Peters plus syndrome mutations affect the function and stability of human β1,3-glucosyltransferase

Peters Plus Syndrome (PTRPLS OMIM #261540) is a severe congenital disorder of glycosylation where patients have multiple structural anomalies, including Peters anomaly of the eye (anterior segment dysgenesis), disproportionate short stature, brachydactyly, dysmorphic facial features, developmental delay, and variable additional abnormalities. PTRPLS patients and some Peters Plus-like (PTRPLS-like) patients (who only have a subset of PTRPLS phenotypes, have mutations in the gene encoding β1,3-glucosyltransferase [B3GLCT]). B3GLCT catalyzes the transfer of glucose to O-linked fucose on thrombospondin type-1 repeats. Most B3GLCT substrate proteins belong to the ADAMTS superfamily and play critical roles in extracellular matrix. We sought to determine whether the PTRPLS or PTRPLS-like mutations abrogated B3GLCT activity. B3GLCT has two putative active sites, one in the N-terminal region and the other in the C-terminal glycosyltransferase domain. Using sequence analysis and in vitro activity assays, we demonstrated that the C-terminal domain catalyzes transfer of glucose to O-linked fucose. We also generated a homology model of B3GLCT and identified D421 as the catalytic base. PTRPLS and PTRPLS-like mutations were individually introduced into B3GLCT, and the mutated enzymes were evaluated using in vitro enzyme assays and cell-based functional assays. Our results demonstrated that PTRPLS mutations caused loss of B3GLCT enzymatic activity and/or significantly reduced protein stability. In contrast, B3GLCT with PTRPLS-like mutations retained enzymatic activity, although some showed a minor destabilizing effect. Overall, our data supports the hypothesis that loss of glucose from B3GLCT substrate proteins is responsible for the defects observed in PTRPLS patients, but not for those observed in PTRPLS-like patients.     

Isolation and characterization of a lead (Pb) tolerant <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> strain HF5 for decolorization of reactive red-120 and other azo dyes

Presence of heavy metals including lead (Pb) in the textile effluents is a crucial factor affecting the growth and potential of the dye decolorizing bacterial strains. This work was planned to isolate and characterize a bacterial strain exhibiting the potential to decolorize a range of azo dyes as well as the resistance to Pb. In this study, several Pb tolerant bacteria were isolated from effluents of textile industry. These bacterial isolates were screened for their potential of decolorizing the reactive red-120 (RR120) azo dye with presence of Pb (50 mg L^?1). The most efficient isolate was further characterized for its potential to resist Pb and decolorize different azo dyes under varying cultural and incubation conditions. Out of the total 82 tested bacterial isolates, 30 bacteria were found to have varying potentials to resist the presence of lead (Pb) and carry out decolorization of an azo dye reactive red-120 (RR120) in the medium amended with Pb (50 mg L^?1). The most efficient selected bacterium, Pseudomonas aeruginosa strain HF5, was found to show a good potential not only to grow in the presence of considerable concentration of Pb but also to decolorize RR120 and other azo dyes in the media amended with Pb. The strain HF5 completely (>?90%) decolorized RR120 in mineral salt medium amended with 100 mg L^?1 of Pb and 20 g L^?1 NaCl. This strain also considerably (>?50%) decolorized RR120 up to the presence of 2000 mg L^?1 of Pb and 50 g L^?1 of NaCl but with reduced rate. The optimal decolorization of RR120 by HF5 was achieved when the pH of the Pb amended (100 mg L^?1) mineral salt media was adjusted at 7.5 and 8.5. Interestingly, this strain also showed the tolerance to a range of metal ions with varying MIC values. The Pseudomonas aeruginosa strain HF5 harboring the unique potentials to grow and decolorize the azo dyes in the presence of Pb is envisaged as a potential bioresource for devising the remediation strategies for treatment of colored textile wastewaters loaded with Pb and other heavy metal ions.     

High frequency conversion of non-embryogenic synseeds and assessment of genetic stability through ISSR markers in <Emphasis Type="Italic">Gymnema sylvestre</Emphasis>

The present study describes the first attempt of exploiting encapsulation technology for high plantlet recovery, short-term storage and conservation of Gymnema sylvestre—an antidiabetic liana. Indole-3-butyric acid (IBA) pretreated nodal segments (NS) were encapsulated in sodium alginate (Na₂-alginate) matrix and the optimal culture conditions were evaluated in terms of maximum conversion capacity of synseeds into complete plantlets. Highest conversion frequency of synseeds was obtained on Murashige and Skoog’s (MS) medium supplemented with 5.0 µM 6-benzyladenine (BA). Augmentation of Na₂-alginate matrix with plant growth regulators (PGRs) and additive further improved in vitro conversion rates and the synthetic endosperm composed of 3% Na₂-alginate in MS?+?2.5 µM BA?+?2.5 µM gibberellic acid (GA₃)?+?50 µM adenine sulphate (AdS) stimulated maximum recovery (88.2?±?0.48%) of complete plantlets from synseeds. Studies on short term cold storage of synseeds showed that nutrient encapsulation maintains the viability of NS for a storage period of 8 weeks. Ex-vitro conversion of synseeds was also carried out on soilrite and vermicompost (3:1) mixture under culture room conditions. Monomorphic DNA profiles produced through Inter-Simple Sequence Repeat (ISSR) markers confirmed the genetic uniformity between synseed derived and mother plantlets.     

Synthesis,structure-activity relationship and molecular docking studies of 3-O-flavonol glycosides as cholinesterase inhibitors

The prime objective of this research work is to prepare readily soluble synthetic analogues of naturally occurring 3-O-flavonol glycosides and then investigate the influence of various substituents on biological properties of synthetic compounds. In this context, a series of varyingly substituted 3-O-flavonol glycosides have been designed, synthesized and characterized efficiently. The structures of synthetic molecules were unambiguously corroborated by IR, ¹H, ¹³C NMR and ESI-MS spectroscopic techniques. The structure of compound 22 was also analyzed by X-ray diffraction analysis. All the synthetic compounds (21–30) were evaluated for in vitro inhibitory potential against cholinesterase enzymes. The results displayed that most of the derivatives were potent inhibitors of acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) with varying degree of IC₅₀ values. The experimental results were further encouraged by molecular docking studies in order to explore their binding behavior with the active pocket of AChE and BChE enzymes. The experimental and theoretical results are in parallel with one another.     

Mitochondrial Genome Analysis of Wild Rice (Oryza minuta) and Its Comparison with Other Related Species

Oryza minuta (Poaceae family) is a tetraploid wild relative of cultivated rice with a BBCC genome. O. minuta has the potential to resist against various pathogenic diseases such as bacterial blight (BB), white backed planthopper (WBPH) and brown plant hopper (BPH). Here, we sequenced and annotated the complete mitochondrial genome of O. minuta. The mtDNA genome is 515,022 bp, containing 60 protein coding genes, 31 tRNA genes and two rRNA genes. The mitochondrial genome organization and the gene content at the nucleotide level are highly similar (89%) to that of O. rufipogon. Comparison with other related species revealed that most of the genes with known function are conserved among the Poaceae members. Similarly, O. minuta mt genome shared 24 protein-coding genes, 15 tRNA genes and 1 ribosomal RNA gene with other rice species (indica and japonica). The evolutionary relationship and phylogenetic analysis revealed that O. minuta is more closely related to O. rufipogon than to any other related species. Such studies are essential to understand the evolutionary divergence among species and analyze common gene pools to combat risks in the current scenario of a changing environment.     

Post-exposure temperature influence on the toxicity of conventional and new chemistry insecticides to green lacewing Chrysoperla carnea (Stephens) (Neuroptera: Chrysopidae)

Chrysoperla carnea (Stephens) is an important biological control agent currently being used in many integrated pest management (IPM) programs to control insect pests. The effect of post-treatment temperature on insecticide toxicity of a spinosyn (spinosad), pyrethroid (lambda cyhalothrin), organophosphate (chlorpyrifos) and new chemistry (acetamiprid) to C. carnea larvae was investigated under laboratory conditions. Temperature coefficients of each insecticide tested were evaluated. From 20 to 40 °C, toxicity of lambda cyhalothrin and spinosad decreased by 2.15- and 1.87-fold while toxicity of acetamiprid and chlorpyrifos increased by 2.00 and 1.79-fold, respectively. The study demonstrates that pesticide effectiveness may vary according to environmental conditions. In cropping systems where multiple insecticide products are used, attention should be given to temperature variation as a key factor in making pest management strategies safer for biological control agents. Insecticides with a negative temperature coefficient may play a constructive role to conserve C. carnea populations.     

Enhanced shoot organogenesis in Cassia angustifolia Vahl. — a difficult-to-root drought resistant medicinal shrub

In vitro regeneration was achieved through callus culture derived from cotyledon explants of Cassia angustifolia Vahl. on MS (Murashige and Skoog, 1962) medium. Calli were induced from cotyledon explants excised from aseptic 14?days old seedlings on MS medium containing 2,4-D (2,4-dichlorophenoxy acetic acid) and 2,4,5-T (2,4,5-trichlorophenoxy acetic acid) at different concentrations with 3% sucrose and 0.8% agar. Optimal growth of callus was obtained at 5.0???M 2,4-D, which was proved to be the best for shoot regeneration when sub cultured onto MS medium supplemented with cytokinins either alone or in combination with an auxin. Maximum number of shoots (23.2?±?1.4) were produced at 5.0???M 6-benzylaminopurine (BA) and 0.4???M ??-naphthalene acetic acid (NAA). Regenerated shoots produced prominent roots when transferred to half strength MS medium supplemented with 1.0???M indole-3-butyric acid (IBA) and 5.0???M phloroglucinol (PG). Rooted plantlets thus developed were hardened and successfully established in the soil. This protocol yielded an average of 23 plants per cotyledon explant over a period of 4?months.     

Synthesis of new sulfonamides as lipoxygenase inhibitors

The present study describes a convenient method for the synthesis of new lipoxygenase inhibitors, 4-(toluene-4-sulfonylamino)-benzoic acids from p-amino benzoic acid. Reaction of p-amino benzoic acid with p-toluenesulfonyl chloride provided thirteen N- and O-alkylation products 4a-4m in moderate to good yields. Lipoxygenase inhibition of newly formed sulfonamide derivatives was investigated and some of these compounds 4m, 4g, 4e, 4f and 4j showed good lipoxygenase inhibitory activities with IC(50) values ranged between 15.8 ± 0.57 and 91.7 ± 0.61 μmol whilst all other compounds exhibited mild anti-lipoxygenase activities with IC(50) values ranged between 139.2 ± 0.75 and 232.1 ± 0.78 μmol. N-alkylated products were more active against the enzyme than O-alkylated or both N- and O-alkylated ones. All synthesized sulfonamides were recrystallized in chloroform to give these title compounds which were characterized using FTIR, (1)H NMR, (13)C NMR, elemental analysis and single crystal X-ray diffraction techniques.     

Constrained (l-)-S-adenosyl-l-homocysteine (SAH) analogues as DNA methyltransferase inhibitors

Potent SAH analogues with constrained homocysteine units have been designed and synthesized as inhibitors of human DNMT enzymes. The five membered (2S,4S)-4-mercaptopyrrolidine-2-carboxylic acid, in 1a, was a good replacement for homocysteine, while the corresponding six-member counterpart was less active. Further optimization of 1a, changed the selectivity profile of these inhibitors. A Chloro substituent at the 2-position of 1a, compound 1d, retained potency against DNMT1, while N⁶ alkylation, compound 7a, conserved DNMT3b2 activity. The concomitant substitutions of 1a at both 2- and N⁶ positions reduced activity against both enzymes.