Studies on the biotin-binding sites of avidin and streptavidin. Tyrosine residues are involved in the binding site.

1. Rat pancreatic islets were isolated and then maintained in culture for 2-4 days before being incubated in groups of 100 in the presence of different glucose (0-20 mM) or CaCl2 (1.2-4.2 mM) concentrations, or with uncoupler. 2. Increases in extracellular glucose concentration resulted in increases in the amount of active, non-phosphorylated, pyruvate dehydrogenase in the islets, with half-maximal effects around 5-6 mM-glucose. Increasing extracellular glucose from 3 to 20 mM resulted in a 4-6-fold activation of pyruvate dehydrogenase within 2 min. 3. The total enzyme activity was unchanged, and averaged 0.4 m-unit/100 islets at 37 degrees C. 4. These changes in active pyruvate dehydrogenase were broadly similar to changes in insulin secretion by the islets. 5. Increasing extracellular Ca2+ or adding uncoupler also activated pyruvate dehydrogenase to a similar degree, but only the former was associated with increased insulin secretion.     

Effects in vitro of alloxan on the glucose metabolism of mouse pancreatic B-cells

To facilitate detailed studies of the B-cytotoxic action of alloxan we developed a model using isolated pancreatic islets of normal mice. An essential feature of this model is the low temperature employed during exposure to alloxan, which minimizes the degradation of the drug. The islets were incubated with alloxan for 30min at 4 degrees C and subsequently various aspects of their metabolism were studied. The O(2) consumption was measured by the Cartesian-diver technique. Islets exposed to 2mm-alloxan and control islets had the same endogenous respiration, whereas the O(2) uptake of the alloxan-treated islets was inhibited and that of the control islets stimulated when they were incubated with 28mm-glucose as an exogenous substrate. The islet glucose oxidation was estimated by measurement of the formation of (14)CO(2) from [U-(14)C]glucose at 37 degrees C. Compared with the controls, alloxan-treated islets showed a decrease in the glucose-oxidation rate in a dose-dependent manner. Pretreatment of the islets with 28mm-glucose for 30min at 37 degrees C completely protected against this effect, whereas preincubations at glucose concentrations below 16.7mm failed to exert any protective effect. The glucose utilization was estimated as the formation of (3)H(2)O from [5-(3)H]glucose. Alloxan (2mm) failed to affect islet glucoseutilization rate in the presence of either 2.8 or 28mm-glucose. In contrast, islets exposed to 5 or 10mm-alloxan exhibited lowered glucose utilization. It is concluded that in vitro alloxan has an acute inhibitory effect on the islet glucose metabolism, and that this action can be prevented by previous exposure to a high glucose concentration. The results are consistent with the idea that the B-cytotoxicity of alloxan reflects an interaction with intracellular sites involved in the oxidative metabolism of the B-cell.     

Sequential cold-sensitive mutations in Aspergillus fumigatus.

Mutants of thermotolerant fungus Aspergillus fumigatus I-21 (ATCC 32722) unable to grow at 37 degrees C were sought. Cold-sensitive mutants were enriched from progeny spores of gamma-irradiated conidia by two or more incubations at various nonpermissive temperatures alternating with filtrations through chessecloth. The approximate minimum, optimum, and maximum growth temperatures of the parent were 12, 40, and 50 degrees C, respectively. Mutants unable to grow at 37 degrees C were not successfully isolated directly from the wild type. A mutant unable to grow at 25 degrees C was isolated and mutations further increasing the cold sensitivity by increments of 3-5 degrees C were found to occur. Mutants completely unable to grow at 37 degrees C were obtained by five sequential mutations. All mutants grew as fast as the wild-type parent at 45 degrees C and higher. Each mutant produced revertants able to grow not only at the nonpermissive temperature used for its isolation but also at lower temperatures.     

Degradation of asialoglycoproteins mediated by the galactosyl receptor system in isolated hepatocytes. Evidence for two parallel pathways

The ability of rat hepatocytes to degrade internalized surface-bound 125I-asialoorosomucoid (ASOR) was determined by measuring the appearance of acid-soluble radioactivity at 37 degrees C. The degradation kinetics were biphasic in cells previously equilibrated at 37 degrees C for 1 h or cultured for 24 h. Degradation began immediately and was linear for at least 20 min after which the rate increased to a steady state value 3-4 times greater than the initial rate. We previously showed that hepatocytes have two functionally distinct populations of galactosyl receptors that mediate ligand dissociation by two kinetically different pathways (Weigel, P. H., Clarke, B. L., and Oka, J. A. (1986) Biochem. Biophys. Res. Commun. 140, 43-50). The activity of one receptor population, designated State 2 galactosyl receptors, can be reversibly modulated by incubating cells between 22 and 37 degrees C and is not expressed on the surface of freshly isolated cells. When 125I-ASOR was prebound to freshly isolated cells at 4 degrees C and degradation was assessed subsequently at 37 degrees C, the kinetics were monophasic, not biphasic. Degradation of the surface-bound 125I-ASOR began immediately and was greater than 90% complete by 6 h. Freshly isolated cells were incubated at temperatures between 22 and 37 degrees C, chilled to 4 degrees C, allowed to pre-bind 125I-ASOR, and then incubated at 37 degrees C. As the State 2 galactosyl receptor population increased, the kinetics of degradation became progressively more biphasic and the rate of the delayed degradation process increased. This effect could be reversed in cells in culture or in suspension by down-modulating surface receptor activity at temperatures below 37 degrees C; only the degradation process appearing after a 20-min lag was affected. Degradation in both pathways is an apparent first order process with identical rate constants (kappa = 0.006 min-1, t1/2 = 116 min). We conclude that there are two separate pathways by which asialoglycoproteins are degraded. The major "classic" pathway mediated by State 2 galactosyl receptors occurs after a 20-min lag and the minor pathway mediated by State 1 galactosyl receptors begins immediately with no detectable lag.     

Thawing and processing of cryopreserved bovine spermatozoa at various temperatures and their effects on sperm viability, osmotic shock and sperm membrane functional integrity

The objective of this study was to evaluate the effects of thawing and processing temperatures on post-thaw sperm viability, occurrence of osmotic shock and sperm membrane functional status. The occurrence of osmotic shock, characterized by increased spermatozoa with coiled tails, eventually results in reduced sperm viability and sperm membrane integrity. The effects of different thawing temperatures were assessed by thawing frozen specimens at 37, 21 or 5 degrees C for 1 to 2-min, followed by processing at these temperatures. A subset of frozen specimens were thawed at 37 degrees C for 10 to 15-sec and transferred to a water bath at 21 or 5 degrees C for 1 to 2-min to complete thawing, followed by processing at these temperatures. Sperm processing (washing) consisted of dilution, centrifugation and resuspension to remove glycerol from the medium and to gradually return the spermatozoa to isotonic conditions. Post-thawed specimens (0.5 mL) were slowly diluted 1:1 (v/v) at a rate of 0.1 mL/min, centrifuged, and resuspended to 0.5 mL (37 degrees C). Diluted specimens were equilibrated for 1 to 2-min after dilution and for 5-min after resuspension. The specimens were then incubated for 2-h (37 degrees C) and assessed at 60-min intervals for the percentage of motility, for progressive motility (Grades 0 to 4), for the percentage of spermatozoa with coiled tails, and for the percentage of swollen spermatozoa. The percentage of swollen spermatozoa (measurement of sperm membrane integrity) was assessed by exposing spermatozoa to a modified hypoosmotic swelling (HOS) test. The results obtained seem to indicate that physiological thawing and processing temperatures (37 degrees C) are required to maintain sperm motility. However, thawing and processing at lower temperatures (< 37 degrees C) seems to prevent the occurrence of osmotic shock and to maintain sperm membrane functional integrity. In this study, thawing at 37 degrees C (10 to 15-sec) and transfer to a water bath at 21 degrees C (1-min) to complete thawing, followed by processing at 21 degrees C, yielded better results in terms of increased sperm viability, reduced occurrence of osmotic shock and higher reactivity to the HOS test.     

High-density lipoprotein binding to guinea-pig hepatic membranes. Comparison of guinea-pig and human ligands

Human and guinea-pig apo-E-free HDL were incubated with isolated guinea-pig hepatic membranes at 4 and 37 degrees C to determine the effects of temperature and heterologous systems on the equilibrium parameters of HDL binding. Receptor mediated HDL binding was highest at 37 degrees C for both lipoproteins. At 4 degrees C, a higher affinity constant (Kd) was observed when guinea-pig HDL was the ligand relative to human HDL; in contrast, both HDL preparations had similar Kd values at 37 degrees C. Calculated binding and receptor number (Bmax) were higher at both temperatures when guinea-pig HDL was the ligand. These results demonstrate a significant species difference in HDL binding to hepatic membrane which is partially temperature-dependent.     

Partial purification and characterization of phospholipase C from Yersinia enterocolitica

About 34% of the strains of Yersinia enterocolitica isolated from raw milk were found to produce lecithinase. A selected strain produced phospholipase C at 22 degrees C and 37 degrees C; production was optimum at 37 degrees C in the stationary phase (14-16 h). A decrease in phospholipase C activity at various storage temperatures (-5 degrees C, 4 degrees C, 37 degrees C) was also observed, although the enzyme was active over a wide range of temperature (5-65 degrees C) and pH (3.5-7.5). The phospholipase C was partially purified by ammonium sulphate precipitation and Sephadex column chromatography, and characterized.     

Characterization of ligand binding and processing by bombesin receptors in an insulin-secreting cell line.

Bombesin is a tetradecapeptide which stimulates insulin secretion in vivo by isolated islets and by HIT-T15 cells, a clonal line of hamster pancreatic-islet cells. In the present study we have used [125I-Tyr4]bombesin to characterize bombesin receptors in HIT-T15 cells. [125I-Tyr4]Bombesin binding was time- and temperature-dependent: maximum binding occurred after 45 min, 90 min and 10 h at 37, 22 and 4 degrees C respectively. Thereafter, cell-associated radioactivity declined at 37 degrees C and 22 degrees C but not at 4 degrees C. Scatchard analysis of [125I-Tyr4]bombesin binding measured at 4 degrees C showed that HIT-T15 cells contain a single class of binding sites (approximately equal to 85000/cell) with an apparent Kd of 0.9 +/- 0.11 nM. Structurally unrelated neuropeptides did not compete for [125I-Tyr4]bombesin binding. However, the relative potencies of bombesin and four bombesin analogues in inhibiting the binding of [125I-Tyr4]bombesin correlated with their ability to stimulate insulin release. Receptor-mediated processing of [125I-Tyr4]bombesin was examined by using an acid wash (0.2 M-acetic acid/0.5 M-NaCl, pH 2.5) to dissociate surface-bound peptide from the cells. Following [125I-Tyr4]bombesin binding at 4 degrees C, more than 85% of the cell-associated radioactivity could be released by acid. When the temperature was then increased to 37 degrees C, the bound radioactivity was rapidly (t1/2 less than 3 min) converted into an acid-resistant state. These results indicate that receptor-bound [125I-Tyr4]bombesin is internalized in a temperature-dependent manner. In fact, the entire ligand-receptor complex appeared to be internalized, since pretreatment of cells with 100 nM-bombesin for 90 min at 37 degrees C decreased the subsequent binding of [125I-Tyr4]bombesin by 90%. The chemical nature of the cell-associated radioactivity was determined by reverse-phase chromatography of the material extracted from cells after a 30 min binding incubation at 37 degrees C. Although 70% of the saturably bound radioactivity was co-eluted with intact [125I-Tyr4]bombesin 90% of the radioactivity subsequently dissociated from cells chromatographed as free iodide. At least some of the degradation of receptor-bound [125I-Tyr4]bombesin appeared to occur in lysosomes, since chloroquine increased the cellular accumulation of [125I-Tyr4]bombesin at 37 degrees C and slowed the release of radioactivity.(ABSTRACT TRUNCATED AT 400 WORDS)     

Virulence and resistance markers in clinical and environmental Aeromonas strains isolated in Romania

The virulence and resistance (R) features of 37 Aeromonas strains from diarrheal cases and 150 from the aquatic environment (isolated during cold and warm season) were tested at different incubation temperatures (4 degrees C, 28 degrees C and 37 degrees C). When incubated at 4 degrees C temperature, the Aeromonasstrains isolated during the cold season expressed the highest number of virulence factors by comparison with the strains isolated during warm season and from diarrhoeal cases, the virulence spectrum increasing simultaneously with the incubation temperature (i.e. 28 degrees C and 37 degrees C) for all strains. Mucinase was the unique virulence factor constantly present in all three categories of strains at all three incubation temperatures. The aquatic as well as clinical strains exhibited similar R levels to ampicillin and colistin, while for the other tested antibiotics, the aquatic strains generally proved higher R levels than clinical strains, excepting cephtazidime. Plasmids of molecular weights ranging between 1904-21226 bp, were isolated in 36.5% of Aeromonas strains, some of them being correlated with specific R patterns. The large virulence spectrum correlated with high R in Aeromonas strains isolated from the aquatic medium is pleading for the significant role of these bacteria in the pathogenic potential of the natural reservoir possibly implicated in human pathology.     

Protein Arp and protein H from group A streptococci. Ig binding and dimerization are regulated by temperature.

Cell surface proteins that bind to the Fc part of Ig are expressed by many strains of group A streptococci, an important human pathogen. Two such bacterial strains, AP4 and AP1, were shown to bind IgA and IgG, respectively, in a temperature-dependent manner. The binding of radiolabeled Ig to the bacterial cells was lower at 37 degrees C than at 22 and 4 degrees C. Similarly, protein Arp, the IgA-binding protein isolated from strain AP4, and protein H, the IgG-binding protein isolated from strain AP1, displayed a strong Ig-binding at 22 degrees C and lower temperatures, and virtually no binding at all at 37 degrees C. The effect was reversible: lowering of the temperature restored the binding and vice versa. A gradual shift between binding and nonbinding took place between 27 and 37 degrees C. Gel chromatography and velocity sedimentation centrifugation showed that protein Arp and protein H appeared as noncovalently associated dimers at 10 and 22 degrees C, and as monomers at 37 degrees C. These results strongly suggest that the dimerization of protein Arp and protein H, rather than the low temperature itself, yielded the strong Ig-binding of the proteins at 10 and 22 degrees C. Indeed, after covalent cross-linking of the dimers at 10 degrees C by incubation with low concentrations of glutaraldehyde, full Ig-binding was achieved even at 37 degrees C. A carboxyl-terminal proteolytic fragment of protein Arp, which completely lacked the IgA-binding capacity at any temperature, showed the same temperature-dependent dimerization as intact protein Arp, suggesting that the Ig-binding part of the protein is not required for dimerization. The implications of these results for the function of Ig-binding group A streptococcal proteins, and their role in the host-parasite relationship are discussed.     

The diisopropylfluorophosphate inhibitable step in antigen-induced histamine release from human leukocytes.

DFP inhibits early events in antigen-induced histamine release from human leukocytes. If added to cells 5 min or more after antigen it is ineffective. If added with antigen it can be removed at 5 min but release will still be inhibited. In contrast, ethylenediaminetetraacetate (EDTA) and 2 deoxyglucose (2DG) still inhibit the reactions when added 5 min after antigen. During incubation of leukocytes for 90 to 120 min at 0 degrees C they react with specific antigen since they subsequently release significant quantities of histamine after washing and reincubation at 37 degrees C without addition of antigen. Such priming at 0 degrees C is at least equivalent to priming for 2 to 4 min at 37 degrees C. During antigen priming at 0 degrees C the cells are not activated beyond the step in the release sequence which is inhibited by diisopropylfluorophosphate (DFP). This is apparent from the undiminished inhibitory activity of DFP on these cells. Furthermore, cells primed with antigen at 0 degrees C in the presence of DFP release as much histamine after washing and incubation at 37 degrees D as control cells primed in the absence of DFP. Incubation of leukocytes with specific antigen at 37 degrees C for 3 min resulted in significant but not quite complete priming for subsequent histamine release in the absence of antigen. Most of these primed cells were not activated beyond the step inhibitable by DFP. However, some had completed the entire sequence including the release of histamine while others had not released their histamine but were not inhibited by DFP from subsequent release. After 5 min incubation with antigen at 37 degrees C almost all leukocytes had progressed beyond the stage which is inhibited by DFP. Incubation of leukocytes at 37 degrees C with DFP but without antigen for up to 15 min followed by washing did not impair subsequent antigen-induced histamine release by these cells. Thus, DFP was inhibitory under these conditions only after antigen activation of leukocytes.     

Microbial spoilage of pre-cooked potato-topped pies

The ecological succession of bacteria which developed in pre-cooked potato-topped pies stored at two different temperatures was examined. Bacillus, Streptococcus and Staphylococcus-Micrococcus spp. were the predominant organisms isolated from freshly prepared pies and those stored at 4 degrees and 37 degrees C. None of these groups of bacteria caused significant biodeterioration of pies held at 4 degrees C, but all groups grew well in pies stored at 37 degrees C and achieved counts of ca 10(8)/g of sample. Bacillus spp. were the first group to grow, followed by Streptococcus and Staphylococcus-Micrococcus spp. Growth which occurred at 37 degrees C did so at the expense of glucose, lactate accumulated and the pH of pie components decreased. Amylase activity detected in all pie components during storage was associated with the growth of Bacillus spp. and probably supplemented glucose already present in pies, by hydrolytic cleavage of potato, flour or binder starches. Spoilage caused by growth and activity of the bacteria isolated was not associated with visual signs of biodeterioration, nor production of 'off' odour usually associated with spoilage of meats. These results suggest that pre-cooked potato-topped pies held at inappropriate temperatures represent a potential public health risk.     

Cryopreservation of mouse pancreatic islets: effects of different glucose concentrations in the post-thaw culture medium on islet recovery

It was the aim of this study to investigate the influence of the glucose concentration of the post-thaw culture medium on islet B-cell survival after cryopreservation by the combined assessments of islet recovery, islet DNA and insulin contents, and insulin release. Collagenase isolated mouse islets were kept in culture for 3 days in the presence of 11.1 mM glucose and then transferred to freezing ampoules containing Hanks' solution supplemented with 10% calf serum and 2 M dimethyl sulfoxide. After a 20-min incubation at 0 degrees C the islets were cooled at a rate of 25 degrees C/min to -70 degrees C and subsequently plunged into liquid nitrogen. After 2 hr the frozen islets were rapidly thawed at 37 degrees C, transferred to culture dishes, and cultured for another 3 days in the presence of 2.8, 5.6, 11.1, 16.7, or 28 mM glucose. Nonfrozen control islets were treated identically after a preceding 3-day culture at 11.1 mM glucose. The percentage recovery of cryopreserved islets was decreased compared to that of nonfrozen islets, but was increased when higher glucose concentrations were used in the post-thaw culture medium. Since the DNA content of the cryopreserved islets was slightly decreased, the overall survival rate of the cryopreserved B-cells, when cultured at the higher glucose concentrations after thawing, was found to be about 75%. The insulin content of the cryopreserved islets was decreased but the glucose-stimulated insulin release was essentially the same as that of the nonfrozen islets.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparisons of the effects of temperature on the liver fatty acid binding proteins from hibernator and nonhibernator mammals.

Hibernating mammals rely heavily on lipid metabolism to supply energy during hibernation. We wondered if the fatty acid binding protein from a hibernator responded to temperature differently than that from a nonhibernator. We found that the Kd for oleate of the liver fatty acid binding protein (1.5 microM) isolated from ground squirrel (Spermophilus richardsonii) was temperature insensitive over 5-37 degrees C, while the rat liver fatty acid binding protein was affected with the Kd at 37 degrees C being about half (0.8 microM) that found at lower temperatures. This same trend was observed when comparing the specificity of various fatty acids of differing chain length and degree of unsaturation for the two proteins at 5 and 37 degrees C. At the lower temperature, ground squirrel protein bound long-chain unsaturated fatty acids, particularly linoleate and linolenate, at least as well as at the higher temperature and matched requirements for these fatty acids in the diet. The most common long-chain fatty acid, palmitate, was a more effective ligand for ground squirrel liver fatty acid binding protein at 5 degrees C than at 37 degrees C, with the opposite occurring in the eutherm. Rat protein was clearly not adapted to function optimally at temperatures lower than the animal's body temperature.     

Anomeric specificity of the stimulatory effect of D-glucose on D-fructose phosphorylation by human liver glucokinase

D-Glucose was recently reported to stimulate d-fructose phosphorylation by human B-cell glucokinase. The present study aims at investigating the anomeric specificity of such a positive cooperativity. The alpha-anomer of D-glucose was found to increase much more markedly than beta-D-glucose the phosphorylation of D-fructose by human liver glucokinase. Such an anomeric preference diminished at high concentrations of the D-glucose anomers, i.e. when the effect of the aldohexose upon d-fructose phosphorylation became progressively less marked. A comparison between the effects of the two anomers of D-glucose and those of equilibrated D-glucose upon D-fructose phosphorylation by human liver glucokinase indicated that the results obtained with the equilibrated aldohexose were not significantly different from those expected from the combined effects of each anomers of D-glucose. In isolated rat islets incubated for 60 min at 4 degrees C, alpha-D-glucose (5.6 mm), but not beta-D-glucose (also 5.6 mm), augmented significantly the conversion of D-[U-(14)C]fructose (5.0 mm) to acidic radioactive metabolites. Likewise, in islets prelabeled with (45)Ca and perifused at 37 degrees C, D-fructose (20.0 mm) augmented (45)Ca efflux and provoked a biphasic stimulation of insulin release from islets exposed to alpha-D-glucose (5.6 mm), while inhibiting (45)Ca efflux and causing only a sluggish and modest increase in insulin output from islets exposed to beta-D-glucose (also 5.6 mm). The enhancing action of D-glucose upon D-fructose phosphorylation by glucokinase thus displays an obvious anomeric preference for alpha-D-glucose, and such an anomeric specificity remains operative in intact pancreatic islets.     

Survival and growth of Campylobacter fetus subsp. jejuni on meat and in cooked foods.

Twelve strains of Campylobacter fetus subsp. jejuni isolated from humans and animals grew at temperatures ranging from 34 to 45 degrees C and pH minima between 5.7 and 5.9. Only one strain grew at pH 5.8 with lactic acid present at a concentration similar to that in meat. All strains had decimal reduction times of less than 1 min at 60 degrees C. Further examination of a typical strain showed that it grew at 37 degrees C on high-pH meat but not at 37 degrees C on normal-pH meat. Bacterial numbers on both high (6.4)-pH and normal (5.8)-pH inoculated meat declined at a similar rate when the meat was stored at 25 degrees C. At -1 degree C, the rate of die-off was somewhat slower on normal-pH meat but was very much slower on high-pH meat. The initial fall in bacterial numbers that occurred when meat was frozen was also greater for normal-pH meat than for high-pH meat. The organism exhibited a long lag phase (1 to 2 days) when grown in cooked-meat medium at 37 degrees C and died in meat pies stored at 37 or 43 degrees C. Evaluation of the risk of Campylobacter contamination of red-meat carcasses to human health must take into account the limited potential of the organism to grow or even survive on fresh meats and in warm prepared foods.     

Preservation of the effects of pregnancy on rat islets of Langerhans in tissue culture.

Islets of Langerhans, isolated from normal or 19-day pregnant rats, were cultured for 20 h at 37 degrees C in tissue culture medium 199. When islets were cultured in medium containing low glucose (5.5 mM), the higher adenylate cyclase activity and insulin secretory responses characteristic of islets from pregnant rats were maintained during the test period of 29 h. Islets from normal and pregnant rats were also cultured for 20 h in medium containing a very high glucose concentration (83.3 mM) in order to load the B cells with glycogen. It was found, after glycogen loading, that, while adenylate cyclase activity increased to a greater extent in islets from pregnant rats than controls, this activity was not increased in proportion to the striking changes in insulin release rate observed in pregnant rat islets. The results show that the difference in insulin secretory response between islets from normal and pregnant rats may be preserved when the islets are cultured for 20 h, and that these differences are enhanced for a variety of reasons after culture of islets in 83.3 mM glucose.     

Characterization of the asialoglycoprotein receptor on isolated rat hepatocytes

Rat hepatocytes, freshly isolated by a collagenase perfusion technique, bound [3H]asialo-orosomucoid in a sugar-specific and calcium-dependent manner as expected for the hepatic asialoglycoprotein receptor. At least 90% of the total cell surface-bound [3H]asialo-orosomucoid represented specific binding and could be removed by washing with EDTA. Freshly isolated cells had about 7 x 10(4) surface receptors per cell. However, when cells were incubated at 37 degrees C, the number of surface receptors per cell rapidly increased 2- to 3-fold to about 2.2 x 10(5). This increase in receptor number occurred in the absence of serum and began within minutes, depending on the particular conditions used to keep the cells in suspension. (The maximal rate of appearance of new receptors at 37 degrees C was about 70 receptors per cell per s.) When cells were first exposed to a brief EDTA treatment at 4 degrees C, before measuring the binding of [3H]asialo-orosomucoid, the number of surface receptors per cell was found to increase by about 45%. Therefore, about 30% of the surface receptors on freshly isolated cells have already bound endogenous asialoglycoproteins or are present in the membrane in a cryptic form. At 4 degrees C the binding of [3H]asialo-orosomucoid was rapid (kon greater than or equal to 1.8 x 10(4) M-1s-1), whereas the dissociation of bound [3H]asialo-orosomucoid, measured in the presence of excess nonradioactive glycoprotein, was extremely slow (koff less than or equal to 0.9 x 10(-5) s-1). The association constant calculated from these data (Ka = 2.0 x 10(9) M-1) agreed well with that obtained from equilibrium binding experiments (Ka = 2.4 x 10(9) M-1) using untreated cells or cells which had first been treated with EDTA or incubated at 37 degrees C. In all cases, when the concentration of [3H]asialo-orosomucoid was higher than about 600 ng/ml, the Scatchard plots were curvilinear. The data are, however, consistent with the conclusion that there is a single high affinity receptor on the hepatocyte surface. The additional receptors that appear on the surface when cells are incubated at 37 degrees C or exposed to EDTA are identical with those on untreated cells,     

Comparison of formation, activation, and nuclear translocation of receptor . estradiol (R . E2) complex at 0 degrees C and 37 degrees C in intact uterine cells.

The present study was undertaken to establish whether molecular events leading to binding, transformation-activation, and nuclear translocation of cytoplasmic uterine estrogen receptor described for cell-free systems also occur in intact uterine cells. Cell suspensions were incubated at 0 degrees C or 37 degrees C with estradiol (E2) and specific binding to intracellular receptors was measured. The data demonstrate that saturation of specific estrogen binding sites occurs within 60 min at 37 degrees C and within 22 h at 0 degrees C, with a total of approximately 24,000 to 30,000 receptor sites per cell. At equilibrium, the total number and subcellular distribution of receptor . estradiol (R . E2) complexes formed in cells incubated at 0 degrees C or 37 degrees C were identical. Scatchard analysis of the equilibrium binding data yielded the same association constants for cytoplasmic and nuclear R . E2 formed in intact cells incubated at either temperature. Sucrose density gradient analysis of nuclear and cytoplasmic R . E2 formed in intact cells at 0 degrees C or 37 degrees C showed that at both temperatures, the nuclear R . E2 had a 5 S sedimentation coefficient; at both temperatures, a 5 S cytosol R . E2 was detected; only in the 0 degrees C incubation, an additional 4 S cytosol R . E2 was found. These results suggest that the molecular interactions regulating the dynamics of estrogen binding in the intact cell are similar at both physiological and low temperatures.     

Isolation and characterization of thermotolerant Gluconobacter strains catalyzing oxidative fermentation at higher temperatures

Thermotolerant acetic acid bacteria belonging to the genus Gluconobacter were isolated from various kinds of fruits and flowers from Thailand and Japan. The screening strategy was built up to exclude Acetobacter strains by adding gluconic acid to a culture medium in the presence of 1% D-sorbitol or 1% D-mannitol. Eight strains of thermotolerant Gluconobacter were isolated and screened for D-fructose and L-sorbose production. They grew at wide range of temperatures from 10 degrees C to 37 degrees C and had average optimum growth temperature between 30-33 degrees C. All strains were able to produce L-sorbose and D-fructose at higher temperatures such as 37 degrees C. The 16S rRNA sequences analysis showed that the isolated strains were almost identical to G. frateurii with scores of 99.36-99.79%. Among these eight strains, especially strains CHM16 and CHM54 had high oxidase activity for D-mannitol and D-sorbitol, converting it to D-fructose and L-sorbose at 37 degrees C, respectively. Sugar alcohols oxidation proceeded without a lag time, but Gluconobacter frateurii IFO 3264T was unable to do such fermentation at 37 degrees C. Fermentation efficiency and fermentation rate of the strains CHM16 and CHM54 were quite high and they rapidly oxidized D-mannitol and D-sorbitol to D-fructose and L-sorbose at almost 100% within 24 h at 30 degrees C. Even oxidative fermentation of D-fructose done at 37 degrees C, the strain CHM16 still accumulated D-fructose at 80% within 24 h. The efficiency of L-sorbose fermentation by the strain CHM54 at 37 degrees C was superior to that observed at 30 degrees C. Thus, the eight strains were finally classified as thermotolerant members of G. frateurii.