Biochemically defined HIV-1 envelope glycoprotein variant immunogens display differential binding and neutralizing specificities to the CD4-binding site

HIV-1 gp120 binds the primary receptor CD4. Recently, a plethora of broadly neutralizing antibodies to the gp120 CD4-binding site (CD4bs) validated this region as a target for immunogen design. Here, we asked if modified HIV-1 envelope glycoproteins (Env) designed to increase CD4 recognition might improve recognition by CD4bs neutralizing antibodies and more efficiently elicit such reactivities. We also asked if CD4bs stabilization, coupled with altering the Env format (monomer to trimer or cross-clade), might better elicit neutralizing antibodies by focusing the immune response on the functionally conserved CD4bs. We produced monomeric and trimeric Envs stabilized by mutations within the gp120 CD4bs cavity (pocket-filling; PF2) or by appending heterologous trimerization motifs to soluble Env ectodomains (gp120/gp140). Recombinant glycoproteins were purified to relative homogeneity, and ligand binding properties were analyzed by ELISA, surface plasmon resonance, and isothermal titration microcalorimetry. In some formats, the PF2 substitutions increased CD4 affinity, and importantly, PF2-containing proteins were better recognized by the broadly neutralizing CD4bs mAbs, VRC01 and VRC-PG04. Based on this analysis, we immunized selected Env variants into rabbits using heterologous or homologous regimens. Analysis of the sera revealed that homologous inoculation of the PF2-containing, variable region-deleted YU2 gp120 trimers (ΔV123/PF2-GCN4) more rapidly elicited CD4bs-directed neutralizing antibodies compared with other regimens, whereas homologous trimers elicited increased neutralization potency, mapping predominantly to the gp120 third major variable region (V3). These results suggest that some engineered Env proteins may more efficiently direct responses toward the conserved CD4bs and be valuable to elicit antibodies of greater neutralizing capacity.     

Activation of both Mos and Cdc25 is required for G2-M transition in perch oocyte

Resumption of meiosis from diplotene arrest during the first meiotic prophase in vertebrate oocytes is universally controlled by MPF, a heterodimer of Cdk1 and cyclin B. Activation of MPF depends on the withdrawal of Cdk1 inhibition by Wee1/Myt1 kinase on the one hand and the activation of Cdk1 by Cdc25 phosphatase on the other. It is relevant to know whether both these pathways are necessary to rescue diplotene arrest or if either one of them is sufficient. In MIH (17alpha, 20beta dihydroxy-4-pregnen-3-one) incubated perch (Anabas testudineus) oocytes we have examined these possibilities. Perch oocyte extract following MIH incubation showed a significant increase in Myt1 phosphorylation from 12 to 16 hr indicating its progressive deactivation. MIH induced Mos expression markedly increased at 16 hr effecting 95% GVBD. Cycloheximide inhibited MIH induced Mos expression and its phosphorylation, which in turn reduced Myt1 phosphorylation and GVBD. Myt1 phosphorylation was blocked in Mos immunodepleted oocytes. All these suggest the involvement of Mos in Myt1 phosphorylation. Oocytes incubated in MIH for 16 hr activated Cdc25, but such activation could not rescue the inhibition of GVBD due to Myt1 in Mos immunodepleted oocytes. Blocking Cdc25 with an antisense oligo significantly inhibited GVBD even though Myt1 remained deactivated during this period. Taken together, our findings indicate that MIH requires both pathways for perch oocyte maturation: the expression and activation of Mos, which is linked to Myt1 deactivation on the one hand, and the activation of Cdc25 on the other, as blocking either pathway compromised G2-M transition in perch oocytes.     

The Prevalence of Depression in White-European and South-Asian People with Impaired Glucose Regulation and Screen-Detected Type 2 Diabetes Mellitus

Background

There is a clear relationship between depression and diabetes. However, the directionality of the relationship remains unclear and very little research has considered a multi-ethnic population. The aim of this study was to determine the prevalence of depression in a White-European (WE) and South-Asian (SA) population attending a community diabetes screening programme, and to explore the association of depression with screen-detected Type 2 diabetes mellitus (T2DM) and impaired glucose regulation (IGR).

Methodology/Principal Findings

Participants were recruited from general practices in Leicestershire (United Kingdom) between August 2004 and December 2007. 4682 WE (40–75 years) and 1327 SA participants (25–75 years) underwent an Oral Glucose Tolerance Test, detailed history, anthropometric measurements and completed the World Health Organisation-Five (WHO-5) Wellbeing Index. Depression was defined by a WHO-5 wellbeing score ≤13. Unadjusted prevalence of depression for people in the total sample with T2DM and IGR was 21.3% (21.6% in WE, 20.6% in SA, p = 0.75) and 26.0% (25.3% in WE, 28.9% in SA, p = 0.65) respectively. For people with normal glucose tolerance, the prevalence was 25.1% (24.9% in WE, 26.4% in SA, p = 0.86). Age-adjusted prevalences were higher for females than males. Odds ratios adjusted for age, gender, and ethnicity, showed no significant increase in prevalent depression for people with T2DM (OR = 0.95, 95%CI 0.62 to 1.45) or IGR (OR = 1.17, 95%CI 0.96 to1.42).

Conclusions

Prior to the knowledge of diagnosis, depression was not significantly more prevalent in people with screen detected T2DM or IGR. Differences in prevalent depression between WE and SA people were also not identified. In this multi-ethnic population, female gender was significantly associated with depression.     

Structure-Based Stabilization of HIV-1 gp120 Enhances Humoral Immune Responses to the Induced Co-Receptor Binding Site

The human immunodeficiency virus type 1 (HIV-1) exterior envelope glycoprotein, gp120, possesses conserved binding sites for interaction with the primary virus receptor, CD4, and also for the co-receptor, generally CCR5. Although gp120 is a major target for virus-specific neutralizing antibodies, the gp120 variable elements and its malleable nature contribute to evasion of effective host-neutralizing antibodies. To understand the conformational character and immunogenicity of the gp120 receptor binding sites as potential vaccine targets, we introduced structure-based modifications to stabilize gp120 core proteins (deleted of the gp120 major variable regions) into the conformation recognized by both receptors. Thermodynamic analysis of the re-engineered core with selected ligands revealed significant stabilization of the receptor-binding regions. Stabilization of the co-receptor-binding region was associated with a marked increase in on-rate of ligand binding to this site as determined by surface plasmon resonance. Rabbit immunization studies showed that the conformational stabilization of core proteins, along with increased ligand affinity, was associated with strikingly enhanced humoral immune responses against the co-receptor-binding site. These results demonstrate that structure-based approaches can be exploited to stabilize a conformational site in a large functional protein to enhance immunogenic responses specific for that region.     

Effect of harmaline on rat intestinal brush border sucrase activity

The effect of harmaline, a plant alkaloid has been studied on rat intestinal brush border sucrase activity. Stimulation of sucrase activity by Na+ was found to be pH-dependent. At neutral pH, 20 mM Na+ stimulated sucrase activity by reducing K(m) by 30%, while at acidic pH (5.2), the activity increased 4-fold compared to Na+-free enzyme. At 1.0 mM, harmaline markedly inhibited (67%) the enzyme activity at pH 5.2 in the absence of Na+. However, inhibition was reduced in presence of 20 mM sodium, whereas 4.0 mM harmaline was required to inhibit the enzyme activity by 65%. In the absence of Na+ ions, harmaline inhibition of sucrase activity was of competitive type, but it changed to non-competitive type in presence of 20 mM Na+ at pH 5.2. Sucrase-harmaline interactions as a function of pH, both in presence and absence of Na+ revealed a shift in pH optima of the enzyme towards a higher pH in presence of 4 mM and 1 mM harmaline respectively. The observed inhibition was reversible in nature and was only partially overcome by sodium, lithium, potassium, cesium, rubidium and ammonium ions. These findings suggest that harmaline also inhibits rat brush border sucrase and that the presence of Na+ site is not a pre-requisite for the inhibition.     

A new furanoditerpenoid marker for the distinction between the seeds of two species of Caesalpinia

A new cassane furanoditerpene, 17-methylvouacapane-8(14),-9(11)-diene (1), has been isolated from the seed kernels of Caesalpinia crista and its structure determined by mass, IR and NMR spectrometry. The 1H- and 13C-NMR spectra were assigned using a combination of 1H-1H COSY, HSQC and HMBC two-dimensional NMR experiments. An HPTLC method has been developed to quantify 1 in seed material. The furanoditerpene can serve as a marker chemically to differentiate C. crista from the synonymous C. bonduc.     

High-yield functional expression of human sodium/d-glucose cotransporter1 in Pichia pastoris and characterization of ligand-induced conformational changes as studied by tryptophan fluorescence

Studies on the structure-function relationship of transporters require the availability of sufficient amounts of the protein in a functional state. In this paper, we report the functional expression, purification, and reconstitution of the human sodium/d-glucose cotransporter1 (hSGLT1) in Pichia pastoris and ligand-induced conformational changes of hSGLT1 in solution as studied by intrinsic tryptophan fluorescence. hSGLT1 gene containing FLAG tag at position 574 was cloned into pPICZB plasmid, and the resulting expression vector pPICZB-hSGLT1 was introduced into P. pastoris strain GS115 by electroporation. Purification of recombinant hSGLT1 by nickel-affinity chromatography yields about 3 mg of purified recombinant hSGLT1 per 1-liter of cultured Pichia cells. Purified hSGLT1 migrates on SDS-PAGE with an apparent mass of 55 kDa. Kinetic analysis of hSGLT1 in proteoliposomes revealed sodium-dependent, secondary active, phlorizin-sensitive, and stereospecific alpha-methyl-d-glucopyranoside transport, demonstrating its full catalytic activity. The position of the maximum intrinsic tryptophan fluorescence and titration with hydrophilic collisional quenchers KI, acrylamide, and trichloroethanol suggested that most of Trps in hSGLT1 in solution are in a hydrophobic environment. In the presence of sodium, sugars that have been identified earlier as substrate for the transporter increase intrinsic fluorescence in a saturable manner by a maximum of 15%. alpha-Methyl-d-glucopyranoside had the highest affinity (K(d) = 0.71 mM), followed by d-glucose, d-galactose, d-mannose, and d-allose which showed a much lower affinity. l-Glucose was without effect. d-Glucose also increased the accessibility of the Trps to hydrophilic collisional quenchers. On the contrary phlorizin, the well-established inhibitor of SGLT1, decreased intrinsic fluorescence by a maximum of 50%, and induced a blue shift of maximum (5 nm). Again, the effects were sodium-dependent and saturable and a high affinity K(d) of 5 muM was observed. In addition the surface of hSGLT1 was labeled with 1-anilinonaphthalene-8-sulfonic acid, a reporter molecule for the surface hydrophobicity. In the presence of sodium, addition of d-glucose decreased ANS fluorescence whereas phlorizin increased ANS fluorescence. Thus three conformational states of SGLT1 could be defined which differ in their packing density and hydrophobicity of their surface. They reflect properties of the empty carrier, the d-glucose loaded carrier facing the outside of membrane and the complex of the outside-orientated carrier with phlorizin.     

Purification and characterisation of alkaline cellulase produced by a novel isolate,<Emphasis Type="Italic"> Bacillus sphaericus</Emphasis> JS1

A novel strain of Bacillus sphaericus JS1 producing thermostable alkaline carboxymethyl cellulase (CMCase; endo-1,4--glucanase, E.C. 3.2.1.4) was isolated from soil using Horikoshi medium at pH 9.5. CMCase was purified 192-fold by (NH₄)₂SO₄ precipitation, ion exchange and gel filtration chromatography, with an overall recovery of 23%. The CMCase is a multimeric protein with a molecular weight estimated by native-PAGE of 183 kDa. Using SDS-PAGE a single band is found at 42 kDa. This suggests presence of four homogeneous polypeptides, which would differentiate this enzyme from other known alkaline cellulases. The activity of the enzyme was significantly inhibited by bivalent cations (Fe³⁺ and Hg²⁺, 1.0 mM each) and activated by Co²⁺, K⁺ and Na⁺. The purified enzyme revealed the products of carboxymethyl cellulose (CMC) hydrolysis to be CM glucose, cellobiose and cellotriose. Thermostability, pH stability, good hydrolytic capability, and stability in the presence of detergents, surfactants, chelators and commercial proteases make this enzyme potentially useful in laundry detergents.     

Assessment of genetic diversity and population structure in gladiolus (<Emphasis Type="Italic">Gladiolus hybridus</Emphasis> Hort.) by ISSR markers

ISSR (Inter simple sequence repeat) markers were used to assess the genetic diversity and population structure in 53 indigenous and exotic genotypes of gladiolus (Gladiolus hybridus Hort.). Molecular markers analysis showed PIC ranges from 0.42 (ISSR 861) to 0.99 (ISSR 855, ISSR 856 and ISSR 889) with an average 0.812, marker index ranged from 0.99 (ISSR 889) to 9.26 (ISSR 851) with an average 4.66 and resolving power of the primers ranged from 0.03 (ISSR 889) to 11.58 (ISSR 861) with an average value 3.80. The dendrogram based UPGMA clustering showed that all the 53 genotypes grouped into three main clusters. Nei’s gene diversity (Na) varied from 0.929 to 1.717, effective number of alleles (Ne) varied from 1.262 to 1.369, Shannon’s information index (I) ranged from 0.251 to 0.359 and gene diversity (He) was in the range from 0.167 to 0.229. Population structure analysis revealed three groups in which 32 genotypes were admixture types.     

Induction of Metallothionein in Rat Liver by Zinc Exposure: A Dose and Time Dependent Study

Metallothioneins (MTs) are low molecular weight ubiquitous metalloproteins with high cysteine (thiol) content. The intracellular concentration of zinc (Zn) is tightly regulated and MT plays a crucial role in it. The present study investigates the relationship between the Zn status (as a function of Zn concentration and time) in the rat liver and the occurrence of hepatic MT. For dose dependent study, four experimental groups, one control and three receiving different levels of metal supplementation, were chosen [Group 1 control and Group 2, Group 3, Group 4 receiving subcutaneous dose of 10, 50 and 100 mg of Zn/kg body weight (in the form of ZnSO₄·7H₂O), respectively]. For the time dependent expression of MT, again four experimental groups, i.e. Group 5 control and Group 6, Group 7, Group 8 receiving 50 mg of Zn/kg body weight (in the form of ZnSO₄·7H₂O) subcutaneously and sacrificed at different time intervals after last injection i.e. 6, 18, 48 h, respectively were chosen. Isolation of MT was done by using combination of gel filtration and ion exchange chromatography while characterization of MT fraction was carried in the wavelength range 200–400 nm. Expression of MT was studied by using Western blot analysis. The results revealed that the MT expression increases with increasing the dose of Zn administered and maximum at 18 h after last Zn injection. Accumulation of MT with increase dose would help in maintaining the intracellular Zn concentration by its sequestration which further reduces the possibility of undesirable binding of Zn to other proteins significantly and maintains Zn homeostasis. The maximum expression of MT at 18 h is indicative of its half life.