Immobilization of Xylan-Degrading Enzymes from Scytalidium thermophilum on Eudragit L-100

Summary Xylanase from Scytalidium thermophilum was immobilized on Eudragit L-100, a pH sensitive copolymer of methacrylic acid and methyl methacrylate. The enzyme was non-covalently immobilized and the system expressed 70% xylanase activity. The immobilized preparation had broader optimum temperature of activity between 55 and 65 °C as compared to 65 °C in case of free enzyme and broader optimum pH between 6.0 and 7.0 as compared to 6.5 in case of free enzyme. Immobilization increased the t_1/2 of enzyme at 60 °C from 15 to 30 min with a stabilization factor of 2. The K_m and V_max values for the immobilized and free xylanase were 0.5% xylan and 0.89 μmol/ml/min and 0.35% xylan and 1.01 μmol/ml/min respectively. An Arrhenius plot showed an increased value of activation energy for immobilized xylanase (227 kcal/mol) as compared to free xylanase (210 kcal/mol) confirming the higher temperature stability of the free enzyme. Enzymatic saccharification of xylan was also improved by xylanase immobilization.     

Proline-Rich Peptide from the Coral Pathogen Vibrio shiloi That Inhibits Photosynthesis of Zooxanthellae

The coral-bleaching bacterium Vibrio shiloi biosynthesizes and secretes an extracellular peptide, referred to as toxin P, which inhibits photosynthesis of coral symbiotic algae (zooxanthellae). Toxin P was produced during the stationary phase when the bacterium was grown on peptone or Casamino Acids media at 29°C. Glycerol inhibited the production of toxin P. Toxin P was purified to homogeneity, yielding the following 12-residue peptide: PYPVYAPPPVVP (molecular weight, 1,295.54). The structure of toxin P was confirmed by chemical synthesis. In the presence of 12.5 mM NH₄Cl, pure natural or synthetic toxin P (10 μM) caused a 64% decrease in the photosynthetic quantum yield of zooxanthellae within 5 min. The inhibition was proportional to the toxin P concentration. Toxin P bound avidly to zooxanthellae, such that subsequent addition of NH₄Cl resulted in rapid inhibition of photosynthesis. When zooxanthellae were incubated in the presence of NH₄Cl and toxin P, there was a rapid decrease in the pH (pH 7.8 to 7.2) of the bulk liquid, suggesting that toxin P facilitates transport of NH₃ into the cell. It is known that uptake of NH₃ into cells can destroy the pH gradient and block photosynthesis. This mode of action of toxin P can help explain the mechanism of coral bleaching by V. shiloi.     

Modulation of gene expression in autoimmune disease and aging by food restriction and dietary lipids

Several recent observations carried out by many investigators have offered some clues in understanding the mechanism of how food restriction (FR) acts in the prolongation of life-span, but the precise mechanisms involved in modulating the immune system have not been clearly understood. Our own ongoing studies indicate that FR may act at the molecular level and may extend the life-span by modulating functional activities of several genes in various target tissues. For instance, while cytochrome P-450 IIB1 and IIB2 expression is known to decline with age in ad libitum-fed rats, FR prevented the loss of (drug-inducible) P-450 enzymes in liver tissues. In addition, both alpha 2u-globulin and senescence marker protein 2 expressions, which are regulated by hormones, were also modulated during aging by FR in Fischer 344 male rats. In short-lived autoimmune-prone mice, both FR and omega-3 (n-3) fatty acids diet lowered the severity of autoimmune disease both in lupus-prone (NZB x NZW)F1 mice and in mice prone to develop lymphoproliferative and renal diseases, whereas saturated (n-9) and polyunsaturated (n-6) dietary lipids not only exacerbated autoimmune disease, but also significantly enhanced expression of several oncogenes in lymphoid tissues. FR and omega-3 fatty acids decreased the expression of certain oncogenes. Both FR and omega-3 fatty acids may modulate the aging and autoimmune disease processes by not only altering the fatty acid composition, membrane fluidity, and signal transduction, but also by modulating the lymphokine hormone receptors and their functions and thereby modulating expression of several genes in various tissues during the aging process.     

One-step purification of glucoamylase by affinity precipitation with alginate.

It was found that alginate binds to glucoamylase, presumably through the recognition of starch binding domain of the latter. The present work exploits this for purification of glucoamylases from commercial preparation of Aspergillus niger and crude culture filtrate of Bacillus amyloliquefaciens by affinity precipitation technique in a single-step protocol. Glucoamylase is selectively precipitated using alginate as macroaffinity ligand and later eluted with 1.0 M maltose. In the case of A. niger, 81% activity is recovered with 28-fold purification. The purified glucoamylase gave a single band on SDS-PAGE corresponding to 78 kDa molecular weight. The developed affinity precipitation process also works efficiently for purification of Bacillus amyloliquefaciens glucoamylase from its crude culture filtrate, giving 78% recovery with 38-fold purification. The purified preparation showed a major band corresponding to 62 kDa and a faint band about 50 kDa on SDS-PAGE. The latter corresponds to the molecular weight for alpha-amylase of Bacillus amyloliquefaciens.     

A pregnane ester diglycoside from Sarcostemma brevistigma

A pregnane ester diglycoside, brevinine, has been isolated from the dried twigs of Sarcostemma brevistigma. Its chemical and spectroscopic data are consistent with the structure 11-O-benzoyl-sarcogenin-3-O-α-L-diginopyranosyl-(1 → 4)-α-L-diginopyranoside.     

Multi-Pronged Interactions Underlie Inhibition of α-Synuclein Aggregation by β-Synuclein

The intrinsically disordered protein β-synuclein is known to inhibit the aggregation of its intrinsically disordered homolog, α-synuclein, which is implicated in Parkinson's disease. While β-synuclein itself does not form fibrils at the cytoplasmic pH?7.4, alteration of pH and other environmental perturbations are known to induce its fibrilization. However, the sequence and structural determinants of β-synuclein inhibition and self-aggregation are not well understood. We have utilized a series of domain-swapped chimeras of α-synuclein and β-synuclein to probe the relative contributions of the N-terminal, C-terminal, and the central non-amyloid-β component domains to the inhibition of α-synuclein aggregation. Changes in the rates of α-synuclein fibril formation in the presence of the chimeras indicate that the non-amyloid-β component domain is the primary determinant of self-association leading to fibril formation, while the N- and C-terminal domains play critical roles in the fibril inhibition process. Our data provide evidence that all three domains of β-synuclein together contribute to providing effective inhibition, and support a model of transient, multi-pronged interactions between IDP chains in both processes. Inclusion of such multi-site inhibitory interactions spread over the length of synuclein chains may be critical for the development of therapeutics that are designed to mimic the inhibitory effects of β-synuclein.     

Stalk-dependent and Stalk-independent Signaling by the Adhesion G Protein-coupled Receptors GPR56 (ADGRG1) and BAI1 (ADGRB1)

The adhesion G protein-coupled receptors (aGPCRs) are a large yet poorly understood family of seven-transmembrane proteins. A defining characteristic of the aGPCR family is the conserved GAIN domain, which has autoproteolytic activity and can cleave the receptors near the first transmembrane domain. Several aGPCRs, including ADGRB1 (BAI1 or B1) and ADGRG1 (GPR56 or G1), have been found to exhibit significantly increased constitutive activity when truncated to mimic GAIN domain cleavage (ΔNT). Recent reports have suggested that the new N-terminal stalk, which is revealed by GAIN domain cleavage, can directly activate aGPCRs as a tethered agonist. We tested this hypothesis in studies on two distinct aGPCRs, B1 and G1, by engineering mutant receptors lacking the entire NT including the stalk (B1- and G1-SL, with “SL” indicating “stalkless”). These receptors were evaluated in a battery of signaling assays and compared with full-length wild-type and cleavage-mimicking (ΔNT) forms of the two receptors. We found that B1-SL, in multiple assays, exhibited robust signaling activity, suggesting that the membrane-proximal stalk region is not necessary for its activation. For G1, however, the results were mixed, with the SL mutant exhibiting robust activity in several signaling assays (including TGFα shedding, activation of NFAT luciferase, and β-arrestin recruitment) but reduced activity relative to ΔNT in a distinct assay (activation of SRF luciferase). These data support a model in which the activation of certain pathways downstream of aGPCRs is stalk-dependent, whereas signaling to other pathways is stalk-independent.     

A novel pentasaccharide from immunostimulant oligosaccharide fraction of buffalo milk.

A processed oligosaccharide mixture of buffalo milk induced significant stimulation of antibody, delayed-type hypersensitivity response to sheep red blood cells in BALB/c mice. This also stimulated non-specific immune response of the animals measured in terms of macrophage migration index. A novel pentasaccharide has been isolated from the oligosaccharide containing fraction having immunostimulant activity of buffalo milk. This compound was isolated by a combination of gel filtration chromatography, silica gel column chromatography of derivatised oligosaccharides while the homogeneity was confirmed by high performance liquid chromatography. The results of structural analyses, i.e. proton nuclear magnetic resonance, fast atom bombardment mass spectrometry, chemical transformations and degradations are consistent with the following structure: GlcNAcbeta(1-->3)Galbeta(1-->4)GlcNAcbeta(1-->3)Gal beta(1-->4)Glc     

Dual treatment with kynurenine pathway inhibitors and NAD+ precursors synergistically extends life span in Drosophila

Tryptophan catabolism is highly conserved and generates important bioactive metabolites, including kynurenines, and in some animals, NAD⁺. Aging and inflammation are associated with increased levels of kynurenine pathway (KP) metabolites and depleted NAD⁺, factors which are implicated as contributors to frailty and morbidity. Contrastingly, KP suppression and NAD⁺ supplementation are associated with increased life span in some animals. Here, we used DGRP_229 Drosophila to elucidate the effects of KP elevation, KP suppression, and NAD⁺ supplementation on physical performance and survivorship. Flies were chronically fed kynurenines, KP inhibitors, NAD⁺ precursors, or a combination of KP inhibitors with NAD⁺ precursors. Flies with elevated kynurenines had reduced climbing speed, endurance, and life span. Treatment with a combination of KP inhibitors and NAD⁺ precursors preserved physical function and synergistically increased maximum life span. We conclude that KP flux can regulate health span and life span in Drosophila and that targeting KP and NAD⁺ metabolism can synergistically increase life span.     

Purification and stability characteristics of an alkaline serine protease from a newly isolated Haloalkaliphilic bacterium sp. AH-6

An alkaline protease secreting Haloalkaliphilic bacterium (Gene bank accession number EU118361) was isolated from the Saurashtra Coast in Western India. The alkaline protease was purified by a single step chromatography on phenyl sepharose 6 FF with 28% yield. The molecular mass was 40 kDa as judged by SDS-PAGE. The enzyme displayed catalysis and stability over pH 8–13, optimally at 9–11. It was stable with 0–4 M NaCl and required 150 mM NaCl for optimum catalysis at 37 °C; however, the salt requirement for optimal catalysis increased with temperature. While crude enzyme was active at 25–80 °C (optimum at 50 °C), the purified enzyme had temperature optimum at 37 °C, which shifted to 80 °C in the presence of 2 M NaCl. The NaCl not only shifted the temperature profile but also enhanced the substrate affinity of the enzyme as reflected by the increase in the catalytic constant (K _cat). The enzyme was also calcium dependent and with 2 mM Ca⁺², the activity reached to maximum at 50 °C. The crude enzyme was highly thermostable (37–90 °C); however, the purified enzyme lost its stability above 50 °C and its half life was enhanced by 30 and sevenfold at 60 °C with 1 M NaCl and 50 mM Ca⁺², respectively. The activity of the enzyme was inhibited by PMSF, indicating its serine type. While the activity was slightly enhanced by Tween-80 (0.2%) and Triton X-100 (0.05%), it marginally decreased with SDS. In addition, the enzyme was highly stable with oxidizing-reducing agents and commercial detergents and was affected by metal ions to varying extent. The study assumes significance due to the enzyme stability under the dual extremities of pH and salt coupled with moderate thermal tolerance. Besides, the facts emerged on the enzyme stability would add to the limited information on this enzyme from Haloalkaliphilic bacteria.