Isolation of hydroquinone (benzene-1,4-diol) metabolite from halotolerant <Emphasis Type="Italic">Bacillus methylotrophicus</Emphasis> MHC10 and its inhibitory activity towards bacterial pathogens

A halotolerant bacterial isolate-MHC10 with broad spectrum antibacterial activity against clinical pathogens was isolated from saltpans located in Tuticorin and Chennai (India). 16S rRNA gene analysis of MHC10 revealed close similarity to that of Bacillus methylotrophicus. The culture conditions of B. methylotrophicus MHC10 strain were optimized for antibacterial production using different carbon and nitrogen sources, as well as varying temperature, pH, sodium chloride (NaCl) concentrations and incubation periods. The maximum antibacterial activity of B. methylotrophicus MHC10 was attained when ZMB was optimized with 1 % (w/v) glucose, 0.1 % (w/v) soybean meal which corresponded to a C/N ratio of 38.83, temperature at 37 °C, pH 7.0 and 8 % NaCl. The activity remained stable between 72 and 96 h and then drastically decreased after 96 h. Solvent extraction followed by chromatographic purification steps led to the isolation of hydroquinone (benzene-1,4-diol). The structure of the purified compound was elucidated based on FTIR, ¹H NMR, and ¹³C NMR spectroscopy. The compound exhibited efficient antibacterial activity against both Gram-positive and Gram-negative bacterial pathogens. The minimum inhibitory concentration (MIC) for Gram-positive pathogens ranged from 15.625 to 62.5 µg/mL^?1, while it was between 7.81 and 250 µg/mL^?1 for Gram-negative bacterial pathogens. This is the first report of hydroquinone produced by halotolerant B. methylotrophicus exhibiting promising antibacterial activity.     

Selection and molecular characterization of a lipoxygenase-free soybean mutant line induced by gamma irradiation

Key message

A lipoxygenase-free soybean mutant line (H70) induced by gamma ray was selected and its detailed information about the lipoxygenase was analyzed by comparison of DNA sequence.

Abstract

Soybean seeds contain three lipoxygenase enzymes, which induce a beany or grassy flavor. The elimination of lipoxygenases can reduce the poor stability and off-flavors of soybean oil and protein products. In this study, we selected a soybean mutant (H70) in which the three lipoxygenases had been mutated using gamma rays. To obtain detailed information about the lipoxygenase, we investigated the sequences of the Lox1, Lox2 and Lox3 genes in H70 compared to the original cultivar, Hwanggum. Comparisons of the sequences of the Lox1 and Lox2 genes in H70 with those in a line with normal lipoxygenase (HG) showed that the mutations in these genes affected a highly conserved group of six histidine residues necessary for enzymatic activity. Lox1 in H70 contained a 74 bp deletion in exon 8, creating a stop codon that prematurely terminates translation. A single point mutation (T-A) in exon 8 of Lox2 changed histidine (H532, one of the iron-binding ligands essential for Lox2 activity) to glutamine. The mutation in the Lox3 gene in H70 was a single-point mutation in exon 6 (A-G), which changed the amino acid from histidine to arginine. This amino acid alteration in Lox3 was located in the N-terminal barrel, which might play a role in molecular recognition during catalysis and/or proteolysis. These results suggest that gene analysis based on DNA sequencing could be useful for elucidating the lipoxygenase content in soybean mutant lines. Additionally, the soybean mutant line selected in this study could be used to develop soybean cultivars with improved flavor.     

3β-Hydroxylup-20(29)-ene-27,28-dioic acid dimethyl ester,a novel natural product from Plumbago zeylanica inhibits the proliferation and migration of MDA-MB-231 cells

Plumbago zeylanica, a traditional Indian herb is being used for the therapy of rheumatism and has been approved for anti-tumor activity. However, the molecular mechanisms involved in the biological action are not very well understood. In this study, the anti-invasive activities of P. zeylanica methanolic extract (PME) and pure compound 3β-hydroxylup-20(29)-ene-27,28-dioic acid (PZP) isolated from it are investigated in vitro. PME and PZP were noted to have the ability to induce apoptosis as assessed by flow cytometry. Further, the molecular mechanism of apoptosis induced by PME and PZP was found by the loss of mitochondrial membrane potential with the down regulation of Bcl-2, increased expression of Bad, release of cytochrome c, activation of caspase-3 and cleavage of PARP leading to DNA fragmentation. Importantly, both PME and PZP were observed to suppress MDA-MB-231 cells adhesion to the fibronectin-coated substrate and also inhibited the wound healing migration and invasion of MDA-MB-231 cells through the reconstituted extracellular matrix. Gelatin zymography revealed that PME and PZP decreased the secretion of matrix metalloproteinases-2 (MMP-2) and metalloproteinases-9 (MMP-9). Interestingly both PME and PZP exerted an inhibitory effect on the protein levels of p-PI3K, p-Akt, p-JNK, p-ERK1/2, MMP-2, MMP-9, VEGF and HIF-1α that are consistent with the observed anti-metastatic effect. Collectively, these data provide the molecular basis of the anti-proliferative and anti-metastatic effects of PME and PZP.     

Urokinase receptor expression involves tyrosine phosphorylation of phosphoglycerate kinase

The interaction of urokinase-type plasminogen activator (uPA) with its receptor, uPAR, plays a central role in several pathophysiological processes, including cancer. uPA induces its own cell surface receptor expression through stabilization of uPAR mRNA. The mechanism involves binding of a 51 nt uPAR mRNA coding sequence with phosphoglycerate kinase (PGK) to down regulate cell surface uPAR expression. Tyrosine phosphorylation of PGK mediated by uPA treatment enhances uPAR mRNA stabilization. In contrast, inhibition of tyrosine phosphorylation augments PGK binding to uPAR mRNA and attenuates uPA-induced uPAR expression. Mapping the specific peptide region of PGK indicated that its first quarter (amino acids 1–100) interacts with uPAR mRNA. To determine if uPAR expression by uPA is regulated through activation of tyrosine residues of PGK, we mutated the specific tyrosine residue and tested mutant PGK for its ability to interfere with uPAR expression. Inhibition of tyrosine phosphorylation by mutating Y76 residue abolished uPAR expression induced by uPA treatment. These findings collectively demonstrate that Y76 residue present in the first quarter of the PGK molecule is involved in lung epithelial cell surface uPAR expression. This region can effectively mimic the function of a whole PGK molecule in inhibiting tumor cell growth.     

3β-taraxerol of Mangifera indica, a PI3K dependent dual activator of glucose transport and glycogen synthesis in 3T3-L1 adipocytes

Background

The present study focuses on identifying and developing an anti-diabetic molecule from plant sources that would effectively combat insulin resistance through proper channeling of glucose metabolism involving glucose transport and storage.

Methods

Insulin-stimulated glucose uptake formed the basis for isolation of a bioactive molecule through column chromatography followed by its characterization using NMR and mass spectroscopic analysis. Mechanism of glucose transport and storage was evaluated based on the expression profiling of signaling molecules involved in the process.

Results

The study reports (i) the isolation of a bioactive compound 3β-taraxerol from the ethyl acetate extract (EAE) of the leaves of Mangifera indica (ii) the bioactive compound exhibited insulin-stimulated glucose uptake through translocation and activation of the glucose transporter (GLUT4) in an IRTK and PI3K dependent fashion. (iii) the fate of glucose following insulin-stimulated glucose uptake was ascertained through glycogen synthesis assay that involved the activation of PKB and suppression of GSK3β.

General significance

This study demonstrates the dual activity of 3β-taraxerol and the ethyl acetate extract of Mangifera indica as a glucose transport activator and stimulator of glycogen synthesis. 3β-taraxerol can be validated as a potent candidate for managing the hyperglycemic state.     

Molecular characterization of India Ginseng Withania somnifera (L) using ISSR markers

Molecular Biology Reports - Ashwagandha (Withania somnifera (L.) Dunal), popularly known as Indian ginseng or winter cherry is a multipurpose plant of immense therapeutic value in the ayurvedic and...     

Optimization of Multiple Pathogen Detection Using the TaqMan Array Card: Application for a Population-Based Study of Neonatal Infection

Identification of etiology remains a significant challenge in the diagnosis of infectious diseases, particularly in resource-poor settings. Viral, bacterial, and fungal pathogens, as well as parasites, play a role for many syndromes, and optimizing a single diagnostic system to detect a range of pathogens is challenging. The TaqMan Array Card (TAC) is a multiple-pathogen detection method that has previously been identified as a valuable technique for determining etiology of infections and holds promise for expanded use in clinical microbiology laboratories and surveillance studies. We selected TAC for use in the Aetiology of Neonatal Infection in South Asia (ANISA) study for identifying etiologies of severe disease in neonates in Bangladesh, India, and Pakistan. Here we report optimization of TAC to improve pathogen detection and overcome technical challenges associated with use of this technology in a large-scale surveillance study. Specifically, we increased the number of assay replicates, implemented a more robust RT-qPCR enzyme formulation, and adopted a more efficient method for extraction of total nucleic acid from blood specimens. We also report the development and analytical validation of ten new assays for use in the ANISA study. Based on these data, we revised the study-specific TACs for detection of 22 pathogens in NP/OP swabs and 12 pathogens in blood specimens as well as two control reactions (internal positive control and human nucleic acid control) for each specimen type. The cumulative improvements realized through these optimization studies will benefit ANISA and perhaps other studies utilizing multiple-pathogen detection approaches. These lessons may also contribute to the expansion of TAC technology to the clinical setting.     

The identification of candidate radio marker genes using a coexpression network analysis in gamma‐irradiated rice

Plant physiological and biochemical processes are significantly affected by gamma irradiation stress. In addition, gamma‐ray (GA) differentially affects gene expression across the whole genome. In this study, we identified radio marker genes (RMGs) responding only to GA stress compared with six abiotic stresses (chilling, cold, anoxia, heat, drought and salt) in rice. To analyze the expression patterns of differentially expressed genes (DEGs) in gamma‐irradiated rice plants against six abiotic stresses, we conducted a hierarchical clustering analysis by using a complete linkage algorithm. The up‐ and downregulated DEGs were observed against six abiotic stresses in three and four clusters among a total of 31 clusters, respectively. The common gene ontology functions of upregulated DEGs in clusters 9 and 19 are associated with oxidative stress. In a Pearson's correlation coefficient analysis, GA stress showed highly negative correlation with salt stress. On the basis of specific data about the upregulated DEGs, we identified the 40 candidate RMGs that are induced by gamma irradiation. These candidate RMGs, except two genes, were more highly induced in rice roots than in other tissues. In addition, we obtained other 38 root‐induced genes by using a coexpression network analysis of the specific upregulated candidate RMGs in an ARACNE algorithm. Among these genes, we selected 16 RMGs and 11 genes coexpressed with three RMGs to validate coexpression network results. RT‐PCR assay confirmed that these genes were highly upregulated in GA treatment. All 76 genes (38 root‐induced genes and 38 candidate RMGs) might be useful for the detection of GA sensitivity in rice roots.     

Serine substitution for cysteine residues in levansucrase selectively abolishes levan forming activity

Levansucrase is responsible for levan formation during sucrose fermentation of Zymomonas mobilis, and this decreases the efficiency of ethanol production. As thiol modifying agents decrease levan formation, a role for cysteine residues in levansucrase activity has been examined using derivatives of Z. mobilis levansucrase that carry serine substitutions of cysteine at positions 121, 151 or 244. These substitutions abolished the levan forming activity of levansucrase whilst only halving its activity in sucrose hydrolysis. Thus, polymerase and hydrolase activities of Z. mobilis levansucrase are separate and have different requirements for the enzyme's cysteine residues.