Continuous production of galacto-oligosaccharides from lactose using immobilized β-galactosidase from Bacillus circulans

Summary -Galactosidase-2 (-d-galactoside galactohydrolase, EC 3.2.1.23) from Bacillus circulans was purified using hydroxyapatite gel chromatography and immobilized onto Duolite ES-762 (phenolformaldehyde resin) and Merckogel (controlled pore silica gel) for continuous production of galacto-oligosaccharides using lactose as the substrate. The maximum amount of ologosaccharides produced by the immobilized enzyme was 35–40% of the total sugar during hydrolysis of 4.56% lactose. Partially purified -galactosidase from B. circulans was also immobilized onto various supports for the same purpose. The stability of the immobilized -galactosidase-2 or partially purified enzyme during a continuous reaction depended on their supports and specific activity. Of the supports tested, Merckogel was best for operational stability. With this support, the enzyme was quite stable with specific activity up to 15 units/g of wet gel; it was reversibly inactivated with more.     

The Cerebral Neurosecretory System,Secretory End-Foot System and Infracerebral Gland—a Probable Neuroendocrine Complex in Nephtys (Annelida; Polychaeta)

Abstract The brain of Nephtys contains four neurosecretory cell types with distinctive cytoplasmic inclusions, a cells are located uniquely in a single pair of ganglionic nuclei and b cells are represented by a single pair of cells, whereas c cells and d cells have a scattered distribution. Their axons form two types of secretory release structure. First, possible axon collaterals synapse upon slender “dentritic twigs” in the core of the brain. Secondly, two tracts descend to the brain floor to form a “neurosecretory neuropile” (or storage and release complex) in contact with the inner surface of the brain capsule. Other neurosecretory fibres penetrate through the capsule, branch extensively, and terminate in contact with its ventral surface in close association with the “infracerebral gland”. The gland is derived from the pericapsular epithelium and exhibits signs of specialization for glandular function. In contrast to certain other polychaetes, it does not contain secretory neuron perikarya. The secretory end-foot system is poorly developed. Its terminals are located adjacent to the neurosecretory neuropile, which they encircle. The cell bodies are probably represented by four e cells which, like the terminals, contain many mitochondria.     

Pandemic strains of O3:K6 Vibrio parahaemolyticus in the aquatic environment of Bangladesh

A total of 1500 environmental strains of Vibrio parahaemolyticus, isolated from the aquatic environment of Bangladesh, were screened for the presence of a major V. parahaemolyticus virulence factor, the thermostable direct haemolysin (tdh) gene, by the colony blot hybridization method using a digoxigenin-labeled tdh gene probe. Of 1500 strains, 5 carried the tdh sequence, which was further confirmed by PCR using primers specific for the tdh gene. Examination by PCR confirmed that the 5 strains were V. parahaemolyticus and lacked the thermostable direct haemolysin-related haemolysin (trh) gene, the alternative major virulence gene known to be absent in pandemic strains. All 5 strains gave positive Kanagawa phenomenon reaction with characteristic beta-haemolysis on Wagatsuma agar medium. Southern blot analysis of the HindIII-digested chromosomal DNA demonstrated, in all 5 strains, the presence of 2 tdh genes common to strains positive for Kanagawa phenomenon. However, the 5 strains were found to belong to 3 different serotypes (O3:K29, O4:K37, and O3:K6). The 2 with pandemic serotype O3:K6 gave positive results in group-specific PCR and ORF8 PCR assays, characteristics unique to the pandemic clone. Clonal variations among the 5 isolates were analyzed by comparing RAPD and ribotyping patterns. Results showed different patterns for the 3 serotypes, but the pattern was identical among the O3:K6 strains. This is the first report on the isolation of pandemic O3:K6 strains of V. parahaemolyticus from the aquatic environment of Bangladesh.     

Effect of Ascorbic Acid on the Degradation of Cyanocobalamin and Hydroxocobalamin in Aqueous Solution: A Kinetic Study

The degradation kinetics of 5 × 10⁻⁵ M cyanocobalamin (B₁₂) and hydroxocobalamin (B_12b) in the presence of ascorbic acid (AH₂) was studied in the pH range of 1.0–8.0. B₁₂ is degraded to B_12b which undergoes oxidation to corrin ring cleavage products. B_12b alone is directly oxidized to the ring cleavage products. B₁₂ and B_12b in degraded solutions were simultaneously assayed by a two-component spectrometric method at 525 and 550 nm without interference from AH₂. Both degrade by first-order kinetics and the values of the rate constants at pH 1.0–8.0 range from 0.08 to 1.05 × 10⁻⁵ s⁻¹ and 0.22–7.62 × 10⁻⁵ s⁻¹, respectively, in the presence of 0.25 × 10⁻³ M AH₂. The t_1/2 values of B₁₂ and B_12b range from 13.7 to 137.5 h and 2.5–87.5 h, respectively. The second-order rate constants for the interaction of AH₂ with B₁₂ and B_12b are 0.05–0.28 × 10⁻² and 1.10–30.08 × 10⁻² M⁻¹ s⁻¹, respectively, indicating a greater effect of AH₂ on B_12b compared to that of B₁₂. The k_obs–pH profiles for both B₁₂ and B_12b show the highest rates of degradation around pH 5. The degradation of B₁₂ and B_12b by AH₂ is affected by the catalytic effect of phosphate ions on the oxidation of AH₂ in the pH range 6.0–8.0.KEY WORDS: ascorbic acid, cyanocobalamin, degradation, hydroxocobalamin, kinetics, two-component spectrometry     

Ethnobotany of the Balti community,Tormik valley,Karakorum range,Baltistan, Pakistan

Background

Limited health facilities and malnutrition are major problems in the Karakorum Range of Northern Pakistan, often resulting in various human disorders. Since centuries, however, local communities in these areas have developed traditional methods for treating various ailments and local foods capes that can be significant for devising public health and nutritional policies. This study was intended to document the ethnobotanical knowledge of the local peoples in the Tormik Valley, especially in the medical and food domains.

Methods

Field trips were undertaken in 14 different villages of the study area from 2010 to 2012. Ethnobotanical data were gathered using semi-structured interviews and group conversation with 69 informants. Details about local uses of plant species were recorded along with demographic characteristics of the visited communities. Relative frequency citation index (RFCi) and preference ranking index (PRi) tools were applied to determine the cultural significance of the reported species.

Results

Sixty-three plant species, with a predominance of Asteraceae and Fabaceae family members, as well as their detailed folk uses were documented. Forty-three percent of the species were used to treat various diseases, 21 % were consumed as wild fruits and vegetables and 53 % of the species had multipurpose applications. Thymus linearis Benth, Hippophae rhamnoides ssp. turkestanica L. and Convolvulus arvensis L. were found to be the most utilized medicinal plant species, i.e. those with significant RFCi values (0.54, 0.51 and 0.48, respectively). Betula utilis D. Don was the most versatile taxon (seven different ways of utilization); being this species a common and easily accessible subalpine tree and then under anthropogenic pressure, the implementation of concrete strategies aimed at its in-situ and ex-situ conservation is strongly recommended.

Conclusion

The valleys in the Karakorum Mountains in the Northern Pakistan host significant Traditional Knowledge on local food and medicinal plant species, which need to be reconsidered and cautiously re-evaluated by ethnopharmacologists, and public health/nutrition actors. Furthermore, germane trans-disciplinary investigations are suggested to ensure the dynamic conservation of precious local knowledge systems, as well as plant diversity in Pakistani mountain regions.

     

H1 RNA polymerase III promoter-driven expression of an RNA aptamer leads to high-level inhibition of intracellular protein activity

Aptamers offer advantages over other oligonucleotide-based approaches that artificially interfere with target gene function due to their ability to bind protein products of these genes with high affinity and specificity. However, RNA aptamers are limited in their ability to target intracellular proteins since even nuclease-resistant aptamers do not efficiently enter the intracellular compartments. Moreover, attempts at expressing RNA aptamers within mammalian cells through vector-based approaches have been hampered by the presence of additional flanking sequences in expressed RNA aptamers, which may alter their functional conformation. In this report, we successfully expressed a ‘pure’ RNA aptamer specific for NF-κB p50 protein (A-p50) utilizing an adenoviral vector employing the H1 RNA polymerase III promoter. Binding of the expressed aptamer to its target and subsequent inhibition of NF-κB mediated intracellular events were demonstrated in human lung adenocarcinoma cells (A549), murine mammary carcinoma cells (4T1) as well as a human tumor xenograft model. This success highlights the promise of RNA aptamers to effectively target intracellular proteins for in vitro discovery and in vivo applications.     

EGFR and EphA2 are host factors for hepatitis C virus entry and possible targets for antiviral therapy

Hepatitis C virus (HCV) is a major cause of liver disease, but therapeutic options are limited and there are no prevention strategies. Viral entry is the first step of infection and requires the cooperative interaction of several host cell factors. Using a functional RNAi kinase screen, we identified epidermal growth factor receptor and ephrin receptor A2 as host cofactors for HCV entry. Blocking receptor kinase activity by approved inhibitors broadly impaired infection by all major HCV genotypes and viral escape variants in cell culture and in a human liver chimeric mouse model in vivo. The identified receptor tyrosine kinases (RTKs) mediate HCV entry by regulating CD81-claudin-1 co-receptor associations and viral glycoprotein-dependent membrane fusion. These results identify RTKs as previously unknown HCV entry cofactors and show that tyrosine kinase inhibitors have substantial antiviral activity. Inhibition of RTK function may constitute a new approach for prevention and treatment of HCV infection.     

Observations on the floristic,life-form,leaf-size spectra and habitat diversity of vegetation in the Bhimber hills of Kashmir Himalayas

Vegetation analysis provides the prerequisites to understand the overall community structure and function of any ecosystem and is a fundamental requirement for the precise evaluation of biodiversity. Although many studies have assessed floristic attributes of specific areas, there are still unexplored regions, as is the case of the mountain region in the Kashmir Himalayas. Current research highlighted the recent findings of the scientific characterization of floristic and ecological aspects on the forest flora found in the Bhimber hills, Pakistan. Floristically, a total of 93 species belonging to 80 genera in 41 families were recorded. The species distribution patterns across the families were disproportionate with half of the species contributed by 8 families and 25 families were monotypic. Based on the floristic analysis, Asteraceae was the largest family with 12% of species followed by Poaceae with (11%) species. PAST software, a multivariate ecological community analysis was used to classify the species similarities and differences among the different habitat types. According to the habitat wise distribution, 21% of species were growing in the natural forest habitat, while 15% of species were dispersed in highly distributed habitats along roadsides and 8% on pedestrians. In terms of functional diversity, the herbaceous growth form was dominant (58%). The biological spectrum revealed therophytes as the dominant life form as it indicates the disturbed habitat vegetation. The phytogeographical analysis revealed that the maximum (69%) species were native, while the minimum (31%) species were exotic. Thus, the study of these functional and habitat diversity patterns can significantly improve our understanding of the ecological aspects of the flora in the geographical location. This information may additionally be useful in devising management plans to ensure sustainable utilization and better management of forest landscapes in this Himalayan region.     

Carbonic Anhydrase Inhibitors: Schiff's Bases of Aromatic and Heterocyclic Sulfonamides and their Metal Complexes

Schiff's bases were obtained from aromatic/heterocyclic sulfonamides and amino-sulfonamide derivatives, such as sulfanilamide, homosulfanilamide, 4-aminoethyl-benzenesulfonamide and 5-amino-1,3,4-thiadiazole-2-sulfonamide. Metal complexes of some of these Schiff's bases, incorporating Zn(II), Co(II), Ni(II) and Cu(II) ions, were also prepared and tested as inhibitors of the zinc enzyme carbonic anhydrase (CA), and more specifically the red blood cell isozymes I and II. The Schiff's bases behaved as medium potency CA I and CA II inhibitors, whereas their metal complexes showed a highly enhanced potency, with several low nanomolar CA II inhibitors detected.