LYMPH (cell culture media from cultured mononuclear cells from healthy volunteers), or LAK (cell culture media from cultured lymphocytes harvested from patients receiving IL 2/LAK in the presence of 1,500 U/ml IL 2) . The cells were removed before perfusion (n = 9) . Cardiac output and coronary flow were measured at 20-min intervals with preload constant (afterload varied or afterload constant (preload varied) . The results indicate a significant depression in cardiac function in hearts treated with LAK . This depression was evident at 20 min and was more pronounced at 60 min . Washout of the KHB plus LAK reversed this depression . Thus, IL 2-stimulated/cultured human mononuclear cells produce a soluble factor that produces a reversible severe depression of cardiac function. J Cell Physiol, 1990 Sep, 144(3), 505 - 10 Synergism between gangliosides and basic fibroblastic growth factor in favouring survival, growth, and motility of capillary endothelium; De Cristan G et al.; The experiments reported were motivated by the observation that in vivo gangliosides promoted angiogenesis when the dose of the angiogenic factor was too low to be effective (Ziche et al.: Laboratory Investigation 61:629-634, 1989) . As an approach to understanding the mechanism of this modulatory effect, we analysed the influence that gangliosides have on survival, growth, and migration of capillary endothelium when an angiogenesis factor like basic fibroblast growth factor (bFGF) was present in the culture medium . Clones of bovine capillary endothelium were cultivated in media unable to sustain survival over a 72 h period . With this experimental approach, cell survival was evaluated after addition of either bFGF or gangliosides or both to the medium . The Boyden chamber procedure was utilized to measure the influence of bFGF or gangliosides on cell mobilization across a micropore filter . Low doses of both molecules, ineffective when added singly to the culture media, improved all three parameters when added in combination . A synergic effect between bFGF and the gangliosides (GM1, GD1b, GT1b) was observed for the improvement of survival or growth and for the acceleration of endothelial cell migration . The removal of sialic acid from the ganglioside molecule prevented any effect on all three parameters . The addition of sialic acid alone to cultures was also totally ineffective . In the adult organism most angiogenic events occur under conditions of tissue damage . The synergism between gangliosides and bFGF can be interpreted as the initial phase of a process for which endothelial cell survival is the indispensable first step in the formation of a new vascular network. Exp Clin Endocrinol, 1990 Sep, 96(1), 64 - 72 Impaired production of interleukin-2(IL2) in patients with Graves' disease by newly developed IL2 radioimmunoassay; Hirooka Y et al.; We have developed a sensitive, reproducible and specific radioimmunoassay for human interleukin-2 . Using 125I-labeled interleukin-2 and polyclonal rabbit antisera raised against recombinant human interleukin-2, a competitive inhibition assay was described which could detect 1 U/ml of human interleukin-2 . Substances such as interleukin-1 alpha, interferon beta, nerve growth factor, tissue necrotizing factor, various hormones, peptides and lectins did not affect the assay . Interleukin-2 was measured in supernatants of culture media of stimulated human blood mononuclear cells . Kinetics of interleukin-2 production in seven normal lymphocytes revealed that in both PHA- and Con A-stimulations, the peak levels of interleukin-2 were seen at the end of 72 hours (113.9 +/- 54.4 U/ml, 111.6 +/- 37.3 U/ml, respectively) and then declined . Interleukin-2 levels in PHA- and Con A-stimulations of untreated Graves' disease were significantly lower (14.5 +/- 15.5 U/ml, 12.3 +/- 12.7 U/ml, respectively) than normal controls . However, the improvement of decreased interleukin-2 production in methimazole-treated patients with Graves' disease was observed (38.2 +/- 28.1 U/ml, 48.0 +/- 35.6 U/ml, respectively) . The present study demonstrates the usefulness of quantitating human interleukin-2 produced by human blood mononuclear cells and that there exists an impaired production of interleukin-2 in Graves' disease. Biol Reprod, 1990 Sep, 43(3), 457 - 65 Identification and characterization of bovine oviductal glycoproteins synthesized at estrus; Boice ML et al.; Published reports indicate that in several mammalian species the oviduct synthesizes and secretes specific glycoproteins which are components of the luminal fluids at the time of ovulation and fertilization . The present study characterized the secretory glycoproteins synthesized by the bovine oviduct at estrus . Oviducts obtained from four crossbred cows in estrus were flushed with saline, and segments of the ampullary and isthmic regions were fixed for immunocytochemical analyses . The remainder of the tissue was subjected to explant culture for 24 h in medium containing either 3H-leucine or 3H-glucosamine . Analysis of culture media by one- and two-dimensional SDS-PAGE followed by fluorography indicated that both the ampullary and isthmic regions synthesized a major class of Mr 97,000 glycoproteins with isoelectric points ranging from 5.5 to 8.1 . A polyclonal antibody was generated to the glycoproteins after their isolation by gel filtration followed by electrophoretic separation . Western blot analysis of oviductal culture media indicated that the antisera cross-reacted with a doublet at Mr 97,000 and to a lesser extent with two additional bands at Mr greater than 200,000 . Immunoreactive antigens were not identified in serum or in culture media of ovary, uterus, and nonreproductive tract tissues . The Mr 97,000 glycoproteins were present in oviductal flushings obtained from cows in estrus . They were also detected to a lesser degree in oviductal flushings obtained from cows at Days 5, 10, 15, and 18 of the estrous cycle, with the least amount of immunoreactivity being observed in Day 10 samples.(ABSTRACT TRUNCATED AT 250 WORDS) Rev Infect Dis, 1990 Sep-Oct, 12(5), 824 - 8 Pneumonia due to Legionella micdadei in bone marrow transplant recipients; Schwebke JR et al.; Legionella micdadei has previously been described as a cause of nosocomial pneumonia, particularly in kidney transplant recipients . Cell-mediated immunity is the principal host defense against this pathogen . A common clinical scenario in the immunocompromised host is that of septic pulmonary embolus, but asymptomatic infections have also been reported . The organism is weakly acid-fast in clinical specimens but loses this property when grown on solid media . We report two cases of L . micdadei pneumonia, differing markedly in clinical severity and outcome, in bone marrow transplant recipients . Additionally, we note the growth of the organism in liquid culture media with preservation of its acid-fast property. Mol Endocrinol, 1990 Sep, 4(9), 1408 - 15 Association of proenkephalin transcripts with polyribosomes in the heart; Low KG et al.; Previous results have shown that the relative abundance of proenkephalin mRNA in the rat heart is comparable to the levels found in the brain; however, the extractable enkephalin-containing peptide levels are much lower in the heart . This lack of correspondence between the levels of transcript and peptide could arise from either the inefficient translation of proenkephalin transcripts or the translation of proenkephalin transcripts into peptides that are rapidly secreted or degraded . To distinguish between these possibilities, the translational status of proenkephalin mRNA in the rat heart was established by Northern blot analysis of sucrose density gradient-sedimented polysomal fractions and compared to the striatum, which is known to efficiently translate proenkephalin transcripts . In both tissues, we detected 1.5-kilobase transcripts, but an additional larger transcript of approximately 3.6 kilobases was detected in the heart . Both transcripts were associated primarily with polyribosomes, suggesting active translation of proenkephalin mRNA in the rat heart . RIA of the culture media and extracts from primary cultures of neonatal rat cardiomyocytes indicated the presence of immunoreactive Met-enkephalin-Arg6-Phe7, which was stimulated by 8-(4-chlorophenylthio)cAMP . These results suggest that proenkephalin transcripts are translated in the heart and that detectable levels of immunoreactive Met-enkephalin-Arg6-Phe7 are present in the media and cell extracts of primary cultures of neonatal rat cardiomyocytes. J Electron Microsc Tech, 1990 Sep, 16(1), 25 - 36 Lectin binding patterns to plasmalemmal glycoconjugates of goblet cells undergoing differentiation in vitro; Frisch EB et al.; The plasmalemmal glycoconjugates of the HT29-18N2 (N2) cell line were characterized on cells grown as 1) undifferentiated multilayers in glucose-containing culture media and 2) monolayers of columnar cells acquiring the goblet cell phenotype in glucose-free media . Lectins were unable to bind sheets of detached N2 cells in the absence of fixation . Following fixation with aldehydes, a dramatic unmasking of lectin binding sites was seen . When fixed monolayers were stained prior to embedding, biotinylated lectins, visualized by the avidin-biotin-complexed peroxidase technique, were more efficient than collodial gold-coupled lectins . Lectin binding sites could also be detected by using collodial gold-coupled lectins to stain monolayers embedded in LR White, Lowicryl K4M, and Lowicryl HM20 . The binding of 5 lectins (wheat germ, Dolichos bifluros, peanut, soybean, and Ulex europeus) was found to be independent of the stage of differentiation; "pre-differentiated" columnar cells which had prominent microvilli and no or few mucous secretory granules had identical staining patterns as well-differentiated goblet cells with large numbers of secretory granules . Ricinus communis I was the only lectin whose binding was influenced by the stage of differentiation; it intensely labeled undifferentiated multilayers of N2 cells but only weakly labeled basolateral membranes of differentiated monolayers . Canavalia ensiformas (ConA) caused a moderate and even labeling of both apical and basolateral membranes of fixed monolayers stained prior to embedding, but post-embedding labeling revealed heavy labeling along the lateral margins of all columnar cells and weak to moderate binding along the apical and basal cell surface. Ann Soc Belg Med Trop, 1990 Sep, 70(3), 237 - 41 {Comparison of 2 culture media for the isolation of Mycobacterium tuberculosis}; Pauwels P et al.; In Kinshasa, Zaire, 392 positive cultures were used to compare the isolation of mycobacteria on two different culture media (Lowenstein-Jensen and Ogawa (1%) with egg yolk) . Although the Ogawa medium is reknown for being well adapted to the growth of atypical mycobacteria, we did not isolate more atypical mycobacteria from the tuberculous population of Kinshasa on Ogawa than were isolated in the previous years on Lowenstein-Jensen . However, significantly more M . tuberculosis were isolated on Ogawa (95% versus 88.4% on Lowenstein-Jensen) . Other factors permitting, the use of the Ogawa medium is recommended. In Vitro Cell Dev Biol, 1990 Sep, 26(9), 857 - 64 Expression of alpha- and beta-human chorionic gonadotropin subunits in cultured human cells; Goldstein S et al.; We surveyed several human cell lines for production of alpha- and beta-human chorionic gonadotropin (hCG) under a variety of conditions known to induce gene expression . alpha- and beta-hCG subunits were monitored in culture media by specific radioimmunoassays and were shown to be quite sensitive to serum refeeding and growth state of all cell types studied . The permanent line JEG-3 secreted both alpha- and beta-subunits whereas HeLa cells secreted only the alpha-subunit . Production of both subunits was augmented in these permanent cell lines, for each growth state, by pretreating cells with 5-azacytidine; in contrast, spontaneous beta-hCG production by normal human fibroblasts (four of six strains) was only rarely increased after 5-azacytidine treatment, and more often was suppressed by 30 to 40% . Three of five strains from inherited chromosomal breakage syndromes produced immunoassayable beta-hCG spontaneously, two of which increased secretion upon treatment with either UV or mitomycin C . Surprisingly, one normal cell strain of fetal origin was induced to secrete alpha-hCG, but not beta-hCG, after UV irradiation . JEG-3 and HeLa cells produced detectable cognate mRNA for alpha- or beta-hCG subunits or both by Northern and S1 nuclease protection analyses, whereas such transcripts from untransformed human fibroblasts were consistently below detectable levels . Quantitation of beta-hCG mRNA by RNA:RNA annealing kinetics indicates that even the fibroblast strain producing the highest secreted beta-hCG levels contained cognate mRNAs at only approximately 0.1 per cell . We conclude that hCG expression in human fibroblasts is strongly repressed at the transcriptional level, although a variety of conditions (growth state, serum refeeding, cell senescence, or DNA damage) can affect the level of "leaky" expression, at least in some responding fraction of cells. Lab Invest, 1990 Sep, 63(3), 298 - 306 Sequential loss of suppressor genes for three specific functions during in vivo carcinogenesis; Moroco JR et al.; As normal cells undergo neoplastic transformation, multiple suppressor gene functions are lost or inactivated . However, the relative contribution that individual suppressor gene defects play in the sequential evolution of solid tumors has not yet been evaluated in a readily analyzable experimental model of carcinogenesis . The present study was undertaken to: (a) document the loss of suppressor genes implicated in the control of angiogenic activity, anchorage and serum growth requirements, and proliferative life span, in populations of hamster buccal pouch (HBP) keratinocytes (Kr) initiated in vivo with the chemical carcinogen 7,12 dimethylbenz(a)anthracene and (b), to determine what combination of defective suppressor genes are necessary for tumorigenesis . Kr were isolated from HBPs at various times after treating the mucosal surfaces in vivo with 7,12 dimethylbenz(a)anthracene . Cells or their conditioned culture media were evaluated for: (a) angiogenic activity in vivo and in vitro, (b) anchorage independent growth, (c) growth in low serum, (d) immortality, and (e) tumorigenicity . Angiogenic activity and immortality were the first two phenotypes detected with anchorage independence and tumorigenesis emerging late in the carcinogenic process . Hybrids generated between Kr which were angiogenic, but otherwise negative for all other phenotypic traits, and a transformed and tumorigenic HBP carcinoma cell line, E1-1, were suppressed for all phenotypes except angiogenic activity and none of the hybrids were tumorigenic . In contrast, Kr positive for angiogenic activity, anchorage independence, immortality, and tumorigenicity and hybrids generated between these cells and E1-1 carcinoma, were tumorigenic . However, hybrids between a nontumorigenic, anchorage independent, immortal but nonangiogenic Kr, and the E1-1 line were not tumorigenic . These results suggest that (a) angiogenic activity is an early phenotypic trait that emerges as a result of the loss of a suppressor gene function and (b) loss of this function is essential but not sufficient for the development of HBP tumors. J Biol Chem, 1990 Aug 15, 265(23), 13635 - 40 Post-transcriptional regulation of cAMP-dependent protein kinase activity by cAMP in GH3 pituitary tumor cells . Evidence for increased degradation of catalytic subunit in the presence of cAMP; Richardson JM et al.; The effects of cyclic AMP treatment on total cAMP-dependent protein kinase activity in GH3 pituitary tumor cells have been studied . Incubation of cells for 24 h with 1 microM forskolin resulted in a 50% decrease in total cAMP-dependent protein kinase activity which was reversible upon removal of forskolin from culture media . A similar response was observed in GH3 cells treated with 5 ng/ml cholera toxin and 0.5 mM dibutyryl cAMP but not 0.5 mM dibutyryl cGMP . Northern blot analysis demonstrated that the steady-state level of the mRNA for each of the six kinase subunit isoforms studied was not detectably altered after treatment with 1 microM forskolin for 24 h . The concentration of catalytic subunit was also assessed by binding studies using a radiolabeled heat-stable protein kinase inhibitor . Treatment of GH3 cells with 1 microM forskolin for 24 h reduced protein kinase inhibitor binding activity by 50%, consistent with the observed forskolin-induced decrease in total kinase activity . Analysis of endogenous heat-stable protein kinase inhibitor activity in GH3 cell extracts showed no significant difference between forskolin-treated cells and cells maintained under control conditions . To assess possible effects on catalytic subunit degradation, pulse-chase experiments were performed and radiolabeled catalytic subunit was isolated by affinity chromatography . The results demonstrated that treatment of cells with chlorophenylthio-cAMP detectably increased the apparent degradation of radiolabeled catalytic subunit . The increased degradation of the catalytic subunit was sufficient to account for the observed decreases in kinase activity . These results suggest that relatively long term cAMP treatment can alter total cAMP-dependent protein kinase activity through effects to alter the degradation of the catalytic subunit of the enzyme. J Steroid Biochem, 1990 Aug 14, 36(5), 473 - 8 Ethanol-induced inhibition of testosterone biosynthesis in rat Leydig cells: role of L-glutamate and pyruvate; Orpana AK et al.; The mechanisms by which ethanol (EtOH) inhibits testicular testosterone biosynthesis were studied with isolated rat Leydig cells in vitro comparing the effects of EtOH in six different culture media . The actual sites of inhibition by EtOH, identified by measuring the steroidogenic precursors, varied depending on the medium used . In Krebs-Ringer bicarbonate buffer, EtOH inhibited both the conversion of pregnenolone to progesterone and androstenedione to testosterone . In the pyruvate (Pyr) supplemented Dulbecco's Modified Eagle medium, the decreased progesterone concentrations in the presence of EtOH were reflected to all successive steroids 17-OH-progesterone, androstenedione and testosterone . The presence of L-glutamate (Glu) in the medium elevated testosterone production, but EtOH still inhibited the conversion of pregnenolone to progesterone, and also the androstenedione/testosterone ratio was elevated because of the decreased testosterone concentrations . In the presence of both Glu and Pyr in the medium the EtOH-induced decreases in the steroid concentrations were fully recovered in isolated Leydig cells . These results demonstrate that both Pyr and Glu supplementations are essential for the maintenance of maximal rate of testosterone synthesis in vitro in the presence of EtOH. Int J Androl, 1990 Aug, 13(4), 287 - 96 Changes in human sperm chromatin stability during preparation for in-vitro fertilization; Rosenborg L et al.; This study was designed to define the effects of sperm preparation on sperm chromatin stability in relation to in-vitro fertilization (IVF) . Semen samples used for IVF-embryo transfer (ET) in the treatment of infertility due to tubal factors were studied . Cases with semen variables below reference limits in previous samples were excluded . Sperm were prepared by a swim-up technique employing either of two different tissue culture media, Ham's F-10 or Earle's balanced salt solution . Sperm chromatin stability was tested by exposure both to sodium dodecyl sulphate (SDS) only and SDS together with a zinc-chelating agent, disodium ethylene diamine tetraacetate (SDS-EDTA) . Sperm head swell scores were defined under different experimental conditions and the relationship to sperm motility, morphology, fertilization rate and pregnancy occurrence was tested . No differences were seen between the chromatin stability of sperm from the original sample and that after swim-up preparation, neither immediately after completion of the swim-up procedure, nor at the time of insemination of ova . With time, the chromatin became more stable, which occurred to a similar extent both in the original sample and in swim-up preparations using Ham's F-10 . Otherwise, sperm chromatin stability was unaffected by either of the two media used for swim-up . At higher incubation temperatures, decondensation in SDS was enhanced . Altogether, no correlation was found between sperm chromatin stability or enhancement of decondensation by temperature and the success of IVF treatment expressed in fertilization rates or pregnancies . The results are reassuring in that only small changes in sperm chromatin stability occurred during the preparation for IVF . As long as semen of presumably good quality is used, these changes in chromatin stability do not seem to be of clinical importance. Fertil Steril, 1990 Aug, 54(2), 328 - 32 High potassium concentration and the cumulus corona oocyte complex stimulate the fertilizing capacity of human spermatozoa; Roblero LS et al.; Progressively motile spermatozoa were incubated for 24 hours in culture media containing 4.7 or 25 mM K, in the presence or absence of hamster cumulus oophorus . The percentage of spermatozoa with progressive motility was significantly higher at 24 hours in the presence of cumulus corona oocyte complexes, irrespective of K concentration . A significant decrease in sperm mortality was observed with the association of 25 mM K and cumulus cells . A higher percentage of acrosome reaction was observed in spermatozoa incubated in 25 mM K when compared with 4.7 mM K, irrespective of time and the presence or absence of cumulus . The percentage of penetrated oocytes at 2 and 5 hours of incubation was higher when sperm had been incubated in 25 mM K than in 4.7 mM K . The presence of cumulus in the culture medium induced an additional significant increase in the percentage of penetrated oocyte . Although at 24 hours of incubation the percentage of acrosome reaction was higher than at 2 and 5 hours, the percentage of penetrated oocytes did not increase proportionally. Cancer Res, 1990 Aug 1, 50(15), 4685 - 91 Comparative metabolism of N'-nitrosonornicotine and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone by cultured F344 rat oral tissue and esophagus; Murphy SE et al.; The metabolism and DNA binding of N'-nitrosonornicotine (NNN) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) by cultured F344 rat oral tissue and esophagus were investigated over a range of concentrations . The metabolites present in the culture media were separated by high performance liquid chromatography and were identified by comparison to standards . alpha-Hydroxylation of NNN, an esophageal carcinogen, was the major pathway for metabolism of this nitrosamine in both tissues . The metabolites formed from 2'-hydroxylation were between 3.0 and 3.9 times those formed from 5'-hydroxylation . 2'-Hydroxylation results in a pyridyloxobutylating species . DNA from esophagus cultured with {5-3H}NNN contained a pyridyloxobutylated adduct which upon acid hydrolysis released 3.8 pmol {5-3H}-4-hydroxy-1-(3-pyridyl)-1-butanone/mumol guanine . DNA from oral tissue cultured under the same conditions, where the extent of metabolism was the same, contained no measurable {5-3H}NNN DNA adduct . This suggests that factors, as yet unknown, cause the DNA of oral cavity tissue to be protected from pyridyloxobutylation by NNN . The metabolism of NNK by alpha-hydroxylation was as much as 10-fold less than the metabolism of NNN by this pathway in both tissues . alpha-Hydroxylation of NNK results in either a methylating species or a pyridyloxobutylating species . DNA from oral tissue cultured with {C3H3}NNK contained between 1.7 and 4.3 pmol 7-methylguanine/mumol guanine, respectively . No pyridyloxobutylated DNA (less than 0.2 pmol/mumol guanine) was detected in oral tissue incubated with {5-3H}NNK . The DNA from esophagi incubated with {C3H3}NNK contained no 7-methylguanine (less than 0.4 pmol/mumol guanine) . The level of pyridyloxobutylation of DNA from esophagi incubated with {5-3H}NNK was 0.17 pmol/mumol guanine . The ability of the esophagus to metabolize NNN to a greater extent than NNK to a reactive species which pyridyloxobutylates DNA may be important in determining the carcinogenicity of NNN in the esophagus . In contrast, the metabolism of NNK to a methylating species by oral cavity tissue suggests that this tobacco-specific nitrosamine is important in tobacco-related oral cavity carcinogenesis. Oncogene, 1990 Aug, 5(8), 1173 - 8 Characterization of monoclonal antibodies specific to the activated ras p21 with aspartic acid at position 13; La Vecchio JA et al.; The ras proto-oncogenes encode membrane bound proteins (p21) which are structurally distinct from the proteins encoded by the activated transforming ras genes . These activated ras genes have been identified in various human tumors as well as their preneoplastic lesions such as colorectal tumors (20-40%), pancreatic carcinomas (95%), lung carcinomas (20-30%), myelodysplasia (40%) and acute myeloid leukemia (30%) . The activation of ras p21 is due to amino acid substitutions at positions 12, 13 or 61 of the p21 protein . This report describes two monoclonal antibodies designated D129 and D146 raised against a synthetic peptide corresponding to amino acids 5-16 of ras p21 activated by the substitution of aspartic acid for glycine at position 13 . D129 and D146 react specifically with the peptide with the aspartic acid substitution at position 13, but not with the peptide with valine at position 13 or the peptide containing the normal glycine at position 13 . Western blot analysis demonstrates that D129 and D146 react specifically with p21 extracted from transformed NIH3T3 fibroblast lines containing aspartic acid at position 13 . These studies also demonstrate that D146 is able to detect the activated p21 with aspartic acid at position 13 that is shed into the culture media . Studies demonstrate that MAb D146 specifically immunoprecipitates the cellular p21 with aspartic acid at position 13 from transformed NIH3T3 cells, whereas D129 cannot immunoprecipitate the activated p21 . Using a sandwich ELISA format, D146 is able to detect the p21 with position 13 aspartic acid from cell extracts and culture fluids . The ability of D146 to function in the ELISA format raises the possibility that this assay maybe a quick and effective way of determining the presence of activated p21 with aspartic acid at position 13 in human fluids and tissues. J Cell Physiol, 1990 Aug, 144(2), 222 - 8 Continuously applied compressive pressure induces bone resorption by a mechanism involving prostaglandin E2 synthesis; Imamura K et al.; In previous research, we devised a specific culture chamber to examine the effect of continuously applied compressive pressure (CCP) on bone formation and resorption . The chamber was infused with compressed mixed gases with different O2 and CO2 composition to maintain the pO2, pCO2, and pH in the culture medium under pressures of +0.5 atm (1.5 atm total) to +2.0 atm (3.0 atm total) at the same levels as those at the ordinary pressure (1 atm) . Using the specific culture chamber, we demonstrated that CCP greatly suppressed the differentiation of mouse osteoblast-like MC3T3-E1 cells . The inhibition by CCP appeared to be mediated by prostaglandin E2 (PGE2) . In the present study, we examined the effect of CCP on osteoclastic bone resorption . CCP treatment of mouse bone marrow culture markedly increased both the PGE2 production and the number of tartrate-resistant acid phosphatase (TRACP)-positive mononuclear cells (possibly precursors of multinucleated osteoclasts) . An autoradiographic study using {125I}-salmon calcitonin showed clearly that those TRACP-positive cells had calcitonin receptors . The CCP effect was the greatest at +1.0 atm (2.0 atm total) . Isobutylmethylxanthine potentiated the production of TRACP-positive cells induced by CCP . Adding indomethacin completely inhibited both the TRACP-positive cell formation and the PGE2 production induced by CCP . CCP also increased the release of 45Ca from prelabeled mouse calvaria during later stages (2-6 days) of the 6-day culture period . CCP markedly increased PGE2 but not interleukin 1 in the culture media of mouse calvaria . These results indicate that, besides inhibiting osteoblast differentiation, CCP stimulates bone resorption by generating new osteoclasts through a mechanism involving PGE2 production. Cancer Res, 1990 Aug 1, 50(15), 4522 - 7 Potent growth inhibition of human tumor cells in culture by arginine deiminase purified from a culture medium of a Mycoplasma-infected cell line; Miyazaki K et al.; Two kinds of growth-inhibitory substances were found in culture of a Rous sarcoma virus-transformed rat liver cell line, RSV-BRL . The two substances were purified from the serum-free culture medium and identified as transforming growth factor beta 1 and Mycoplasma-derived arginine deiminase (EC 3.5.3.6), respectively . The arginine deiminase was an acid-labile but dithiothreitol-resistant protein with a molecular weight of 45,000 and pI 4.7 . Its Km value for L-arginine was 0.3 mM, which is about 30 times lower than that of bovine liver arginase . It was stable and active under culture conditions . When added into cultures, the arginine deiminase inhibited the growth of various human cancer cell lines at a dose of 5 ng/ml or higher by depleting L-arginine in the culture media . This effective dose was about 1000 times lower than that of bovine liver arginase . These results suggested the possibility of chemotherapeutic use of arginine deiminase for human cancers. Br J Haematol, 1990 Aug, 75(4), 598 - 602 Parasite uptake of desferroxamine: a prerequisite for antimalarial activity; Scott MD et al.; Desferroxamine has been shown to exhibit potent antimalarial activity . However, it is unclear as to whether desferroxamine functions by the chelation of extracellular, intra-erythrocytic, or parasite-associated iron . In order to determine desferroxamine's site of action, we have employed a large molecular weight dextran derivative of desferroxamine (70 kDa) and a reversible osmotic lysis technique by which erythrocytes were intracellularly loaded with this chelator . The desferroxamine-dextran derivative has virtually identical iron-binding characteristics to desferroxamine but, unlike desferroxamine, it is unable to cross the erythrocyte membrane . As previously shown, desferroxamine added to culture media exhibited potent antimalarial activity (mean effective inhibitory dose (ED50) approximately 6 microM) . However, extracellular desferroxamine-dextran showed antimalarial activity only at very high doses (ED50 greater than or equal to 180 microM), indicating that extracellular iron chelation is not involved in the antimalarial activity of desferroxamine . The intra-erythrocytic entrapment of the desferroxamine-dextran derivative also had no significant effect, except at very high concentrations, demonstrating that desferroxamine does not remove a non-haem iron source necessary for malarial replication . The results of this study clearly suggests that the antimalarial activity of desferroxamine is directly related to its ability to enter the parasitic compartment and not due to the chelation of extra- or intra-erythrocytic iron pools necessary for malarial growth. Agric Biol Chem, 1990 Aug, 54(8), 1905 - 14 Molecular cloning of the glucoamylase gene of Aspergillus shirousami and its expression in Aspergillus oryzae; Shibuya I et al.; The glucoamylase enzyme (GAase) gene from Aspergillus shirousami was cloned and sequenced from genomic and cDNA libraries . The genomic gene was located in the 5.4 kb EcoRI fragment . The deduced amino acid sequence of GAase contained 639 amino acid residues with a relative molecular mass of approximately 68,000 daltons (non-glycosylated form) . The genomic gene of A . shirousami GAase was introduced into Aspergillus oryzae . These transformants had increased GAase and raw starch degradation (RSD) activity in culture media and in rice-koji extracts . Analysis by Southern, Northern, SDS-PAGE, and Western blot techniques confirmed the foreign gene was correctly transcribed, translated, and expressed in A . oryzae. Vet Pathol, 1990 Jul, 27(4), 230 - 4 Experimental transmission of a dermal sarcoma in fingerling walleyes (Stizostedion vitreum vitreum); Martineau D et al.; Dermal sarcoma is a benign skin tumor of adult walleyes (Stizostedion vitreum vitreum) with a suspected viral etiology . A laboratory study was initiated to determine if the tumor could be experimentally transmitted by inoculating young walleyes with materials prepared from tumors from adult fish . Eighty walleye fingerlings were divided into four groups of 20 fish each . Two groups were inoculated intramuscularly at 4 months of age either with live tumor cells or with cell-free filtrates of sonicated tumor cells . The two other groups were used as controls and were inoculated either with cultured cells from normal walleye fry or with tissue culture media . Neoplasms, similar to the dermal sarcoma affecting adult walleyes, were observed after 4 months only in fingerlings inoculated with cell-free filtrates of sonicated tumor cells . Like the tumor affecting wild adult walleyes, the transmitted tumors were restricted to the dermis and originated from the superficial surface of scales . They never invaded locally and never metastasized . The transmitted tumors differed from tumors of adult walleyes in their severity and the absence of osteoid . The multicentric origin of transmitted walleye dermal sarcoma suggests that the virus spreads systemically and that tumor cells are polyclonal . This successful transmission of the lesion, along with the presence of C-type virus particles budding from tumor cells in two of seven tumor-bearing fingerlings, supports a retroviral etiology. J Membr Biol, 1990 Jul, 117(1), 91 - 9 Identification of riboflavin transport by MDCK cells using quantitative fluorescence video microscopy; Lowy RJ et al.; MDCK cells, when examined by low-light level video microscopy displayed an endogenous fluorescence with two differing patterns . A low intensity emission which was punctate and associated with cell organelles was observed with emission and excitation conditions generally used to observe either fluorescein (450-500 nm excitation/greater than 510 nm emission) or rhodamine (514 nm excitation/greater than 530 emission) type dyes . A second 5- to 10-fold brighter emission for 450-500 nm excitation was observed, which was unusual in that each cell appeared to be outlined . Evidence obtained from spectroscopy and from using culture media of altered composition supported the conclusion that the water-soluble vitamin riboflavin accumulated in the basolateral spaces and fluid-filled "domes" and was the source of this fluorescent emission . Quantitative measurements showed that exposure to cultures to 10 microM riboflavin resulted in accumulation in domes of 565 +/- 80 microM . The transport rate was calculated to be 189 +/- 30 pmol/min-cm2 . One mM probenecid, a known inhibitor of riboflavin transport in vivo, reduced transport to 54% of control, while 10 mM nearly abolished the uptake . The results demonstrate that removal of riboflavin reduces MDCK cell fluorescence to levels compatible with low-light level imaging . Furthermore, these cells actively transport riboflavin and provide a new in vitro model for this process. J Hepatol, 1990 Jul, 11(1), 65 - 9 Detection of malotilate toxicity in vitro with peripheral blood mononuclear cells as targets . A preliminary report; Nomura F et al.; An in vitro assessment of susceptibility to malotilate was made with peripheral blood mononuclear cells (PBMCs) as targets . PBMCs were incubated with malotilate in the presence or absence of the NADPH generating system and hepatic microsomes . Malotilate cytotoxicity to PBMCs, assessed by trypan blue dye exclusion and lactate dehydrogenase (LDH) release into the culture media, was found to be markedly increased by the addition of the NADPH generating system, indicating that metabolites play a significant role in toxicity . Once the significant role of malotilate metabolites in cytotoxicity was established, we applied this system to patients with suspected malotilate-induced liver injury . It was demonstrated that cytotoxicity was greater in PBMCs which were obtained from patients with chronic active hepatitis with a parallel malotilate-induced liver injury than in those from two different control groups; normal volunteers and patients with chronic active hepatitis who had had long-term malotilate treatment without any adverse reactions . This in vitro system seems to be of value in predicting potential malotilate toxicity in subjects who might be susceptible to this drug. Hepatology, 1990 Jul, 12(1), 48 - 58 Cytotoxic interactions of cardioactive cationic amphiphilic compounds in primary rat hepatocytes in culture; Bandyopadhyay S et al.; Hepatocytes from adult male Sprague-Dawley rats were isolated by the two-stage collagenase perfusion technique; 1 x 10(6) cells/plate were incubated in primary cell culture in Leibovitz's L-15 medium for 24 hr with or without various concentrations (12.5 to 400 mumol/L) of cardioactive cationic amphiphilic compounds such as propranolol, verapamil, sotalol, atenolol and procainamide . Propranolol and verapamil caused a significant release of lactate dehydrogenase (used as cytotoxic index in this study) in the culture media in a concentration-dependent manner, with LC50 values of 220 +/- 10 and 224 +/- 7 mumol/L, respectively . Atenolol, sotalol and procainamide had no effect on lactate dehydrogenase release . Electron microscopy of the hepatocytes showed that subtoxic concentrations of propranolol (12.5 to 125 mumol/L) and verapamil (12.5 to 100 mumol/L) induced multilamellar inclusion bodies after 24 hr of incubation . The two higher concentrations of propranolol (50 and 125 mumol/L) and 100 mumol/L of verapamil produced a significant decrease in the percentage of volume density of the mitochondria as quantitated by morphometrical analysis . An unusual feature of the electron microscopical changes with propranolol and verapamil was the presence of mitochondria within the multilamellar inclusion bodies . When these two drugs were used together or with subtoxic concentrations of amiodarone or desethylamiodarone, release of lactate dehydrogenase was significantly enhanced . No correlation was evident between the cytotoxic response and the volume density of cellular inclusions in hepatocytes treated with different concentrations of propranolol, verapamil, amiodarone or desethylamiodarone . Sotalol, atenolol and procainamide in concentrations up to 400 mumol/L did not produce any ultrastructural changes in hepatocytes after 24 hr of incubation . These results show that (a) cationic amphiphilic structure per se is not the only requirement for induction of multilamellar inclusions, (b) propranolol and verapamil can induce the formation of multilamellar inclusion bodies and cause a concentration-dependent release of lactate dehydrogenase from hepatocytes and (c) combination of different cationic amphiphiles in subtoxic concentrations can enhance cytotoxicity and increase the volume density of multilamellar inclusions. Am J Pathol, 1990 Jul, 137(1), 121 - 34 The formation of asbestos bodies by mouse peritoneal macrophages . An in vitro study; Koerten HK et al.; For studies on the mechanism of asbestos body formation, Union Internationale Contre Cancer (UICC) crocidolite asbestos fibers were added to a culture of mouse peritoneal macrophages . Small asbestos fibers were totally ingested by the macrophages, but fibers too long to be taken up completely remained as a consequence extracellular . These long asbestos fibers became the basis for asbestos body formation . The basic mechanism underlying asbestos body formation was found to be the exocytotic activity of macrophages . The number of iron-rich inclusion bodies was dependent on the availability of iron in the culture media, and the same holds for the amount of iron in the asbestos body coat . This means that asbestos body formation is a phenomenon that occurs accidentally when macrophages come into contact with long fibers in an iron-rich environment . A time-dependent increase in the number, average size, and rate of segmentation of the asbestos bodies was observed . The present report is the first to describe asbestos body formation in vitro. Trop Geogr Med, 1990 Jul, 42(3), 283 - 5 Kala azar in an adult Libyan and review of visceral leishmaniasis in Libya; Jain S et al.; Less than 30 confirmed cases of kala azar have been reported from Libya in this c