A three-state model for connexin37 gating kinetics

The gating behavior of human connexin 37 (hCx37) is unaffected by the nature of the bathing monovalent (for Na, K, Rb). It is modified by [Mg] in the millimolar range. For fitting the kinetics, we propose a simple extension to three states of the canonical 2-state model of the hemichannel. The extra closed state allows for some immobilization of a hemichannel at high transjunctional voltages. The model is reasonably efficient at fitting data at various voltage protocols. Interpreting the fits of the data at different [Mg] is consistent with a binding site for Mg.     

Functional expression and biophysical properties of polymorphic variants of the human gap junction protein connexin37

Connexin37 (Cx37) forms gap junction channels between endothelial cells, and two polymorphic Cx37 variants (Cx37-S319 and Cx37-P319) have been identified with a possible link to atherosclerosis. We studied the gap junction channel properties of these hCx37 polymorphs by expression in stably transfected communication-deficient cells (N2A and RIN). We also expressed a third, truncated variant (Cx37-fs254Delta293) and Cx37 constructs containing epitope tags added to their amino or carboxyl termini. All Cx37 constructs were produced by the transfected cells as demonstrated by RT-PCR and immunoblotting and trafficked to appositional surfaces between cells as demonstrated by immunofluorescence microscopy. Dual whole cell patch-clamping studies demonstrated that Cx37-P319, Cx37-S319, and Cx37-fs254Delta293 had large unitary conductances ( approximately 300 pS). However, addition of an amino terminal T7 tag (T7-Cx37-fs254Delta293) produced a single channel conductance of 120-145 pS with a 24-30 pS residual state. Moreover, the kinetics of the voltage-dependent decline in junctional current for T7-Cx37-fs254Delta293 were significantly slower than for the wild type, implying a destabilization of the transition state. These data suggest that the amino terminus of Cx37 plays a significant role in gating as well as conductance. The carboxyl terminal tail has lesser influence on unitary conductance and inactivation kinetics.     

The Role of Exciton Delocalization in the Major Photosynthetic Light-Harvesting Antenna of Plants

In the major peripheral plant light-harvesting complex LHCII, excitation energy is transferred between chlorophylls along an energetic cascade before it is transmitted further into the photosynthetic assembly to be converted into chemical energy. The efficiency of these energy transfer processes involves a complicated interplay of pigment-protein structural reorganization and protein dynamic disorder, and the system must stay robust within the fluctuating protein environment. The final, lowest energy site has been proposed to exist within a trimeric excitonically coupled chlorophyll (Chl) cluster, comprising Chls a610-a611-a612. We studied an LHCII monomer with a site-specific mutation resulting in the loss of Chls a611and a612, and find that this mutant exhibits two predominant overlapping fluorescence bands. From a combination of bulk measurements, single-molecule fluorescence characterization, and modeling, we propose the two fluorescence bands originate from differing conditions of exciton delocalization and localization realized in the mutant. Disruption of the excitonically coupled terminal emitter Chl trimer results in an increased sensitivity of the excited state energy landscape to the disorder induced by the protein conformations. Consequently, the mutant demonstrates a loss of energy transfer efficiency. On the contrary, in the wild-type complex, the strong resonance coupling and correspondingly high degree of excitation delocalization within the Chls a610-a611-a612 cluster dampens the influence of the environment and ensures optimal communication with neighboring pigments. These results indicate that the terminal emitter trimer is thus an essential design principle for maintaining the efficient light-harvesting function of LHCII in the presence of protein disorder.     

Recombinant renewable polyclonal antibodies

Only a small fraction of the antibodies in a traditional polyclonal antibody mixture recognize the target of interest, frequently resulting in undesirable polyreactivity. Here, we show that high-quality recombinant polyclonals, in which hundreds of different antibodies are all directed toward a target of interest, can be easily generated in vitro by combining phage and yeast display. We show that, unlike traditional polyclonals, which are limited resources, recombinant polyclonal antibodies can be amplified over one hundred million-fold without losing representation or functionality. Our protocol was tested on 9 different targets to demonstrate how the strategy allows the selective amplification of antibodies directed toward desirable target specific epitopes, such as those found in one protein but not a closely related one, and the elimination of antibodies recognizing common epitopes, without significant loss of diversity. These recombinant renewable polyclonal antibodies are usable in different assays, and can be generated in high throughput. This approach could potentially be used to develop highly specific recombinant renewable antibodies against all human gene products.     

Comparison of different delivery systems of DNA vaccination for the induction of protection against tuberculosis in mice and guinea pigs

The great challenges for researchers working in the field of vaccinology are optimizing DNA vaccines for use in humans or large animals and creating effective single-dose vaccines using appropriated controlled delivery systems. Plasmid DNA encoding the heat-shock protein 65 (hsp65) (DNAhsp65) has been shown to induce protective and therapeutic immune responses in a murine model of tuberculosis (TB). Despite the success of naked DNAhsp65-based vaccine to protect mice against TB, it requires multiple doses of high amounts of DNA for effective immunization. In order to optimize this DNA vaccine and simplify the vaccination schedule, we coencapsulated DNAhsp65 and the adjuvant trehalose dimycolate (TDM) into biodegradable poly (DL-lactide-co-glycolide) (PLGA) microspheres for a single dose administration. Moreover, a single-shot prime-boost vaccine formulation based on a mixture of two different PLGA microspheres, presenting faster and slower release of, respectively, DNAhsp65 and the recombinant hsp65 protein was also developed. These formulations were tested in mice as well as in guinea pigs by comparison with the efficacy and toxicity induced by the naked DNA preparation or BCG. The single-shot prime-boost formulation clearly presented good efficacy and diminished lung pathology in both mice and guinea pigs.     

Bio-sequestration of carbon dioxide using carbonic anhydrase enzyme purified from <Emphasis Type="Italic">Citrobacter freundii</Emphasis>

The increase in the atmospheric concentrations of one of the vital green house gasses, carbon dioxide, due to anthropogenic interventions has led to several undesirable consequences such as global warming and related changes. In the global effort to combat the predicted disaster, several CO₂ capture and storage technologies are being deliberated. One of the most promising biological carbon dioxide sequestration technologies is the enzyme catalyzed carbon dioxide sequestration into bicarbonates which was endeavored in this study with a purified C. freundii SW3 β-carbonic anhydrase (CA). An extensive screening process for biological sequestration using CA has been defined. Six bacteria with high CA activity were screened out of 102 colonies based on plate assay and presence of CA in these bacteria was further emphasized by activity staining and Western blot. The identity of selected bacteria was confirmed by 16S rDNA analysis. CA was purified to homogeneity from C. freundii SW3 by subsequent gel filtration and ion exchange chromatography which resulted in a 24 kDa polypeptide and this is in accordance with the Western blot results. The effect of host on metal ions, cations and anions which influence activity of the enzyme in sequestration studies suggests that mercury and HCO₃ ⁻ ion almost completely inhibit the enzyme whereas sulfate ion and zinc enhances carbonic anhydrase activity. Calcium carbonate deposition was observed in calcium chloride solution saturated with carbon dioxide catalyzed by purified enzyme and whereas a sharp decrease in calcium carbonate formation has been noted in purified enzyme samples inhibited by EDTA and acetazolamide.     

Functional interplay between two Xanthomonas oryzae pv,. oryzae secretion systems in modulating virulence on rice

The type II (T2S) and type III (T3S) secretion systems are important for virulence of Xanthomonas oryzae pv. oryzae, causal agent of bacterial leaf blight of rice. The T3S of gram-negative bacterial plant pathogens has been shown to suppress host defense responses, including programmed cell death reactions, whereas the T2S is involved in secreting cell-wall-degrading enzymes. Here, we show that a T3S-deficient (T3S-) mutant of X. oryzae pv. oryzae can induce a basal plant defense response seen as callose deposition, immunize rice against subsequent X. oryzae pv. oryzae infection, and cause cell-death-associated nuclear fragmentation. A T2S- T3S- double mutant exhibited a substantial reduction in the ability to evoke these responses. We purified two major effectors of the X. oryzae pv. oryzae T2S and characterized them to be a cellulase (ClsA) and a putative cellobiosidase (CbsA). The purified ClsA, CbsA, and lipase/esterase (LipA; a previously identified T2S effector) proteins induced rice defense responses that were suppressible by X. oryzae pv. oryzae in a T3S-dependent manner. These defense responses also were inducible by the products of the action of these purified proteins on rice cell walls. We further show that a CbsA- mutant or a ClsA- LipA- double mutant are severely virulence deficient. These results indicate that the X. oryzae pv. oryzae T2S secretes important virulence factors, which induce innate rice defense responses that are suppressed by T3S effectors to enable successful infection.     

Synthesis of silver nanoparticles using haloarchaeal isolate Halococcus salifodinae BK3

Numerous bacteria, fungi, yeasts and viruses have been exploited for biosynthesis of highly structured metal sulfide and metallic nanoparticles. Haloarchaea (salt-loving archaea) of the third domain of life Archaea, on the other hand have not yet been explored for nanoparticle synthesis. In this study, we report the intracellular synthesis of stable, mostly spherical silver nanoparticles (AgNPs) by the haloarchaeal isolate Halococcus salifodinae BK₃. The culture on adaptation to silver nitrate exhibited growth kinetics similar to that of the control. NADH-dependent nitrate reductase was involved in silver tolerance, reduction, synthesis of AgNPs, and exhibited metal-dependent increase in enzyme activity. The AgNPs preparation was characterized using UV–visible spectroscopy, XRD, TEM and EDAX. The XRD analysis of the nanoparticles showed the characteristic Bragg peaks of face-centered cubic silver with crystallite domain size of 22 and 12 nm for AgNPs synthesized in NTYE and halophilic nitrate broth (HNB), respectively. The average particle size obtained from TEM analysis was 50.3 and 12 nm for AgNPs synthesized in NTYE and HNB, respectively. This is the first report on the synthesis of silver nanoparticles by haloarchaea.