Influence of chemical modification of cysteine and histidine side chains upon subunit reassembly of alpha crystallin

Alpha crystallin, the important multimeric structural protein of mammalian eye lens, is an assembly composed of 30 alpha-A and 10 alpha-B subunits. The influence of either partial or complete chemical modification of two important amino acid side chains, cysteine and histidine, upon the integrity of native alpha crystallin assembly and also upon the mode of subunit reassembly has been investigated. It has been found that chemical modification of surface-exposed cysteine and histidine side chains does not affect the subunit-subunit interactions stabilizing the native aggregate. Cysteine modifications, either partial or complete, unlike histidine modifications, do not seem to affect the backbone conformation of the subunits refolded after denaturation. Both cysteine and histidine modifications, however, affect the packing of the refolded structural elements forming the tertiary structure of the subunits and also the mode of oligomeric reorganization. The most striking effect of histidine modification is the considerable increase in size of the aggregates upon reassociation of the modified subunits. The chaperone activity, however, has been found to remain almost unaffected in spite of these chemical modifications.     

Staff Perception on Biomedical or Health Care Waste Management: A Qualitative Study in a Rural Tertiary Care Hospital in India

Background

Health care or biomedical waste, if not managed properly, can be of high risk to the hospital staff, the patients, the community, public health and the environment, especially in low and middle income settings where proper disposal norms are often not followed. Our aim was to explore perceptions of staff of an Indian rural tertiary care teaching hospital on hospital waste management.

Method

A qualitative study was conducted using 10 focus group discussions (FGDs), with different professional groups, cleaning staff, nurses, medical students, doctors and administrators. The FGD guide included the following topics: (i) role of Health Care Waste Management (HCWM) in prevention of health care associated infections, (ii) awareness of and views about HCWM-related guidelines/legislation, (iii) current HCWM practices, (iv) perception and preparedness related to improvements of the current practices, and (v) proper implementation of the available guidelines/legislation. The FGDs were recorded, transcribed verbatim, translated to English (when conducted in Hindi) and analysed using content analysis.

Results

Two themes were identified: Theme (A), ‘Challenges in integration of HCWM in organizational practice,’ with the categories (I) Awareness and views about HCWM, (II) Organizational practices regarding HCWM, and (III) Challenges in Implementation of HCWM; and Theme (B), ‘Interventions to improve HCWM,’ with three categories, (I) Educational and motivational interventions, (II) Organizational culture change, and (III) Policy-related interventions.

Conclusion

A gap between knowledge and actual practice regarding HCWM was highlighted in the perception of the hospital staff. The participants suggested organizational changes, training and monitoring to address this. The information generated is relevant not merely to the microsystem studied but to other institutions in similar settings.     

Statistical optimization of <Emphasis Type="Italic">Bacillus alcalophilus</Emphasis> α-amylase immobilization on iron-oxide magnetic nanoparticles

We report the statistical optimization of the immobilization of alkaline α-amylase [E.C. 3.2.1.1] from Bacillus alcalophilus onto nano-sized supermagnetic ironoxide nanoparticles (MNPs) for augmenting the cost effective industrial application of MNP-bound α-amylase. Both Plackett-Burman factorial design and response surface methodology (RSM) were employed to screen the influence of different parameters and the central effect of response on the α-amylase-iron oxide MNP binding process. The high coefficient of determination (R2) and analysis of variance (ANOVA) of the quadratic model indicated the competence of the proposed model. The size of the MNPs was confirmed by X-ray diffraction and scanning electron microscope analyses in which Fourier transform infrared spectroscopy suggested immobilization of the enzyme on iron-oxide MNPs. A significant improvement (∼ 26-fold) in specific activity, thermal and storage stability, and reusability of α-amylase after binding with iron-oxide MNP reinforced the improved biotechnological potential of the α-amylase iron-oxide MNP bioconjugate compared to free α-amylase. These results open new avenues for applying this MNP immobilized enzyme in different industrial sectors, notably in the paper and brewing industries.     

Baculovirus expression system: An alternative for producing catalytically active human PTP-1B

Protein tyrosine phosphatases (PTPs) play multiple roles in many physiological processes. Over-expression of the PTPs has been shown to be associated with cellular toxicity, which may also lead to the deletion of the respective gene from stable cell clones. We also observed that PTP-1B over-expression in CHO and HEK293 stable cell clones led to cytotoxicity and low revival rates during clone generation and maintenance. To address these issues, bacmid transposition technology was utilized to generate recombinant PTP-1B baculovirus, and Spodoptera frugiperda (Sf9 and Sf21) insect cell lines were infected with the virus. The data obtained on expression and activity of the PTP-1B highlights clear advantage of the recombinant baculovi-rus-insect cell expression system over the mammalian cell line technique due to increase in enzyme activity, strongly inhibited by phosphatase specific inhibitor RK682. Possible application of the expression system for producing active enzymes in bulk quantity for a new drug discovery is also discussed.     

Antisense expression of a gene encoding a calcium-binding protein in transgenic tobacco leads to altered morphology and enhanced chlorophyll

Entamoeba histolytica contains a novel calcium-binding protein like calmodulin, which was discovered earlier, and we have reported the presence of its homologue(s) and a dependent protein kinase in plants. To understand the functions of these in plants, a cDNA encoding a calcium-binding protein isolated from Entamoeba histolytica (EhCaBP) was cloned into vector pBI121 in antisense orientation and transgenic tobacco plants were raised. These plants showed variation in several phenotypic characters, of which two distinct features, more greenness and leaf thickness, were inherited in subsequent generations. The increase in the level of total chlorophyll in different plants ranged from 60% to 70%. There was no major change in chloroplast structure and in the protein level of D1, D2, LHCP and RuBP carboxylase. These morphological changes were not seen in antisense calmodulin transgenic tobacco plants, nor was the calmodulin level altered in EhCaBP antisense plants. The results of this paper have been granted US Patent No. 6,791,009.