Effect of Available Water on Thermal Resistance of Three Nonsporeforming Species of Bacteria

Cells of Escherichia coli, Pseudomonas fluorescens, and Staphylococcus aureus, previously grown in Trypticase Soy Broth (TSB) at a high level of available moisture (a(w) 0.994) and at low levels produced by addition of NaCl or glucose, were heated in neutral phosphate buffer, and in this buffer adjusted to low levels of available moisture by means of NaCl or glucose. Glucose in the heating medium was more protective than NaCl for E. coli and P. fluorescens, but hastened the thermal destruction of S. aureus. Added protection was given P. fluorescens during heating in glucose-buffer solution at a(w) 0.97 by previous growth in TSB adjusted to that a(w) value with glucose. Added protection was given E. coli during heating in NaCl-buffer solution at a(w) 0.98 by previous growth in TSB adjusted to that value with NaCl. With S. aureus, however, previous growth in TSB plus NaCl or glucose had little effect on heat resistance, but the solute in the heating medium had great influence, in that NaCl was very protective and glucose destructive. Opportunity may have been given during tempering of the cell suspension at 30 C in the heating medium prior to heating for the NaCl and glucose to diffuse into the staphylococcal cells.     

EPSP synthase: binding studies using isothermal titration microcalorimetry and equilibrium dialysis and their implications for ligand recognition and kinetic mechanism.

Isothermal titration calorimetry measurements are reported which give important new binding constant (Kd) information for various substrate and inhibitor complexes of Escherichia coli EPSP synthase (EPSPS). The validity of this technique was first verified by determining Kd's for the known binary complex with the substrate, shikimate 3-phosphate (S3P), as well as the herbicidal ternary complex with S3P and glyphosate (EPSPS.S3P.glyphosate). The observed Kd's agreed very well with those from previous independently determined kinetic and fluorescence binding measurements. Further applications unequivocally demonstrate for the first time a fairly tight interaction between phosphoenolpyruvate (PEP) and free enzyme (Kd = 390 microM) as well as a correspondingly weak affinity for glyphosate (Kd = 12 mM) alone with enzyme. The formation of the EPSPS.PEP binary complex was independently corroborated using equilibrium dialysis. These results strongly suggest that S3P synergizes glyphosate binding much more effectively than it does PEP binding. These observations add important new evidence to support the hypothesis that glyphosate acts as a transition-state analogue of PEP. However, the formation of a catalytically productive PEP binary complex is inconsistent with the previously reported compulsory binding order process required for catalysis and has led to new studies which completely revise the overall EPSPS kinetic mechanism. A previously postulated ternary complex between S3P and inorganic phosphate (EPSPS.S3P.Pi, Kd = 4 mM) was also detected for the first time. Quantitative binding enthalpies and entropies were also determined for each ligand complex from the microcalorimetry data. These values demonstrate a clear difference in thermodynamic parameters for recognition at the S3P site versus those observed for the PEP, Pi, and glyphosate sites.     

Stimulation of membrane phospholipid metabolism by agents that increase acidification in toad urinary bladder

Previous reports have indicated that metabolic acidosis stimulates H+ excretion, and this excretion is accompanied by an increased turnover of phospholipids (PL) in toad urinary bladder. The purpose of this experiment was to determine if other known stimulators of H+ excretion [insulin, deoxycorticosterone acetate (DOCA), epinephrine, parathyroid hormone, and CO2] might also stimulate PL turnover in the toad urinary bladder. Quarter bladders from normal toads were removed, weighed, and then incubated with [32P]orthophosphate for 2 hr at 25 degrees C. PL were extracted, separated, and detected using thin layer chromatography and autoradiography, and quantitated by liquid scintillation counting. Results were expressed in cpm (100 mg bladder)-1 (hr)-1. One quarter bladder received insulin (100 milliunits/ml), DOCA (10(-6) M), epinephrine (50 mM), parathyroid hormone (100 micrograms/ml), or 5% CO2 during the incubation, whereas the paired quarter bladder received no treatment. Phosphatidylcholine (PC) and phosphatidylinositol turnover were increased by insulin (P less than 0.025 and less than 0.05, respectively). DOCA had no effect on PL turnover, but stimulated the percentage fraction of PC (P less than 0.05) expressed as percentage fraction of total lipids. Five percent CO2 in the bath resulted in an increased rate of turnover of the PL fractions phosphatidylinositol (P less than 0.05), and the phosphatidic acid plus phosphatidyl-serine (P less than 0.01). Epinephrine and parathyroid hormone were both without effect on PL metabolism. We conclude that insulin, DOCA, and CO2 may stimulated H+ excretion in toad bladder in part by increasing turnover of membrane PL, PC, and phosphatidylinositol, and in the case of CO2, phosphatidic acid plus phosphatidylserine as well, but not PC.     

Structure and evolution of somatostatin genes

A bovine pancreatic preprosomatostatin cDNA clone has been isolated and sequenced. Although it encodes a predicted 116 amino acid preprosomatostatin that is very similar in primary structure to those deduced from other mammalian preprosomatostatin cDNAs, there are some differences in amino acid composition. Hybridization of this clone to Northern blots of fetal bovine pancreatic poly(A+) RNA reveals a mRNA of 700 nucleotides. Evolution of the preprosomatostatin genes was studied by statistical analysis of anglerfish, catfish, bovine, rat, and human cDNA sequences. The results suggest that the two somatostatin genes present in both anglerfish and catfish were the result of a gene duplication event in a common ancestor of anglerfish and catfish.     

Insulin-mediated H+ and NH+4 excretion in the urinary bladder of Bufo marinus

This study was done to determine if insulin mediates H+ and NH+4 excretion in the urinary bladder of Bufo marinus. Acidosis was induced by gavaging with 10 ml of 120 mM NH4Cl 3X daily for 2 days. Hemibladders were mounted between Lucite chambers. Insulin (porcine) was added to the serosal solution of the experimental bladder (10(2) mU/ml). After a 15-min equilibration the flux was measured for 2 hr. H+ excretion was measured from change in pH of the mucosal fluid and the NH+4 measured colorimetrically. The excretion was normalized for weight of bladder and reported in units of nanomoles (100 mg bladder)-1(min)-1. Plasma insulin was determined by radioimmunoassay and glucose by the glucose oxidase method. In 14 control bladders H+ excretion was 8.75 +/- 1.28 and experimental was 16.35 +/- 2.50 (P less than 0.025), while NH+4 excretion in control bladder was 3.29 +/- 0.95 and experimental was 6.58 +/- 1.89 (P less than 0.01). This response was absent when the insulin was heat inactivated (P greater than 0.2 and P greater than 0.3 respectively). Plasma insulin-like levels in 10 normal toads was 0.57 +/- 0.16 ngm/ml and in acidotic toads 1.25 +/- 0.16 ng/ml (P less than 0.025). Plasma glucose levels in 10 normal toads were 22.0 +/- 3.5 mg/dl and in 12 acidotic toads 17.8 +/- 0.75 mg/dl (P less than 0.025). We conclude that plasma insulin is increased in acidosis and that insulin stimulates excretion of H+ and NH+4 in the toad urinary bladder.     

Restriction fragment length polymorphisms as genetic markers in soybean,Glycine max (L.) merrill

Summary Restriction Fragment Length Polymorphisms (RFLP) have been identified between widely distant cultivars (Minsoy and Noir 1 ) of soybean Glycine max (L.) Merrill. Using as probes randomly chosen clones of DNA, one in five probes revealed a polymorphism. More than half of these polymorphisms appear to result from rearrangements of the genomic DNA. Twenty seven markers were analyzed for linkage in F₂ plants. Eleven of these markers were contained in four linkage groups. Five cultivars were compared in a search for new alleles. When RFLP markers corresponding to low copy DNA were used to analyze three other cultivars — Sooty, Forrest and Mandarin (Ottawa) — few new alleles were found. Using these probes, five different markers could be used to differentiate the five cultivars. Complex probes, which correspond to repeated DNA, revealed different polymorphisms in different cultivars and a single such probe could be used to distinguish the five cultivars from each other.     

Poly(2-amino-8-methyldeoxyadenylic acid): contrasting effects in deoxy- and ribopolynucleotides of 2-amino and 8-methyl substituents

Poly(2-amino-8-methyldeoxyadenylic acid) interacts readily with pyrimidine polynucleotides to form double helices only slightly less stable than those in which the purine polymer lacks the 8-Me group. In the ribo series, by contrast, complexes formed with poly(2-amino-8-methyladenylic acid) are very strongly destabilized by the 8-Me group, despite a larger stabilizing effect of the 2-NH2 group in the ribo series. These results are interpreted in terms of a smaller steric interference of the 8-Me group with 2'-CH2 than with 2'-CHOH, leading to a smaller population of syn structures in the deoxy chain and a consequent lower interference with homopolymer duplex formation. UV, circular dichroism (CD), and IR spectra of the new polymer and its complexes are reported and related to structural and energetic characteristics of the molecules. Since direct synthesis of 2-amino-8-methyldeoxyadenosine was not feasible, the corresponding riboside was prepared, the 3'- and 5'-positions were protected with a disilyloxy group, and a 2'-[(imidazol-1-yl)thiocarbonyl] group was introduced. Reduction with tributyltin hydride followed by deprotection gave the nucleoside, which was then converted to the triphosphate by standard methods. The homopolymer was prepared with terminal deoxynucleotidyl transferase.     

Amino acid sequence of kappa-flavitoxin: establishment of a new family of snake venom neurotoxins

The complete amino acid sequence of kappa-flavitoxin, a neurotoxin isolated from the venom of Bungarus flaviceps, has been determined by automated Edman analysis of the intact protein and of peptides derived from digests with trypsin and chymotrypsin. kappa-Flavitoxin consists of a single 66-residue polypeptide chain which is completely devoid of methionine. The amino acid sequence of kappa-flavitoxin demonstrates that although the toxin is related to the alpha-neurotoxin family, it displays a much higher degree of homology with kappa-bungarotoxin. The conserved structural features of the kappa-neurotoxins and their pharmacological profiles, which are distinct from those of all known alpha-neurotoxins, provide evidence for a new, structurally and functionally unique family of snake venom neurotoxins.     

Evolution of glucagon genes

Statistical analyses of DNA sequences of the preproglucagon genes from bovine, human, hamster, and anglerfish suggest that a gene duplication creating two anglerfish genes (AF I and II) occurred about 160 Myr ago, long after the separation of fish and mammals. The analyses further suggest that the internal duplication producing the glucagon and glucagon-like peptide II (GLP-II) regions occurred about 1.2 billion years ago, which would indicate that the GLP-II region was present in the ancestral anglerfish sequence but was silenced or deleted before the gene duplication separating AF I and II. The glucagon-like peptide I (GLP-I) was derived from a duplication of the ancestral glucagon region about 800 Myr ago. The rate of synonymous substitution in these genes is approximately 4.3 x 10(-9) substitutions per year per synonymous site. The rate of nonsynonymous substitution in the signal peptide region is about 1.1 x 10(-9) substitutions per year per nonsynonymous site, a high rate comparable to that in the C-peptide region of preproinsulin. The rate of nonsynonymous substitution in the glicentin-related pancreatic polypeptide (GRPP) region is 0.63 x 10(-9) for the comparisons between mammalian species and 1.8 x 10(-9) for the comparisons between fish and mammals; the moderate rate in mammals suggests a physiological role for GRPP. The glucagon region is extremely conservative; no nonsynonymous substitution is observed in the mammalian genes, and a nonsynonymous rate of 0.18 x 10(-9) was obtained from the comparisons between fish and mammals. In the GLP-I region, the rate of nonsynonymous substitution was estimated to be 0.08 x 10(-9) for the comparisons between mammalian species and 0.30 x 10(- 9) for the comparisons between fish and mammals. In the GLP-II region, the rate was estimated to be 0.25 x 10(-9) for the comparisons between mammalian species. Thus, GLP-I and II are also very conservative, which suggests an important physiological role for these peptides.