Suppression of lymphocyte proliferation by proteins secreted by cultured Sertoli cells.

Secreted proteins from cultured rat Sertoli cells were assessed for effects on phytolectin-stimulated rat splenic lymphocytes. Sertoli cell proteins (SCP) suppressed DNA, RNA and protein synthesis in stimulated rat splenic lymphocytes whether added at 0, 4, 24 and 48 h after culture initiation. SCP preparations were not toxic to cells. SCP suppressive activity was heat stable but was not associated with the carbohydrate component of SCP preparations. SCP also suppressed the proliferation of lymphoid and non-lymphoid cell lines from several different animal species but did not inhibit proliferation-independent lysis of YAC-1 target cells by rat natural killer cells. These results suggest that Sertoli cells synthesize inhibitory factors that might be secreted into seminal plasma. Furthermore, our results demonstrate that one mode of action of these factors is suppression of cell proliferation.

Bovine interleukin 2: regulatory mechanisms.

A cDNA clone of the bovine interleukin-2 (IL-2) gene has been isolated and demonstrated to produce a functional bovine IL-2 protein when transfected into either CV-1 or COS-1 monkey cells. Homology comparisons of both the nucleotide and predicted amino acid sequences of bovine IL-2 with those of the human and mouse show extensive regions of sequence conservation between the species. The amino acid sequence of the mature bovine IL-2 protein shares about 60-63% homology with those of the human and mouse, but the 3' untranslated regions of the human and mouse gene share as much, if not greater, sequence homology with the 3' untranslated regions of the human and mouse genes. In particular, a tandemly repeated sequence (TATT), n, found in the 3' untranslated tail of the bovine IL-2 clone is also found in the 3' untranslated region of a large group of cytokine genes and other inducible genes of the lymphoid and immune response systems. This sequence may serve a specific regulatory function in the immune system.

Metal ion binding to tetrameric lima bean lectin.

The binding of Mn2+ and Ca2+ to tetrameric lima bean lectin has been examined by equilibrium dialysis and magnetic resonance techniques. Demetalized lectin prepared by acid treatment binds either 1 Mn2+ or 2 Ca2+/monomer. When demetalized lectin is presaturated with Ca2+, only 1 Mn2+ binds per dimer. Water proton relaxation rate enhancements and Mn2+ electron spin resonance spectra were used to monitor metal ion association processes. Following Mn2+ binding to demetalized lectin, a conformational change with activation energy of 16 kcal/mol was detected; this is similar in magnitude to that observed for a conformational change with the lectin concanavalin A. The pH dependence suggests that a histidine residue is involved. ESR spectroscopy shows clearly that 1 Mn2+ binds to each demetalized subunit, but that Ca2+ induces dissociation of half the Mn2+; this result is in agreement with the equilibrium dialysis studies.

Role of second metal ion in establishing active conformations of concanavalin A.

The stoichiometry of Mn2+ binding to concanavalin A was found to be influenced by temperature, pH, and the presence or absence of saccharide. Demetalized concanavalin A binds one Mn2+ (S1 site) at 5 degrees C, pH 6.5, and two Mn2+ at 25 degrees C (S1 and S2 sites). The association constants for Mn2+ are 6.2 x 10(5) and 3.7 x 10(4) M-1 for the S1 and S2 sites, respectively, at 25 degrees C. Concanavalin A with one Mn2+ bound per monomer remains in an open conformation and exhibits a relatively high water proton relaxation rate. Concanavalin A with two Mn2+ ions remains in a closed conformation characterized by a lower relaxation rate. The rate of binding of the second Mn2+ to concanavalin A as determined by ESR and the rate of conversion of open form to closed form (folding over) as determined by proton relaxation rate measurements gave an identical rate constant of 80.0 +/- 5.8 M-1 h-1 at 17 degrees C. Ca2+, Sr2+, and high levels of methyl alpha-D-mannopyranoside also induce folding of concanavalin A. Ca2+ is not catalytic but stoichiometric in causing the folding. Mn2+ in the S1 site can be displaced by Ni2+, Co2+, and Zn2+, and Mn2+ in the S2 site can be displaced by Ca2+ and Sr2+. Concanavalin A with Ni2+, Co2+, Zn2+, or Mn2+ in the S1 site and Ca2+ or Sr2+ in the S2 site has a higher affinity for methylumbelliferyl alpha-D-mannopyranoside than Ni-Mn-, Co-Mn-, Zn-Mn-, and Cd-Cd-concanavalin A.

Light-induced, carrier-mediated transport of tetracycline by Rhodopseudomonas sphaeroides.

Tetracycline accumulation by the phototrophic bacterium Rhodopseudomonas sphaeroides has been studied, using the fluorescence properties of the antibiotic and measuring uptake of [7- 3H]tetracycline. Accumulation was carrier mediated, with a Km of approximately 300 micronM. Efflux also appeared to be carried mediated, with a Km of 25 mM. Chlorotetracycline competitively inhibited tetracycline transport. The transport was energy dependent. Efflux occurred during the influx process, and an energy-requiring steady state was reached when influx balanced efflux. Transport was inhibited by metabolic inhibitors such as antimycin A, cyanide, and iodoacetate. Proton conductors such as carbonylcyanide m-chlorophenyl hydrazone were strongly inhibitory. Efflux was not energy dependent. Efflux is partially blocked by mercuric ions and completely blocked by an external pH of 9 to 11. Although efflux rates increased continuously with lowering of the pH, influx rates have a sharp maximum at pH 7.

Light-induced tetracycline accumulation by Rhodopseudomonas sphaeroides.

Light has been used as a primary energy source in studies of tetracycline transport by Rhodopseudomonas sphaeroides. Accumulation of the antibiotic occurs in light, while efflux occurs in dark. Both fluorescence enhancement and radioisotopic tracing have been used to monitor transport. Km's obtained from both techniques are similar. Light-induced accumulation of tetracyclines is inhibited by a variety of inhibitors, including antimycin A, N-ethylmaleimide, carbonylcyanide m-chlorophenylhydrazone, and 2,4-dinitrophenol. A rapid efflux is observed after loading when cells are placed in the dark or treated with inhibitors.

Production and characterization of bovine interleukin-2.

A lymphokine analogous to interleukin-2 from other species was generated from bovine suprapharyngeal lymph nodes and characterized biochemically. The isolated material can support the long-term growth of concanavalin A (Con-A) or mixed lymphocyte reaction (MLR)-activated bovine T cells. The material elutes from DEAE-Sephadex at a low salt concentration, approximately 0.075 M, and exhibits molecular weight of approximately 25,000 on Sephadex G-100. When subjected to analysis by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), three peaks of activity were observed, corresponding to molecular weights of 14,400, 16,800 and 20,200. Finally, the isolated material exhibited a marked heterogeneity when subjected to chromatofocussing. Three major peaks of activity, with pIs of approximately 5.95, 5.41 and 5.0 are present with peaks of lesser activity at pIs of 5.82, 5.70, 4.73 and 4.20.

Mn2+ and Ca2+ binding to the lima bean lectins.

We have purified the two lectins from lima beans (Phaseolus lunatus) and studied the binding of Ca2+ and Mn2+ ions to each. The 120,000-dalton tetrameric lima bean lectin (LBL4) and the 240,000-dalton octamer (LBL8) were purified by chromatography on Ultrogel and sulfopropyl Sephadex. Using equilibrium dialysis, we have found that demetallized LBL4 (apo-lbl4) will bind either 2 Mn2+ or 4 Ca2+ ions. The Ca2+ binding is strongly cooperative while the Mn2+ binding is not. If apo-LBL4 is presaturated with Mn2+ (Mn2+-LBL4), it will bind only 2 Ca2+ ions in a noncooperative manner. Apo-LBL4 presaturated with Ca2+ (Ca2+-LBL4) does not bind Mn2+. The metal stoichiometry for LBL8 is double that for LBL4 under all of the above conditions.

Concept and progress of a regional effort to improve blood lead reporting to six Western States by incorporating electronic laboratory reporting.

While electronic laboratory reporting (ELR) has the potential to be both more timely and complete than non-electronic data transmission and direct electronic data transfer can also reduce data input errors, these benefits are often underutilized. A survey of states in HHS Regions IX and X (Alaska, Arizona, California, Hawaii, Oregon, Washington) led to collaborative efforts to maximize ELR benefits on a regional scale. Collaboration outcomes included the ratification of a regional blood lead HL7 message format and the formation of a multi-state committee to address reporting discrepancies by the large regional labs to multiple states in the regions.

Trypanosoma brucei: a fluorescence and spin label study of the membranes of the bloodstream form.

Fluidity of the plasma membrane of Trypanosoma brucei brucei has been examined with fluorescence and electron spin resonance spectroscopy. Fluorescent probes 1,6-diphenyl-1,3,5-hexatriene and 8-anilino-1-naphthalene sulfonate and the spin label probe 5-doxyl stearate have been employed to examine fluidity under a variety of conditions. The temperature dependence of 8-anilino-1-naphthalene sulfonate polarization and of the order parameter S for 5-doxyl stearate reveals phase alterations near 30 C. 1,6-Diphenyl-1,3,5-hexatriene polarization shows that proteolysis of the surface glycoprotein with trypsin increases fluidity but treatment with human serum which is trypanocidal produces no detectable change in membrane fluidity.

The role of tumor-cell surface carbohydrate in experimental metastasis.

Quantitative studies on the binding of concanavalin A (Con A) and wheat-germ agglutinin (WGA) to a series of rat hepatocarcinoma metastatic variants revealed a positive correlation between the amount of cell-surface-bound lectin and lung colonization potential. Scatchard analysis of Con A and WGA binding to 10 individual clones isolated from a subcutaneous (s.c.) tumor transplant and to tumor-cell isolates from 10 individual spontaneous lung metastases from the same animal showed diverse binding characteristics for these cell populations. Nevertheless, the expression of Con A receptor sites accurately predicted the lung colonization potential of 3 isolates from the lung metastases. Higher lectin binding curves were observed for the clones from the subcutaneous tumor than for the isolates from lung metastases. These data suggest that a high Con-A binding potential is indicative of a high lung colonization potential for these hepatocarcinoma cells, but that this phenotype may be rapidly lost during tumor outgrowth in the lungs. The binding of tumor cells to vascular endothelial cell monolayers was inhibited in the presence of Con A; however, no inhibition was observed with 2 other lectins. Attachment of tumor cells to endothelial cell monolayers was also inhibited by the monosaccharides methyl alpha-D-mannopyranoside and N-acetyl-D-galactosamine. Other monosaccharides tested did not alter the attachment of tumor cells to endothelial cell monolayers.

Decreased osmotic fragility in heritable canine myopathy.

Osmotic fragility was examined in red blood cells from dogs with a heritable muscle disorder that clinically resembles a muscular dystrophy. Several erythrocyte abnormalities have been reported in patients with certain forms of muscular dystrophy and it is thought that these changes reflect genetically induced alterations in the plasma membrane. It is believed that the examination of erythrocytes may eventually lead to the understanding of membrane involvement in muscle disorders. In this study, the mean osmotic fragility was found to be significantly lower in affected cells than in normal cells. These differences were maintained regardless of changes in incubation temperature (5 degrees, 20 degrees, or 35 degrees C) and pH (6.5, 7.0, 7.5, or 8.0). Quantitative analysis of glycolytic metabolites and adenine nucleotide concentrations revealed little variance between erythrocytes from normal and affected animals. Similarly, the pattern of membrane protein phosphorylation in intact erythrocytes from affected animals did not differ from that observed when erythrocytes from normal animals were examined. Of the red cell indices measured, the erythrocyte count in affected animals was moderately increased, but both the mean corpuscular volume and mean corpuscular hemoglobin content were significantly reduced. From these data it is concluded that the decrease in osmotic fragility cannot be explained by differences in cell metabolism or energy production. However, the decrease in affected cell mean corpuscular volume and mean corpuscular hemoglobin content may be correlated with the decrease in osmotic fragility in a manner similar to that observed in the hemolytic disorder of beta-thalassemia.

Kinetic analysis of tetracycline accumulation by Streptococcus faecalis.

Tetracycline transport by Streptococcus faecalis occurs by an energy-dependent, carrier-mediated process. The Michaelis constant for transport was unchanged, but the maximal velocity was increased when an energy source, glucose, was present. Accumulation levels, sometimes 25-fold greater than the external concentration, were controlled by the transport system.

Membrane lateral phase separations and chlortetracycline transport by Bacillus megaterium.

Chlortetracycline, a fluorescent probe of its own active transport, has been used to study lateral phase separations of membrane lipid in Bacillus megaterium cells. Arrhenius plots of initial accumulation rates are triphasic, with transitions or characteristic temperatures of 20 degrees and 9.5 degrees . At the higher temperature, the mobility of the chloretracycline, as measured by fluorescence polarization, is markedly altered. Chlortetracycline transport exhibits saturation kinetics, and fluorescence energy transfer from protein to bound antibiotic can be observed. N-Phenyl-1-naphthylamine, a lipophilic fluorescent probe, responds to changes in the hydrophobic regions of the membrane that are distinct from membrane protein. The fluorescent properties of N-phenylnaphthylamine in partitioning and polarization experiments are altered most significantly at the lower characteristic temperature. No fluorescence energy transfer between N-phenylnaphthylamine and membrane protein or bound tetracycline can be detected. In correlative electron spin resonance experiments on the partitioning of a lipid-soluble spin label, the same characteristic temperatures detected in the fluorescence studies were measured. These data suggest that different probes may respond to either or both of the characteristic temperatures describing the lateral phase separation. Between these characteristic temperatures the chlortetracycline transport system is most intimately associated with relatively immobile lipids that are surrounded by a more mobile lipid phase.

Streptococcus faecalis proton gradients and tetracycline transport.

The transport of chlortetracycline by Streptococcus faecalis is energy dependent. Addition of glucose to energy-depleted cells enhances both the transport rates and accumulation levels. Transport rates can be altered independently of glucose by treating cells with ionophores that increase or decrease the proton gradient. The transport of the antibiotic is linked only to the transmembrane pH difference, delta pH, and not the transmembrane electrical potential, delta psi. This conclusion was verified by quantitative measurements of delta pH, delta psi, and tetracycline accumulation levels. A linear correlation between delta pH and the tetracycline electrochemical potential was observed. Tetracycline most likely accumulates by the symport of protons in which the protons are bound to an anionic form of the antibiotic to form an uncharged molecule.

Calorimetric study of carbohydrate binding to concanavalin A.

Flow microcalorimetry has been used to examine the delta H of binding of two types of saccharides, a series of simple monosaccharides and a series of alpha-(1----4)-linked glucosides, to the lectin Concanavalin A. It has been found that the delta H decreases with any change in the stereochemistry of a hydroxyl group relative to methyl alpha-D-mannopyranoside. The data have allowed the calculation of the relative contribution of two of the hydroxyl groups. The delta H's of binding for the alpha-(1----4)-linked glucosides are approximately 31 kJ/mol, and the apparent association constants vary insignificantly with increasing length. This result indicates that only one glucose residue binds to concanavalin A by hydrogen bonds, and that the additional glucose residues have no interaction either by hydrogen bonds or by nonspecific hydrophobic interactions. This result confirms the absence of an extended binding site for alpha-(1----4)-linked glucopyranosides, in contrast to that proposed for alpha-(1----2)-linked mannopyranosides which show an increase in apparent association constants with increasing length.

A comparison of the interactions of the mitogenic and nonmitogenic lima bean lectins with human lymphocytes.

Binding and mitogenic characteristics of the 2 Phaseolus lunatus (lima bean) lectins have been examined with human peripheral blood lymphocytes. Chemical cross-linking of the nonmitogenic lima bean lectin produced a species that stimulated human lymphocytes as well as or better than the mitogenic lima bean lectin, which is a T lymphocyte mitogen with requirement for monocyte participation. The maximal stimulation and the dose response to the cross-linked lima bean lectin did not significantly differ from that observed with the mitogenic lectin. We have used fluorescein-labeled lima bean lectins to show that both lectins share mutually exclusive binding sites on lymphocytes. Our results strongly support the concept that multiple valence of lectins is important in inducing mitogenesis. Both the mitogenic and nonmitogenic lectins demonstrated selective binding by labeling only 70% od human peripheral blood lymphocytes. The fraction not binding lectin is a population of T lymphocytes. Competitive binding studies with the lima bean lectins and other N-acetyl-D-galactosamine-specific lectins suggest that the cell surface receptors for these various lectins may be quite different. We have also studied the response of human lymphocytes to the lima bean lectins and soybean agglutinin after neuraminidase treatment. As previously demonstrated (22), neuraminidase treatment of the cells drastically altered the binding and mitogenic response to soybean agglutinin. Lima bean lectin binding to the treated cells was also markedly increased, but the mitogenic response was essentially unaffected.

Microflora of partially processed lettuce.

Bacteria, yeasts, and molds isolated from partially processed iceberg lettuce were taxonomically classified. The majority of bacterial isolates were gram-negative rods. Pseudomonas, Erwinia, and Serratia species were commonly found. Yeasts most frequently isolated from lettuce included members of the genera Candida, Cryptococcus, Pichia, Torulaspora, and Trichosporon. Comparatively few molds were isolated; members of the genera Rhizopus, Cladosporium, Phoma, Aspergillus, and Penicillium were identified.

Evidence for two discrete phases of IL-2 production in bovine lymphocytes.

Regulation of IL-2 production in Con A-stimulated bovine lymph node cells was studied by following the time course of IL-2 synthesis and secretion over a 24-h period. A biphasic time course curve of IL-2 secretion was observed after stimulation with Con A alone or with Con A plus PMA. Both phases of IL-2 production were confirmed by three separate biochemical techniques: IL-2 protein bioassay, mRNA Northern blots, and anti-IL-2 antibody detection. Bovine IL-2 transcripts were detected as early as 2 h after Con A stimulation. The early phase of IL-2 protein secretion was initially detected 3 h after Con A stimulation, and the late phase occurred between 10 and 16 h. Both phases of IL-2 production were enhanced when PMA was added to the Con A stimulation. Each phase of IL-2 protein secretion was preceded by the accumulation of IL-2 mRNA.