TORC1 and TORC2 work together to regulate ribosomal protein S6 phosphorylation in Saccharomyces cerevisiae

Nutrient-sensitive phosphorylation of the S6 protein of the 40S subunit of the eukaryote ribosome is highly conserved. However, despite four decades of research, the functional consequences of this modification remain unknown. Revisiting this enigma in Saccharomyces cerevisiae, we found that the regulation of Rps6 phosphorylation on Ser-232 and Ser-233 is mediated by both TOR complex 1 (TORC1) and TORC2. TORC1 regulates phosphorylation of both sites via the poorly characterized AGC-family kinase Ypk3 and the PP1 phosphatase Glc7, whereas TORC2 regulates phosphorylation of only the N-terminal phosphosite via Ypk1. Cells expressing a nonphosphorylatable variant of Rps6 display a reduced growth rate and a 40S biogenesis defect, but these phenotypes are not observed in cells in which Rps6 kinase activity is compromised. Furthermore, using polysome profiling and ribosome profiling, we failed to uncover a role of Rps6 phosphorylation in either global translation or translation of individual mRNAs. Taking the results together, this work depicts the signaling cascades orchestrating Rps6 phosphorylation in budding yeast, challenges the notion that Rps6 phosphorylation plays a role in translation, and demonstrates that observations made with Rps6 knock-ins must be interpreted cautiously.     

Discovery of a unique Mycobacterium tuberculosis protein through proteomic analysis of urine from patients with active tuberculosis

Identification of pathogen-specific biomarkers present in patients' serum or urine samples can be a useful diagnostic approach. In efforts to discover Mycobacterium tuberculosis (Mtb) biomarkers we identified by mass spectroscopy a unique 21-mer Mtb peptide sequence (VVLGLTVPGGVELLPGVALPR) present in the urines of TB patients from Zimbabwe. This peptide has 100% sequence homology with the protein TBCG_03312 from the C strain of Mtb (a clinical isolate identified in New York, NY, USA) and 95% sequence homology with Mtb oxidoreductase (MRGA423_21210) from the clinical isolate MTB423 (identified in Kerala, India). Alignment of the genes coding for these proteins show an insertion point mutation relative to Rv3368c of the reference H37Rv strain, which generated a unique C-terminus with no sequence homology with any other described protein. Phylogenetic analysis utilizing public sequence data shows that the insertion mutation is apparently a rare event. However, sera from TB patients from distinct geographical areas of the world (Peru, Vietnam, and South Africa) contain antibodies that recognize a purified recombinant C-terminus of the protein, thus suggesting a wider distribution of isolates that produce this protein.     

Tissue-specific oxidative imbalance and mitochondrial dysfunction during Trypanosoma cruzi infection in mice

In this study, we examined the tissue specificity of inflammatory and oxidative responses and mitochondrial dysfunction in mice infected by Trypanosoma cruzi. In acute mice, parasite burden and associated inflammatory infiltrate was detected in all tissues (skeletal muscle>heart>stomach>colon). The extent of oxidative damage and mitochondrial decay was in the order of heart>stomach>skeletal muscle>colon. In chronic mice, a low level of parasite burden and inflammation continued in all tissues; however, oxidant overload and mitochondrial inefficiency mainly persisted in the heart tissue (also detectable in stomach). Further, we noted an unvaryingly high degree of oxidative stress, compromised antioxidant status, and decreased mitochondrial respiratory complex activities in peripheral blood of infected mice. A pair-wise log analysis showed a strong positive correlation in the heart-versus-blood (but not other tissues) levels of oxidative stress markers (malonyldialdehyde, glutathione disulfide), antioxidants (superoxide dismutase, MnSOD, catalase), and mitochondrial inhibition of respiratory complexes (CI/CIII) in infected mice. T. cruzi-induced acute inflammatory and oxidative responses are widespread in different muscle tissues. Antioxidant/oxidant status and mitochondrial function are consistently attenuated in the heart, and reflected in the peripheral-blood of T. cruzi-infected mice. Our results provide an impetus to investigate the peripheral-blood oxidative responses in relation to clinical severity of heart disease in chagasic human patients.     

Characterization of a β-1,4-mannanase from a newly isolated strain of Pholiota adiposa and its application for biomass pretreatment

A highly efficient β-1,4-mannanase-secreting strain, Pholiota adiposa SKU0714, was isolated and identified on the basis of its morphological features and sequence analysis of internal transcribed spacer rDNA. P. adiposa β-1,4-mannanase was purified to homogeneity from P. adiposa culture supernatants by one-step chromatography on a Sephacryl gel filtration column. P. adiposa β-1,4-mannanase showed the highest activity toward locust bean gum (V _max = 1,990 U/mg protein, K _m = 0.12 mg/mL) ever reported. Its internal amino acid sequence showed homology with hydrolases from the glycoside hydrolase family 5 (GH5), indicating that the enzyme is a member of the GH5 family. The saccharification of commercial mannanase and P. adiposa β-1,4-mannanase-pretreated rice straw by Celluclast 1.5L (Novozymes) was compared. In comparison with the commercial Novo Mannaway^® (113 mg/g-substrate), P. adiposa β-1,4-mannanase-pretreated rice straw released more reducing sugars (141 mg/g-substrate). These properties make P. adiposa β-1,4-mannanase a good candidate as a new commercial β-1,4-mannanase to improve biomass pretreatment.     

Hypochlorous acid-induced heme degradation from lactoperoxidase as a novel mechanism of free iron release and tissue injury in inflammatory diseases

Lactoperoxidase (LPO) is the major consumer of hydrogen peroxide (H(2)O(2)) in the airways through its ability to oxidize thiocyanate (SCN(-)) to produce hypothiocyanous acid, an antimicrobial agent. In nasal inflammatory diseases, such as cystic fibrosis, both LPO and myeloperoxidase (MPO), another mammalian peroxidase secreted by neutrophils, are known to co-localize. The aim of this study was to assess the interaction of LPO and hypochlorous acid (HOCl), the final product of MPO. Our rapid kinetic measurements revealed that HOCl binds rapidly and reversibly to LPO-Fe(III) to form the LPO-Fe(III)-OCl complex, which in turn decayed irreversibly to LPO Compound II through the formation of Compound I. The decay rate constant of Compound II decreased with increasing HOCl concentration with an inflection point at 100 μM HOCl, after which the decay rate increased. This point of inflection is the critical concentration of HOCl beyond which HOCl switches its role, from mediating destabilization of LPO Compound II to LPO heme destruction. Lactoperoxidase heme destruction was associated with protein aggregation, free iron release, and formation of a number of fluorescent heme degradation products. Similar results were obtained when LPO-Fe(II)-O(2), Compound III, was exposed to HOCl. Heme destruction can be partially or completely prevented in the presence of SCN(-). On the basis of the present results we concluded that a complex bi-directional relationship exists between LPO activity and HOCl levels at sites of inflammation; LPO serve as a catalytic sink for HOCl, while HOCl serves to modulate LPO catalytic activity, bioavailability, and function.     

Cellular Immune Responses to Cell Wall Peptidoglycan Associated Protein Antigens in Tuberculosis Patients and Healthy Subjects

We have isolated cell wall peptidoglycan associated proteins (CW-Pr) of Mycobacterium tuberculosis H₃₇Ra by chemical treatment with trifluoromethanesulfonic acid:anisole (2:1), which further resolved into 71, 60 and 45 kDa proteins on SDS-PAGE. A study was carried out to investigate the immunoreactivity of these proteins with blood samples from 4 categories, including 15 tuberculous patients (TB), 5 tuberculous patients on ATT (TBT), 10 PPD non-reactive healthy controls (HPPD^?) and 11 PPD reactive healthy controls (HPPD⁺). Comparing the proliferative responses to cell wall protein antigens, it was observed that the 71 kDa protein gave maximum stimulation with PBMCs from the TB and HPPD⁺ groups. The adherent PBMCs from the TB group also demonstrated enhanced phagocytosis, particularly in the presence of 71 and 45 kDa proteins, and the phagocytic index was significantly higher (P < 0.05) than the TBT group. However, PBMCs from of the groups recognized the 60 kDa cell wall antigen. Our results suggest that the 71 kDa protein from the cell wall of M. tuberculosis is highly immunogenic.     

Recent trends and advancements in microbial tannase-catalyzed biotransformation of tannins: a review

The outburst of green biotechnology has facilitated a substantial upsurge in the usage of enzymes in a plethora of industrial bioconversion processes. The tremendous biocatalytic potential of industrial enzymes provides an upper edge over chemical technologies in terms of safety, reusability, and better process control. Tannase is one such enzyme loaded with huge potential for bioconversion of hydrolysable tannins to gallic acid. Tannins invariably occur in pteridophytes, gymnosperms, and angiosperms and predominately cumulate in plant parts like fruits, bark, roots, and leaves. Furthermore, toxic tannery effluents from various tanneries are loaded with significant levels of tannins in the form of tannic acid. Tannase can be principally employed for debasing the tannins that predominately occur in the toxic tannery effluents thus providing a relatively much cheaper measure for their biodegradation. Over the years, microbial tannase-catalyzed tannin degradation has gained momentum. The plentious availability of tannin-containing agro- and industrial waste paves a way for efficient utilization of microbial tannase for tannin degradation eventually resulting into gallic acid production. Gallic acid has received a great deal of attention as a molecule of enormous therapeutic and indusrial potential. The current worldwide demand of gallic acid is 8000 t per annum. As a matter of fact, bioconversion of tannins into gallic acid through fermentation has not been exploited completely. This necessitates further studies for development of more efficient, economical, productive processes and improved strains for gallic acid production so as to meet its current demand.