A lactate biosensor based on lactate dehydrogenase/nictotinamide adenine dinucleotide (oxidized form) immobilized on a conducting polymer/multiwall carbon nanotube composite film

An amperometric lactate biosensor was developed based on a conducting polymer, poly-5,2′-5′,2′′-terthiophene-3′-carboxylic acid (pTTCA), and multiwall carbon nanotube (MWNT) composite on a gold electrode. Lactate dehydrogenase (LDH) and the oxidized form of nicotinamide adenine dinucleotide (NAD⁺) were subsequently immobilized onto the pTTCA/MWNT composite film. The modified electrode was characterized by quartz crystal microbalance (QCM), scanning electron microscopy (SEM), and electrochemical experiments. The detection signal was amplified by the pTTCA/MWNT assembly onto which a sufficient amount of enzyme was immobilized and stabilized by the covalent bond formation between the amine groups of enzyme and the carboxylic acid groups of the pTTCA/MWNT film. Experimental parameters affecting the sensor responses, such as applied potential, pH, and temperature, were assessed and optimized. Analytical performances and dynamic ranges of the sensor were determined, and the results showed that the sensitivity, stability, and reproducibility of the sensor improved significantly using pTTCA/MWNT composite film. The calibration plot was linear (r² = 0.9995) over the range of 5 to 90 μM. The sensitivity was approximately 0.0106 μA/μM, with a detection limit of 1 μM, based on a signal/noise ratio of 3. The applicability of the sensor for the analysis of l-lactate concentration in commercial milk and human serum samples was demonstrated successfully.     

Spectroscopic studies on the comparative interaction of cationic single-chain and gemini surfactants with human serum albumin

To gain insights into the comparative effect of single-chain/gemini surfactants on proteins and the possible implications, the interaction of human serum albumin (HSA) with cationic single-chain surfactant cetyltrimethylammonium bromide (CTAB) and its gemini counterpart bis(cetyldimethylammonium)butane dibromide with spacer -(CH(2))(4)- (designated as G4) using turbidity measurements, far-UV and near-UV circular dichroism (CD), intrinsic fluorescence and extrinsic fluorescence spectroscopy at pH 7.0 are reported in this contribution. A decrease of 33.5% alpha-helical content at 22.5 microM G4 was monitored compared to a 15% decrease at 2,250 microM CTAB. Against a 3.5% increase at 11,250 microM CTAB, a rise of 21.1% in the alpha-helical content was observed 375 microM G4. The result is related to the stronger electrostatic and hydrophobic forces in G4, owing to the presence of two charged headgroups and two hydrophobic hydrocarbon tails that make it to bind strongly to the protein compared to its single chain counterpart, CTAB, resulting in larger unfolding. The stabilization at higher concentrations is attributed to the highly hydrophobic microdomain of the G4 aggregates formed at such concentrations. The results of the multi-technique approach are consistent with the fact that the gemini surfactants are more efficient than their conventional single-chain counterparts and hence may be used more effectively in the renaturation of proteins produced in the genetically engineered cells via the artificial chaperone protocol, as solubilizing agents to recover proteins from insoluble inclusion bodies and in drug delivery.     

Rechargeable biocidal cellulose: Synthesis and application of 3-(2,3-dihydroxypropyl)-5,5-dimethylimidazolidine-2,4-dione

An N-halamine precursor containing two hydroxyl groups (diol) has been synthesized. The N-halamine diol precursor was coated onto cotton fabrics with the assistance of the cross-linking agent BTCA. The surface of the coated cotton could be rendered biocidal by exposure to dilute hypochlorite solutions. Syntheses routes, characterization data, and antimicrobial test results will be presented. The durability and rechargeability of chlorine on the coated cotton was determined according to an AATCC standard washing test. The chlorinated cotton swatches were challenged with Staphylococcus aureus (ATCC 6538) and Escherichia coli O157:H7 (ATCC 43895) and showed excellent efficacy against these two bacterial species within a brief time of contact. Over 70% of the chlorine lost after repeated washing or UVA irradiation could be regained upon rechlorination.     

A Putative Leucine-Rich Repeat Receptor-Like Kinase of Jute Involved in Stress Response

A putative leucine-rich repeat receptor-like protein kinase (LRR-RLK) gene together with its 5′ and 3′ untranslated regions of jute (Corchorus olitorius L.) has been identified and sequenced. The gene is 3,371 bp long containing two exons and one intron. The coding sequence of the gene is 2,879 bp long encoding a peptide of 957 amino acids. The predicted protein contains several domains and motifs characteristic of a transmembrane protein kinase. It is complete with domains for an N-terminal leucine-rich repeat and a protein kinase core, an active site for serine/threonine protein kinase, an ATP binding conserved site and a transmembrane region. Expression of the gene is induced by low temperature, high salt concentration, dehydration, abscisic acid treatment, and fungal infection, suggesting the involvement of the gene in multiple stress response pathways in jute (C. olitorius L.). A possible mechanism of the role of the gene in signal transduction and environmental stress response is discussed. To date, LRR-RLK is the only jute gene which has been completely sequenced and characterized.     

Novel missense mutation in FHIT gene: interpreting the effect in HPV-mediated cervical cancer in Indian women

Human papillomavirus (HPV) is considered to be a major etiological factor but is not sufficient for the development of cervical cancer. Other host factors including altered tumor suppressor gene activities might contribute to the carcinogenic process. Fragile Histidine Triad (FHIT) has been shown to play a pivotal role in carcinogenesis. Therefore, we made an attempt to find out point mutation of FHIT gene in HPV mediated cervical cancer in Indian women. 112 cases of cervical carcinoma tissue biopsies and 38 cervical scrapes samples of normal cytology were employed for this study. Herein, we report a novel mutation identified at nucleotide position 655, at codon 98 from CAT → CGT with ultimate replacement of amino acid Histidine by Arginine in cervical cancer cases. Molecular modeling was performed to predict the effect of this mutation in disease pathology. We predict that this change, His to Arg substitution in substrate-binding domain may generate catalytically inactive protein with loss of tumor suppressor activity.     

Naturally arising HIV-1 Nef variants conferring escape from cytotoxic T lymphocytes influence viral entry co-receptor expression and susceptibility to superinfection

HIV-1 Nef is a key factor for pathogenesis and is known to down-regulate functionally important molecules, including viral entry co-receptor CCR5 and CXCR4, from the surface of HIV-infected cells. Some of these Nef activities are mediated by the well-conserved proline-rich region of Nef, and this region is highly targeted by cytotoxic T lymphocytes (CTLs). In the present study, we asked whether Nef variants selected under CTL-mediated selective pressure in vivo may constrain these important Nef activities. The analysis of autologous nef sequences isolated from a cohort of total 235 subjects in Japan revealed that the subjects showing amino acid variations, such as Arg75Thr and Tyr85Phe, located within the proline-rich region were significantly over-represented by those having HLA-B*3501. CTL assays corroborated that these mutations conferred escape from HLA-B(?)3501-restricted CTLs. The Arg75Thr variant Nef selectively impaired CCR5, but not CXCR4, down-regulation activity from the cell surface; whereas the Tyr85Phe variant Nef affected neither CCR5 nor CXCR4 down-regulation activity. Moreover, the cells expressing the Arg75Thr variant Nef significantly impaired protection from superinfection by CCR5-tropic, but not CXCR4-tropic, viruses. These results highlighted the importance of certain Nef-specific CTLs in modulation of viral co-receptor down-regulation activity and protection from HIV-1 superinfection, providing us with additional insight into vaccine design.     

Vitamin E as a novel enhancer of macroautophagy in rat hepatocytes and H4-II-E cells

Autophagy is an intracellular bulk degradation process induced by nutrient starvation, and contributes to macromolecular turnover and rejuvenation of cellular organelles. We demonstrated that vitamin E was a novel nutritional enhancer of autophagy in freshly isolated rat hepatocytes and rat hepatoma H4-II-E cells. Supplementation of fresh hepatocytes with vitamin E (up to 100 μM) increased proteolysis significantly in the presence or absence of amino acids in a dose-dependent manner. The cytosolic LC3 ratio, a newly established index of autophagic flux, was significantly increased by vitamin E, strongly suggesting that the possible site of action is the LC3 conversion step, an early step in autophagosome formation. A typical antioxidant, α-lipoic acid, exerted autophagy suppression, while H₂O₂ stimulated autophagy. It is conceivable that autophagy was stimulated by oxidative stress and this stimulation was cancelled by cellular antioxidative effects. However, in our studies, vitamin E could have enhanced autophagy over-stimulation by H₂O₂, rather than suppress it. From these results, using a new cytosolic LC3 ratio, vitamin E increases autophagy by accelerating LC3 conversion through a new signaling pathway, emerging as a novel enhancer of autophagy.     

Nanoscale fabrication of a peptide layer in cell chip to detect effects of environmental toxins on HEK293 cells

A cysteine-terminated C(RGD)₄ peptide film was fabricated on a gold electrode for improving the attachment of cells. The electrochemical signals of cyclic voltammogram from cells on a C(RGD)₄ deposited electrode was enhanced from 0.27 to 0.49 μA compared to a bare electrode. The developed cell-based sensor determined the effect of bisphenol-A (BPA) and dichlorodiphenyltrichloroethane (DTT) on the viability of HEK-293 cells by detecting decrease of reduction peaks (1.12–0.15 μA for BPA and 0.81–0.29 μA for DDT) after the treatment of environmental chemicals. This developed system can be a powerful tool for the monitoring of environmental toxicants.