Am J Clin Pathol, 1985 Aug, 84(2), 159 - 65 Hematopoietic cell surface markers on metastatic small cell carcinoma detected with monoclonal antibodies; Patterson BJ et al.; Using a panel of monoclonal antibodies, cells from lymph node biopsies have been examined in three patients with small cell carcinoma presenting with cervical lymphadenopathy . Two patients had small (oat) cell carcinoma of the lung; in the third patient, a primary tumor was not found . Two lymph node biopsies showed typical small (oat) cell carcinoma, and one was an intermediate cell variant; in the last, lung biopsy showed small (oat) cell carcinoma . Electron microscopy demonstrated desmosomes in all three tumors . In each case, lymph node cell suspensions were examined by indirect immunofluorescence with the use of a panel of monoclonal antibodies to antigens usually associated with lymphoid or myeloid cells . In two of the three cases malignant cells were positive with the lymphoid marker BA-2; in two cases malignant cells were positive with OK1a1, a marker for the Ia-like antigen (HLA-DR); and in one case malignant cells were positive with My-1 . Caution is needed in the interpretation of cell surface marker studies in the differential diagnosis of small round cell tumors. Mol Pharmacol, 1985 Aug, 28(2), 100 - 6 Expression of both alpha 1- and alpha 2-adrenoceptors in an insulin-secreting cell line . Parallel studies of cytosolic free Ca2+ and insulin release; Ullrich S et al.; Changes in intracellular free Ca2+, {Ca2+}i, and immunoreactive insulin release in response to alpha-adrenergic agents were measured in RINm5F cell suspensions . Cells were loaded with the fluorescent indicator quin 2 for monitoring {Ca2+}i . Epinephrine (1 microM), which inhibited alanine-stimulated insulin release by 73%, evoked a transient rise in {Ca2+}i . This rise is in part due to Ca2+ mobilization, since it is still present in the absence of extracellular Ca2+ . The alpha 2-adrenergic agonist clonidine (10 microM) mimicked the epinephrine effect on insulin release without any change in {Ca2+}i . In contrast, the alpha 1-adrenergic agonist phenylephrine (10 and 100 microM) raised {Ca2+}i, albeit to a lesser extent than epinephrine . Phenylephrine enhanced basal, but had no effect on alanine-stimulated insulin release . To examine further the nature of the effect of epinephrine, specific alpha-adrenergic blocking agents were employed . The epinephrine-induced increase in {Ca2+}i could be inhibited by the alpha 1-adrenergic antagonists BE2254 (0.1 microM) and prazosin (0.01 microM) . In the presence of these blockers, epinephrine was still able to inhibit insulin release . When alpha 2-adrenergic receptors were blocked by the addition of idazoxan (0.1 and 1 microM), epinephrine still raised {Ca2+}i . At the higher concentration of idazoxan, the epinephrine inhibition of insulin release was completely overcome . The alpha-adrenergic agonists did not attenuate the alanine-induced rise in {Ca2+}i . This study shows that both subtypes of alpha-adrenergic receptors are present in the insulin-secreting cell line RINm5F . The activation of alpha 1-adrenergic receptors leads to an increase in {Ca2+}i . In contrast, the inhibition of insulin release due to epinephrine is mediated through alpha 2-adrenergic receptors . The alpha 2-adrenergic mechanism does not involve changes in {Ca2+}i, but is rather exerted at a later step in the secretory process. Exp Parasitol, 1985 Aug, 60(1), 63 - 72 Entamoeba histolytica: impedance measurements and cytotoxicity in the presence of bepridil, verapamil, and cytochalasin D; Ravdin JI et al.; Entamoeba histolytica, and invasive enteric protozoa, kills mammalian target cells by sequential adherence and cytolytic events . Using platinum plate electrodes with an alternating current source placed in a Wheatstone bridge circuit, the impedance (resistance to ion flow) of a cell suspension of axenic amebae (strain HM1-IMSS) was measured . The impedance of the amebic cell suspension, expressed as resistivity (in ohm-cm), was significantly greater than the test solution and increased with decreasing temperature or greater cell packing (P less than 0.01), indicating that the resistivity measurements reflected the impedance of the amebic surface membrane . Cytochalasin D (10 micrograms/ml), a microfilament inhibitor which inhibited amebic in vitro adherence and cytolysis of target Chinese hamster ovary (CHO) cells (P less than 0.001), also increased resistivity of the amebic suspension (P less than 0.01) . Exposure of amebae to bepridil (10(5) M), a slow-channel blocker, inhibited amebic killing of target cells (P less than 0.01) and also increased the resistivity of the amebic suspension (P less than 0.01), but both to a lesser degree than cytochalasin D (P less than 0.001) . In contrast, exposure of amebae to verapamil followed by washing had no effect on amebic killing of target cells or resistivity of the amebic suspension . The increased resistivity measured in cytochalasin D or following exposure to bepridil was not due to a change in cell density of the amebic suspension . These studies indicate that changes in impedance of the amebic surface membrane are produced by bepridil and cytochalasin D . The effect of these agents on membrane impedance may contribute directly to the concurrent observed alteration in amebic cytopathogenic capacity or may serve as a parallel marker for the cell membrane alterations induced by such pharmacologic agents which inhibit amebic microfilament function or calcium flux. J Reprod Immunol, 1985 Aug, 8(1), 25 - 31 Suppression of mixed lymphocyte reaction by cells of human first trimester pregnancy endometrium; Nakayama E et al.; In order to identify an immunological role for decidual tissue in pregnancy we have prepared single cell suspensions from the tissues of normal pregnant women and examined the effects of these cells on one-way mixed-lymphocyte reactions (MLR) . The separated cells were heterogeneous, containing classical decidual cells, glandular epithelial cells, granular endometrial cells, macrophages and small lymphoid cells . {3H}Thymidine incorporation at day 6 of the MLR was suppressed by addition of the cells at the initiation of the cultures and the degree of suppression was inversely correlated to the gestational age of the decidual tissue, apparently through inhibition of the antigen recognition phase of the MLR . These findings support the view that the cells of the human first trimester pregnancy endometrium may play an important role in protecting the feto-placental unit from rejection, at least in the early phase of pregnancy. Mol Pharmacol, 1985 Aug, 28(2), 191 - 9 alpha-Fluoromethylhistidine . Kinetics of uptake and inhibition of histamine synthesis in basophil (2H3) cell cultures; WoldeMussie E et al.; Labeled histidine was taken up into rat leukemic basophil 2H3 cells by a system with high affinity for histidine and then decarboxylated to form histamine . Uptake was partially inhibited and decarboxylation was completely blocked by alpha-fluoromethylhistidine (alpha-FMH) at concentrations of 10-100 microM . alpha-FMH appeared to be co-transported by a histidine uptake system but the affinity of the system for alpha-FMH was lower than that for histidine (Km 130 and 24 microM, respectively) . The drug rapidly penetrated into and became highly localized within the cells . By 60 min the apparent IC50 for inhibition of histamine synthesis in intact cell suspensions was 0.2 microM compared to an IC50 of 1-2 microM alpha-FMH for inhibition of soluble histidine decarboxylase preparations . Turnover of histidine decarboxylase activity in 2H3 cells was rapid (t1/2, 37 min), and biphasic effects were noted after 24-h exposure of 2H3 cells to drug . At low concentrations (greater than 0.1 microM), decarboxylase activity was increased (up to 134 +/- 9% of control values) . Higher concentrations of the drug (0.1-10 microM) were inhibitory, and inhibition was related to drug concentration . No detectable decarboxylase activity was observed with 10 microM alpha-FMH after 4 days . Histamine levels increased (up to 232 +/- 2% of control values) or decreased in parallel with decarboxylase activity . Even in cultures devoid of histamine or decarboxylase activity (with 10 microM alpha-FMH) cell division and growth were not affected . Thus the drug appeared to inhibit specifically histamine synthesis without impairing essential cellular metabolic processes . However, kinetics of drug uptake and perturbation of enzyme turnover are additional factors to be considered in the action of alpha-FMH in intact cell systems. Immunology, 1985 Aug, 55(4), 721 - 8 Intestinal mucosal mast cells: isolation from rat lamina propria and purification using unit gravity velocity sedimentation; Lee TD et al.; Mucosal mast cell (MMC) suspensions obtained from the rat intestinal lamina propria by collagenase digestion (35.2 +/- 3.2% MMC) were enriched to 65.5 +/- 5.2% MMC by the use of a discontinuous gradient (30%/80%) of Percoll . Further purification to 95.7 +/- 1.3% MMC was achieved using velocity sedimentation at unit gravity (Sta-Put) . Analysis of the cells throughout the purification procedure confirms that the purified MMC are representative of the MMC in the initial isolated cell suspension . No differences were seen in terms of size, histamine content, protease content and responsiveness to secretagogues among the initial isolated population, the Percoll-enriched population and the Sta-Put-purified population . This study represents a major advance in mast cell research in that, for the first time, mast cells isolated from a homogeneous in vivo mucosal source have been obtained at levels of purity sufficient for specific biochemical characterization . Such characterization will aid in the interpretation of the role of MMC in disease and will provide a firm basis of knowledge of the form and function of intestinal MMC for comparison with mast cells derived from other mucosal sites or cultured in vitro from various organs. J Immunol, 1985 Aug, 135(2), 1046 - 52 Decidual cell-specific surface antigen(s) recognized by monoclonal antibodies: tissue and species distribution; Kearns M et al.; Decidual cells are direct descendants of endometrial stromal cells and the ultimate progeny of bone marrow-derived precursors . In view of their bone marrow genealogy and demonstrated immunoregulatory role during pregnancy, this study attempted to identify a lineage-specific differentiation marker(s) on murine decidual cells with the hope of tracing their developmental pathway and exploring their familial relationship to other lymphomyeloid cells . Two protein A-binding, IgG2b isotype monoclonal antibodies (secreted by clones 16F12 and 2G4F8) were raised by immunizing virgin CBA mice with syngeneic decidual cells . The presence and the density of the antigenic marker(s) recognized by these antibodies were examined by radioautography on various cell types in single cell suspensions of the decidua, placenta, and lymphomyeloid organs after a sandwich labeling with hybridoma supernatants followed by 125I-protein A . Both antibodies appeared to recognize antigen(s) unique for the decidual cell lineage in mice, humans, and rats . The incidence of antigen-bearing decidual cells increased with gestational age in CBA, C3H, and CD1 mice between days 8 and 14, and in humans between 6 and 10.5 wk; in rats, however, some decline was noted between days 8 and 14 . The binding was always higher with 16F12 than with 2G4F8 supernatants . No significant binding of either antibody to trophoblast cells of the placenta or leukocytes within the decidua was noted in any of the above mouse strains or species . Little or no labeling of any cell type was seen on lymphomyeloid cells of the virgin or pregnant CBA mice, but a consistent labeling of a rare blast-type cell in the blood was observed with both antibodies, raising the possibility that this cell may represent the circulating precursor of the decidual cell lineage . It remains to be investigated whether these antibodies are recognizing the same or different differentiation antigen(s) on the decidual cells, and whether a conservation of this antigen(s) during speciation signifies its functional importance. Biochim Biophys Acta, 1985 Jul 30, 846(1), 127 - 34 The assimilation of tri- and tetrapeptides by human erythrocytes; Vandenberg JI et al.; Evidence is presented that tripeptides enter human erythrocytes via saturable transport system(s) at rates similar to those previously described for dipeptides (King, G.F . and Kuchel, P.W . (1985) Biochem . J . 227, 833-842) but that the transmembrane flux rates for tetrapeptides are considerably less . 1H spin-echo NMR spectroscopy was used to monitor the coupled uptake and hydrolysis of peptides by red cells, since it enabled the simultaneous measurement of the levels of substrates and products of peptidase-catalysed reactions in suspensions with haematocrits similar to those found in vivo . Weighted non-linear least-squares regression of the integrated Michaelis-Menten equation onto progress curves obtained from the hydrolysis of Tyr-Gly-Gly and Gly-Gly-Gly in RBC lysates gave Km = 2.11 +/- 0.08 and 23.4 +/- 0.9 mmol/l and Vmax = 307 +/- 3 and 905 +/- 22 mmol/h per 1 packed cells, respectively . In whole cell suspensions, the rate of hydrolysis was considerably less and was dominated by the transmembrane flux of tripeptide . Progress curve analysis thus yielded the steady-state kinetic parameters for peptide transport; the values were Km = 11.6 +/- 1.1 and 56 +/- 18 mmol/l and Vmax = 12.9 +/- 3.0 and 36.4 +/- 3.2 mmol/h per 1 packed cells, respectively, for the previously mentioned peptides . The rate of transport of the tetrapeptide Gly-Gly-Gly-Gly was considerably less than either of the tripeptides . The above mentioned steady-state kinetic parameters were used in computer simulations of the coupled uptake and hydrolysis of tripeptides by human erythrocytes under physiological conditions; these simulations revealed certain similarities between the rates of peptide uptake by erythrocytes and the intestine in vivo. Biochim Biophys Acta, 1985 Jul 30, 846(1), 14 - 20 Endothelial binding of transferrin in fractionated liver cell suspensions; Kishimoto T et al.; Several studies using crude liver cell suspensions incubated with labeled transferrin have led to a conclusion that hepatocytes have transferrin receptors . When a visual probe, which permits evaluation of transferrin binding to individual cells, was used, the binding was unexpectedly found to be limited to endothelial cells in liver cell suspensions . Neither hepatocytes nor Kupffer cells contained transferrin receptors . In the present study, we fractionated liver cell suspensions using metrizamide gradients and centrifugal elutriation to obtain hepatocytes, Kupffer cell and endothelial cell fractions of high purity . Incubation of these fractions with 125I- or 59Fe-labeled transferrin led to exclusive binding to endothelial cells but not hepatocytes nor Kupffer cells . Kinetic analysis demonstrated Kd of 1.9 X 10(-7) M, Bmax of 3.1 pmol/10(6) cells per min, corresponding to 2.1 X 10(5) molecules/cell per min . At 4 degrees C, the binding reached a steady-state plateau within 5 min . Comparison of our data with those of previous investigators demonstrates a consistency if we consider that crude liver cell suspensions are contaminated with 2-3% endothelial cells . Thus, the previously reported findings may be entirely due to the contamination of crude liver cell suspensions with a small number of endothelial cells. J Biol Chem, 1985 Jul 15, 260(14), 8330 - 5 Ca2+-dependent, temperature-sensitive regulation of Na+ channels in tight epithelia . A study using membrane vesicles; Garty H et al.; Na+ fluxes were measured in toad bladder microsomes . Under favorable conditions, 60-90% of the tracer uptake was blocked by amiloride (Ki = 2.3 X 10(-8) M), i.e . mediated by the apical Na+-specific channels . Vesicles derived from cells maintained at 0 degrees C exhibited relatively small amiloride-sensitive fluxes . However, incubating the scraped cells at 25 degrees C prior to homogenization induced a nearly 5-fold increase of the amiloride-blockable flux in vesicles . This activation was fairly slow (t 1/2 = 5-10 min), irreversible, and strongly dependent on the incubation temperature . On the other hand, the Na+-specific apical conductance measured in mounted bladders was only slightly affected by the incubation temperature . The above activation process could be observed only in Ca2+-free EGTA-containing solutions . Adding Ca2+ (1 mM) to the cell suspension and subsequently removing it before homogenization blocked almost completely the amiloride-sensitive tracer uptake in the vesicles . The data are compatible with the model that the epithelial Na+ channels are down-regulated by a Ca2+-dependent reaction . The incubation of scraped, somewhat permeabilized cells in a Ca2+-free solution releases channels from this down-regulation and increases the Na+ conductance in a temperature-dependent process . The regulation of channels appears to involve a cytoplasmic factor which induces a stable modification of the apical membrane, preserved by the isolated vesicle. Mycopathologia, 1985 Jul, 91(1), 29 - 33 Pathogenicity of Exophiala jeanselmei for ddY mice; Nishimura K et al.; The pathogenicity of three cultures isolated as Phialophora jeanselmei was compared with that of three cultures of Phialophora gougerotii using ddY mice . One hundred and twenty mice were used . They were divided into 6 groups consisting of 20 each . Each culture was evaluated in 20 mice . Mice were inoculated intravenously with 0.2 ml of a 1% (wet weight/vol.) yeast-like cell suspension and sacrificed at adequate intervals until the 30th day . As results, 1) the virulence of the three cultures each of P . jeanselmei and P . gougerotii to ddY mice was mild . 2) These cultures were not neurotropic . 3) P . gougerotii survived longer in the mice than P . jeanselmei . 4) There were no major differences in histopathology of the lesions in the mice inoculated with the two taxa. J Biochem (Tokyo), 1985 Jul, 98(1), 9 - 17 Purification and some properties of chalcone synthase from a carrot suspension culture induced for anthocyanin synthesis and preparation of its specific antiserum; Ozeki Y et al.; Chalcone synthase was purified to homogeneity by polyacrylamide gel electrophoresis from cell suspension cultures of carrot in which anthocyanin synthesis was induced by transferring the cells from a medium containing 2,4-dichlorophenoxy-acetic acid (2,4-D) to one lacking it . A molecular weight of 80,000-85,000 for the enzyme was determined by gel filtration and disc-gel polyacrylamide electrophoresis, and one of about 40,600 for the subunit by SDS slab-gel electrophoresis . The primary reaction product was chalcone and the pH optimum of the reaction was 8.0 . The Km values for 4-coumaroyl-CoA and malonyl-CoA were 5.7 microM and 18 microM, respectively . These properties of carrot chalcone synthase were discussed in comparison to those of that from cell cultures of parsley reported previously . Antiserum against chalcone synthase from carrot was obtained from mice bred under specific pathogen free conditions . Crossreactivity was examined by Western-blotting, and the high specificity of the antiserum against chalcone synthase was demonstrated. J Neurol Sci, 1985 Jul, 69(3), 207 - 21 Adhesive interactions between normal and dystrophic human skin fibroblasts; Pizzey JA et al.; The adhesive properties of skin fibroblasts from patients with Duchenne muscular dystrophy (DMD) were studied by analysing cell aggregate formation in suspensions consisting of normal and DMD fibroblasts . By the use of aggregation kinetics and fluorescent labelling, the genotypic composition of aggregates in mixed-cell suspensions could be visualised . The distribution of normal and DMD cells within these aggregates could then be compared to theoretical binomial distributions which assume no difference in the specific adhesiveness between the two genotypes . Analysis of the 3- and 4-cell aggregates which were produced by co-aggregating normal and DMD cells demonstrate that there is no qualitative (specific) difference in the adhesiveness between normal and DMD fibroblasts . However, quantitative changes in the cell-cell adhesion of DMD fibroblasts may be present, and this is supported by the relatively small proportion of intermediate size heterotypic aggregates which were formed in mixed-genotype cell suspensions . In such mixtures, fewer aggregates consisting of 5 or more cells were formed compared to fibroblast suspensions derived from pairs of normal individuals . Furthermore, cell suspensions from pairs of DMD patients produced even less greater than or equal to 5-cell aggregates than were found in the mixed-genotype experiments . These findings are considered in relation to previous reports of abnormal cell adhesiveness and other adhesion-related mechanisms in DMD cells. Geburtshilfe Frauenheilkd, 1985 Jul, 45(7), 477 - 81 {Drug sensitivity testing of gynecologic tumors using Volm's test and stem cell assay}; Eidtmann H et al.; A variety of test systems have been developed for predicting the efficacy of cytotoxic drugs in the treatment of individual malignant human tumors . The present paper reports on the author's experience with Volm's short-term chemosensitivity test and Hamburger and Salmon's stem-cell assay . In the Volm test the influence of adriamycin on the incorporation of radioactively labelled uridine in an individual tumor cell suspension was investigated . Comparison with a cytostatic-free control permitted conclusions to be drawn with regard to the proliferation-related chemosensitivity . The stem-cell assay is based on the capacity of certain tumor cells, the so-called stem cells, to form colonies in a bilaminar soft agar system . The growth of the colony of pre-incubated cytostatics was evaluated in relation to that of untreated tumor cells . The Volm test was successful in 63 (95%) out of a total of 66 tests conducted . Twelve tumors were chemosensitive in the test and 51 chemoresistant . In the stem-cell assay, growth of a colony which permitted chemosensitivity to be tested was only found in 27 out of 183 tests . The criterion of chemosensitivity with a reduction of at least 50% in the number of cells in the colony by at least one cytostatic was satisfied by 14 (29%) of the 49 stem-cell assays which could be evaluated, there being no differences between breast and ovarian carcinomas . The two test systems indicated the chemosensitivity correctly in less than 50% of 49 retrospectively evaluated courses of disease . In contrast, resistance was predicted correctly in 90%.(ABSTRACT TRUNCATED AT 250 WORDS) Immunology, 1985 Jul, 55(3), 531 - 8 Origin and immunological hyporeactivity of canine alveolar lymphocytes; Kirby J et al.; Autochthonous canine thoracic duct lymphocytes were isolated, labelled with 111indium and injected intravenously . Direct sampling showed that the label passed from blood to lymph within 25 hr . By gamma camera imaging, the initial accumulation of 111In the liver and spleen was followed by a striking increase in lymph node associated activity between 24-36 hr . Although initial lung-associated counts were low, a bicarbonate-induced non-specific inflammation of the right lung induced a rapid and selective accumulation of labelled lymphocytes (ratio right to left lung 8:1) . Cell suspensions lavaged from the alveolar surface showed only a 9% response to phytohaemagglutinin (PHA) compared to peripheral blood lymphocytes (PBL) . The addition of unfractionated lavage cells to PBL caused a macrophage-dependent suppression of mitogen responsiveness . However, macrophage depletion of alveolar lavage cells did not restore the lymphocyte response to PHA . Similarly, the frequency of alloreactive precursors of cytolytic lymphocytes (CTL) was almost 10-fold less in alveolar lymphocytes compared to PBL . Thus, bronchoalveolar lavage provides a technique by which viable cells may be recovered in significant numbers from the alveoli and should be invaluable for the investigation of lung allograft rejection. Cytometry, 1985 Jul, 6(4), 321 - 6 DNA analysis and sorting of rat testis cells using two-parameter flow cytometry; van Kroonenburgh MJ et al.; By use of two-parameter flow cytometry of rat testis cell suspensions stained with mithramycin for DNA (the peak amplitude of the fluorescence signal versus total fluorescence intensity integrated over time), eight cell compartments could be distinguished without pre-enrichment of the samples . Cells in these compartments were identified by sorting and subsequent microscopic examination. Cytometry, 1985 Jul, 6(4), 281 - 5 Differential of light-scattering detection in an arc-lamp-based epi-illumination flow cytometer; Steen HB et al.; Light-scattering histograms of blood cell suspensions were recorded for various ranges of scattering angles by means of an arc-lamp-based flow cytometer (AL-FCM) . The results were compared with those obtained with a conventional, laser-based flow cytometer (L-FCM) with forward scattering (2-20 degrees) and scattering at right angles . Measuring with the AL-FCM in the angle range upward from 13 degrees, the relative light-scattering intensities of lymphocytes, monocytes, and granulocytes were essentially independent of scattering angle and closely similar to the values measured as right-angle scattering in the L-FCM . With a range of scattering angles upward from 2 degrees the AL-FCM yielded histograms similar although not identical to that of the forward-scattering detector in the L-FCM . Differentiation between live and dead cells in this mode of operation was similar in the two instruments. Arthritis Rheum, 1985 Jul, 28(7), 742 - 52 IgG and IgM rheumatoid factor synthesis in rheumatoid synovial membrane cell cultures; Wernick RM et al.; The detection of rheumatoid factors (RFs) in synovial membranes and fluids of patients with rheumatoid arthritis (RA) has suggested that local production of these antiimmunoglobulin autoantibodies may have a role in the pathogenesis of synovitis . To quantitate RF synthesis in the rheumatoid synovial membrane, 12 synovial specimens were obtained from patients with seropositive RA, 5 from patients with seronegative RA, and 6 from patients with other arthritides . Single cell suspensions were cultured, and supernatants were analyzed for IgG, IgM, IgG-RF, and IgM-RF by solid-phase radioimmunoassays . IgM-RF was detected in all of the 12 seropositive culture supernatants, and IgG-RF was detected in 8 of the 12 . Addition of cycloheximide to the cultures resulted in a greater than or equal to 40% decreased in the amount of IgM-RF . A similar decrease in IgG-RF occurred in the 4 cultures in which the largest amounts of IgG-RF were detected . IgM-RF synthesis represented 7.3 +/- 0.7% (mean +/- SEM) of the total IgM produced, and IgG-RF represented 2.6 +/- 1.1% (mean +/- SEM) of the IgG synthesized in those cultures with detectable IgG-RF . Cultures of synovial membrane cells (SMC) from seronegative RA patients or patients with other arthritides did not contain detectable amounts of IgM-RF or IgG-RF . Selective synthesis of RF by seropositive synovium was suggested by the observation that the fractions of synthesized IgM with RF activity were greater in the SMC supernatants than in paired sera in all cases, and the fractions of IgG with RF activity were greater in the SMC supernatants of 3 of the 4 cases in which substantial amounts of IgG-RF were produced . Comparison of the percentages of newly synthesized IgM with RF activity in paired cultures of SMC and peripheral blood mononuclear cells similarly indicated selective synthesis of IgM-RF by the synovium . These results demonstrate active and selective synthesis of both IgG-RF and IgM-RF by seropositive SMC . However, RFs account for only a minor fraction of the total Ig produced. Arch Biochem Biophys, 1985 Jul, 240(1), 265 - 72 Chalcone synthase from cell suspension cultures of Daucus carota L; Hinderer W et al.; Chalcone synthase (CHS) has been partially purified about 35-fold . Withdrawal of 2-mercaptoethanol after precipitation with ammonium sulfate led to higher stability during further purification steps . In order to determine CHS activity, two procedures {according to Schroder et al . (1979) Plant Sci . Lett . 14, 281-286} were applied . The radioactivity extracted with ethyl acetate from the assay mixture (total products) was compared to 14C-labeled flavanone purified by TLC . The activity of CHS increased with bovine serum albumin (BSA) or 2-mercaptoethanol in the assay . Both effects were synergistic, but BSA did not promote "side products" as 2-mercaptoethanol did . BSA (10 mg ml-1) and 2-mercaptoethanol (1.4 mM) were components of the standard assay . Under these conditions, the CHS from Daucus carota had different pH optima for naringenin formation (7.9) and eriodictyol formation (6.8) . The apparent Km values were 0.6 microM for 4-coumaroyl-CoA (pH 7.9), 7.7 microM for caffeoyl-CoA (pH 6.8), and 3.0 microM for malonyl-CoA (pH 7.9) . Substrate inhibition was observed with 4-coumaroyl-CoA (greater than 10 microM) and malonyl-CoA (greater than 50 microM) . The inhibitory activity of various flavonoids and related compounds (100 microM) was investigated . Naringenin and naringenin-chalcone inhibited eriodictyol formation totally and naringenin formation by 50% . In contrast, eriodictyol and eriodictyol-chalcone inhibited only eriodictyol formation by 40% . It was shown that the inhibition with naringenin was fully uncompetitive . These in vitro data support the view that the true substrate of CHS in D . carota is 4-coumaroyl-CoA. Endocrinology, 1985 Jul, 117(1), 47 - 54 Estrogen receptors and estrogen sensitivity of fetal thymocytes are restricted to blast lymphoid cells; Gulino A et al.; We have observed {3H}estradiol binding sites in the cytosol of lymphoid cell suspensions obtained from the fetal thymus of guinea pig . Scatchard analysis {dissociation constant (Kd), 0.5 +/- 0.02 (SE) nM}, binding specificity, and diethylaminoethyl chromatography of these {3H}estradiol binding sites are similar to those described for estrogen receptors . Estrogen receptor levels in the thymic lymphoid cell population have been studied after fractionating cells by Percoll discontinuous density gradients into large and low density cells (alpha-cells) and small and high density cells (beta-cells) . Estrogen receptor levels are higher in alpha-cells {1002 +/- 200 (SE) sites per cell} than beta-cells {61 +/- 6 (SE) sites per cell} . Electron microscopy shows that the ultrastructural characteristics of 95% alpha-cells are consistent with their lymphoblastoid nature, whereas 90% of the beta-cells represented here can be categorized as typical small lymphocytes . To study whether cellular estrogen receptor expression was related to blastogenesis, fetal thymocyte suspensions were cultured throughout 48 h in the presence of the mitogen Concanavalin A . A significant increase in estrogen receptor levels was observed in thymic lymphoid cells cultured in the presence of the mitogen {1500 +/- 193 (SE) sites per cell} with respect to cells cultured in the absence of the mitogen {400 +/- 54 (SE) sites per cell} . One-day and 3-day in vivo estrogen treatments decrease significantly the {3H}thymidine uptake {by 40 +/- 1% (SE) and 66 +/- 5% (SE), respectively}, the mitotic index {by 82 +/- 8% (SE) and 96 +/- 2% (SE), respectively} and the frequency of lymphoblastoid cells {by 15 +/- 4% (SE) and 50 +/- 2% (SE), respectively} in fetal thymocyte suspensions . A 29 +/- 0.7% (SE), 31 +/- 0.8% (SE), and 35 +/- 2% (SE) decrease of {3H}thymidine incorporation in the DNA of alpha-cells cultured throughout 24 h, 48 h, and 72 h, respectively, in the presence of estradiol (10 nM) was observed with respect to untreated alpha-cells (P less than 0.01), while no effect of estradiol was observed on {3H}thymidine uptake by beta-cells . The mitotic index of cultured alpha-cells was significantly decreased {by 67 +/- 5% (SE)} 48 h after the addition of estradiol to the culture medium . It is concluded that: 1) estrogen receptors and estrogen sensitivity are restricted to lymphoblastoid thymic cells, and 2) estrogen receptor levels are increased after lymphoid blast transformation. Cancer Res, 1985 Jul, 45(7), 3058 - 63 Distribution of a new B-cell-associated surface antigen (BL7) detected by a monoclonal antibody in human leukemic disorders; Al-Katib A et al.; A murine monoclonal antibody (anti-BL7) was raised by immunization of BALB/c mice with a precursor B-cell line (Josh-7) which detects a heat-stable, nonimmunoprecipitable antigen . The expression of BL7 was investigated in peripheral blood and/or bone marrow leukemic cell suspensions stained by indirect immunofluorescence and analyzed by flow cytometry . Lymphoblasts from 43 of 43 cases of "null" acute lymphoblastic leukemia were BL7- . Five cases of T-acute lymphoblastic leukemia and 5 cases of terminal deoxynucleotidyl transferase-positive blastic chronic myelogenous leukemia were also BL7- . All 63 cases of B-cell chronic lymphocytic leukemia were BL7+ . Neoplastic cells in 22 of 28 cases of B-cell non-Hodgkin's lymphomas in leukemic phase were also BL7+ . Expression of BL7 showed some correlation with Rappaport's histological classification . Four cases of multiple myeloma and plasma cell leukemia were BL7- . Twenty-three cases of acute nonlymphocytic leukemias were also analyzed . Of these, only the acute promyelocytic (M3,4 cases) and acute myelomonocytic (M4, one case) varieties expressed BL7 on a small proportion (approximately 15%) of the leukemic cells . All other subgroups were BL7- . The reactivity of anti-BL7 was compared to other B-cell antibodies on selected samples and was shown to be different from B1, B2, and the BA antibodies . Anti-BL7 is a unique monoclonal antibody useful in the study of B-cell cancers. Immunology, 1985 Jul, 55(3), 489 - 500 Differential expression of HLA class II antigens on human fetal and adult lymphocytes and macrophages; Edwards JA et al.; A panel of monoclonal antibodies to monomorphic determinants of the MHC class II subregion locus products: DP, DR and DQ, was used to investigate the expression of these antigens on early lymphocytes and macrophages from human fetal liver (13-20 weeks), placenta (16 weeks and term) and cord blood, in relation to the class II phenotype of cells from adult tonsil and peripheral blood . Fetal liver sections and cell suspensions showed differential expression of class II antigens . DP was expressed at a higher frequency (11.0% of nucleated cells) than DR on lymphoid cells and macrophages from fetal liver, and DQ was either absent or expressed on less than 0.3% of nucleated cells . Consistent with this finding, DP but not DR or DQ antigens were observed on vascular elements and macrophages in the villi of 16-week placenta . At term, all three subregion locus products were expressed . Adult tonsil and peripheral blood B lymphocytes expressed DP, DR and DQ antigens with similar frequency; however, DQ was expressed at a lower frequency than DP and DR on cord blood B lymphocytes . In contrast, 30-50% macrophages from cord blood and adult peripheral blood expressed DP and DR, but fewer (5% and 18%, respectively) expressed DQ . These data suggest that class II antigens are expressed in the sequence DP, DR, DQ on developing lymphocytes . A similar sequence is suggested for macrophages. J Invest Dermatol, 1985 Jul, 85(1), 20 - 4 The role of epidermal cells in the induction of delayed-type hypersensitivity to alloantigens; Tamaki K et al.; Recent studies suggest that skin graft rejection and delayed-type hypersensitivity (DTH) are different manifestations of the same mechanism . In order to investigate the alloantigenicity of epidermal cells and epidermal Langerhans cells (LC), we used epidermal cell suspensions; experiments were performed to try to induce DTH to alloantigens by the subcutaneous administration of epidermal cells . Our study establishes the precise conditions for the induction of DTH response to alloantigens using epidermal cells . Transfer experiments have shown that effector cells were Thy-1+, Lyt-1+, and Lyt-2-cells . Furthermore, the experiments using congenic strains of mice and grafted skin revealed the contribution of gene products coded by MHC and non-MHC, and also the epidermal LC in the induction of DTH response. Mutat Res, 1985 Jul, 146(1), 33 - 42 Photoreactivation of UV damage in Escherichia coli uvrA6: lethality is more effectively reversed than either premutagenic lesions or SOS induction; Yamamoto K et al.; The effect of cyclobutyl pyrimidine dimers on cytotoxicity, induction of synthesis of the RecA and UmuC proteins, and mutagenesis was studied in Escherichia coli uvrA6 cells possessing excess amounts of photoreactivating enzyme . Exposure of 254 nm ultraviolet-irradiated (10 J/m2) cells to radiation from daylight fluorescent lamps reduced the amounts of thymine-containing dimers in a photoreactivating fluence-dependent manner, up to about 90% reduction at 5 min exposure . Of the lethal ultraviolet damage, 85% was photoreactivable (i.e . cyclobutyl pyrimidine dimers) and 15% was non-photoreactivable . An incident fluence of 1 J/m2 resulted in approximately a 5-fold increase in the synthesis of the RecA and UmuC proteins, as compared to the spontaneous level . If the UV-irradiated cell suspensions were illuminated with a fluorescent lamp at a dose which resulted in the full photoreactivation of viability, the yields of both proteins were reduced to 60% of the non-photoreactivated control cells . Furthermore, photoreactivation was shown to be more effective in the repair of lethal damage than in the repair of premutational damage . These experiments suggest that, among lethal damages, non-photoreactivable damage plays a more important role in both induction of the SOS functions and mutagenesis in uvrA6 cells than do cyclobutyl pyrimidine dimers. Proc Natl Acad Sci U S A, 1985 Jul, 82(14), 4823 - 7 Differentiation of normal marrow and HL60 cells induced by antithymocyte globulin; Hunter RF et al.; Antithymocyte globulin (ATG) therapy is an important treatment alternative for patients with acquired aplastic anemia . The mechanism by which it exerts its effects on hematopoiesis is unknown . In this report, we describe the ability of horse ATG to induce growth and differentiation of normal bone marrow . A single cell suspension of normal human bone marrow was cultured in methylcellulose medium and examined for the growth and maturation after incubation with ATG (10 micrograms/ml) . After 3-4 days of culture, spherical colonies containing mature myeloid elements were found in cultures containing ATG but not in cultures containing medium or preimmunization horse IgG . The addition of 10% colony-stimulating factor increased growth by 40% . The number of spherical colonies is not dependent on the presence of macrophages or T lymphocytes . This property of ATG may be relevant to the mechanism behind the hematologic recovery in some patients with acquired aplastic anemia . We also describe the ability of ATG to induce terminal differentiation in the HL60 leukemic cell line . ATG binds to HL60 cells and at concentrations between 10 and 100 micrograms/ml, 50% of the cells become mature granulocytes, acquire the ability to reduce nitroblue tetrazolium, and lose their proliferative capacity in the clonogenic assay . These new observations of ATG-induced differentiation of normal marrow myeloid elements and terminal differentiation of the HL60 cell line point to different avenues for future search of differentiation-inducing agents. Sci Sin {B}, 1985 Jul, 28(7), 736 - 44 Establishment of a model of transplantable myelocytic leukemia (L801) in LACA mice; Zhao NK et al.; A transplantable myelocytic leukemia model of LACA mice, designated by the name of L801, was established by intravenous injection of spleen cell suspension from mice with radiation-induced myelocytic leukemia into mice of the same strain . Until now, for more than three years, the L801 has maintained stable and rapid growth and has been reproduced for over 130 serial passages . The incidence of leukemia in inoculated animals was approximately 100% and mean survival time was 10.9 +/- 2.1 days . The L801 is of myelocytic type which has been determined by cytological, cytochemical, pathological and ultrastructural observations . Its karyotype was hypodiploid, characterized by modal number of 39, loss of Y chromosome and an abnormal huge marker chromosome . The cell cycle duration of the L801 was 16 h . C-type viral particles were observed under the electron-microscope . The L801 was sensitive, to varying extents, to various anti-tumor agents . We presume that the L801 is a useful tool in studies on mechanism of leukemogenesis, anti-tumor agent screening and treatment of experimental tumors. JPEN J Parenter Enteral Nutr, 1985 Jul-Aug, 9(4), 477 - 9 Effect of the dietary fatty acid component on the release of 14C-taurocholate, 14C-bilirubin, alkaline phosphatase, and aspartate transaminase by isolated rat liver cells; Anderson FH et al.; Rats were fed liquid diets for 7 days containing either triolein or Liposyn, which is rich in linoleic acid, as fat sources, and liver cell suspensions were prepared following collagenase perfusion . The release from isolated cells of alkaline phosphatase and aspartate transaminase during a 3-hr incubation did not differ . The uptake and release of 14C-taurocholate during a brief incubation was lower but not significantly in Liposyn-fed rats (0.1 greater than p greater than 0.05): the uptake was 9.74 +/- 1.58 vs 16.7 +/- 3.3 nmol/mg protein in triolein-fed rats; the release was 3.17 +/- 0.65 vs 5.35 +/- 1.01 nmol/mg protein in triolein-fed rats . The uptake of 14C-aminolevulinic acid was similar in both groups, but release of 14C-bilirubin during a 30-min incubation was 5,420 +/- 1010 in the Liposyn group vs 12,030 +/- 2,200 dpm/mg protein in the triolein group (p = 0.02) . It is concluded that a diet high in linoleic acid decreases bilirubin release in isolated liver cells consistent with the ability of this diet to cause cholestasis in vivo. J Invest Dermatol, 1985 Jul, 85(1 Suppl), 21s - 26s The mixed epidermal cell-lymphocyte reaction . II . Epidermal Langerhans cells are responsible for the enhanced allogeneic lymphocyte-stimulating capacity of normal human epidermal cell suspensions; Sontheimer RD; Earlier studies carried out in our laboratory which demonstrated that disaggregated human epidermal cells isolated from normal flexor forearm skin produced a greater degree of primary allogeneic lymphocyte blastogenic response than did autologous peripheral blood mononuclear cells have since been confirmed by others . We have now completed a series of additional studies designed to determine the basis for this difference . Blocking studies with anti-HLA-DR antibodies revealed that the allogeneic response triggered by epidermal cells was completely dependent on the presence of unbound HLA-DR molecules eliminating the possibility that nonspecific mitogenic effects produced by the epidermal cell suspension might be responsible for the difference . In addition we were unable to demonstrate that epidermal keratinocytes were supplying a nonspecific helper or growth-promoting effect to the interaction between stimulating HLA-DR bearing Langerhans cells and responding T lymphocytes . Since it has recently been suggested that the entire alloantigen-presenting capacity of unfractionated peripheral blood mononuclear cells can be attributed to a small population of HLA-DR antigen-bearing dendritic cells, we have considered the possibility that the greater degree of allogeneic lymphocyte response produced by epidermal cells could be due to the presence of a greater percentage of HLA-DR positive dendritic cells present in epidermal cell suspensions (i.e., Langerhans cells) . Peripheral blood dendritic cell-enriched fractions and epidermal cell suspensions that contained similar percentages of dendritic cells produced equivalent amounts of allogeneic lymphocyte stimulation, whereas peripheral blood dendritic cell-enriched fractions that contained greater percentages of dendritic cells than were present in epidermal cell suspensions produced greater amounts of allogeneic stimulation . We therefore conclude that the enhanced mixed lymphocyte reaction stimulating capacity of human epidermal cell suspensions could be explained by the fact that epidermal cell suspensions contain a greater percentage of HLA-DR bearing alloantigen-presenting dendritic cells than do unfractionated peripheral blood mononuclear cell suspensions. Cell Immunol, 1985 Jul, 93(2), 303 - 14 Characterization of immune effector cells present in early murine decidua; Gambel P et al.; It has been suggested that murine decidual cells act as an important immunoregulatory population localized to the pregnant uterus . We have examined early murine decidua to determine if immune effector cells occur in the decidual environment in proximity to the conceptus . High levels of natural killer (NK) cell activity were found consistently in decidual cell suspensions with peak activity occurring on Day 6.5 of gestation . NK activity declined as pregnancy proceeded and was not significant by Day 12.5 of gestation . Decidual cell suspensions did not appear to contain significant numbers of functional B or T effector cells . No antipaternal T-cell response could be demonstrated even in the decidua of immune mice . Lack of T-cell responses was attributed to the absence of T cells from decidua rather than to their inactivation because precursors of cytotoxic T lymphocytes (pCTL) could not be detected in decidual cell suspensions . Furthermore, the levels of pCTL detectable in spleen cell suspensions could not be reduced by mixing spleen cells with 7.5-day decidual cells . These results suggest that B cells and T cells may not occur in early decidua while NK cells are present and regulated independently. Blood, 1985 Jul, 66(1), 128 - 34 Induction of immunoglobulin secretion in follicular non-Hodgkin's lymphomas: role of immunoregulatory T cells; Braziel RM et al.; B cell neoplasms are clonal expansions of B lymphocytes thought to be frozen at various points along the normal B cell differentiation pathway . We studied cell suspensions from lymph nodes involved by follicular (nodular) non-Hodgkin's lymphoma to determine the capacity of the malignant B cells to secrete immunoglobulin (Ig) . Neoplastic B cells from all 14 follicular lymphomas secreted monoclonal immunoglobulin in culture when appropriate signals were provided . In most cases, maximal Ig secretion occurred when autologous T cells were removed by E rosette depletion, replaced with allogeneic normal T cells, and the cultures were exposed to 12-O-tetradecanoylphorbol-13-acetate . Autologous T cells exerted a suppressor effect on Ig secretion in 8/8 cases studied, diminishing the response of the malignant B cells to allogeneic T cells . This suppressor effect did not correlate with the percentage of cells staining with anti-Leu-2a or with "helper-suppressor" (Leu-3a-Leu-2a) ratios of the lymph node T cells . Our findings demonstrate that the arrested differentiation of most follicular lymphomas is reversible and implicate a T cell-mediated host immunoregulatory mechanism affecting Ig secretion in vivo . An additional contribution of our results is the demonstration of a cell culture system for synthesis of sufficient monoclonal Ig for use as an immunogen in production of anti-idiotype antibodies. Avian Dis, 1985 Jul-Sep, 29(3), 662 - 71 Comparison of a kinetic-based enzyme-linked immunosorbent assay (KELISA) and virus-neutralization test for infectious bursal disease virus . I . Quantitation of antibody in white Leghorn hens; Solano W et al.; The kinetics of the enzyme-substrate reaction served to evaluate a single-serum-dilution indirect enzyme-linked immunosorbent assay (ELISA) for quantitating antibody in white leghorn hens inoculated with infectious bursal disease virus (IBDV) vaccines . The antibody profiles of primary and secondary responses induced by two IBDV immunization regimens were compared using a kinetic-based ELISA (KELISA) and the virus-neutralization (VN) test . KELISA was standardized with an IBDV-infected VERO cell suspension . Antigen was capable of binding minute quantities of sample (5 microliter) without requiring dilutions . Conjugate consisted of immunoglobulin G fraction of goat antiserum against chicken IgG bound to horseradish peroxidase . Neither test revealed a difference in antibody profiles between the two immunized groups . The KELISA was as efficient as the VN test in detecting antibody after vaccination and one log 2 unit more sensitive . The KELISA was suitable for testing large numbers of samples (n = 60) in a microplate compared with a conventional ELISA (n = 8). J Clin Invest, 1985 Jul, 76(1), 40 - 51 Antigenic characterization of human hepatocellular carcinoma . Development of in vitro and in vivo immunoassays that use monoclonal antibodies; Carlson RI et al.; Several libraries of monoclonal antibodies have been produced by immunization of Balb/c mice with single cell suspensions of nontrypsin-treated human hepatocellular carcinoma cell (HCC) lines in order to study the antigenic properties of transformed hepatocytes . The antibodies were characterized with regards to specificity for hepatoma-associated antigens and their capability for use as reagents in radioimmunoassays (RIAs) and tumor localization in vivo . Three such antibodies namely, P215457, PM4E9917, P232524 of the IgG2a, IgG2a, and IgG1 isotypes, respectively, not only recognized separate and distinct antigenic determinants on four human hepatoma cell lines but also reacted with epitopes present on chemically induced rat hepatoma cell lines . In contrast, only 1 of 38 other human malignant and transformed cell lines demonstrated reactivity with the three antibodies; normal human tissues were also found to be unreactive . Monoclonal antibody P215457 densely stained the plasma membrane by indirect immunofluorescence, showed rapid binding activity to HCC cells in suspension, and precipitated a 50,000-mol wt cell surface protein; antibody PM4E9917 also stained the plasma membrane and precipitated a 65,000-mol wt protein, whereas P232534 recognized cytoplasmic antigenic determinants . With these antibodies "simultaneous sandwich" RIAs were established that detect soluble hepatoma-associated antigens in culture supernatants . Finally, the Fab fragment of P215457 was found to be useful in tumor localization in vivo . This antibody fragment when labeled with 131I was shown to localize by radionuclide-imaging studies in human hepatoma grown in nude mice . Thus, these investigations demonstrate that monoclonal antibodies may be produced against epitopes that reside almost exclusively on transformed hepatocytes and such antibodies may be successfully employed in the development of in vitro and in vivo immunoassays. Cytometry, 1985 Jul, 6(4), 334 - 41 Flow cytometric evaluation of cell dispersion from human head and neck tumors; Bijman JT et al.; The preparation of single-cell suspensions from 25 human head and neck tumors is described . Dispersal was performed overnight at 4 degrees C under slight agitation of the tissue suspensions using various combinations of enzymes and additives . The cell suspensions were examined for number of cells released, viability, amount of debris, and DNA distribution by means of flow cytometry (FCM) . It was shown that both trypsin/dithioerythritol (TD) and collagenase/D Nase (CDse) were of value in dispersing single cells from tumor tissue . In contrast to CDse, incubation with TD appeared to be cytolytic to normal lymphocytes . In a number of cases, DNA-FCM revealed ploidy abnormalities in a TD-suspension, which were not discernible in the concurrent CDse-suspension . Cell culture of primary cell suspensions corroborated the reliability of the DNA-FCM measurements . Pretreatment with CDse improved tumor disaggregation by TD and indicated a different dispersal capacity . Addition of Ca2+ and Mg2+ ions to the dispersal mixtures and preincubation of tumor slices in complete medium for 1 day before initiation of cell dispersion influenced favorably the quality of the cell suspension. Am J Physiol, 1985 Jul, 249(1 Pt 2), F74 - 83 Phosphate uptake by proximal cells isolated from rabbit kidney: role of dexamethasone; Poujeol P et al.; A single cell suspension was prepared from rabbit kidney cortex by gentle mechanical dissociation . The isolated cells were functionally intact and metabolically active, as indicated by exclusion of eosin dye, respiratory measurement, and ATP content . The isolated cells were shown to possess long microvilli, and their proximal origin was confirmed by enzymatic and glucose production studies . The uptakes of 0.1 mM phosphate (Pi) and 0.05 mM D-glucose at 37 degrees C were strongly dependent on the extracellular Na+ . Incubation of the cells with parathyroid hormone (10 U/ml) for 20 min reduced 20-s Pi uptake by 20% . The addition of dibutyryl cAMP decreased Pi uptake, with a maximal effect at 10(-6) M . Incubation of cells with gluconeogenic substrates, under conditions in which glucose production was increased, did not promote any change in Pi accumulation . When incubated for 60 min at 37 degrees C in the presence of dexamethasone (Dex), the Na+-dependent Pi uptake was decreased by 29% at 10(-9) M and by 36% at 10(-7) M without modification of the glucose uptake . Replacing Dex by aldosterone (10(-7) M) remained without effect on Pi uptake . It is concluded that: renal cells isolated by our preparative method are mainly of proximal origin; isolated cells are a good model for studying the regulation of Pi uptake in the proximal tubule; and glucocorticoids have an acute specific effect on Pi transport detectable in vitro and this effect does not seem to be related to modifications of renal gluconeogenesis. Endocrinology, 1985 Jul, 117(1), 23 - 30 Corticotropin-releasing factor stimulation of adrenocorticotropin and beta-endorphin release: effects of acute and chronic stress; Young EA et al.; The effects of acute and chronic stress on the release of ACTH and beta-endorphin in response to stimulation by ovine corticotropin-releasing factor (CRF) and arginine vasopressin were examined . Pituitaries were removed from rats who had received either acute stress, chronic stress daily for 14 days with the last stress occurring 24 h before decapitation, or chronic stress followed by an acute stress immediately before decapitation (chronic stress-acute stress) . Pituitaries from naive unstressed animals were used as the control group . After processing into single cell suspensions, the pituitaries were incubated with various doses of CRF (10(-11) M to 10(-9) M) and AVP (10(-10) M to 10(-8) M) . Release of ACTH and beta-endorphin into the medium was measured by RIA . A clear dose-dependent response to both releasers was seen in control pituitaries . In acute stress, a decreased responsiveness to arginine vasopressin and CRF was seen . This same blunted response was not seen in chronic stress even if the animals are stressed immediately before decapitation . At higher doses of CRF (10(-9) M) a substantially increased release of ACTH and beta-endorphin was seen in the chronically stressed rats . When the content of the anterior pituitary lobe was assayed in these animals, both chronic stress groups show increased content of ACTH and beta-endorphin, which may indicate an increase amount of ACTH and beta-endorphin in the releasable pools in chronic stress . In addition, the failure of further stress to alter the response to CRF in the chronic stress-acute stress group may indicate a down-regulation of the steroid feedback on the pituitary . However, it is clear that no down-regulation of the CRF receptor occurs in this chronic stress paradigm. J Immunol, 1985 Jul, 135(1), 215 - 22 Immunoactive products of placenta . IV . Impairment by placental cells and their products of CTL function at effector stage; Chaouat G et al.; Trophoblast-enriched cell suspensions prepared by collagenase digestion from midterm murine placentae were found resistant to CTL-mediated lysis . Treatment of such cells by trypsin or neuraminidase rendered these cells susceptible to such lytic effectors . Collagenase-prepared cell suspensions could impair CTL action, whereas neuraminidase- or trypsin-treated cells did not retain this property . This effect was also observed with extracts . These results indicate that soluble factors (which we will characterize in another paper) released by trophoblast cells (in fact, spongiotrophoblast) can interfere in a dose-dependent fashion with the action of lytic effectors . We suggest that such active mechanisms are physiologic components of the placental barrier and might be defective in some cases of immunologic abortions. Can J Microbiol, 1985 Jul, 31(7), 629 - 34 Pathway of ammonium assimilation in Streptomyces venezuelae examined by amino acid analyses and 15N nuclear magnetic resonance spectroscopy; Laycock M et al.; To obtain information on the route(s) of ammonium assimilation in Streptomyces venezuelae, cell suspensions transferred to fresh medium lacking nitrogen were pulsed with {15N2}ammonium sulphate . Cells and extracellular fluids were examined by nuclear magnetic resonance and amino acid analysis to assess changes in amino acid pools and the disposition of {15N}ammonium . Following addition of {15N}ammonium, glutamate--glutamine pools of low cell density replacement cultures expanded rapidly and became progressively labelled with 15N, whereas the alanine pool size increased much more slowly and became labelled with 15N to a much lesser extent . These results are consistent with the assimilation of ammonium via glutamate dehydrogenase or glutamine synthetase--glutamate synthase rather than alanine dehydrogenase . Under anaerobic conditions, S . venezuelae assimilates ammonium into alanine rather than glutamate--glutamine . Alanine dehydrogenase may thus function as a vehicle to regenerate NAD+ to maintain substrate-level phosphorylation during periods of anaerobiosis. Immunology, 1985 Jul, 55(3), 391 - 7 Immunobiology of the oral mucosa in the mouse; Deslauriers N et al.; The incidence of immunoglobulin (Ig)-synthesizing cells, Thy 1-positive cells and macrophages in the murine oral mucosa was investigated . Immunofluorescence studies of frozen tissue sections showed that IgA-, IgM- and IgG-containing cells and Thy 1-bearing cells were closely associated with the minor salivary glands . A quantitative analysis was then undertaken using single cell suspensions of the tissue . After mechanical disruption or enzymatic digestion of the mucosa, lymphoid cells were recovered almost exclusively from the mucosa of the posterior soft palate where we observed a dense accumulation of minor salivary glands . Thy 1-bearing cells were found at a higher frequency (25% of recovered cells) than membrane Ig-positive B lymphocytes (6-7%) in these suspensions . Cytoplasmic Ig+ cells accounted for about 6% of recovered cells, whereas plaque-forming cells (Ig-secreting cells) occurred at the same frequency as in the spleen (0.1%) . Plasma cells of the IgA and IgM isotypes predominated over IgG-secreting cells (A:M:G ratio = 1:1:0.2); this distribution did not directly correlate with the isotype distribution of salivary Igs (A:M:G ratio = 1:0.003:0.07) . In addition, about 10-14% of the cells in our preparations were esterase-positive mononuclear cells . Present data indicate that the murine oral mucosa contains both effector and regulatory cells required for the development and expression of local antibody responses. J Immunol, 1985 Jul, 135(1), 483 - 91 Isolation and characterization of human intestinal mucosal mast cells; Fox CC et al.; Study of the role of mast cells in the human gastrointestinal tract has suffered from the inability to examine these cells in vitro . In addition, work in rodent systems suggests that there are substantial differences between intestinal mast cells and those from other tissues, making extrapolation of data from other sources difficult . We report a method for producing mast cell-containing single cell suspensions from human intestinal tissue by mechanical and enzymatic dispersion . This method yields 4.5 X 10(5) mast cells per gram of tissue in purity of 3.1 +/- 2.1% . These mast cells were functionally intact as assessed by survival in short-term culture, low spontaneous release, and appropriate IgE-mediated histamine release . They were morphologically intact on electron microscopy and conformed to published descriptions of human lung mast cells . The intestinal mucosal mast cells were also indistinguishable from human lung mast cells in histamine content, goat anti-human IgE dose-response curves, kinetics of histamine release, unresponsiveness to f-met peptide, and production of arachidonic acid metabolites, prostaglandin D2, and leukotriene C4 . This procedure produces human intestinal mast cell suspensions in sufficient numbers to make pharmacologic characterization and further purification of this cell feasible. J Histochem Cytochem, 1985 Jul, 33(7), 672 - 6 Characterization and cell cycle kinetics of hepatocyte populations isolated from adult liver tissue by a nonenzymatic procedure; Higgins PJ et al.; Suspensions of liver cells enriched in lobular parenchymal hepatocytes were isolated from adult mouse hepatic tissue by nonenzymatic dispersion in chelating buffer and sedimentation of the released cells at unit g . Single cell suspensions so obtained were suitable for flow cytometric measurements of hepatic ploidy class distributions . The more quickly sedimenting cell population consisted of 88% albumin/transferrin-positive epithelial hepatocytes, the nuclei of which were bimodally distributed with respect to RNA content . This dual G1 population was observed in 2C DNA content liver nuclei prepared by several methods and appears to be a general cytochemical characteristic of adult liver parenchymal cells. Toxicol Appl Pharmacol, 1985 Jun 30, 79(2), 283 - 95 Mechanism of chemical-induced toxicity . I . Use of a rapid centrifugation technique for the separation of viable and nonviable hepatocytes; Fariss MW et al.; A major obstacle in defining the mechanism of chemical-induced toxicity has been the inability to distinguish between events that cause cell death and those that result from cell death . This problem results from measuring biochemical parameters in tissues or cell pellets containing both viable and nonviable cells . In the present study, we described a method for the rapid separation of viable hepatocytes from nonviable cells and medium prior to biochemical analysis . Separation of viable hepatocytes was accomplished in a microcentrifuge tube by layering a sample of isolated hepatocyte suspension over a dibutyl phthalate oil layer and centrifuging for several seconds . As a result, greater than 90% of the hepatocytes centrifuged through dibutyl phthalate were viable while greater than 90% of the cells recovered above the oil layer were nonviable . The separation of viable hepatocytes by the dibutyl phthalate method was not affected by the presence of the hepatotoxins, adriamycin (ADR) in combination with 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) or ethyl methanesulfonate (EMS), though the ratio of viable to nonviable cells in the suspension was drastically reduced . The metabolic and morphological integrity of hepatocytes centrifuged through dibutyl phthalate was altered after cell suspensions were treated with the ADR-BCNU or EMS . These chemically treated viable hepatocytes showed degenerative ultrastructural changes and a greater than 80% reduction in intracellular K+ and glutathione concentrations . Because centrifugation through dibutyl phthalate does not significantly alter the concentration of intracellular constituents nor the ultrastructure of control hepatocytes, the signs of reversible injury observed in hepatocytes centrifuged through oil resulted from the chemical treatment . These data indicate that the dibutyl phthalate separation technique offers the advantage of monitoring only viable hepatocytes for changes in membrane integrity or metabolic performance during a toxic chemical insult. J Immunol Methods, 1985 Jun 25, 80(2), 155 - 62 A flow cytometric method for the detection of adenosine deaminase in mononuclear cells; SenGupta S et al.; The purpose of this study was to develop a flow cytometric method for the detection of adenosine deaminase (ADA) in a single cell suspension of mononuclear cells . Anti-human ADA antibody was purified by affinity chromatography on a column of Sepharose 4B to which calf ADA was covalently linked . This antibody was used for indirect immunofluorescent staining of cells fixed in 4% paraformaldehyde . The specificity of staining was proved by substitution of anti-human ADA with normal rabbit IgG and by absorption experiments . The fluorescence profile of the cells was then analyzed by flow cytometry . Two groups of cells were studied: (a) thymocytes, tonsil cells and peripheral blood mononuclear cells (PBMC), (b) ADA-positive and ADA-deficient cell lines . In each of these populations of cells a peak of specific immunofluorescence staining for the enzyme could be easily distinguished from weak background staining of control preparations . Within each group, the cell population with higher ADA activity also displayed a greater intensity of cell fluorescence . Flow cytometry provides a means for quantitation of ADA in individual mononuclear cells. J Biol Chem, 1985 Jun 25, 260(12), 7226 - 33 Receptor-mediated inhibition of adenylate cyclase and stimulation of arachidonic acid release in 3T3 fibroblasts . Selective susceptibility to islet-activating protein, pertussis toxin; Murayama T et al.; Thrombin exhibited diverse effects on mouse 3T3 fibroblasts . It (a) decreased cAMP in the cell suspension, (b) inhibited adenylate cyclase in the Lubrol-permeabilized cell suspension in a GTP-dependent manner, increased releases of (c) arachidonic acid and (d) inositol from the cell monolayer prelabeled with these labeled compounds, (e) increased 45Ca2+ uptake into the cell monolayer, and (f) increased 86Rb+ uptake into the cell monolayer in a ouabain-sensitive manner . Most of the effects were reproduced by bradykinin, platelet-activating factor, and angiotensin II . The receptors for these agonists are thus likely to be linked to three separate effector systems: the adenylate cyclase inhibition, the phosphoinositide breakdown leading to Ca2+ mobilization and phospholipase A2 activation, and the Na,K-ATPase activation . Among the effects of these agonists, (a), (b), (c), and (e) were abolished, but (d) and (f) were not, by prior treatment of the cells with islet-activating protein (IAP), pertussis toxin, which ADP-ribosylates the Mr = 41,000 protein, the alpha-subunit of the inhibitory guanine nucleotide regulatory protein (Ni), thereby abolishing receptor-mediated inhibition of adenylate cyclase . The effects (a), (c), (d), and (e) of thrombin, but not (b), were mimicked by A23187, a calcium ionophore . The effects of A23187, in contrast to those of receptor agonists, were not affected by the treatment of cells with IAP . Thus, the IAP substrate, the alpha-subunit of Ni, or the protein alike, may play an additional role in signal transduction arising from the Ca2+-mobilizing receptors, probably mediating process(es) distal to phosphoinositide breakdown and proximal to Ca2+ gating. Brain Res, 1985 Jun 17, 336(2), 302 - 7 Transplanted septal neurons make viable cholinergic synapses with a host hippocampus; Segal M et al.; Cell suspensions from the fetal septal region were injected stereotaxically into the hippocampus of fornix-fimbria-transected adult rats . The host rats were sacrificed up to 3 months after the operation and the hippocampus sliced into 350 microns transverse slices . Intracellular recording was made from CA1 neurons adjacent to the graft . Electrical stimulation of the graft produced a voltage-dependent depolarization in some recorded neurons . This was associated with an increase in spontaneous and anodal break action potential discharges . In addition, a slow after-hyperpolarization (AHP) which typically follows a burst discharge was blocked during the depolarization indicating that the stimulation may block a Ca2+-dependent K+ current . The effects of the stimulation were antagonized by atropine . A response to the stimulation was seen 2 weeks but not 1 week after grafting . Over time, cells that were located away from the graft became activated by the stimulation . This was correlated with the extent of proliferation of acetylcholinesterase-containing fibers around the graft . These results suggest that grafted septal neurons make viable cholinergic connections with a host hippocampus. Biochemistry, 1985 Jun 4, 24(12), 2819 - 22 Fluorescence-detected circular dichroism of ethidium in vivo and bound to deoxyribonucleic acid in vitro; Lamos ML et al.; Fluorescence-detected circular dichroism (FDCD) spectra are reported for ethidium in Escherichia coli cells and bound to E . coli DNA in vitro . FDCD bands are observed at 325 and 385 nm . These bands change amplitude as the ethidium to DNA ratio changes . Spectra are similar for in vivo and in vitro measurements . However, the bands at 325 and 385 nm disappear when ethidium binds to macromolecules without intercalating between base pairs . The results demonstrate that FDCD spectra can be measured in cell suspensions and indicate that ethidium binds to nucleic acids in E . coli cells by intercalation. Eur J Biochem, 1985 Jun 3, 149(2), 411 - 9 L-Phenylalanine ammonia-lyase from Phaseolus vulgaris . Characterisation and differential induction of multiple forms from elicitor-treated cell suspension cultures; Bolwell GP et al.; L-Phenylalanine ammonia-lyase (EC 4.3.1.5) has been purified over 200-fold from cell cultures of bean (phaseolus vulgaris L.) exposed to elicitor heat-released from the cell walls of the phytopathogenic fungus Colletotrichum lindemuthianum . Four forms of the enzyme, with identical Mr but differing apparent pI values of 5.4, 5.2, 5.05 and 4.85, were observed following the final chromatofocussing stage of the purification . A preparation (purified 43-fold by ammonium sulphate precipitation, gel-filtration and ion-exchange chromatography) containing all four forms exhibited apparent negative rate cooperativity with respect to substrates . However, the individual forms displayed normal Michaelis-Menten kinetics, with Km values of 0.077 mM, 0.122 mM, 0.256 mM and 0.302 mM in order of decreasing apparent pI value . A preparation purified 200-fold and containing all four forms was used to immunise rabbits for the production of anti-(phenylalanine ammonia-lyase) serum . The antiserum was characterised by: immunotitration experiments; solid phase enzyme-linked immunosorbent assays; comparison of immunoprecipitates of 35S-labelled phenylalanine ammonia-lyase subunits (synthesized both in vivo and in vitro) on both one-dimensional and two-dimensional polyacrylamide gels after immunoprecipitation with the bean antiserum or antisera raised against pea and parsley phenylalanine ammonia-lyase preparations and immune blotting . SDS/polyacrylamide gels and SDS/polyacrylamide gel electrophoresis followed by immune blotting, indicated that the Mr of newly synthesized (in vivo and in vitro) bean phenylalanine ammonia-lyase subunits is 77000; a 70000-Mr form is readily generated as a partial degradation product during purification . Immunoprecipitates of bean phenylalanine ammonia-lyase synthesized both in vivo and in vitro showed the presence of multiple subunit types of identical Mr but differing in pI . Furthermore, treatment of bean cultures with Colletotrichum elicitor resulted in a 10-fold increase in phenylalanine ammonia-lyase extractable activity within 8 h, and chromatofocussing analysis indicated that this was associated with differential increased appearance of the high-pI, low-Km forms as compared to the two higher Km forms . This differential induction was further confirmed by immune blotting of crude extracts subjected to isoelectric focussing. Cancer Lett, 1985 Jun, 27(2), 171 - 9 Environmental temperature and metastatic spread of melanoma in the crested newt; Zavanella T; The influence of environmental temperature on tumor growth in newts injected subcutaneously with melanoma cell suspensions was studied . No signs of tumor growth were observed in animals kept at 4 degrees C, even after 1 year, whereas animals kept at 30 degrees C died after 2-4 weeks of widespread metastatic disease . In newts kept at both 17 degrees C and 27 degrees C and killed 25 days after the tumor grafting, blood-borne tumor emboli were often found . However, widespread metastases were present only in those kept at 27 degrees C . These findings suggest that by operating at a proper environmental temperature to slow down tumor growth, melanoma of the crested newt could serve as a useful experimental model for study of the different steps of metastatic spread. J Invest Dermatol, 1985 Jun, 84(6), 465 - 8 Immunogold technique applied to simultaneous identification of T6 and HLA-DR antigens on Langerhans cells by electron microscopy; Dezutter-Dambuyant C et al.; A double-labeling immunogold technique in electron microscopy and specific monoclonal antibodies to surface antigens of Langerhans cells (OKT6 and BL2) were applied to assess directly the coexpression of two cell surface antigens (T6 and HLA-DR antigens) in a heterogeneous epidermal cell suspension . Electron microscopic examination of double-labeled cells revealed that all Birbeck granule-containing Langerhans cells bound OKT6 and BL2 . The preparation of markers with colloidal gold particles and the procedure for double labeling are described . Several problems related to the steric hindrance and current artifacts are illustrated by micrographs and also discussed. J Natl Cancer Inst, 1985 Jun, 74(6), 1215 - 21 Effect of radiation on the production of interleukins and T-lymphocyte activities; Manori I et al.; The effect of 0-400 rad 60Co gamma-ray doses on distinct steps in the process of murine T-cell activation by concanavalin A (Con A) was investigated . When C57BL/6 spleen cells were stimulated immediately after irradiation, production of interleukin-1 (IL-1) and interleukin-2 (IL-2) was not impaired . Concomitantly, the display of IL-2 receptors in Con A-induced reactivity to IL-2 was not affected . The proliferative response was markedly diminished by increasing doses of radiation . The effect of radiation was found to depend not only on the delivered dose but also on the time interval between irradiation and stimulation of the lymphoid cultures . When the mitogenic stimulus was delayed for 24 hours following irradiation, IL-1 production was not diminished, whereas IL-2 production was impaired by doses greater than 200 rad . The proliferative response was diminished to a markedly higher degree as compared to the degree it was diminished in cell cultures stimulated by Con A immediately after irradiation . IL-2 production and the proliferative response to Con A of irradiated cell suspensions, cultured without mitogen for 24 hours post irradiation, were also assessed after adjustment for cell death . In this case, an impairment in IL-2 production that was dose dependent was apparent, but still the levels of IL-2 secreted by 400-rad irradiated cells reached high levels . In contrast, the proliferative response to Con A could not be restored . When T-cell growth factor was added concomitantly with Con A to irradiated cell cultures, a radioprotective effect could be observed. J Immunol, 1985 Jun, 134(6), 4001 - 8 Tissue localization and biochemical characteristics of a new thymic antigen recognized by a monoclonal thymocytotoxic autoantibody from New Zealand black mice; Bray KR et al.; Naturally occurring thymocytotoxic autoantibodies (NTA) have been described in both humans and mice with SLE . To define further the role of anti-thymic autoantibodies in murine lupus, we studied the cellular and molecular specificity of a spontaneous monoclonal NTA, designated TC-17, derived from a 4-mo-old New Zealand Black mouse . TC-17, an IgM autoantibody, has been shown previously to be unreactive with Lyt-1, Lyt-2, and L3T4 (T helper) antigens . We have shown further that it is also unreactive with Thy-1 . TC-17 recognizes a new thymic antigen that appears to mark a distinct subpopulation of cortisol-sensitive cortical thymocytes . The antigen consists of a single glycoprotein chain with an apparent m.w . of 88,000 . TC-17 shows reduced binding to thymocytes treated with tunicamycin, indicating either that glycosylation of TC-17 antigen is necessary for TC-17 to bind to it or that glycosylation is required for expression of the antigen on the cell surface . TC-17 uniquely reacts with two of 17 murine lymphoid tumor cell lines of intermediate cellular maturity . The thymocytotoxic activity of TC-17 is absorbed by single cell suspensions of murine stomach, small intestine, large intestine, kidney, and thymus . Moreover, the specific binding of TC-17 to gut tissue of normal and germfree mice can be demonstrated by indirect immunofluorescence, suggesting antigenic cross-reactions between thymic and gut tissue . TC-17 reacts with rat thymocytes as well as it does with murine cells, indicating moderate evolutionary conservation of the TC-17 antigen . The expression of this glycoprotein by a discrete thymocyte subset may prove to be a valuable probe for the study of murine T cell differentiation. J Anat, 1985 Jun, 140 ( Pt 4), 669 - 78 Sheet preparations of the stratum granulosum from mammalian skin and oral epithelium; Hume WJ et al.; Acetic acid has been used to obtain sheet preparations of the stratum granulosum from epidermis and oral epithelium of mouse, rat, hamster and man . Clear differences exist in the appearance of nuclei and keratohyalin granules and in the extent of cell overlapping . In the ventral surface of mouse tongue, the number of granular cells in a sheet preparation is relatively constant (average +/- S.D . = 1633 +/- 160/mm2) . Cell suspensions show a greater variation in the area and maximum diameter of the cells in ventral tongue compared to mouse ear epidermis . As might be expected, this is reflected in the pattern of stacking of suprabasal cells into discrete columns in ear, but not in ventral tongue epithelium . The ratio of granular to basal cells for different sites and species is fairly constant, with an average of approximately 10:1 . The pattern of nuclear breakdown can be followed, beginning in the middle of the nucleus and gradually extending outwards, often in an irregular manner until the entire nucleus is removed. J Bacteriol, 1985 Jun, 162(3), 1100 - 5 Cadmium uptake in Escherichia coli K-12; Laddaga RA et al.; 109Cd2+ uptake by Escherichia coli occurred by means of an active transport system which has a Km of 2.1 microM Cd2+ and a Vmax of 0.83 mumol/min X g (dry weight) in uptake buffer . 109Cd2+ accumulation was both energy dependent and temperature sensitive . The addition of 20 microM Cd2+ or Zn2+ (but not Mn2+) to the cell suspensions preloaded with 109Cd2+ caused the exchange of Cd2+ . 109Cd2+ (0.1 microM) uptake by cells was inhibited by the addition of 20 microM Zn2+ but not Mn2+ . Zn2+ was a competitive inhibitor of 109Cd2+ uptake with an apparent Ki of 4.6 microM Zn2+ . Although Mn2+ did not inhibit 109Cd2+ uptake, the addition of either 20 microM Cd2+ or Zn2+ prevented the uptake of 0.1 microM 54Mn2+, which apparently occurs by a separate transport system . The inhibition of 54Mn2+ accumulation by Cd2+ or Zn2+ did not follow Michaelis-Menten kinetics and had no defined Ki values . Co2+ was a competitive inhibitor of Mn2+ uptake with an apparent Ki of 34 microM Co2+ . We were unable to demonstrate an active transport system for 65Zn2+ in E . coli. Gen Comp Endocrinol, 1985 Jun, 58(3), 386 - 93 The endocrine activity of isolated follicular cells of the carp ovary in primary culture; Stoklosowa S et al.; Oocytes were isolated from the ovaries of carp females of one breed . Interstitial tissue was mechanically removed in order to obtain single oocytes surrounded only by the follicular envelope . Such a preparation was trypsinized at room temperature and also with greater success at 37 degrees C . The follicular cell suspension contained both cells and cell clumps . It was the mixture of theca (T) and granulosa (G) cells . The temperature of 37 degrees C was preferable for the successful growth of the cells in tissue culture . The cells were grown as monolayers in Leighton tubes in Medium 199 supplemented with calf serum . Activity of delta 5, 3 beta-hydroxysteroid dehydrogenase (delta 5, 3 beta-HSD) was detected in the growing cells by a histochemical test . Culture media were analyzed for estrogen, androgen, and progesterone content by appropriate radioimmunoassays . Follicular cells prior to and in culture exhibited activity of the delta 5, 3 beta-HSD and secreted steroid hormones into the culture medium. Proc Natl Acad Sci U S A, 1985 Jun, 82(12), 4268 - 9 Failure to demonstrate pluripotential hemopoietic stem cells in mouse brains; Hoogerbrugge PM et al.; Hemopoietic stem cells as defined by the capacity to produce spleen colonies in lethally irradiated recipients were reported by P . F . Bartlett {(1982) Proc . Natl . Acad . Sci . USA 79, 2722-2725} to be present in high frequencies in mouse brain . He also reported similar numbers of colony-forming units, spleen (CFU-s), in the brains of Wf/Wf mice, the bone marrow of which lacks detectable spleen colony-forming cells . To verify these observations, single cell suspensions were produced from murine brains by incubation with trypsin and DNase, followed by removal of myelin by Percoll gradient centrifugation . Two to 13 CFU-s were detected per brain . This low number suggested contamination of the brains by either blood or bone marrow leaking from the skull bones during dissection . When the isolated, intact brains were washed carefully in balanced salt solution, the recovered number of CFU-s decreased to 0.1-0.4 per brain . No CFU-s could be detected in the brains of W/Wv mice . It is concluded that the CFU-s observed by Bartlett in preparations of mouse brain did not originate from the brain tissue. Br J Cancer, 1985 Jun, 51(6), 865 - 75 Lung tumour cell lines synthesizing peptide hormones established from tumours of four histological types: characterization of the cell lines and analysis of their peptide hormone production; Luster W et al.; Thirty permanent and more than 60 primary tumour cell lines were established from pleural and pericardial exudates or wedge biopsies from human bronchial carcinoma . The permanent cell lines have their origin in 6 small cell, 5 large cell, 9 squamous and 5 adeno carcinomas of the lung . Tumour cells were purified from non tumour cells by direct cloning in fluid cultures or by soft agar cloning . In vitro secretion of ACTH, bombesin, calcitonin, and neurotensin was demonstrated for lung tumour cells belonging to the four major histological types . Cell suspensions of peptide hormone secreting permanent cell cultures were grown to solid tumours after xenotransplantation into nude mice . Comparative ultrastructural examination of the primary tumour and of cells grown in tissue culture and in xenografts demonstrated the preservation of most tumour type specific structural criteria in the ex vivo/in vitro systems . The present data show that not only tumour cells from small cell carcinoma but also from other histological types are capable of synthesizing a broad spectrum of immunoreactive peptide hormones . This result might be interpreted as indicating a common expression of hormone biosynthesis and secretion by all lung tumours. J Med Microbiol, 1985 Jun, 19(3), 391 - 400 Effect of prednisolone on the toxicity of Bordetella pertussis for mice; Parton R; Prednisolone, given orally or intraperitoneally before challenge, protected mice against the lethal effect of a crude cell extract of Bordetella pertussis containing heat-labile toxin (HLT) as the major toxic component . Prednisolone did not diminish the lethal toxicity of heated B . pertussis cell suspensions containing pertussis toxin and endotoxin but devoid of HLT . This suggests that the protective effect of the steroid was directed against the HLT . When live bacteria were injected intraperitoneally, prednisolone showed a protective effect against the initial toxaemia . By day 7, however, the protection was no longer evident and the steroid promoted the survival of the organisms within the peritoneal cavity . These findings are discussed in the light of reports of the beneficial effects of corticosteroids in the treatment of whooping cough and in relation to a possible role for HLT in the pathogenesis of the disease. Cancer Res, 1985 Jun, 45(6), 2560 - 6 Induction of highly immunogenic variants of Lewis lung carcinoma tumor by ultraviolet irradiation; Peppoloni S et al.; This study was undertaken to determine whether in vitro treatment of Lewis lung carcinoma (3LL) cells with ultraviolet (UV) radiation could increase their immunogenicity . Tumor cells were irradiated with UV light from a germicidal lamp (254 nm; UV-C) at a dose of 720 J/sq m . After 2 weeks of culture, the surviving cell population was cloned by limiting dilution . Cell suspensions of each clone were injected intrafootpad in C57BL/6 mice at a dose of 2.5 X 10(5) cells per mouse . Eighty independent clones were tested . Fifty-one clones showed decreased tumorigenicity and failed to grow in 20 to 95% of immunocompetent mice, whereas they produced tumors in 100% of irradiated (550 R) and athymic nude mice . These clones were designated "tum-" (nontumorigenic) clones . In contrast, all 25 clones selected from the untreated parental 3LL induced progressively growing tumors in 100% of the mice . After two courses of UV treatment, the uncloned 3LL population was rejected in 45% of inoculated mice . Mice rejecting an inoculum of a tum- clone were completely resistant to subsequent challenge with higher doses of the same or unrelated tum- clones . This resistance was fully expressed even after irradiation of immune mice with 550 R . Mice immune to a tum- clone also were able to prevent the growth of various tum+ clones or untreated 3LL tumor cells . When tum- and tum+ clone cells were simultaneously inoculated intrafootpad in opposite legs, rejection of tum- clone resulted also in the prevention of the growth of tum+ clone . Spleen cells of immune mice caused rapid elimination of radiolabeled 3LL tumor cells from the place of their inoculation (intrafootpad) and prevented tumor growth . In an in vitro cytotoxic assay, spleen cells after in vivo and in vitro immunization with tum- clones demonstrated high cytotoxic activity against various tum+ clones and parental 3LL cells, as well as against tum- clones . In addition, parental 3LL tumor cells and tum- cells were similarly able to inhibit cytotoxic activity in the cold target inhibition assay . However, in contrast to tum- cells, 3LL cells were less efficient in in vitro restimulation of cytotoxic activity of immune spleen cells . Therefore, these data suggest that tum-, tum+, and parental 3LL cells share a common antigenic specificity, which is not immunogenic in 3LL cells . UV treatment presumably converted the antigenic determinants present in the 3LL cells into an immunogenic form. Mech Ageing Dev, 1985 May 31, 30(3), 251 - 9 Impairment of T immunoregulatory activities in the induction of antibody specific response in aged humans; Antonaci S et al.; T helper (Th) and T suppressor (Ts) functions on the induction of specific antibody response have been studied in 80 aged individuals by means of a plaque-forming cell assay . Of the subjects 45.2% exhibited a reduction of Ts activity on Ig production by adding Concanavalin A (Con A) to cultures on day 0, while 35.7% of aged donors showed a decrease of Th functions by supplementation of Con A on day 2 . A small number of individuals displayed a combined deficit (Th + Ts) . Furthermore, these defects seem to be related to soluble suppressive factors which might adhere to cell surface . In fact, preincubation of peripheral blood mononuclear cells (PBMC) before their addition to cultures and resuspension in fresh medium normalized the immunoregulatory defects . On the other hand, overnight supernatants from old PBMC transferred to young PBMC cultures induced the same deficit observed in the aged cell suspensions . Finally, Zinc chloride supplementation to cultures was able to correct the deficient Th activity only . These data suggest an additional defect of immunoregulation in the elderly. Biochim Biophys Acta, 1985 May 28, 815(3), 477 - 85 Effects of preparative procedures on the volume and content of resealed red cell ghosts; Nash GB et al.; The effects of variations in preparative procedures on the volume and content of resealed red cell ghosts have been investigated . Following hypotonic lysis at 0 degrees C, and after a variable delay time (td), concentrated buffer was added to restore isotonicity; resealing was then induced by incubation at 37 degrees C for one hour . Using this procedure, both the resealed ghost volume and the residual hemoglobin (Hb) content decreased for increasing td . If ghosts were maintained at 0 degree C (i.e., no 37 degrees C incubation), they remained nearly spherical until isotonicity was restored . Their volume then fell abruptly, but subsequently increased toward an intermediate level . The fall in volume was greater and the final level achieved was smaller for longer delay times . At 0 degree C, return to isotonicity also halted the otherwise gradual loss of residual Hb from unsealed ghosts . In addition, ghosts with internal osmolality of 40 to 300 mosmol/kg were prepared by adding different amounts of concentrated buffer before resealing for one hour at 37 degrees C . Under these conditions, the final ghost volume was inversely related to the resealing osmolality (i.e., lower osmolality yielded a larger volume) . Ghost volume also increased, along with Hb content, if the quantity or concentration of the red cell suspension added to the lysing medium was increased . We conclude that resealed ghost volume is influenced by the ratio of lysate to resealing medium osmolality and by the colloid osmotic pressure of the residual ghost Hb . These data indicate methods by which ghosts with desired characteristics can be prepared, and have potential application for studies of ghost mechanical and biophysical behavior. J Immunol Methods, 1985 May 23, 79(2), 195 - 204 A filter immuno-plaque assay for the detection of antibody-secreting cells in vitro; Moller SA et al.; A plaque assay has been developed that is based on enzyme immunoassay principles and capable of screening several hundred samples in one day . Single cell suspensions of in vivo or in vitro immunized mouse splenocytes are incubated on antigen-coated nitrocellulose membranes in microfilter plates or in petri dishes . The antibody production of individual cells is detected using a horse radish peroxidase-labeled second antibody, and the insoluble products of the enzymatic reaction are visualized as blue plaques on the membranes . The nitrocellulose membrane of the microfilter plates, which readily absorb a variety of antigens, and the filtration unit used for the washing steps greatly facilitates the plaque assay . Furthermore, this procedure only needs small amounts of antigen for the enumeration of isotype-specific antibody-secreting cells in a defined medium containing low protein levels or in a completely serum-free medium . The plaque assay may be used to evaluate the optimal conditions required for in vitro immunizations. Anal Biochem, 1985 May 15, 147(1), 75 - 9 Elimination of the settling artifact in the nuclear magnetic resonance spectroscopy of living cells; Labotka RJ et al.; Nuclear magnetic resonance spectroscopy has become a powerful tool for metabolic investigations on living cell suspensions . However, unless mechanical means are used to maintain the cells in dispersion, settling occurs during the NMR experiment . Because high packed-cell volumes are generally used to produce maximum NMR signals, settling may be inapparent to the eye, leading to unrecognized artifactual changes in NMR spectra . Such artifacts include time-dependent loss of signal intensity when the sample volume approximates the sensitive volume of the NMR probe, and time-dependent increase in signal intensity when the sample volume exceeds the sensitive volume . Through the addition of the polysaccharide arabinogalactan, increasing the buoyant density of the suspending medium to approach that of the cells, we have eliminated cell settling and improved the quality of 31P NMR spectra of human erythrocytes. Anal Biochem, 1985 May 15, 147(1), 197 - 209 Nucleotide profiles of normal human blood cells determined by high-performance liquid chromatography; de Korte D et al.; An anion-exchange high-performance liquid chromatography method has been used to quantitate the intracellular purine and pyrimidine nucleotides in extracts of pure lymphocytes, monocytes, neutrophils, eosinophils, erythrocytes, and platelets isolated from the blood of healthy human donors . For accurate and reproducible measurements of the nucleotide profiles in different types of pure leukocytes, the cell suspensions have to be free of platelets and erythrocytes . Incubation of the purified leukocytes for 1 h at 0 degrees C did not alter the nucleotide concentrations but reduced the interdonor variation to 10% . Incubation of purified lymphocytes for 1 h at 37 degrees C caused considerable changes in the relative concentrations of the adenine, guanine, uracil, and cytosine nucleotides . During this incubation the cell viability, the cell number, and the ATP:ADP ratio decreased . Incubation of monocytes and granulocytes for 1 h at 37 degrees C caused considerable loss of cells and/or cell death . For erythrocytes and platelets reproducible nucleotide concentrations were obtained after extraction of freshly isolated cells . During storage of erythrocytes, both at 0 degrees C and at 37 degrees C, a decrease in the ATP:ADP ratio was detected . In all cell types the predominant nucleotides were purine nucleotides, especially adenosine triphosphate . The relative concentrations of the adenine, guanine, uracil, and cytosine nucleotides were very reproducible per cell type and appeared to be characteristic for each cell type . The total nucleotide content was nearly the same for all cell types except erythrocytes, when expressed per microgram of protein . The described methods for purification and storage of blood cells will be useful for comparison of blood cells from healthy donors with those of patients, for example, leukemia patients, in which deviations of the purine and pyrimidine metabolic enzymes have already been described. Thromb Res, 1985 May 15, 38(4), 341 - 51 Fibrinogen synthesis by megakaryocyte rich human marrow cell concentrates; Belloc F et al.; A rapid method is described for the production of a human marrow cell suspension highly enriched in megakaryocytes . These concentrates were incubated with radiolabelled amino-acids, and cell lysates were then analysed for fibrinogen synthesis . Neosynthesized proteins were detected by immunoprecipitation, immuno-affinity chromatography and electrophoresis . Fluorography of the electrophoresis gels showed three radioactive bands corresponding to the three chains of cold fibrinogen . Immunoblotting and autoradiography of bidimensional, nonreduced-reduced electrophoresis gels showed that these three proteins were joined by disulfide bonds in the cell . These results suggest that megakaryocytes synthesize fibrinogen, and imply that platelet fibrinogen is of megakaryocytic origin. Biochim Biophys Acta, 1985 May 14, 815(2), 223 - 32 Lead-induced activation and inhibition of potassium-selective channels in the human red blood cell; Shields M et al.; The selective increase of net K+ permeability in human red cells brought about by either Ca2+ or lead was studied using a light scattering technique to measure net K+ fluxes in cell suspensions and the patch-clamp technique to study K+ transport in individual K+-selective channels of the red cell membrane . Using ultrapure solutions it was demonstrated that the effect of lead is neither the indirect consequence of a lead-induced increase of the accessibility of the receptor sites of the K+-selective channels to traces of Ca2+ that are present as contamination in analytical grade reagents nor to the release of Ca2+ from intracellular Ca2+ stores . It is further shown that in cell-free membrane patches low concentrations of lead (10 microM) in Suprapur solutions evoke the same single-channel events as added Ca2+ and that this activity can be inhibited by high concentrations of lead (100 microM), similar to the net KCl efflux measured by means of the light scattering technique . It is concluded, therefore, that both Ca2+ and lead independently activate the same K+-selective channels in the red cell membrane. J Immunol Methods, 1985 May 10, 79(1), 79 - 88 Flow cytometry sorting of unlabelled epidermal Langerhans cells using forward and orthogonal light scatter properties; Cordier G et al.; A variety of techniques based on the presence of specific markers has been proposed to enrich Langerhans cells from epidermal cell suspensions . Computer analysis of multiparameter flow cytometry records involving forward angle and orthogonal scattered light and immunofluorescence of epidermal cells allowed us to determine the scattering properties of Langerhans cells . Unlabelled cells sorted according to these properties were shown to be Langerhans cells by electron microscopy and/or subsequent labelling by anti-HLA-DR monoclonal antibody . The relevance of this method is discussed to sorting viable Langerhans cells which may be used in functional studies and for establishing long-term culture. Eur J Biochem, 1985 May 2, 148(3), 571 - 8 Metabolic changes in elicitor-treated bean cells . Enzymic responses associated with rapid changes in cell wall components; Bolwell GP et al.; Treatment of cell suspension cultures of bean (Phaseolus vulgaris c.v . Immuna) with an elicitor preparation heat-released from the cell walls of the phytopathogenic fungus Colletotrichum lindemuthianum resulted in rapid changes in the composition of the bean cell walls . These consisted of (a) increases in phenolic material bound to the cellulosic and hemicellulosic fractions of the wall, (b) loss of material (mainly glucose) from the hemicellulosic fraction and (c) an increase in wall-associated hydroxyproline . The increases in wall-bound phenolics were preceded by (a) rapid decreases in the intracellular levels of free hydroxycinnamic acids and (b) transient increases in the extractable activities of L-phenylalanine ammonia-lyase and cinnamic acid 4-hydroxylase . 4-Hydroxycinnamic acid 3-hydroxylase activity was present at a high level in control cultures and was not induced by elicitor . Changes in the levels of cytochrome P-450, as determined by dot blot assays utilising an anti-(P-450) monoclonal antibody, paralleled the changes in cinnamic acid 4-hydroxylase activity . The accumulation of cell wall hydroxyproline was associated with rapid transient increases in the extractable activities of proline 2-oxoglutarate dioxygenase and a protein arabinosyl transferase . An hydroxyproline-rich acceptor protein of Mr 42 500 was the major protein to incorporate {3H}arabinose following elicitation of the bean cells, and the kinetics of the extent of labelling of this protein paralleled the accumulation of hydroxyproline protein in the endomembrane system . The above metabolic changes associated with cell wall components followed rapid kinetics similar to those involved in the formation of the phytoalexin kievitone in the elicited cultures {Robbins, M . P . et al . (1985) Eur . J . Biochem . 148, 563-569} . It is therefore concluded that increased 5-hydroxy-substituted isoflavonoid biosynthesis, wall-bound phenolic synthesis and synthesis of arabinosylated hydroxyproline-rich protein are all early events which are closely linked to the initial interaction between plant cell and fungal elicitor. Eur J Biochem, 1985 May 2, 148(3), 563 - 9 Metabolic changes in elicitor-treated bean cells . Selectivity of enzyme induction in relation to phytoalexin accumulation; Robbins MP et al.; Treatment of cell suspension cultures of Phaseolus vulgaris c.v . Immuna with an elicitor preparation heat-released from the cell walls of the phytopathogenic fungus Colletotrichum lindemuthianum resulted in rapid accumulation of the prenylated 5-hydroxyisoflavanone phytoalexin kievitone followed by later accumulation of the pterocarpan-derived phytoalexin phaseollin . Kievitone formation was preceded by rapid transient increases in the extractable activities of the enzymes L-phenylalanine ammonia-lyase and chalcone synthase . The extractable activities of 15 enzymes were measured in the cell cultures during the period of kievitone accumulation . The results suggest a highly selective induction of enzymes associated directly with the phytoalexin pathway . No induction of enzymes of pathways diverging from or providing substrates for the phenylpropanoid----isoflavonoid pathway was observed . The increase in glutamate dehydrogenase activity in control cultures was prevented by elicitor application . A comparison of enzyme activities in control and Colletotrichum-infected bean hypocotyls provided further evidence of the selective induction of enzymes of phytoalexin synthesis, although peroxidase, glutamate dehydrogenase and glutamate synthase activities were higher in infected than in healthy hypocotyls . It is concluded that the major enzymic changes occurring in elicitor-treated bean cells are probably those directly associated with defence mechanisms such as the formation of isoflavonoid phytoalexins (this paper) or accumulation of phenolic compounds and hydroxyproline-protein in the cell walls {Bolwell, G . P . et al . (1985) Eur . J . Biochem . 148, 571-578}. Eur J Biochem, 1985 May 2, 148(3), 545 - 50 ATP synthesis in Methanobacterium thermoautotrophicum coupled to CH4 formation from H2 and CO2 in the apparent absence of an electrochemical proton potential across the cytoplasmic membrane; Schonheit P et al.; Methanogenic bacteria are considered to couple methane formation with the synthesis of ATP by a chemiosmotic mechanism . This hypothesis was tested with Methanobacterium thermoautotrophicum . Methane formation from H2 and CO2 (2.5 - 3 mumol X min-1 X mg cells-1) by cell suspensions of this organism resulted in the formation of an electrochemical proton potential (delta mu H +) across the cytoplasmic membrane of 230 mV (inside negative) and in the synthesis of ATP up to an intracellular concentration of 5 - 7 nmol/mg . The addition of ionophores at concentrations which completely dissipated delta mu H + without inhibiting methane formation did not result in an inhibition of ATP synthesis . It thus appears that delta mu H + across the cytoplasmic membrane is not the driving force for the synthesis of ATP in M . thermoautotrophicum. Eur J Cell Biol, 1985 May, 37, 111 - 6 Isolation of mouse megakaryocytes . I . Separation of two fractions enriched in different maturational stages; Raha S et al.; Megakaryocytes (MK) were isolated from mouse bone marrow by centrifugation on discontinuous gradients of isotonic albumin or Percoll and characterized by acetylcholinesterase (AChE) staining . The apparent density distributions of MK varied greatly depending upon the nature of the gradient medium and the composition of the cell suspension buffer while the density range of the other bone marrow cells remained largely unchanged . The present findings also indicate that the unusual morphological and functional characteristics of MK may underlie the observed shift in their density profile . Thrombocytopoietic stimulatory factor (TSF) was administered to mice 18 h before killing to elevate the normally low numbers of earlier MK in the bone marrow and to improve the yield of immature MK during the subsequent isolation procedure . Cells belonging to earlier stages in maturation were separated from the more mature ones on discontinuous Percoll density gradients, providing a basis for further investigation of MK development. Scand J Gastroenterol, 1985 May, 20(4), 508 - 11 On the mode of action of the pentagastrin test in the carcinoid syndrome; Gronstad KO et al.; Provocation with pentagastrin (PG) (0.6 micrograms/kg intravenously) causes a release of serotonin (5-hydroxytryptamine (5-HT} in excess of the metabolizing capacity associated with carcinoid symptoms and a moderate fall in systemic arterial blood pressure in patients with midgut carcinoids and hepatic metastases . In this study PG also caused a release of 5-HT into portal circulation of anesthetized cats with stable peripheral levels of 5-HT in whole blood, indicating an effective hepatic metabolization . A similar response was obtained in the same animal when the PG test was repeated after 3 h . PG provocation was also performed in animals before and after adrenalectomy . Adrenalectomy seems to prevent the PG-induced release of 5-HT into portal circulation, indicating involvement of an adrenal mechanism . PG does not induce 5-HT release from cell suspensions of midgut carcinoid tumors, but such release was induced by incubation with adrenoceptor agonists . These findings indicate the presence of adrenoceptors on carcinoid tumor cells . The mode of action of the PG test may therefore be activation of such adrenoceptors by catecholamines, released from the adrenal medulla at the fall of arterial blood pressure at PG provocation. Acta Paediatr Scand, 1985 May, 74(3), 378 - 81 Red cell membrane lipid peroxidation and hemolysis secondary to phototherapy; Ostrea EM Jr et al.; The exposure of red cells to phototherapy light in the presence of a sensitizer (bilirubin) resulted in oxidative injury to the red cell membrane as manifested by a significant increase in the concentration of the products of lipid peroxidation (TBA reactants and diene conjugation) in the membrane and hemolysis . To induce a photo-oxidized membrane injury, the sensitizer (bilirubin) has to be membrane bound . Thus, by altering the availability of free bilirubin in the red cell suspension through changes in the molar concentration ratio of bilirubin to albumin, one is able to regulate the occurrence and extent of the oxidative red cell membrane injury . The clinical implications of these findings are discussed. Scand J Immunol, 1985 May, 21(5), 479 - 84 Biology of human epidermal Langerhans cells: cell cycle studies; Czernielewski J et al.; Langerhans cells (LC) are considered to play an important role in the initiation of the immune response in the skin . This study was performed to analyse the kinetics of LC in normal human epidermis . Using flow cytometry (FCM), we have applied three methods to estimate LC DNA distribution: FCM DNA measurement in LC-enriched suspensions (70-90% purity), FCM-correlated analysis of DNA and OKT6-positive cells in original epidermal cell suspensions, and staining of LC-enriched suspensions by the Feulgen technique on microscopic slides and counter labelling of contaminating keratinocytes with anti-keratin antiserum to eliminate them from the LC DNA estimation . All three methods clearly showed that human LC are a cycling cell population, and it was suggested that LC may represent a stable, self-reproducing cell population in normal epidermis. J Invest Dermatol, 1985 May, 84(5), 424 - 6 Epidermal Langerhans cells--a cycling cell population; Czernielewski J et al.; The limited number of Langerhans cells (LC) in human epidermis and the resultant technical difficulties have left open the question of LC kinetics . In the present study using flow cytometry (FCM) we have applied 3 methods to estimate LC-DNA distribution: (1) FCM-DNA measurement on highly enriched LC suspensions, (2) FCM-correlated analysis of DNA and OKT-6(+) cells in total epidermal cell suspensions, (3) LC-enriched suspensions (70-90%) were FACS (fluorescence-activated cell sorter) sorted on microscopic slides, and stained with the Feulgen technique, and DNA was measured densitometrically . In the latter method, contaminating keratinocytes were counterlabeled with antikeratin serum to eliminate them from LC-DNA estimation . All 3 in vitro analyses clearly showed that human LC are a cycling cell population in the epidermis . The number of LC in S (1.3-3.3%) and G2/M (1.0-2.5%) phase compares with those found for keratinocytes . Assuming that this percentage of keratinocytes in S and G2/M phases is sufficient to maintain the structural integrity of the epidermis, it was suggested that LC may represent a stable, self-reproducing cell population in normal epidermis. Cytometry, 1985 May, 6(3), 238 - 53 Flow cytometric analysis of mouse spermatogenic function following exposure to ethylnitrosourea; Evenson DP et al.; The effects of the mutagenic agent ethylnitrosourea (ENU) on spermatogenic function and sperm chromatin structure were studied by flow cytometry and the results compared with sperm head morphology measurements . Groups of mice received daily exposures ranging from 0 to 75 mg/kg body weight X 5 days and were sacrificed 28 days later . Fresh testicular cell suspensions and epididymal sperm were stained with acridine orange (AO) and measured by flow cytometry . Sperm nuclei were isolated, fixed, rehydrated, and then either subjected to thermal stress or not prior to staining with AO . Body weights were unaffected by the chemical exposure while the testicular weights were reduced by about 50% . Two-parameter (DNA, RNA) flow cytometry measurements showed a dose-response relationship in the loss of certain cell types, particularly the elongated spermatids, from the testes of treated animals . Flow cytometric analysis of both heat-stressed and non-heat-stressed nuclei showed a relationship between dosage and the coefficient of variation of alpha t {red/(red + green fluorescence)} measurements of AO stained nuclei, thereby demonstrating that alterations of chromatin structure occurred in response to ENU . Enzymatic digestions with RNAse, DNAse, and nuclease S1 suggest that the increase in red fluorescence is due to an increase of single-stranded DNA induced by heat or acid treatment of chemically altered chromatin structure . The lowest daily dosage used (5 mg/kg) caused no significant changes in ratios of testicular cell types, a questionable increase in abnormal sperm head morphology and a detectable change in chromatin structure expressed as alpha t . This report shows that our technique for assaying sperm nuclear chromatin structure appears to have the same level of sensitivity to ENU induced nuclear alterations as the sperm head morphology test. Biochem Pharmacol, 1985 May 1, 34(9), 1449 - 55 Cellular activation of diaziquone {2,5-diaziridinyl-3,6-bis (carboethoxyamino)-1,4-benzoquinone} to its free radical species; Gutierrez PL et al.; Human leukemic cell lines K562 and HL60, and the murine leukemic cell line L1210, reduce Diaziquone (AZQ) (NCS182986) to its free radical anion . With all cell lines, the free radical was observed immediately in both aerobic and anaerobic cell suspensions . The steady-state concentration of AZQ free radicals was approximately 1% of the total AZQ concentration . L1210 cells treated with azide reduced AZQ, but cells treated with diamide and N-ethylmaleimide did not . NADPH and L-cysteine reduced AZQ . The latter did so under anaerobic conditions; the former did so under both anaerobic and aerobic conditions. Cancer Res, 1985 May, 45(5), 2128 - 31 Growth kinetics of human colorectal carcinoma; Ota DM et al.; In this study, we investigated the influence of some of the variables of the thymidine labeling index (TLI)