Synthesis of two sequential polypeptides by dispersion in benzene and their circular dichroism spectra in aqueous solution: Poly(L-glu-L-lys-L-ala-gly) and poly(L-ala-D-glu-L-lys-D-ala-gly)

The monomers γ-benzylglutamyl-ε-benzyloxycarbonyl-lysylalanylglycine pentachlorophenyl ester and alanyl-γ-benzyl-D -glutamyl-ε-benzyloxycarbonyllysyl-D -alanyl-glycine pentachlorophenyl ester, were polymerized in dilute solutions of dimethylform-amide (DMF) or as dispersions in the same volume of benzene. After deprotection with hydrogen bromide, the products were either chromatographed on Sephadex G-50 or dialyzed. The polymers derived from the polymerization in benzene were considerably larger than those from DMF. The results in benzene indicated that high monomer to solvent ratios are not necessary for the production of high-molecular-weight sequential polypeptides. Circular dichroism spectra of the polymers and monomers at neutral and acid pH indicated that poly(L -Glu-L -Lys-L -Ala-Gly) exists in a random coil configuration and poly(L -Ala-D -Glu-L -Lys-D -Ala-Gly) exists in a β conformation.     

Helicobacter pylori dampens gut epithelial self-renewal by inhibiting apoptosis, a bacterial strategy to enhance colonization of the stomach

Colonization of the gastric pits in the stomach by Helicobacter pylori (Hp) is a major risk factor for gastritis, gastric ulcers, and cancer. Normally, rapid self-renewal of gut epithelia, which occurs by a balance of progenitor proliferation and pit cell apoptosis, serves as a host defense mechanism to limit bacterial colonization. To investigate how Hp overcomes this host defense, we use the Mongolian gerbil model of Hp infection. Apoptotic loss of pit cells induced by a proapoptotic agent is suppressed by Hp. The ability of Hp to suppress apoptosis contributed to pit hyperplasia and persistent bacterial colonization of the stomach. Infection with WT Hp but not with a mutant in the virulence effector cagA increased levels of the prosurvival factor phospho-ERK and antiapoptotic protein MCL1 in the gastric pits. Thus, CagA activates host cell survival and antiapoptotic pathways to overcome self-renewal of the gastric epithelium and help sustain Hp infection.     

The influence of electrostatic interactions on partition in aqueous polyethylene glycol/dextran biphasic systems: Part II

In aqueous polyethylene glycol/dextran two-phase systems, the hydrophobicity, free volume, surface tension, and interfacial tension of the phases in equilibrium were measured as a function of pH and ionic strength. These parameters were found to change with pH, but the pattern and magnitude cannot explain the unusual partition of charged macromolecules, observed previously. The electrostatic potential difference was determined by a new experimental approach based on the measurement of the pH difference between the phases at equilibrium. In polyethylene glycol/dextran systems containing sodium chloride as ionized species, the electrostatic potential is not constant in the pH range 2 to 11. The partition behavior of charged macromolecules and its dependence on pH can be explained by the combined action of charge and phase potential. This conclusion was tested with poly-L-glutamate, which partitioned as predicted and in a pattern opposite to positively charged macro- molecules. (c) 1995 John Wiley & Sons, Inc.     

Trace Metal Bioaccumulation in Epibenthic Macroinvertebrates (EMI) from the San Pedro Shelf,California

Trace metal/metalloid (As, Ag, Cd, Cr, Cu, Hg, Ni, Pb, Se, Zn) bioaccumulation was measured over a period of ten years (1985–1995) in five species of epibenthic macroinvertebrates (EMI) from the San Pedro Shelf, California. Four of the species are numerical dominants in the Southern California Bight (SCB) and two species are commercially harvested. Among three echinoderms, a predatory sea star yielded highest tissue concentrations compared to a sea cucumber and a sea urchin, and concentrations measured in a mantid shrimp exceeded those from a prawn. Comparison of trace metal bioaccumulation from the study area, including the ocean outfall, and elsewhere indicated that bioaccumulation in local EMI was generally low. Measurements from an outfall station and two reference stations were used to evaluate the spatial and temporal relationship between trace metal bioaccumulation and the wastewater discharge. It was concluded that there was no spatial or temporal relationship between EMI trace metal bioaccumulation and the discharge.     

Deletion of deoxyribonucleic acid binding domain of the vitamin D receptor abrogates genomic and nongenomic functions of vitamin D

The vitamin D hormone 1,25-dihydroxyvitamin D(3) [1,25-(OH)(2)D(3)], the biologically active form of vitamin D, is essential for an intact mineral metabolism. Using gene targeting, we sought to generate vitamin D receptor (VDR) null mutant mice carrying the reporter gene lacZ driven by the endogenous VDR promoter. Here we show that our gene-targeted mutant mice express a VDR with an intact hormone binding domain, but lacking the first zinc finger necessary for DNA binding. Expression of the lacZ reporter gene was widely distributed during embryogenesis and postnatally. Strong lacZ expression was found in bones, cartilage, intestine, kidney, skin, brain, heart, and parathyroid glands. Homozygous mice are a phenocopy of mice totally lacking the VDR protein and showed growth retardation, rickets, secondary hyperparathyroidism, and alopecia. Feeding of a diet high in calcium, phosphorus, and lactose normalized blood calcium and serum PTH levels, but revealed a profound renal calcium leak in normocalcemic homozygous mutants. When mice were treated with pharmacological doses of vitamin D metabolites, responses in skin, bone, intestine, parathyroid glands, and kidney were absent in homozygous mice, indicating that the mutant receptor is nonfunctioning and that vitamin D signaling pathways other than those mediated through the classical nuclear receptor are of minor physiological importance. Furthermore, rapid, nongenomic responses to 1,25-(OH)(2)D(3) in osteoblasts were abrogated in homozygous mice, supporting the conclusion that the classical VDR mediates the nongenomic actions of 1,25-(OH)(2)D(3).     

Synthetic substrate analogues for UDP-GlcNAc: Manα1-6R β(1-2)-N-acetylglucosaminyltransferase II. Substrate specificity and inhibitors for the enzyme

UDP-GlcNAc: Man1-6R (1-2)-N-acetylglucosaminyltransferase II (GlcNAc-T II; EC 2.4.1.143) is a key enzyme in the synthesis of complexN-glycans. We have tested a series of synthetic analogues of the substrate Man1-6(GlcNAc1-2Man1-3)Man-O-octyl as substrates and inhibitors for rat liver GlcNAc-T II. The enzyme attachesN-acetylglucosamine in 1-2 linkage to the 2-OH of the Man1-6 residue. The 2-deoxy analogue is a competitive inhibitor (K _i=0.13mm). The 2-O-methyl compound does not bind to the enzyme presumably due to steric hindrance. The 3-, 4- and 6-OH groups are not essential for binding or catalysis since the 3-, 4- and 6-deoxy and -O-methyl derivatives are all good substrates. Increasing the size of the substituent at the 3-position to pentyl and substituted pentyl groups causes competitive inhibition (K _i=1.0–2.5mm). We have taken advantage of this effect to synthesize two potentially irreversible GlcNAc-T II inhibitors containing a photolabile 3-O-(4,4-azo)pentyl group and a 3-O-(5-iodoacetamido)pentyl group respectively. The data indicate that none of the hydroxyls of the Man1-6 residue are essential for binding although the 2- and 3-OH face the catalytic site of the enzyme. The 4-OH group of the Man-O-octyl residue is not essential for binding or catalysis since the 4-deoxy derivative is a good substrate; the 4-O-methyl derivative does not bind. This contrasts with GlcNAc-T I which cannot bind to the 4-deoxy-Man- substrate analogue. The data are compatible with our previous observations that a bisectingN-acetylglucosamine at the 4-OH position prevents both GlcNAc-T I and GlcNAc-T II catalysis. However, in the case of GlcNAc-T II, the bisectingN-acetylglucosamine prevents binding due to steric hindrance rather than to removal of an essential OH group. The 3-OH of the Man1-3 is an essential group for GlcNAc-T II since the 3-deoxy derivative does not bind to the enzyme. The trisaccharide GlcNAc1-2Man1-3Man-O-octyl is a good inhibitor (K _i=0.9mm). The above data together with previous studies indicate that binding of the GlcNAc1-2Man1-3Man- arm of the branched substrate to the enzyme is essential for catalysis. Abbreviations: GlcNAc-T I, UDP-GlcNAc:Man1-3R (1-2)-N-acetylglucosaminyltransferase I (EC 2.4.1.101); GlcNAc-T II, UDP-GlcNAc:Man1-6R (1-2)-N-acetylglucosaminyltransferase II (EC 2.4.1.143); MES, 2-(N-morpholino)ethane sulfonic acid monohydrate.     

Cryptic dispersal of Cyanidiophytina (Rhodophyta) in non-acidic environments from Turkey

Extremophiles - Cyanidiophytina are a group of polyextremophilic red algae with a worldwide, but discontinuous colonization. They are restricted to widely dispersed hot springs, geothermal...     

Drug susceptibility in Leishmania isolates following miltefosine treatment in cases of visceral leishmaniasis and post kala-azar dermal leishmaniasis

Background

With widespread resistance to antimonials in Visceral Leishmaniasis (VL) in the Indian subcontinent, Miltefosine (MIL) has been introduced as the first line therapy. Surveillance of MIL susceptibility in natural populations of Leishmania donovani is vital to preserve it and support the VL elimination program.

Methodology and Principal Findings

We measured in vitro susceptibility towards MIL and paromomycin (PMM) in L. donovani isolated from VL and PKDL, pre- and post-treatment cases, using an amastigote-macrophage model. MIL susceptibility of post-treatment isolates from cured VL cases (n = 13, mean IC₅₀±SD = 2.43±1.44 µM), was comparable (p>0.05) whereas that from relapses (n = 3, mean IC₅₀ = 4.72±1.99 µM) was significantly higher (p = 0.04) to that of the pre-treatment group (n = 6, mean IC₅₀ = 1.86±0.75 µM). In PKDL, post-treatment isolates (n = 3, mean IC₅₀ = 16.13±2.64 µM) exhibited significantly lower susceptibility (p = 0.03) than pre-treatment isolates (n = 5, mean IC₅₀ = 8.63±0.94 µM). Overall, PKDL isolates (n = 8, mean IC₅₀ = 11.45±4.19 µM) exhibited significantly higher tolerance (p<0.0001) to MIL than VL isolates (n = 22, mean IC₅₀ = 2.58±1.58 µM). Point mutations in the miltefosine transporter (LdMT) and its beta subunit (LdRos3) genes previously reported in parasites with experimentally induced MIL resistance were not present in the clinical isolates. Further, the mRNA expression profile of these genes was comparable in the pre- and post-treatment isolates. Parasite isolates from VL and PKDL cases were uniformly susceptible to PMM with respective mean IC₅₀ = 7.05±2.24 µM and 6.18±1.51 µM.

Conclusion

The in vitro susceptibility of VL isolates remained unchanged at the end of MIL treatment; however, isolates from relapsed VL and PKDL cases had lower susceptibility than the pre-treatment isolates. PKDL isolates were more tolerant towards MIL in comparison with VL isolates. All parasite isolates were uniformly susceptible to PMM. Mutations in the LdMT and LdRos3 genes as well as changes in the expression of these genes previously correlated with experimental resistance to MIL could not be verified for the field isolates.