Molecular identification and characterizations of rhizobacterial isolates collected from lentil rhizosphere of Indo-gangetic plains.

Plant growth promoting rhizobacteria (PGPR) are also known to colonize in the soil rhizosphere and prevent the development of other soil borne pathogens residing in the root surface. These microorganisms play a vital role in growth and development of the plant and also enhances the soil fertility by enriching the soil with different beneficial nutrients. This study was aimed at isolation of different rhizobacteria and their molecular characterization in search of efficient bacterial strains with multiple growth regulating activities. A total 36 bacteria were isolated from lentil root nodule as well as soil from different lentil growing fields with a view to screen/evaluate their plant growth promoting potential. Morphological characterization of isolated rhizobacterial candidates were done by observing the colonies on YEMA and nutrient agar media. Determination of CFU, Congo red test and gram staining tests were done to further screen them according to their morphology. All the isolates were then undergone molecular phylogenetic analysis using the partial sequences of the 16 S rDNA. Based upon the Gram staining test, all the isolates were negative in gram reaction except six Bacillus isolates, PSB2 and AB3. Results of Ribosomal Database Project (RDP) and Basic Local Alignment Search Tool for Nucleotide Sequences (BLASTn) from 16 S rDNA gene sequences showed that these isolates are genetically diverse. A total of 15 isolates of Rhizobium, 6 isolates of Bacillus, 3 isolates of Pseudomonas, 2 isolates of Phosphate Solubilizing Bacteria, 4 isolates of actinomycetes were identified by molecular sequencing of their 16 S rDNA region and comparing them with the other isolates enlisted in the database of NCBI for the similarity percentage, query coverage. The purpose of the present study was to select native rhizosphere bacteria from the lentil nodule and soil of Lentil field and to evaluate their plant growth promoting potential as an alternative of chemical fertilizer for sustainable, environment friendly agriculture and assessment of their phylogenetic characterization.

Characterization of plant growth-promoting bacteria isolated from rhizosphere of lentil (Lens culinaris L.) grown in two different soil orders of eastern India.

Lentil, which is an important grain legume, can be co-inoculated with plant growth-promoting rhizobia and rhizobacteria to boost nitrogen fixation, increase biomass, and a possibility for early nodulation. The goal of the ongoing study was to identify plant growth-promoting rhizobacteria (PGPR) in the rhizosphere of lentil growing soils in eastern India. Sixteen rhizosphere bacteria were isolated from two different soil orders, and their capacity to solubilize phosphate and generate hydrogen cyanide (HCN), siderophore, and indole acetic acid (IAA) was assessed. The three best strains were selected for compatibility study with twenty Rhizobium isolated from lentil root nodules. The isolated rhizobacteria were able to produce ammonia and different mycolytic enzymes. Isolate B3 produced the highest amount of IAA and siderophore; the highest amount of phosphate solubilized by PSB1 strain; and isolates AB1, AB2, B3, PS2, and PSB2 produced considerable amount of HCN gas. Among all the isolates, B3, PSB1, and PS2 performed better based on different plant growth-promoting abilities. These three bacterial isolates showed compatible reaction with most of the Rhizobium strains. Isolates B3, PS2, and PSB1 were identified as Bacillus subtilis (MT729775), Pseudomonas palmensis (MT729782), and Paraburkholderia caribenis (MZ956803), respectively. Lentil shoot weight, root length, nodule number, N uptake, and P uptake were increased in the pot culture experiment when inoculated with these strains. PGPR strain B3 performed best among the three strains in the pot culture experiment. Strain B3 can be used as potential biofertilizer along with compatible Rhizobium species for better production of lentil.

Butyrate limits inflammatory macrophage niche in NASH.

Immune cell infiltrations with lobular inflammation in the background of steatosis and deregulated gut-liver axis are the cardinal features of non-alcoholic steatohepatitis (NASH). An array of gut microbiota-derived metabolites including short-chain fatty acids (SCFA) multifariously modulates NASH pathogenesis. However, the molecular basis for the favorable impact of sodium butyrate (NaBu), a gut microbiota-derived SCFA, on the immunometabolic homeostasis in NASH remains elusive. We show that NaBu imparts a robust anti-inflammatory effect in lipopolysaccharide (LPS) stimulated or classically activated M1 polarized macrophages and in the diet-induced murine NASH model. Moreover, it impedes monocyte-derived inflammatory macrophage recruitment in liver parenchyma and induces apoptosis of proinflammatory liver macrophages (LM) in NASH livers. Mechanistically, by histone deactylase (HDAC) inhibition NaBu enhanced acetylation of canonical NF-κB subunit p65 along with its differential recruitment to the proinflammatory gene promoters independent of its nuclear translocation. NaBu-treated macrophages thus exhibit transcriptomic signatures that corroborate with a M2-like prohealing phenotype. NaBu quelled LPS-mediated catabolism and phagocytosis of macrophages, exhibited a differential secretome which consequently resulted in skewing toward prohealing phenotype and induced death of proinflammatory macrophages to abrogate metaflammation in vitro and in vivo. Thus NaBu could be a potential therapeutic as well as preventive agent in mitigating NASH.

Interaction of a Triantennary Quinoline Glycoconjugate with the Asialoglycoprotein Receptor.

Targeted intracellular delivery is an efficient strategy for developing therapeutics against cancer and other intracellular infections. Nonspecific drug delivery shows limited clinical applications owing to high dosage, cytotoxicity, nonspecific action, high cost, etc. Therefore, targeted delivery of less cytotoxic drug candidates to hepatocytes through ASGPR-mediated endocytosis could be an efficient strategy to surmount the prevailing shortcomings. In the present work, the gene encoding ASGPR-H1-CRD was amplified from Huh7 cells, cloned into pET 11a vector, and the ASGPR-H1-CRD protein was expressed and purified from E. coli. A novel triantennary galactose-conjugated quinoline derivative 4 was synthesized that demonstrates 17-fold higher binding affinity to isolated ASGPR-H1-CRD protein receptor (Kd ∼54 µM) in comparison to D-galactose (Kd ∼900 µM). Moreover, micro-calorimetric studies for the interaction of glycoconjugate 4 with ASGPR protein on live hepatocytes showed notable thermal response in case of ASGPR-containing Huh7 cells, in comparison to non-ASGPR Chang cells. These results might serve as an approach towards targeted delivery of small glycoconjugates to hepatocytes.

Mnemonic Similarity Task to study episodic memory in Parkinson's disease.

INTRODUCTION: Parkinson's disease (PD) patients commonly experience episodic memory impairments, which are associated with an increased risk of dementia. The Mnemonic Similarity Task (MST) is a well-validated test to investigate episodic memory changes in healthy aging and in neurodegenerative diseases but has not been studied in PD patients. METHODS: In the MST task, participants respond during a testing phase whether visualized images are "repeat", "similar", or "new", compared to images previously shown during an encoding phase. We tested 17 PD without cognitive impairment (level-II criteria), both off (PD-OFF) and on (PD-ON) dopaminergic medications; and compared PD-OFF with 17 age- and education-matched healthy controls (HC). RESULTS: We found no influence of dopaminergic medications nor of disease on MST reaction time for any responses ("repeat", "similar", and "new") during the test phase. However, response probabilities showed that the MST is sensitive to subtle PD-related memory impairments. Specifically, PD-OFF responded more frequently with 'repeat', instead of 'similar' during lure trials, compared to HC (p = 0.030). This finding was still significant after correcting for response bias using the Recognition Index (p = 0.005). CONCLUSIONS: PD patients perform the MST without interference from bradykinesia or other PD-related motor symptoms. Our findings suggest that PD patients who do not meet criteria for mild cognitive impairment can have subtle recall or recognition impairments, which can be identified using the MST. We propose the MST as a well-tolerated and sensitive cognitive task in future studies of episodic memory impairment and progressive memory dysfunction in people with PD.

Episodic recognition memory and the hippocampus in Parkinson's disease: A review.

Parkinson's disease is a progressive neurodegenerative disorder of aging. The hallmark pathophysiology includes the development of neuronal Lewy bodies in the substantia nigra of the midbrain with subsequent loss of dopaminergic neurons. These neuronal losses lead to the characteristic motor symptoms of bradykinesia, rigidity, and rest tremor. In addition to these cardinal motor symptoms patients with PD experience a wide range of non-motor symptoms, the most important being cognitive impairments that in many circumstances lead to dementia. People with PD experience a wide range of cognitive impairments; in this review we will focus on memory impairment in PD and specifically episodic memory, which are memories of day-to-day events of life. Importantly, these memory impairments severely impact the lives of patients and caregivers alike. Traditionally episodic memory is considered to be markedly dependent on the hippocampus; therefore, it is important to understand the exact nature of PD episodic memory deficits in relation to hippocampal function and dysfunction. In this review, we discuss an aspect of episodic memory called recognition memory and its subcomponents called recollection and familiarity. Recognition memory is believed to be impaired in PD; thus, we discuss what aspects of the hippocampus are expected to be deficient in function as they relate to these recognition memory impairments. In addition to the hippocampus as a whole, we will discuss the role of hippocampal subfields in recognition memory impairments.

Antiplatelet and antithrombotic activity of a fibrin(ogen)olytic protease from Bacillus cereus strain FF01.

Fibrin(ogen)olytic enzymes offer great promise for the treatment of thrombosis associated disorders. The present study describes the characterization of an extracellular fibrin(ogen)olytic serine protease (named Bacethrombase) purified from the Bacillus cereus strain FF01. The molecular mass of the Bacethrombase was determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis and matrix assisted laser desorption/ionization-time-of-flight-mass spectroscopy analyses at 39.5 kDa and 38,450.51 Da, respectively. The peptide mass fingerprinting and analyses of the composition of the amino acids revealed the similarity of the Bacethrombase to the bacterial serine proteases. The secondary structure of the Bacethrombase was composed of 14% helix, 6.6% beta-sheet, and 79.4% random coil. Bacethrombase was found to contain 48% sialic acid and it preferentially degraded the Aα-chain of fibrinogen, as well as fibrin. The anticoagulant potency of the Bacethrombase was comparable with that of warfarin and heparin, and was corroborated by its fibrinogenolytic activity rather than the inhibition of thrombin, prothrombin or FXa. Bacethrombase demonstrated antiplatelet activity, and dose-dependently inhibited the ADP-induced platelet aggregation. Bacethrombase (10 mg/kg) did not show toxicity after i.v. administration in Wistar rats; however, it revealed an in vivo anticoagulant effect and significantly inhibited the carrageenan-induced in vivo thrombus formation in rats.

Phosphorylation of Leghemoglobin at S45 is Most Effective to Disrupt the Molecular Environment of Its Oxygen Binding Pocket.

In leguminous plants, nitrogenase that catalyzes anaerobic symbiotic nitrogen fixation is protected by the sequestration of O2 by Leghemoglobin (LegH). The modulation of the oxygen binding capacity of Hemoglobin (Hb) by different post-translational modifications is well studied; whereas similar studies on LegH's O2 binding are not yet benchmarked. Our results show that in vitro serine phosphorylation of recombinant LegH from Lotus japonicus, a model legume, by a homologous kinase caused a reduction in its oxygen consumption as determined by Clark type electrode. Although mass spectrometry revealed a few phosphorylated serine residues in the LegH, molecular modeling study showed that particularly S45 is the most critical one, along with S55, however the latter with lesser impact on its molecular environment responsible for oxygen consumption. Separate S45D and S55D mutants of recombinant LegH also corroborated the results obtained from molecular modeling study. Thus, this work lays groundwork for further investigation of structural and functional role of serine phosphorylation as one of the mechanisms by which oxygen consumption by LegH may possibly be regulated during nodulation.

Loss of pattern separation performance in schizophrenia suggests dentate gyrus dysfunction.

Motivated by evidence that the dentate gyrus differentially mediates the pattern separation (PS) component of declarative memory function and that dentate gyrus harbors molecular and cellular pathologies in schizophrenia, we examined whether PS performance is altered in volunteers with schizophrenia (SZV) relative to healthy volunteers (HV). In groups of well-characterized SZV (n=14) and HV (n=15), we contrasted performance on the Behavioral Pattern Separation (BPS) Task, acquiring two outcome measures, a PS parameter and a Recognition Memory (RM) parameter, as well as specific recognition data by stimulus type. The SZVs showed a significant decrement in PS performance relative to HV (mean ± SEM, SZV: 3.1 ± 2.7%; HV: 17.1 ± 5.8%; p=0.039, d'=0.86); whereas SZV and HV did not significantly differ in RM performance (SZV: 50.1 ± 8.1%; HV: 59.3 ± 5.5%; p=0.350, d'=0.36). Moreover, the SZVs showed a selective defect in correctly identifying similar lure items (SZV: 24.0 ± 3.7%; HV: 41.2 ± 4.6%; p<0.05), but demonstrated no impairment in identifying targets and novel foils. These data suggest that the dentate gyrus is dysfunctional in schizophrenia, a feature that could contribute to declarative memory impairment in the disorder and possibly to psychosis, a conclusion consistent with the considerable molecular pathology in the dentate gyrus in schizophrenia.

Cigarette smoke induces p-benzoquinone-albumin adduct in blood serum: Implications on structure and ligand binding properties.

Earlier we had reported that irrespective of the source cigarette smoke (CS) contains substantial amounts of p-benzosemiquinone, which is readily converted to p-benzoquinone (p-BQ) by disproportionation and oxidation by transition metal containing proteins. Here we show that after CS-exposure, p-BQ-protein adducts are formed in the lungs as well as serum albumin of guinea pigs. We also show that serum of human smokers contains p-BQ-albumin adduct. It is known that human serum albumin (HSA) plays a very important role in binding and transport of a variety of ligands, including fatty acids and drugs. We show in vitro that p-BQ forms covalent adducts with free amino groups of all twenty amino acids as well as ɛ-amino groups of lysine residues of HSA in a concentration dependent manner. When HSA is incubated with p-BQ in the molar ratio of 1:1, the number of p-BQ incorporated is 1. At the molar ratio of 1:60, the number of p-BQ incorporated is 40. The formation of HSA-p-BQ adduct has been demonstrated by absorption spectroscopy, MALDI-MS and MALDI-TOF-TOF-MS analyses. Upon complexation with p-BQ, the secondary structure and conformation of HSA are altered, as evidenced by steady state and time-resolved fluorescence, circular dichroism, 8-anilino-1-napthalenesulfonic acid binding and differential scanning calorimetry. Alteration of the structure and conformation of HSA results in impairment of its ligand binding properties with respect to myristic acid, quercitin and paracetamol. This might be one of the reasons why transport and distribution of lipids and drugs are impaired in smokers.

NAD(P)H: quinone oxidoreductase 1 deficiency conjoint with marginal vitamin C deficiency causes cigarette smoke induced myelodysplastic syndromes.

BACKGROUND: The etiology of myelodysplastic syndromes (MDS) is largely unknown. Exposure to cigarette smoke (CS) is reported to be associated with MDS risk. There is inconsistent evidence that deficiency of NAD(P)H-quinone: oxidoreductase 1 (NQO1) increases the risk of MDS. Earlier we had shown that CS induces toxicity only in marginal vitamin C-deficient guinea pigs but not in vitamin C-sufficient ones. We therefore considered that NQO1 deficiency along with marginal vitamin C deficiency might produce MDS in CS-exposed guinea pigs. METHODOLOGY AND PRINCIPAL FINDINGS: Here we show that CS exposure for 21 days produces MDS in guinea pigs having deficiency of NQO1 (fed 3 mg dicoumarol/day) conjoint with marginal vitamin C deficiency (fed 0.5 mg vitamin C/day). As evidenced by morphology, histology and cytogenetics, MDS produced in the guinea pigs falls in the category of refractory cytopenia with unilineage dysplasia (RCUD): refractory anemia; refractory thrombocytopenia that is associated with ring sideroblasts, micromegakaryocytes, myeloid hyperplasia and aneuploidy. MDS is accompanied by increased CD34(+) cells and oxidative stress as shown by the formation of protein carbonyls and 8-oxodeoxyguanosine. Apoptosis precedes MDS but disappears later with marked decrease in the p53 protein. MDS produced in the guinea pigs are irreversible. MDS and all the aforesaid pathophysiological events do not occur in vitamin C-sufficient guinea pigs. However, after the onset of MDS vitamin C becomes ineffective. CONCLUSIONS AND SIGNIFICANCE: CS exposure causes MDS in guinea pigs having deficiency of NQO1 conjoint with marginal vitamin C deficiency. The syndromes are not produced in singular deficiency of NQO1 or marginal vitamin C deficiency. Our results suggest that human smokers having NQO1 deficiency combined with marginal vitamin C deficiency are likely to be at high risk for developing MDS and that intake of a moderately large dose of vitamin C would prevent MDS.

Rhythmic structure of Hindi and English: new insights from a computational analysis.

Much information about speech rhythm is believed to be embedded in low frequency temporal modulations of the speech envelope. Using novel methods of spectral analysis we construct a spectro-temporal modulation spectrum and extract low frequency temporal modulations of spoken utterances to study the rhythmic structure of English and Hindi. The results of our spectral analysis reveal a narrower temporal bandwidth for Hindi as compared to English. We also calculate variability in syllable durations and find that variability in English is greater than Hindi. We relate temporal bandwidth of the modulation spectrum to variability in syllable duration and suggest that narrow bandwidth in the modulation spectrum implies less variability, whereas broad bandwidth implies greater variability in syllable duration. Our results also demonstrate that syllabic information is contained in low frequency temporal modulations of the speech envelope. Our results suggest that the modulation spectrum can be explored as a promising tool to study the temporal structure of language.

Antibodies directed against O-acetylated sialoglycoconjugates accelerate complement activation in Leishmania donovani promastigotes.

BACKGROUND: An enhanced presence of 9-O-acetylated sialoglycoconjugates (9-O-AcSGs) triggers the alternate pathway (AP) in Indian visceral leishmaniasis (VL). Antibodies directed against these epitopes are present in high titers. The biological relevance of these antibodies, with regard to activation of the classical pathway (CP), was investigated. METHODS: Complement activators were affinity purified, complement activation via the CP, AP, and lectin-mediated complement pathway was measured by use of an anti-C3 radio-binding assay, and the number of C3 molecules was quantitated by Scatchard analysis. Cell death induced via the complement pathways was measured by use of MTT (tetrazolium salt 3- [4, 5-dimethylthiazol-2-yl] -2, 5-diphenyltetrazolium bromide) assay, and uptake of propidium iodide (PI) was measured by flow cytometry. RESULTS: Anti-O-AcSGs from both healthy donors and patients with VL elicited C3 deposition as early as 3 min, which triggered parasite lysis, as demonstrated by use of MTT assay and corroborated by the high rate of uptake of PI. Analysis of complement activation by mannan-binding lectin and C-reactive protein demonstrated their negligible contribution during the 3-min time frame. CONCLUSIONS: Anti-O-AcSGs were identified as an important source of CP activation under normal physiological conditions, suggesting that they play a role in conferring host protection against parasite infection.

Protein A--a new ligand for human C-reactive protein.

Phosphorylcholine (PC) is a classical ligand of C-reactive protein (CRP), a clinically important acute phase protein. In search of new ligands, CRPs were affinity-purified from several pathological samples, which exhibited distinct molecular variants induced in different diseases. Both glycosylated and non-glycosylated CRPs showed calcium-independent differential-binding to Staphylococcus aureus cell-surface Protein A. CRP possesses separate binding sites for Protein A and PC with different binding constants. We have demonstrated that Protein A is another ligand in addition to PC establishing an extended definition of CRP. Protein A binding may impart immunomodulatory roles of CRP in combating microorganisms or other foreign materials.

Variations in binding characteristics of glycosylated human C-reactive proteins in different pathological conditions.

C-reactive protein (CRP) is a clinically important classic acute phase pentameric protein. It is thought to play an important role in immunomodulation. Earlier reports convincingly demonstrated that human CRP is differentially glycosylated in different pathological conditions. Although CRP is considered to be a clinically important molecule, changes in binding characteristics with appropriate ligands with respect to glycosylation remain unexplored. In an effort to demonstrate that these glycosylated molecular variants are capable of modulating their binding activity with different ligands, CRPs were affinity purified from six different clinical samples. Variable amounts of linkage-specific sialic acid derivatives were found in these CRPs with varying tryptophan contents. Differential binding patterns with antibodies against human CRP, human IgG, and other ligands like fibronectin, fetuin, and asialofetuin indicated that the purified CRPs differed significantly in their lectin-like interactions. Thus, we have convincingly demonstrated that differentially induced CRPs exhibited variable binding characteristics. These results may have far reaching practical applications for understanding acute phase responses.

Induction of glycosylation in human C-reactive protein under different pathological conditions.

As an acute-phase protein, human C-reactive protein (CRP) is clinically important. CRPs were purified from several samples in six different pathological conditions, where their levels ranged from 22 to 342 microg/ml. Small, but significant, variations in electrophoretic mobilities on native PAGE suggested differences in molecular mass, charge and/or shape. Following separation by SDS/PAGE, they showed single subunits with some differences in their molecular masses ranging between 27 and 30.5 kDa, but for a particular disease, the mobility was the same for CRPs purified from multiple individuals or pooled sera. Isoelectric focusing (IEF) also indicated that the purified CRPs differed from each other. Glycosylation was demonstrated in these purified CRPs by Digoxigenin kits, neuraminidase treatment and binding with lectins. The presence of N-linked sugar moiety was confirmed by N-glycosidase F digestion. The presence of sialic acid, glucose, galactose and mannose has been demonstrated by gas liquid chromatography, mass spectroscopic and fluorimetric analysis. Matrix-assisted laser-desorption ionization analysis of the tryptic digests of three CRPs showed systematic absence of two peptide fragments, one at the N-terminus and the other near the C-terminus. Model-building suggested that the loss of these fragments exposed two potential glycosylation sites on a cleft floor keeping the protein-protein interactions in pentraxins and calcium-dependent phosphorylcholine-binding qualitatively unaffected. Thus we have convincingly demonstrated that human CRP is glycosylated in some pathological conditions.