Reducing the stimulation of CD8+ T cells during infection with intracellular bacteria promotes differentiation primarily into a central (CD62LhighCD44high) subset

During infection with lymphocytic choriomeningitis virus, CD8(+) T cells differentiate rapidly into effectors (CD62L(low)CD44(high)) that differentiate further into the central memory phenotype (CD62L(high)CD44(high)) gradually. To evaluate whether this CD8(+) T cell differentiation program operates in all infection models, we evaluated CD8(+) T cell differentiation during infection of mice with recombinant intracellular bacteria, Listeria monocytogenes (LM) and Mycobacterium bovis (BCG), expressing OVA. We report that CD8(+) T cells primed during infection with the attenuated pathogen BCG-OVA differentiated primarily into the central subset that correlated to reduced attrition of the primed cells subsequently. CD8(+) T cells induced by LM-OVA also differentiated into central phenotype cells first, but the cells rapidly converted into effectors in contrast to BCG-OVA. Memory CD8(+) T cells induced by both LM-OVA as well as BCG-OVA were functional in that they produced cytokines and proliferated extensively in response to antigenic stimulation after adoptive transfer. During LM-OVA infection, if CD8(+) T cells were guided to compete for access to APCs, then they received reduced stimulation that was associated with increased differentiation into the central subset and reduced attrition subsequently. Similar effect was observed when CD8(+) T cells encountered APCs selectively during the waning phase of LM-OVA infection. Taken together, our results indicate that the potency of the pathogen can influence the differentiation and fate of CD8(+) T cells enormously, and the extent of attrition of primed CD8(+) T cells correlates inversely to the early differentiation of CD8(+) T cells primarily into the central CD8(+) T cell subset.     

Computer aided screening and evaluation of herbal therapeutics against MRSA infections

Methicillin resistant Staphylococcus aureus (MRSA), a pathogenic bacterium that causes life threatening outbreaks such as community-onset and nosocomial infections has emerged as 'superbug'. The organism developed resistance to all classes of antibiotics including the best known Vancomycin (VRSA). Hence, there is a need to develop new therapeutic agents. This study mainly evaluates the potential use of botanicals against MRSA infections. Computer aided design is an initial platform to screen novel inhibitors and the data finds applications in drug development. The drug-likeness and efficiency of various herbal compounds were screened by ADMET and docking studies. The virulent factor of most of the MRSA associated infections are Penicillin Binding Protein 2A (PBP2A) and Panton-Valentine Leukocidin (PVL). Hence, native structures of these proteins (PDB: 1VQQ and 1T5R) were used as the drug targets. The docking studies revealed that the active component of Aloe vera, β-sitosterol (3S, 8S, 9S, 10R, 13R, 14S, 17R) -17- [(2R, 5R)-5-ethyl-6-methylheptan-2-yl] -10, 13-dimethyl 2, 3, 4, 7, 8, 9, 11, 12, 14, 15, 16, 17- dodecahydro-1H-cyclopenta [a] phenanthren-3-ol) showed best binding energies of -7.40 kcal/mol and -6.34 kcal/mol for PBP2A and PVL toxin, respectively. Similarly, Meliantriol (1S-1-[ (2R, 3R, 5R)-5-hydroxy-3-[(3S, 5R, 9R, 10R, 13S, 14S, 17S)-3-hydroxy 4, 4, 10, 13, 14-pentamethyl-2, 3, 5, 6, 9, 11, 12, 15, 16, 17-decahydro-1H-cyclopenta[a] phenanthren-17-yl] oxolan-2-yl] -2- methylpropane-1, 2 diol), active compound in Azadirachta indica (Neem) showed the binding energies of -6.02 kcal/mol for PBP2A and -8.94 for PVL toxin. Similar studies were conducted with selected herbal compound based on pharmacokinetic properties. All in silico data tested in vitro concluded that herbal extracts of Aloe-vera, Neem, Guava (Psidium guajava), Pomegranate (Punica granatum) and tea (Camellia sinensis) can be used as therapeutics against MRSA infections.     

Alkali‐based AFEX pretreatment for the conversion of sugarcane bagasse and cane leaf residues to ethanol

Sugarcane is one of the major agricultural crops cultivated in tropical climate regions of the world. Each tonne of raw cane production is associated with the generation of 130 kg dry weight of bagasse after juice extraction and 250 kg dry weight of cane leaf residue postharvest. The annual world production of sugarcane is ～1.6 billion tones, generating 279 MMT tones of biomass residues (bagasse and cane leaf matter) that would be available for cellulosic ethanol production. Here, we investigated the production of cellulosic ethanol from sugar cane bagasse and sugar cane leaf residue using an alkaline pretreatment: ammonia fiber expansion (AFEX). The AFEX pretreatment improved the accessibility of cellulose and hemicelluloses to enzymes during hydrolysis by breaking down the ester linkages and other lignin carbohydrate complex (LCC) bonds and the sugar produced by this process is found to be highly fermentable. The maximum glucan conversion of AFEX pretreated bagasse and cane leaf residue by cellulases was ～85%. Supplementation with hemicellulases during enzymatic hydrolysis improved the xylan conversion up to 95–98%. Xylanase supplementation also contributed to a marginal improvement in the glucan conversion. AFEX‐treated cane leaf residue was found to have a greater enzymatic digestibility compared to AFEX‐treated bagasse. Co‐fermentation of glucose and xylose, produced from high solid loading (6% glucan) hydrolysis of AFEX‐treated bagasse and cane leaf residue, using the recombinant Saccharomyces cerevisiae (424A LNH‐ST) produced 34–36 g/L of ethanol with 92% theoretical yield. These results demonstrate that AFEX pretreatment is a viable process for conversion of bagasse and cane leaf residue into cellulosic ethanol. Biotechnol. Bioeng. 2010;107: 441–450. © 2010 Wiley Periodicals, Inc.     

Long Term Expression of Drosophila melanogaster Nucleoside Kinase in Thymidine Kinase 2-deficient Mice with No Lethal Effects Caused by Nucleotide Pool Imbalances

Mitochondrial DNA depletion caused by thymidine kinase 2 (TK2) deficiency can be compensated by a nucleoside kinase from Drosophila melanogaster (Dm-dNK) in mice. We show that transgene expression of Dm-dNK in Tk2 knock-out (Tk2^−/−) mice extended the life span of Tk2^−/− mice from 3 weeks to at least 20 months. The Dm-dNK^+/−Tk2^−/− mice maintained normal mitochondrial DNA levels throughout the observation time. A significant difference in total body weight due to the reduction of subcutaneous and visceral fat in the Dm-dNK^+/−Tk2^−/− mice was the only visible difference compared with control mice. This indicates an effect on fat metabolism mediated through residual Tk2 deficiency because Dm-dNK expression was low in both liver and fat tissues. Dm-dNK expression led to increased dNTP pools and an increase in the catabolism of purine and pyrimidine nucleotides but these alterations did not apparently affect the mice during the 20 months of observation. In conclusion, Dm-dNK expression in the cell nucleus expanded the total dNTP pools to levels required for efficient mitochondrial DNA synthesis, thereby compensated the Tk2 deficiency, during a normal life span of the mice. The Dm-dNK^+/− mouse serves as a model for nucleoside gene or enzyme substitutions, nucleotide imbalances, and dNTP alterations in different tissues.     

Functional role and affinity of inorganic cations in stabilizing the tetrameric structure of the KcsA K+ channel

Crystal structures of the tetrameric KcsA K+ channel reveal seven distinct binding sites for K+ ions within the central pore formed at the fourfold rotational symmetry axis. Coordination of an individual K+ ion by eight protein oxygen atoms within the selectivity filter suggests that ion-subunit bridging by cation-oxygen interactions contributes to structural stability of the tetramer. To test this hypothesis, we examined the effect of inorganic cations on the temperature dependence of the KcsA tetramer as monitored by SDS-PAGE. Inorganic cations known to permeate or strongly block K+ channels (K+, Rb+, Cs+, Tl+, NH4+, Ba2+, and Sr2+) confer tetramer stability at higher temperatures (T0.5 range = 87 degrees C to >99 degrees C) than impermeant cations and weak blockers (Li+, Na+, Tris+, choline+; T0.5 range = 59 degrees C to 77 degrees C). Titration of K+, Ba2+, and other stabilizing cations protects against rapid loss of KcsA tetramer observed in 100 mM choline Cl at 90 degrees C. Tetramer protection titrations of K+, Rb+, Cs+, Tl+, and NH4+ at 85 degrees C or 90 degrees C exhibit apparent Hill coefficients (N) ranging from 1.7 to 3.3 and affinity constants (K0.5) ranging from 1.1 to 9.6 mM. Ba2+ and Sr2+ titrations exhibit apparent one-site behavior (N congruent with 1) with K0.5 values of 210 nM and 11 microM, respectively. At 95 degrees C in the presence of 5 mM K+, titration of Li+ or Na+ destabilizes the tetramer with K0.5 values of 57 mM and 109 mM, respectively. We conclude that specific binding interactions of inorganic cations with the selectivity filter are an important determinant of tetramer stability of KscA.     

AT4 receptor structure-binding relationship: N-terminal-modified angiotensin IV analogues

The effect of structural changes in the N-terminal amino acid of AIV, with respect to AT₄ receptor binding, was examined by competition with [¹²⁵I]AIV in bovine adrenal membranes. Analogues with modifications of the first residue α-amino group possessed lower affinities than the primary amine-containing parent compound. Peptides with a residue 1 α-carbon in the d conformation exhibited poor affinity for the AT₄ receptor. Modifications of the residue 1 R-group demonstrate that a straight chain aliphatic moiety containing four carbons is optimal for receptor-ligand binding, as evidenced by the extremely high affinity of [Nle¹]AIV (K_i = 3.59±0.51 pM). Replacement of the 1–2 peptide bond of AIV with the methylene bond isostere Ψ (CH₂-NH), increased the K_i approximately fivefold, indicating that the peptide bond may be replaced wihle maintaining relatively high-affinity receptor binding.