Adiabatic compressibility and intrinsic viscosity studies on peptide aggregates

Summary Density and sound velocity measurements and¹H NMR investigations were carried out in aqueous solution at various temperatures for determining the adiabatic compressibility (β) and hydration of the tetrapeptide, TFA. Tyr-Gly-Phe-Ala-Obz I. The present investigation showed changes in the temperature coefficient of adiabatic compressibility at 40°C,¹H NMR studies indicated the inverse temperature transition in the concentration range studied.     

A Sub-population of Group A Streptococcus Elicits a Population-wide Production of Bacteriocins to Establish Dominance in the Host

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Feedback inhibition of ammonium (methylammonium) ion transport in Escherichia coli by glutamine and glutamine analogs.

When cultured with glutamate or glutamine as the nitrogen source, Escherichia coli expresses a specific ammonium (methylammonium) transport system. Over 95% of the methylammonium transport activity in washed cells was blocked by incubation with 100 microM L-glutamine in the presence of chloramphenicol (100 micrograms/ml). The time course for the onset of this glutamine inhibition followed a first-order rate expression with a t1/2 of 2.8 min. The inhibition of transport by L-glutamine was noncompetitive (Ki = 18 microM) with respect to the [14C]methylammonium substrate. D-Glutamine had no significant effect. The glutamine analogs gamma-L-glutamyl hydroxamate (Ki = 360 microM) and gamma-L-glutamyl hydrazide (Ki = 800 microM) were also noncompetitive inhibitors of methylammonium transport, suggesting that glutamine metabolism is not required. The role of the intracellular glutamine pool in the regulation of ammonium transport was investigated by using mutants carrying defects in the operon of glnP, the gene for the glutamine transporter. The glnP mutants had normal rates of methylammonium transport but were refractory to glutamine inhibition. Glycylglycine, a noncompetitive inhibitor of methylammonium uptake in wild-type cells (Ki = 43 microM), was equipotent in blocking transport in glnP mutants. Although ammonium transport is also subject to repression by growth of E. coli in the presence of ammonia, this phenomenon is unrelated to glutamine inhibition. A GlnL RegC mutant which constitutively expressed ammonium transport activity exhibited a sensitivity to glutamine inhibition similar to that of wild-type cells. These findings indicate that ammonium transport in E. coli is regulated by the internal glutamine pool via feedback inhibition.     

Isolation of an ammonium or methylammonium ion transport mutant of Escherichia coli and complementation by the cloned gene.

During nitrogen-limited growth, Escherichia coli expresses a specific ammonium or methylammonium ion transport system (Amt). Strains carrying defects in Amt have been isolated following Tn10 transposon mutagenesis. These mutants have less than 10% of the transport activity of the parental strain. Glutamate, glutamine, arginine, or high levels (20 mM) of ammonium will serve as the sole nitrogen source for growth of these strains, and glutamine synthetase is normally expressed and repressed by the nitrogen regulatory (Ntr) system. When transformed with plasmid pGln84, containing lacZ fused to an Ntr promoter (glnLp), the Amt mutants expressed a normal level of beta-galactosidase. Furthermore, P1 bacteriophage transduction of the amt mutation into an Ntr mutant, normally constitutive for Amt, gave Amt- transductants. Therefore, the mutations are unlikely to lie within genes affecting Ntr elements. Following transformation with plasmid libraries of E. coli genomic DNA constructed in pUC9, two plasmids conferring the Amt+ phenotype on the amt mutants were isolated. These plasmids were unable to complement the Amt- phenotype of Ntr- mutants. Restriction digestion of these plasmids revealed common fragments, and Southern blot analyses indicated that the Amt-complementing sequence and the site of Tn10 insertion in the genome occur in the same 3.4-kilobase HindIII-SalI fragment. Insertion of TnphoA into this fragment produced amt::phoA fusions which gave high levels of alkaline phosphatase under nitrogen-limiting conditions but low levels during ammonia excess. This suggests that the amt product contains domains which are exported to the periplasm.     

Probing possible egress channels for multiple ligands in human CYP3A4: A molecular modeling study

Human cytochrome P450 (CYP) 3A4 extensively contributes to metabolize 50% of the marketed drugs. Recently, a CYP3A4 structure with two molecules of ketoconazole (2KT) was identified. However, channels for egresses of these inhibitors are unexplored. Thus, we applied molecular dynamics simulations followed by channel analyses. Two simulations of empty and 2KT-bound CYP3A4 results revealed the multiple ligand-induced conformational changes in channel forming regions, which appear to be important for the regulation of channels. In addition, we observed that the channel-3 entrance is closed due to the large structural deviation of the key residues from Phe-cluster. F215 and F220 are known as entrance blockers of channel-2 in metyrapone-bound CYP3A4. Currently, F220 blocks the channel-3 along with F213 and F241. Therefore, it suggested that channel-1 and 2 could potentially serve as egress routes for 2KT. It is also supported by the results from MOLAxis analyses, in which the frequency of channel occurrence and bottleneck radius during simulation favor channel-1 and 2. Several bottleneck residues of these channels may have critical roles in 2KT egresses, especially S119. Our modeling study for multiple ligand-channeling of CYP3A4 could be very helpful to gain new insights into channel selectivity of CYP3A4.     

Glutamine in the pathogenesis of acute hepatic encephalopathy

Hepatic encephalopathy (HE) is the major neurological disorder associated with liver disease. It presents in chronic and acute forms, and astrocytes are the major neural cells involved. While the principal etiological factor in the pathogenesis of HE is increased levels of blood and brain ammonia, glutamine, a byproduct of ammonia metabolism, has also been implicated in its pathogenesis. This article reviews the current status of glutamine in the pathogenesis of HE, particularly its involvement in some of the events triggered by ammonia, including mitochondrial dysfunction, generation of oxidative stress, and alterations in signaling mechanisms, including activation of mitogen-activated protein kinases (MAPKs) and nuclear factor-kappaB (NF-κB). Mechanisms by which glutamine contributes to astrocyte swelling/brain edema associated with acute liver failure (ALF) will also be described.     

Use of coastal waters as condenser coolant in electric power plants: Impact on phytoplankton and primary productivity

(1) Studies on phytoplankton entrained in cooling system of a power plant revealed a reduction in biomass and gross primary productivity (GPP) at outfall, compared to intake.     

Lymphocyte toxicity of prion fragments

Prion protein fragments that are extracted from the brains of patients with Gerstmann-Straussler-Scheinker disease are known to have stimulating action on circulating leukocytes. In particular, the amyloidogenic hydrophobic prion peptide HuPrP (113-127) AGAAAAGAVVGGLGG has been reported to be associated with significant cellular toxicity. In this paper we show that the self assembled form of HuPrP (113-127) and its valine rich domains viz. GAVVGGLG [HuPrP (119-126)] and VVGGLGG [HuPrP (121-127)] are toxic to peripheral lymphocytes. To explore the cytotoxic mechanism of these fragments, we studied 3-(4,5-dimethylthiazol-2yl)-2-5-diphenyltetrazolium bromide (MTT) reduction, reactive oxygen species (ROS) generation, calcium influx and raft sequestration of' peptide treated lymphocytes. Langmuir monolayer studies on these peptides showed a maximum lipid perturbing property of HuPrP (121-127) as compared to the other two fragments. MTT reduction assays on lymphocytes treated with peptides indicated that the prion peptide fibrils are relatively more toxic than freshly solubilized peptide preparations. Lymphocytes treated with HuPrP (121-127), HuPrP (113-127) and HuPrP (119-126) fibrils underwent 60%, 30% and 40% cell death, respectively. Abeta(1-42), HuPrP (119-126) and HuPrP (121-127) fibrils caused 4 fold increases in intracellular ROS as compared with control cells. However, HuPrP (113-127) fibrils lacked such a significant ROS generating activity, indicating that a subtle difference in sequence leads to a difference in the toxic mechanism in the cell. HuPrP (119-126) and HuPrP (121-127) fibrils also produced maximum raft sequestration and calcium influx. Taken together, these data suggest that the assemblage of prion fragments has significant toxic activity on peripheral lymphocytes, a finding with implications for controlling reactive lymphocytes in prion infected subjects.