Isolation and characterization of polysaccharides of a hybrid mushroom (backcross mating between PfloVv12 and Volvariella volvacea)

Three polysaccharide fractions (PS-I, PS-II, and PS-III) were isolated from the aqueous extract of a hybrid mushroom obtained through backcross mating of a somatic hybrid mushroom PfloVv12 (Sterile line) with Volvariellavolvacea. PfloVv12 was obtained through protoplast fusion of Pleurotusflorida and V. volvacea. PS-I was identified as 1,6-β glucan. PS-II and PS-III were identified as mannoglucogalactan but differing in molecular weights only. On the basis of total acid hydrolysis, methylation analysis, periodate oxidation, and NMR experiment (¹H, ¹³C, DEPT-135, DQF-COSY, TOCSY, NOESY, ROESY, HMQC, and HMBC) the structures of these polysaccharides were established as;     

Malaria Parasite-Synthesized Heme Is Essential in the Mosquito and Liver Stages and Complements Host Heme in the Blood Stages of Infection

Heme metabolism is central to malaria parasite biology. The parasite acquires heme from host hemoglobin in the intraerythrocytic stages and stores it as hemozoin to prevent free heme toxicity. The parasite can also synthesize heme de novo, and all the enzymes in the pathway are characterized. To study the role of the dual heme sources in malaria parasite growth and development, we knocked out the first enzyme, δ-aminolevulinate synthase (ALAS), and the last enzyme, ferrochelatase (FC), in the heme-biosynthetic pathway of Plasmodium berghei (Pb). The wild-type and knockout (KO) parasites had similar intraerythrocytic growth patterns in mice. We carried out in vitro radiolabeling of heme in Pb-infected mouse reticulocytes and Plasmodium falciparum-infected human RBCs using [4-¹⁴C] aminolevulinic acid (ALA). We found that the parasites incorporated both host hemoglobin-heme and parasite-synthesized heme into hemozoin and mitochondrial cytochromes. The similar fates of the two heme sources suggest that they may serve as backup mechanisms to provide heme in the intraerythrocytic stages. Nevertheless, the de novo pathway is absolutely essential for parasite development in the mosquito and liver stages. PbKO parasites formed drastically reduced oocysts and did not form sporozoites in the salivary glands. Oocyst production in PbALASKO parasites recovered when mosquitoes received an ALA supplement. PbALASKO sporozoites could infect mice only when the mice received an ALA supplement. Our results indicate the potential for new therapeutic interventions targeting the heme-biosynthetic pathway in the parasite during the mosquito and liver stages.     

Phylogenetic analysis, based on EPIYA repeats in the cagA gene of Indian Helicobacter pylori, and the implications of sequence variation in tyrosine phosphorylation motifs on determining the clinical outcome

The population of India harbors one of the world's most highly diverse gene pools, owing to the influx of successive waves of immigrants over regular periods in time. Several phylogenetic studies involving mitochondrial DNA and Y chromosomal variation have demonstrated Europeans to have been the first settlers in India. Nevertheless, certain controversy exists, due to the support given to the thesis that colonization was by the Austro-Asiatic group, prior to the Europeans. Thus, the aim was to investigate pre-historic colonization of India by anatomically modern humans, using conserved stretches of five amino acid (EPIYA) sequences in the cagA gene of Helicobacter pylori. Simultaneously, the existence of a pathogenic relationship of tyrosine phosphorylation motifs (TPMs), in 32 H. pylori strains isolated from subjects with several forms of gastric diseases, was also explored. High resolution sequence analysis of the above described genes was performed. The nucleotide sequences obtained were translated into amino acids using MEGA (version 4.0) software for EPIYA. An MJ-Network was constructed for obtaining TPM haplotypes by using NETWORK (version 4.5) software. The findings of the study suggest that Indian H. pylori strains share a common ancestry with Europeans. No specific association of haplotypes with the outcome of disease was revealed through additional network analysis of TPMs.     

Characterization of SNP and Structural Variations in the Mitochondrial Genomes of Tilletia indica and Its Closely Related Species Formed Basis for a Simple Diagnostic Assay

Tilletia indica causes the disease Karnal bunt in wheat. The disease is under international quarantine regulations. Comparative mitochondrial (mt) genome analysis of T. indica ({"type":"entrez-nucleotide","attrs":{"text":"KX394364","term_id":"1128620031","term_text":"KX394364"}}KX394364 and {"type":"entrez-nucleotide","attrs":{"text":"DQ993184","term_id":"115361453","term_text":"DQ993184"}}DQ993184) and T. walkeri ({"type":"entrez-nucleotide","attrs":{"text":"EF536375","term_id":"144925993","term_text":"EF536375"}}EF536375) has found 325 to 328 SNPs, 57 to 60 short InDels (from 1 to 13 nt), two InDels (30 and 61 nt) and five (>200 nt) presence/absence variations (PAVs) between the two species. The mt genomes of both species have identical gene order. The numbers of SNPs and InDels between the mt genomes of the two species are approximately nine times of the corresponding numbers between the two T. indica isolates. There are eight SNPs between T. indica and T. walkeri that resulted in amino acid substitutions in the mt genes of cob, nad2 and nad5. In contrast, there is no amino acid substitution in the mt genes of the T. indica isolates from the SNPs found. The five PAVs present in T. indica ({"type":"entrez-nucleotide","attrs":{"text":"DQ993184","term_id":"115361453","term_text":"DQ993184"}}DQ993184) are absent in T. walkeri. Four PAVs are more than 1 kb and are not present in every T. indica isolate. Analysis of their presence and absence separates a collection of T. indica isolates into 11 subgroups. Two PAVs have ORFs for the LAGLIDAG endonuclease and two have ORFs for the GIY-YIG endonuclease family, which are representatives of homing endonuclease genes (HEGs). These intron- encoded HEGs confer intron mobility and account for their fluid distribution in T. indica isolates. The small PAV of 221 bp, present in every T. indica isolate and unique to the species, was used as the genetic fingerprint for the successful development of a rapid, highly sensitive and specific loop mediated isothermal amplification (LAMP) assay. The simple procedure of the LAMP assay and the easy detection formats will enable the assay to be automated for high throughput diagnosis.     

Improved production of alkaline protease from a mutant of alkalophilic Bacillus pantotheneticus using molasses as a substrate

An alkalophilic bacterial isolate identified as Bacillus pantotheneticus, isolated from saline-alkali soils of Avadh region of UP, India, was studied for the production of alkaline protease. The mutant of the isolated species showed 44% improved production over the parent strain. Organic nitrogen sources supported better protease production than the inorganic sources. The production of alkaline protease was (242 U/ml) in the medium containing molasses, which was comparable with molasses and wheat bran (285 U/ml) as carbon and nitrogen sources, respectively. Protease production was best at pH 10 and temperature 30 degrees C. The Km (for casein) was 11 mg/ml and Vmax was 380-microg tyrosine/ml/min. The enzyme was stable between pH 7 and 10.7 and temperature between 30 and 60 degrees C with a pH and temperature optimum at 8.4 and 40 degrees C, respectively. The results indicated that molasses was an optimal substrate for alkaline protease production.     

Glycoprotein glycans may not be necessary for the differentiation of skeletal myoblasts

Previous work using glycosylation inhibitors has suggested that high-mannose type but not complex type oligosaccharides on the surface of cells may play a role in the differentiation of skeletal myoblasts. Earlier, we had shown that a concanavalin A-resistant mutant derived from an L6 myoblast line fails to differentiate in a medium containing 10% horse serum. Here we show that one such concanavalin A-resistant mutant (D-1) which was reported to have oligosaccharides of the type Man(3-5)G1cNAc2, shows significant fusion ability when grown in media containing 1% horse serum. Lowering the serum concentration did not alter the dolichol-phosphate mannosyltransferase activity in D-1 which remained at low levels compared to L6. The incorporation of [3H]mannose in D-1 was found to be 60% of L6 in 10% serum whereas in 1% serum the incorporation into D-1 was further reduced to 30% of L6. [3H]mannose-labeled ConA-binding proteins isolated from L6 were quantitatively and qualitatively similar in cells grown in either 10 or 1% serum. However, in D-1 cells a further decrease in the ConA-binding ability of these glycoproteins was observed. Biochemical differentiation also occurs in D-1 upon fusion in 1% serum as seen by the increase in mRNA levels of the muscle-specific markers myosin light chain and troponin T. These results suggest the high-mannose type of oligosaccharides may not be involved in myoblast differentiation.     

Xanthomonas campestris, a novel stress tolerant, phosphate-solubilizing bacterial strain from saline–alkali soils

A total of 198 bacterial strains were isolated from various niches of saline–alkali soils, out of which 85 strains were able to solubilize phosphate on plates at 30 °C. The strain RMLU-26, identified as Xanthomonas campestris, was the most efficient with its ability to solubilize P, subjected to N-methyl-N′-nitro-N-nitrosoguanidine (NTG) for development of mutants. The P solubilizing ability of X. campestris is reported for the first time. The wild type and mutant strains of X. campestris revealed a differential response to various stress factors (high pH, temperature, and salt concentration). The mutant strain revealed maximum P solubilization (67.1%) at 30 °C and pH 8.0 while the wild type strain showed maximum solubilization (41.9%) at 35 °C and pH 7.0. Percent P₂O₅ solubilization by both strains revealed a steep decline in tricalcium phosphate solubilization with an increase in NaCl concentration from 0.5 to 10% along with a concomitant drop in pH of the medium from 8.0 to 4.5 in wild type and 4.0 in mutant strain. However, a 1.5- to 2-fold increase in ‘P’ solubilization was observed in the mutant strain when compared to the wild type strain in the presence of NaCl. The overall improved tolerance of the strains to alkalinity and salinity could be due to accumulation and/or secretion of specific solute (xanthan).     

Small RNA interactome of pathogenic E. coli revealed through crosslinking of RNase E

RNA sequencing studies have identified hundreds of non‐coding RNAs in bacteria, including regulatory small RNA (sRNA). However, our understanding of sRNA function has lagged behind their identification due to a lack of tools for the high‐throughput analysis of RNA–RNA interactions in bacteria. Here we demonstrate that in vivo sRNA–mRNA duplexes can be recovered using UV‐crosslinking, ligation and sequencing of hybrids (CLASH). Many sRNAs recruit the endoribonuclease, RNase E, to facilitate processing of mRNAs. We were able to recover base‐paired sRNA–mRNA duplexes in association with RNase E, allowing proximity‐dependent ligation and sequencing of cognate sRNA–mRNA pairs as chimeric reads. We verified that this approach captures bona fide sRNA–mRNA interactions. Clustering analyses identified novel sRNA seed regions and sets of potentially co‐regulated target mRNAs. We identified multiple mRNA targets for the pathotype‐specific sRNA Esr41, which was shown to regulate colicin sensitivity and iron transport in E. coli. Numerous sRNA interactions were also identified with non‐coding RNAs, including sRNAs and tRNAs, demonstrating the high complexity of the sRNA interactome.     

Distinct role of p38 and c-Jun N-terminal kinases in IL-10-dependent and IL-10-independent regulation of the costimulatory molecule B7.2 in lipopolysaccharide-stimulated human monocytic cells

The costimulatory molecule B7.2 (CD86) plays a vital role in immune activation and development of Th responses. The molecular mechanisms by which B7.2 expression is regulated are not understood. We investigated the role of mitogen-activated protein kinases (MAPK) in the regulation of B7.2 expression in LPS-stimulated human monocytic cells. LPS stimulation of human monocytes resulted in the down-regulation of B7.2 expression that could be abrogated by anti-IL-10 Abs. Furthermore, SB202190, a specific inhibitor of p38 MAPK, inhibited LPS-induced IL-10 production and reversed B7.2 down-regulation, suggesting that LPS-induced B7.2 down-regulation may be mediated, at least in part, via regulation of IL-10 production by p38 MAPK. In contrast to human promonocytic THP-1 cells that are refractory to the inhibitory effects of IL-10, LPS stimulation enhanced B7.2 expression. This IL-10-independent B7.2 induction was not influenced by specific inhibitors of either p38 or p42/44 MAPK. To ascertain the role of the c-Jun N-terminal kinase (JNK) MAPK, dexamethasone, an inhibitor of JNK activation, was used, which inhibited LPS-induced B7.2 expression. Transfection of THP-1 cells with a plasmid expressing a dominant-negative stress-activated protein/extracellular signal-regulated kinase kinase 1 significantly reduced LPS-induced B7.2 expression, thus confirming the involvement of JNK. To study the signaling events downstream of JNK activation, we show that dexamethasone did not inhibit LPS-induced NF-kappaB activation in THP-1 cells, suggesting that JNK may not be involved in NF-kappaB activation leading to B7.2 expression. Taken together, our results reveal the distinct involvement of p38 in IL-10-dependent, and JNK in IL-10-independent regulation of B7.2 expression in LPS-stimulated monocytic cells.