Snake venom hyaluronidase: An evidence for isoforms and extracellular matrix degradation

The present study attempts to establish the isoforms of hyaluronidase enzyme and their possible role in the spreading of toxins during envenomation. Screening of venoms of 15 snakes belonging to three different families revealed varied hyaluronidase activity in ELISA-like assay, but with relatively similar pH and temperature optima. The zymograms of individual venoms showed varied activity banding patterns and indicated the presence of at least two molecular forms of the enzyme. During envenomation, activity of hyaluronidase is considered crucial for the spreading of toxins and is presumed to distort the integrity of extracellular matrix through the degradation of hyaluronic acid in it. This property has been addressed through localization of hyaluronic acid in human skin and muscle tissue sections using the probe, biotinylated hyaluronic acid binding protein. Faint and discontinuous staining pattern of hyaluronidase treated tissue sections over intense staining of untreated tissue sections confirm the selective degradation of hyaluronic acid in extracellular matrix and thus provide an evidence for the spreading property of the enzyme.     

The MUR3 gene of Arabidopsis encodes a xyloglucan galactosyltransferase that is evolutionarily related to animal exostosins

Xyloglucans are the principal glycans that interlace cellulose microfibrils in most flowering plants. The mur3 mutant of Arabidopsis contains a severely altered structure of this polysaccharide because of the absence of a conserved alpha-L-fucosyl-(1-->2)-beta-D-galactosyl side chain and excessive galactosylation at an alternative xylose residue. Despite this severe structural alteration, mur3 plants were phenotypically normal and exhibited tensile strength in their inflorescence stems comparable to that of wild-type plants. The MUR3 gene was cloned positionally and shown to encode a xyloglucan galactosyltransferase that acts specifically on the third xylose residue within the XXXG core structure of xyloglucan. MUR3 belongs to a large family of type-II membrane proteins that is evolutionarily conserved among higher plants. The enzyme shows sequence similarities to the glucuronosyltransferase domain of exostosins, a class of animal glycosyltransferases that catalyze the synthesis of heparan sulfate, a glycosaminoglycan with numerous roles in cell differentiation and development. This finding suggests that components of the plant cell wall and of the animal extracellular matrix are synthesized by evolutionarily related enzymes even though the structures of the corresponding polysaccharides are entirely different from each other.     

Isolation of Enteric Nervous System Progenitor Cells from the Aganglionic Gut of Patients with Hirschsprung’s Disease

Enteric nervous system progenitor cells isolated from postnatal human gut and cultured as neurospheres can then be transplanted into aganglionic gut to restore normal patterns of contractility. These progenitor cells may be of future use to treat patients with Hirschprung’s disease, a congenital condition characterized by hindgut dysmotility due to the lack of enteric nervous system ganglia. Here we demonstrate that progenitor cells can also be isolated from aganglionic gut removed during corrective surgery for Hirschsprung’s disease. Although the enteric nervous system marker calretinin is not expressed in the aganglionic gut region, de novo expression is initiated in cultured neurosphere cells isolated from aganglionic Hirschsprung bowel. Furthermore, expression of the neural markers NOS, VIP and GFAP also increased during culture of aganglionic gut neurospheres which we show can be transplantation into cultured embryonic mouse gut explants to restore a normal frequency of contractility. To determine the origin of the progenitor cells in aganglionic region, we used fluorescence-activated cell sorting to demonstrate that only p75-positive neural crest-derived cells present in the thickened nerve trunks characteristic of the aganglionic region of Hirschsprung gut gave rise to neurons in culture. The derivation of enteric nervous system progenitors in the aganglionic gut region of Hirschprung’s patients not only means that this tissue is a potential source of cells for future autologous transplantation, but it also raises the possibility of inducing the differentiation of these endogenous cells in situ to compensate for the aganglionosis.     

Infarction Distribution Pattern in Acute Stroke May Predict the Extent of Leptomeningeal Collaterals

Objective

The aim of this study was to evaluate whether the distribution pattern of early ischemic changes in the initial MRI allows a practical method for estimating leptomeningeal collateralization in acute ischemic stroke (AIS).

Methods

Seventy-four patients with AIS underwent MRI followed by conventional angiogram and mechanical thrombectomy. Diffusion restriction in Diffusion weighted imaging (DWI) and correlated T2-hyperintensity of the infarct were retrospectively analyzed and subdivided in accordance with Alberta Stroke Program Early CT score (ASPECTS). Patients were angiographically graded in collateralization groups according to the method of Higashida, and dichotomized in 2 groups: 29 subjects with collateralization grade 3 or 4 (well-collateralized group) and 45 subjects with grade 1 or 2 (poorly-collateralized group). Individual ASPECTS areas were compared among the groups.

Results

Means for overall DWI-ASPECTS were 6.34 vs. 4.51 (well vs. poorly collateralized groups respectively), and for T2-ASPECTS 9.34 vs 8.96. A significant difference between groups was found for DWI-ASPECTS (p<0.001), but not for T2-ASPECTS (p = 0.088). Regarding the individual areas, only insula, M1-M4 and M6 showed significantly fewer infarctions in the well-collateralized group (p-values <0.001 to 0.015). 89% of patients in the well-collateralized group showed 0–2 infarctions in these six areas (44.8% with 0 infarctions), while 59.9% patients of the poor-collateralized group showed 3–6 infarctions.

Conclusion

Patients with poor leptomeningeal collateralization show more infarcts on the initial MRI, particularly in the ASPECTS areas M1 to M4, M6 and insula. Therefore DWI abnormalities in these areas may be a surrogate marker for poor leptomeningeal collaterals and may be useful for estimation of the collateral status in routine clinical evaluation.     

Dissection of β‐barrel outer membrane protein assembly pathways through characterizing BamA POTRA 1 mutants of Escherichia coli

BamA of Escherichia coli is an essential component of the hetero‐oligomeric machinery that mediates β‐barrel outer membrane protein (OMP) assembly. The C‐ and N‐termini of BamA fold into trans‐membrane β‐barrel and five soluble POTRA domains respectively. Detailed characterization of BamA POTRA 1 missense and deletion mutants revealed two competing OMP assembly pathways, one of which is followed by the archetypal trimeric β‐barrel OMPs, OmpF and LamB, and is dependent on POTRA 1. Interestingly, our data suggest that BamA also requires its POTRA 1 domain for proper assembly. The second pathway is independent of POTRA 1 and is exemplified by TolC. Site‐specific cross‐linking analysis revealed that the POTRA 1 domain of BamA interacts with SurA, a periplasmic chaperone required for the assembly of OmpF and LamB, but not that of TolC and BamA. The data suggest that SurA and BamA POTRA 1 domain function in concert to assist folding and assembly of most β‐barrel OMPs except for TolC, which folds into a unique soluble α‐helical barrel and an OM‐anchored β‐barrel. The two assembly pathways finally merge at some step beyond POTRA 1 but presumably before membrane insertion, which is thought to be catalysed by the trans‐membrane β‐barrel domain of BamA.     

Production of a highly glucose tolerant β-glucosidase by Paecilomyces variotii MG3: optimization of fermentation conditions using Plackett–Burman and Box–Behnken experimental designs

β-Glucosidases (β-d-glucoside glucohydrolase, 3.2.1.21) are a group of enzymes mainly involved in the hydrolysis of β-glycosidic bonds connecting carbohydrate residues in different classes of β-d glycosides. During cellulose degradation they convert cellobiose and cellooligosaccharides produced by the endo and exoglucanases to glucose. Most of the microbial β-glucosidases are inhibited by glucose. This limits their application in commercial scale cellulose degradation ventures. Solid state fermentation production of a highly glucose tolerant β-glucosidase by a novel isolate of Paecilomyces was optimized using a two step statistical experiment design. In the first step which employed a Plackett–Burman design, the effects of parameters such as moisture, temperature, pH, inoculum concentration, incubation time and different concentrations of (NH₄)₂SO₄, KH₂PO₄, NaCl, peptone and cellobiose were evaluated. The parameters with significant influence on the process were selected and fine tuned in the second step using a Box–Behnken design. The model obtained was validated and a peptone concentration of 2 g/l, inoculum concentration of 1.2 × 10⁶ spores/ml and an incubation period of 96 h were found to be optimum for the maximum production of the enzyme. The optimization resulted in a doubling of the enzyme production by the fungus.     

Bio-ethanol from water hyacinth biomass: An evaluation of enzymatic saccharification strategy

Biomass feedstock having less competition with food crops are desirable for bio-ethanol production and such resources may not be localized geographically. A distributed production strategy is therefore more suitable for feedstock like water hyacinth with a decentralized availability. In this study, we have demonstrated the suitability of this feedstock for production of fermentable sugars using cellulases produced on site. Testing of acid and alkali pretreatment methods indicated that alkali pretreatment was more efficient in making the sample susceptible to enzyme hydrolysis. Cellulase and β-glucosidase loading and the effect of surfactants were studied and optimized to improve saccharification. Redesigning of enzyme blends resulted in an improvement of saccharification from 57% to 71%. A crude trial on fermentation of the enzymatic hydrolysate using the common baker’s yeast Saccharomyces cerevisiae yielded an ethanol concentration of 4.4 g/L.     

Assessing Genetic Differentiation in Geographic Populations of Labeo calbasu Using Allozyme Markers

The population structure of Labeo calbasu from 11 rivers belonging to the Indus, Ganges, Bhima, Mahanadi, and Godavari basins was investigated using allozyme marker systems. Seven out of 20 allozyme loci (35%) were polymorphic (P < 0.99). Both probability and score tests indicated significant deviation of genotype proportions from Hardy–Weinberg expectations at two loci, XDH* (Mahanadi, Bhima, and Godavari) and G6PDH* (Mahanadi). A pairwise genetic homogeneity test and F _ST values indicated a low-to-moderate level (0.0515) of genetic structuring in the wild population of L. calbasu. AMOVA analysis also indicated moderate differentiation among the samples from different river basins. Analysis for genetic bottleneck was performed under the infinite allele model. The study revealed nine genetic stocks of L. calbasu from the natural population across Indian rivers. Evidence of genetic bottlenecks in some rivers was also revealed.