Structures of the iron-sulfur flavoproteins from Methanosarcina thermophila and Archaeoglobus fulgidus

Iron-sulfur flavoproteins (ISF) constitute a widespread family of redox-active proteins in anaerobic prokaryotes. Based on sequence homologies, their overall structure is expected to be similar to that of flavodoxins, but in addition to a flavin mononucleotide cofactor they also contain a cubane-type [4Fe:4S] cluster. In order to gain further insight into the function and properties of ISF, the three-dimensional structures of two ISF homologs, one from the thermophilic methanogen Methanosarcina thermophila and one from the hyperthermophilic sulfate-reducing archaeon Archaeoglobus fulgidus, were determined. The structures indicate that ISF assembles to form a tetramer and that electron transfer between the two types of redox cofactors requires oligomerization to juxtapose the flavin mononucleotide and [4Fe:4S] cluster bound to different subunits. This is only possible between different monomers upon oligomerization. Fundamental differences in the surface properties of the two ISF homologs underscore the diversity encountered within this protein family.     

Protein stabilization by introduction of cross-strand disulfides

Disulfides cross-link residues in a protein that are separated in primary sequence and stabilize the protein through entropic destabilization of the unfolded state. While the removal of naturally occurring disulfides leads to protein destabilization, introduction of engineered disulfides does not always lead to significant stabilization of a protein. We have analyzed naturally occurring disulfides that span adjacent antiparallel strands of beta sheets (cross-strand disulfides). Cross-strand disulfides have recently been implicated as redox-based conformational switches in proteins such as gp120 and CD4. The propensity of these disulfides to act as conformational switches was postulated on the basis of the hypothesis that this class of disulfide is conformationally strained. In the present analysis, there was no evidence to suggest that cross-strand disulfides are more strained compared to other disulfides as assessed by their torsional energy. It was also observed that these disulfides occur solely at non-hydrogen-bonded (NHB) registered pairs of adjacent antiparallel strands and not at hydrogen-bonded (HB) positions as suggested previously. One of the half-cystines involved in cross-strand disulfide formation often occurs at an edge strand. Experimental confirmation of the stabilizing effects of such disulfides was carried out in Escherichia coli thioredoxin. Four pairs of cross-strand cysteines were introduced, two at HB and two at NHB pairs. Disulfides were formed in all four cases. However, as predicted from our analysis, disulfides at NHB positions resulted in an increase in melting temperature of 7-10 degrees C, while at HB positions there was a corresponding decrease of -7 degrees C. The reduced state of all proteins had similar stability.     

Picrotoxin inhibition mechanism of a gamma-aminobutyric acid A receptor investigated by a laser-pulse photolysis technique

The gamma-aminobutyric acid(A) (GABA(A)) receptor, a major inhibitory neurotransmitter receptor, belongs to a family of membrane-bound proteins that regulate signal transmission between approximately 10(12) cells of the nervous system. It plays a major role in many neurological disorders, including epilepsy. It is the target of many pharmacological agents, including the convulsant picrotoxin. Here, we present the mechanism of inhibition by picrotoxin of the rat alpha1beta2gamma2L GABA(A) receptor investigated using rapid kinetic techniques in combination with whole-cell current recordings. The following new results were obtained by using transient kinetic techniques, the cell-flow method and the laser-pulse photolysis (LaPP) technique with a microsecond to millisecond time resolution. (i) The apparent dissociation constant of picrotoxin for the open-channel form of the receptor was approximately 5 times higher than that of the closed-channel form. (ii) Picrotoxin increased the channel-closing rate constant (k(cl)) approximately 4-fold, while the rate constant for channel opening (k(op)) remained essentially unaffected. (iii) The mechanism indicates that picrotoxin binds to an allosteric site of the receptor with higher affinity for the closed-channel form than for the open-channel form and thereby inhibits the receptor by decreasing 4-fold its channel-opening equilibrium constant [Phi(I)(-)(1) = k(op(I))/k(cl(I))]. (iv) The mechanism further indicates that compounds that bind with equal affinity to the picrotoxin-binding site on the open-channel form of the receptor and the closed-channel form will not affect the channel-opening equilibrium and can, therefore, displace picrotoxin and prevent inhibition of the GABA(A) receptor by picrotoxin. Such compounds may be therapeutically useful in counteracting the effects of compounds and diseases that unfavorably affect the channel-opening equilibrium of the receptor channel.     

Treatment with high-dose simvastatin reduces secretion of apolipoprotein B-lipoproteins in patients with diabetic dyslipidemia

HMG-CoA reductase inhibitors (statins) are effective lipid-altering drugs for the treatment of dyslipidemia in patients with type 2 diabetes mellitus. We conducted a randomized, double-blind, placebo-controlled, crossover design trial to determine the effects of simvastatin, 80 mg/day, on plasma lipid and lipoprotein levels and on the metabolism of apolipoprotein B (apoB) in VLDL, intermediate density lipoprotein (IDL), and LDL and of triglycerides (TGs) in VLDL. Simvastatin therapy decreased TG, cholesterol, and apoB significantly in VLDL, IDL, and LDL. These effects were associated with reduced production of LDL-apoB, mainly as a result of reduced secretion of apoB-lipoproteins directly into the LDL density range. Statin therapy also reduced hepatic production of VLDL-TG. There were no effects of simvastatin on the fractional catabolic rates of VLDL-apoB or -TG or LDL-apoB. The basis for decreased VLDL-TG secretion during simvastatin treatment is not clear, but recent studies suggest that statins may activate peroxisomal proliferator-activated receptor alpha (PPARalpha). Activation of PPARalpha could lead to increased hepatic oxidation of fatty acids and less synthesis of TG for VLDL assembly.     

Correlation between molecular markers and adaptively significant genetic variation in Bromus tectorum (Poaceae), an inbreedingannual grass

Single sequence repeat (SSR) and amplified fragment length polymorphic (AFLP) molecular marker genotypes in cheatgrass (Bromus tectorum) were compared to published data on phenotypic variation in seed dormancy, vernalization requirement, and resistance to the pathogen Ustilago bullata. Several features of cheatgrass facilitated this study: it is a recent invader in the western United States, has considerable phenotypic polymorphism, and is an obligate self-pollinator. Forty self-pollinating lines from four populations common to the three phenotypic data sets were analyzed for molecular genetic variation using seven SSR loci and 31 AFLP loci. We examined correlations between distance matrices using the Mantel test for each pair of studies. The two molecular data sets were significantly correlated (r = 0.636). The AFLP markers often distinguished among several lines with identical SSR genotypes. The AFLP data were also significantly correlated with the phenotypic data (r values from 0.4640 to 0.5658), but the SSR data were much more highly correlated (r values from 0.677 to 0.844). The difference between molecular marker systems was especially notable when an outlier population from Potosi Pass, Nevada, was excluded from the analysis. These results suggest that SSR markers may be good surrogates for phenotypic traits in population genetic studies of strongly inbreeding species such as cheatgrass.     

Amblyomma americanum salivary gland homolog of nSec1 is essential for saliva protein secretion

Soluble N-ethylmaleimide-sensitive factor attachment protein receptor proteins assemble in tight core complexes which promote fusion of carrier vesicles with target compartments. Members of this class of proteins are expressed in all eukaryotic cells and distributed in distinct subcellular compartments. All vesicle transport mechanisms known to date have an essential requirement for a member of the Sec1 protein family, including the nSec1 in regulated exocytosis. A homolog of nSec1 was cloned and sequenced from the salivary glands of partially fed female ticks. Double-stranded RNA was used to specifically reduce the amount of nSec1 mRNA and protein in female adult tick salivary glands. This reduction was accompanied by a decrease in anticoagulant protein release by the glands and by abnormalities in feeding by dsRNA treated ticks. We report the efficacy of double-stranded RNA-mediated interference in "knocking down" nSec1 both in vivo and in vitro in tick salivary glands and the applicability of this technique for studying the mechanism of exocytosis in tick salivary glands.     

Functional Characterization of Na<Superscript>+</Superscript>/H<Superscript>+</Superscript> Exchangers in Primary Cultures of Prairie Dog Gallbladder

Gallbladder Na⁺ absorption is linked to gallstone formation in prairie dogs. We previously reported Na⁺/H⁺ exchanger (NHE1-3) expression in native gallbladder tissues. Here we report the functional characterization of NHE1, NHE2 and NHE3 in primary cultures of prairie dog gallbladder epithelial cells (GBECs). Immunohistochemical studies showed that GBECs grown to confluency are homogeneous epithelial cells of gastrointestinal origin. Electron microscopic analysis of GBECs demonstrated that the cells form polarized monolayers characterized by tight junctions and apical microvilli. GBECs grown on Snapwells exhibited polarity and developed transepithelial short-circuit current, I_sc, (11.6 ± 0.5 µA · cm^–2), potential differences, V_t (2.1 ± 0.2 mV), and resistance, R_t (169 ± 12 · cm²). NHE activity in GBECs assessed by measuring dimethylamiloride-inhibitable ²²Na⁺ uptake under a H⁺ gradient was the same whether grown on permeable Snapwells or plastic wells. The basal rate of ²²Na⁺ uptake was 21.4 ± 1.3 nmol · mg prot^–1 · min^–1, of which 9.5 ± 0.7 (~45%) was mediated through apically-restricted NHE. Selective inhibition with HOE-694 revealed that NHE1, NHE2 and NHE3 accounted for ~6%, ~66% and ~28% of GBECs total NHE activity, respectively. GBECs exhibited saturable NHE kinetics (V_max 9.2 ± 0.3 nmol · mg prot^–1 · min^–1; K_m 11.4 ± 1.4 mM Na⁺). Expression of NHE1, NHE2 and NHE3 mRNAs was confirmed by RT-PCR analysis. These results demonstrate that the primary cultures of GBECs exhibit Na⁺ transport characteristics similar to native gallbladder tissues, suggesting that these cells can be used as a tool for studying the mechanisms of gallbladder ion transport both under physiologic conditions and during gallstone formation.     

Whole Genomic analysis of a clinical isolate of Uropathogenic Escherichia coli strain of Sequence Type - 101 carrying the drug resistance NDM-7 in IncX3 plasmid

The emerging NDM-producing Enterobactereciae is a major threat to public health. The association of NDM-7 with sequence type 101 E.coli is identified in very few numbers. Therefore, it is of interest to analyse the whole genome sequence of NDM-producing uropathogenic E. coli XA31 that was found to carry numerous drug resistance genes of different antibiotic classes. The isolate E. coli belongs to ST-101 carrying blaNDM-7 coexisting with several resistance genes blaOXA-1, blaTEM1-A, blaCTX-M15, aac(6'')-Ib-cr, catB3, tetB. Resfinder predicts this and four other plasmid replicons were identified using the Plasfinder in the CGE platform. The high transferable IncX3 plasmid was found to carry the NDM-7 gene. Thus, we the report the combination of NDM-7-ST101-IncX3 in India. The combination of this epidemic clone with NDM-7 is highly required to develop an effective infection control strategy.     

Characterization of HIV-1 envelope gp41 genetic diversity and functional domains following perinatal transmission

Background

HIV-1 envelope gp41 is a transmembrane protein that promotes fusion of the virus with the plasma membrane of the host cells required for virus entry. In addition, gp41 is an important target for the immune response and development of antiviral and vaccine strategies, especially when targeting the highly variable envelope gp120 has not met with resounding success. Mutations in gp41 may affect HIV-1 entry, replication, pathogenesis, and transmission. We, therefore, characterized the molecular properties of gp41, including genetic diversity, functional motifs, and evolutionary dynamics from five mother-infant pairs following perinatal transmission.

Results

The gp41 open reading frame (ORF) was maintained with a frequency of 84.17% in five mother-infant pairs' sequences following perinatal transmission. There was a low degree of viral heterogeneity and estimates of genetic diversity in gp41 sequences. Both mother and infant gp41 sequences were under positive selection pressure, as determined by ratios of non-synonymous to synonymous substitutions. Phylogenetic analysis of 157 mother-infant gp41 sequences revealed distinct clusters for each mother-infant pair, suggesting that the epidemiologically linked mother-infant pairs were evolutionarily closer to each other as compared with epidemiologically unlinked sequences. The functional domains of gp41, including fusion peptide, heptad repeats, glycosylation sites and lentiviral lytic peptides were mostly conserved in gp41 sequences analyzed in this study. The CTL recognition epitopes and motifs recognized by fusion inhibitors were also conserved in the five mother-infant pairs.

Conclusion

The maintenance of an intact envelope gp41 ORF with conserved functional domains and a low degree of genetic variability as well as positive selection pressure for adaptive evolution following perinatal transmission is consistent with an indispensable role of envelope gp41 in HIV-1 replication and pathogenesis.