Functional roles of H98 and W99 and β2α2 loop dynamics in the α-l-arabinofuranosidase from Thermobacillus xylanilyticus

This study is focused on the elucidation of the functional role of the mobile β2α2 loop in the α-l-arabinofuranosidase from Thermobacillus?xylanilyticus, and particularly on the roles of loop residues H98 and W99. Using site-directed mutagenesis, coupled to characterization methods including isothermal titration calorimetry (ITC) and saturation transfer difference nuclear magnetic resonance (STD-NMR) spectroscopy, and molecular dynamics simulations, it has been possible to provide a molecular level view of interactions and the consequences of mutations. Binding of para-nitrophenyl α-l-arabinofuranoside (pNP-α-l-Araf) to the wild-type arabinofuranosidase was characterized by K(d) values (0.32 and 0.16?mm, from ITC and STD-NMR respectively) that highly resembled that of the arabinoxylo-oligosaccharide XA(3) XX (0.21?mm), and determination of the thermodynamic parameters of enzyme?:?pNP-α-l-Araf binding revealed that this process is driven by favourable entropy, which is linked to the movement of the β2α2 loop. Loop closure relocates the solvent-exposed W99 into a buried location, allowing its involvement in substrate binding and in the formation of a functional active site. Similarly, the data underline the role of H98 in the 'dynamic' formation and definition of a catalytically operational active site, which may be a specific feature of a subset of GH51 arabinofuranosidases. Substitution of H98 and W99 by alanine or phenylalanine revealed that mutations affected K(M) and/or k(cat) . Molecular dynamics performed on W99A implied that this mutation causes the loss of a hydrogen bond and leads to an alternative binding mode that is detrimental for catalysis. STD-NMR experiments revealed altered binding of the aglycon motif in the active site, combined with reduced STD intensities of the α-l-arabinofuranosyl moiety for W99 substitutions.     

Isolation and characterization of non-Frankia actinobacteria from root nodules of Alnus glutinosa, Casuarina glauca and Elaeagnus angustifolia

Actinobacterial isolates randomly obtained on nitrogen-free BAP medium from surface sterilized root nodules of Alnus glutinosa, Casuarina glauca and Elaeagnus angustifolia sampled from fields were reported. They were assigned on the basis of partial 16S rRNA sequences to Micromonospora, Nocardia and Streptomyces genera. The isolates have been screened for hydrolytic activities, indole acetic acid (IAA) and siderophores production, phosphate solubilization and antagonistic activities. Results suggest putative traits as plant growth promoting bacteria proprieties of the isolates that occur in unique association in root nodules of the three analysed actinorhizal host species.     

Eichhornia crassipes (Mart) solms: From water parasite to potential medicinal remedy

Water hyacinth, Eichhornia crassipes (Mart) Solms, originating in the amazonian basin, is a warm water aquatic plant. Water hyacinth is considered one of the most productive plants on earth and, accordingly, is considered one of the top ten world''s worst weeds. Water hyacinth spread to other tropical and subtropical regions by humans. It invaded about 62 countries in Africa, Asia and North America, and propagated extremely serious ecological, economical and social problems in the region between 40 degrees north and 45 degrees south. The dense weed of water hyacinth forms dense monocultures that can threaten local native species diversity and change the physical and chemical aquatic environment, thus altering ecosystem structure and function by disrupting food chains and nutrient cycling. We have separated and identified nine active fractions from water hyacinth and showed their promising therapeutic activities. Several compounds (alkaloid, phthalate derivatives, propanoid and phenyl derivatives) were identified in the extract of water hyacinth.Key words: water hyacinth, antimicrobial, anticancer, active compounds     

Implication of PMLIV in Both Intrinsic and Innate Immunity

PML/TRIM19, the organizer of nuclear bodies (NBs), has been implicated in the antiviral response to diverse RNA and DNA viruses. Several PML isoforms generated from a single PML gene by alternative splicing, share the same N-terminal region containing the RBCC/tripartite motif but differ in their C-terminal sequences. Recent studies of all the PML isoforms reveal the specific functions of each. The knockout of PML renders mice more sensitive to vesicular stomatitis virus (VSV). Here we report that among PML isoforms (PMLI to PMLVIIb), only PMLIII and PMLIV confer resistance to VSV. Unlike PMLIII, whose anti-VSV activity is IFN-independent, PMLIV can act at two stages: it confers viral resistance directly in an IFN-independent manner and also specifically enhances IFN-β production via a higher activation of IRF3, thus protecting yet uninfected cells from oncoming infection. PMLIV SUMOylation is required for both activities. This demonstrates for the first time that PMLIV is implicated in innate immune response through enhanced IFN-β synthesis. Depletion of IRF3 further demonstrates the dual activity of PMLIV, since it abrogated PMLIV-induced IFN synthesis but not PMLIV-induced inhibition of viral proteins. Mechanistically, PMLIV enhances IFN-β synthesis by regulating the cellular distribution of Pin1 (peptidyl-prolyl cis/trans isomerase), inducing its recruitment to PML NBs where both proteins colocalize. The interaction of SUMOylated PMLIV with endogenous Pin1 and its recruitment within PML NBs prevents the degradation of activated IRF3, and thus potentiates IRF3-dependent production of IFN-β. Whereas the intrinsic antiviral activity of PMLIV is specific to VSV, its effect on IFN-β synthesis is much broader, since it affects a key actor of innate immune pathways. Our results show that, in addition to its intrinsic anti-VSV activity, PMLIV positively regulates IFN-β synthesis in response to different inducers, thus adding PML/TRIM19 to the growing list of TRIM proteins implicated in both intrinsic and innate immunity.     

Phylogeny of members of the <Emphasis Type="Italic">Frankia</Emphasis> genus based on <Emphasis Type="Italic">gyr</Emphasis>B, <Emphasis Type="Italic">nif</Emphasis>H and <Emphasis Type="Italic">gln</Emphasis>II sequences

To construct an evolutionary hypothesis for the genus Frankia, gyrB (encoding gyrase B), nifH (encoding nitrogenase reductase) and glnII (encoding glutamine synthetase II) gene sequences were considered for 38 strains. The overall clustering pattern among Frankia strains based on the three analyzed sequences varied among themselves and with the previously established 16S rRNA gene phylogeny and they did not reliably reflect clear evolution of the four discerned Frankia clusters (1, 2, 3 and 4). Based on concatenated gyrB, nifH and glnII, robust phylogenetic trees were observed with the three treeing methods (Maximum Likelihood, Parsimony and Neighbor-Joining) and supported by strong bootstrap and posterior probability values (>75%) for overall branching. Cluster 4 (non-infective and/or non-nitrogen-fixing Frankia) was positioned at a deeper branch followed by cluster 3 (Rhamnaceae and Elaeagnaceae infective Frankia), while cluster 2 represents uncultured Frankia microsymbionts of the Coriariaceae, Datiscaceae, Rosaceae and of Ceanothus sp. (Rhamnaceae); Cluster 1 (Betulaceae, Myricaceae and Casuarinaceae infective Frankia) appears to have diverged more recently. The present study demonstrates the utility of phylogenetic analyses based upon concatenated gyrB, nifH and glnII sequences to help resolve previously unresolved or poorly resolved nodes and will aid in describing species among the genus Frankia.     

Variation in antioxidant activity and phenolic content in different organs of two Tunisian cultivars of Olea europaea L.

This paper reports a comparative study based on the antioxidant compounds, total phenol content and antioxidant activities of leaves, stems and fruits from the main Tunisian cultivars, ‘chetoui’ and ‘chemchali’, grown in two different locations, north and south of Tunisia. The repartition of olive phenolic compounds was organ dependant. Therefore, the HPLC analysis indicated that the olive organs from the northern cultivar had the highest level of hydroxybenzoic acids, hydroxycinnamic acids and flavonoid class; which were less in the southern cultivar. Principal component analysis of the phenolic compounds showed discrimination between methanol extracts of the organs olive. Significant differences (p < 0.05) in the levels of phenols, orthodiphenols and flavonoids were found between cultivars and between organs. Antioxidant activities of the methanolic extract from aerial parts of the two studied cultivars were evaluated using 2,2-diphenyl-1-picrylhydrazyl and 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) assays. In all tests, methanolic extracts of different olive parts showed higher antioxidant activity. These results can be used to discriminate and to characterize the ‘chetoui’ and ‘chemchali’ aerial parts.     

Study of the association of adrenomedullin and basic-fibroblast growth factors with the peripheral arterial blood flow and endothelial dysfunction biomarkers in type 2 diabetic patients with peripheral vascular insufficiency

Background

Progressive micro-vascular vaso-degeneration is the major factor in progression of diabetic complications. Adrenomedullin (AM) and basic-Fibroblast growth factor (b-FGF) are strongly correlated with angiogenesis in vascular diseases. This study aims to provide base line data regarding the vascular effects and correlation of AM, and b-FGF with the peripheral blood flow in diabetic patients with peripheral vascular disease (PVD), and their effect on endothelial dysfunction markers. Ninety age- and sex matched females were enrolled in the study: 30 were controls, 30 had diabetes without complications (group II) and 30 had diabetes with PVD (group III) diagnosed by ankle/ brachial index (A/BI). Plasma levels of AM, b-FGF, intercellular adhesion molecule −1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) were measured by indirect enzyme immunoassay (ELISA).

Results

There was a significant increase in plasma AM, VCAM-1and ICAM-1, while a significant decrease in plasma b-FGF in diabetic patients with PVD (p < 0.05). A positive correlation was observed between plasma AM, b-FGF and A/BI and a negative correlation with VCAM −1 and ICAM in diabetic PVD. AM was not a predictor, while b-FG, VCAM-1 and ICAM-1 could be predictors for peripheral blood flow in diabetic PVD.

Conclusion

This study elucidates for the first time that AM and b-FGF are correlated and have a direct impact on the peripheral blood flow, the rise of AM in diabetic PVD may be a consecutive and compensatory vasculo-protective effect as its angiogenic and anti-inflammatory properties act to relief the endothelial insult. Down expression of b-FGF may be a predisposing factor for micro-vascular derangement. It is not clear if the rise of AM and the decline of b- FGF levels may be consequences or predisposing factors for VCAM-1 and ICAM-1 elevation as these endothelial dysfunction biomarkers could reduce peripheral blood flow and vascular integrity. It is optimistic to believe that drug intervention through AM and b-FGF administration together with reversing the endothelial inflammatory process by targeting VCAM and ICAM could reduce the prevalence of diabetic vascular complications, reduce the risk of cerebrovascular and cardiovascular morbidity in diabetes through normalizing vascular endothelium function and peripheral blood flow.     

Serum netrin and VCAM-1 as biomarker for Egyptian patients with type IΙ diabetes mellitus

ObjectiveThis study aimed to evaluate the serum level of netrin and soluble vascular cell adhesion molecule 1 (VCAM-I) in patients with type IΙ diabetes mellitus (T2DM) and evaluate the association of their levels with the development of a diabetic complication.Patients and methodsThis study was carried out on type II diabetic patients with and without complications and healthy individuals served as controls. All subjects were submitted to the estimation of serum lipid profile, serum creatinine, urinary albumin/creatinine ratio (ACR), fasting blood glucose (FBG), glycated hemoglobin (HbA1c), visceral adiposity index (VAI), atherogenic index of plasma (AIP), lipid accumulation product (LAP) and detection of serum level of netrin1 and VCAM1.ResultsDiabetic patients with complications had significantly higher serum levels of creatinine, ACR, cholesterol, Triglyceride, low-density lipoprotein, netrin1, and VCAM1 than diabetic patients without complications. Likewise, the level of VAI and LAP as markers of excessive body fat were significantly higher in diabetic patients with complications than diabetic patients without complications. The netrin1 and VCAM1 were a significant discriminator of T2DM renal complications with a sensitivity of 96%, 90%, and specificity of 82.7%, 91.3% respectively.ConclusionIt can be concluded that serum netrin1 and VCAM1 correlated significantly with markers of excessive body fat, a renal complication in the patient with type 2 diabetes mellitus.     

Defective Initiation of Glycosaminoglycan Synthesis due to B3GALT6 Mutations Causes a Pleiotropic Ehlers-Danlos-Syndrome-like Connective Tissue Disorder

Proteoglycans are important components of cell plasma membranes and extracellular matrices of connective tissues. They consist of glycosaminoglycan chains attached to a core protein via a tetrasaccharide linkage, whereby the addition of the third residue is catalyzed by galactosyltransferase II (β3GalT6), encoded by B3GALT6. Homozygosity mapping and candidate gene sequence analysis in three independent families, presenting a severe autosomal-recessive connective tissue disorder characterized by skin fragility, delayed wound healing, joint hyperlaxity and contractures, muscle hypotonia, intellectual disability, and a spondyloepimetaphyseal dysplasia with bone fragility and severe kyphoscoliosis, identified biallelic B3GALT6 mutations, including homozygous missense mutations in family 1 (c.619G>C [p.Asp207His]) and family 3 (c.649G>A [p.Gly217Ser]) and compound heterozygous mutations in family 2 (c.323_344del [p.Ala108Glyfs^∗163], c.619G>C [p.Asp207His]). The phenotype overlaps with several recessive Ehlers-Danlos variants and spondyloepimetaphyseal dysplasia with joint hyperlaxity. Affected individuals’ fibroblasts exhibited a large decrease in ability to prime glycosaminoglycan synthesis together with impaired glycanation of the small chondroitin/dermatan sulfate proteoglycan decorin, confirming β3GalT6 loss of function. Dermal electron microcopy disclosed abnormalities in collagen fibril organization, in line with the important regulatory role of decorin in this process. A strong reduction in heparan sulfate level was also observed, indicating that β3GalT6 deficiency alters synthesis of both main types of glycosaminoglycans. In vitro wound healing assay revealed a significant delay in fibroblasts from two index individuals, pointing to a role for glycosaminoglycan defect in impaired wound repair in vivo. Our study emphasizes a crucial role for β3GalT6 in multiple major developmental and pathophysiological processes.