Neeta Sehgal

Biomolecules for development of biosensors and their applications

Biosensors are analytical devices incorporating biological materials such as enzymes, tissues, microorganisms, antibodies, cell receptors or biologically derived materials or a biomimic component intimately associated with or integrated within a physicochemical transducer or transducing microsystem which may be either optical, electrochemical, thermometric, piezoelectric or magnetic. The electronic signals produced are proportional to the concentration of specific analyte. A biomaterial may be any material, natural or man-made, that comprises whole or part of a living structure or biomedical device, which performs natural function. An essential component of molecular sensor is reagent layers. Creation of these layers require the immobilization of recognition elements for the detection method. The recognition elements are biomolecules. Laboratory methods of immobilization are numerous, but may not always appropriate for manufacture of biosensors. In the present article, we describe the use of various biomaterials for biosensors as well as their availability.

Adverse effects associated with persistent stimulation of Leydig cells with hCG in vitro

The detrimental effects of persistent stimulation with hCG were investigated in rat Leydig cells in vitro. Significant rise in lipid peroxidation and reactive oxygen species (ROS) with concomitant attenuation in the activities of antioxidant enzymes: superoxide dismutase, catalase, and glutathione‐S‐transferase was observed. Transcripts for catalase and superoxide dismutase were also depleted. Subsequent to each hCG challenge, the total antioxidant capacity in the target cells also declined significantly (P < 0.05). There was an increase in cell apoptosis (23%), which was associated with a rise in caspase‐3 activity, PARP cleavage, and Fas, FasL, caspase‐8 expression. While Bax and Caspase‐9 expression remained unchanged, Bcl‐2 demonstrated a marked decline. Taken together, the above data indicate that persistent hCG stimulation of Leydig cells induced adverse effects leading to oxidative stress and apoptosis which was channeled primarily through the extrinsic pathway. Mol. Reprod. Dev. 76: 1076–1083, 2009.

Morphology, morphogenesis, and molecular phylogeny of Sterkiella tetracirrata n. sp. (Ciliophora, Oxytrichidae), from the Silent Valley National Park, India

The morphology and morphogenesis during cell division of Sterkiella tetracirrata n. sp., isolated from a soil sample collected from the Silent Valley National Park, Kerala, India, were investigated using live observation, protargol staining and scanning electron microscopy. The new species differs from its congeners by the following combination of features: cell size in vivo 85-110×35-50μm, on average 84×37μm in protargol preparations; four ellipsoidal macronuclear nodules; 31 adoral membranelles; 17 frontal-ventral-transverse cirri consisting of three frontal, four frontoventral, one buccal, three ventral, two pretransverse and invariably four transverse cirri; resting cyst with separate macronuclear nodules. Sterkiella tetracirrata differs from the similar species S. terricola in the number of transverse cirri (invariably 4 vs. 3) and in the number of adoral membranelles (24-35 vs. 22 or 23). Morphogenesis resembles that of its congeners S. nova and S. histriomuscorum. Phylogenetic analyses based on SSU rRNA gene sequences consistently place the new species within the stylonychine oxytrichids, clustering closer to Gastrostyla steinii than to either S. cavicola or S. histriomuscorum. The analyses support the morphological evidence (e.g., similarity in the oral apparatus and the dorsal kinety pattern) that Gastrostyla and Pattersoniella evolved from a Sterkiella-like ancestor.

Effect of chronic oestrogen administration on androgen receptor expression in reproductive organs and pituitary of adult male rat

Following chronic (15 or 30 days) treatment with oestradiol 3‐benzoate (75 μg rat−1 day−1 in 100 μl of olive oil) to adult rats, androgen receptor (AR) expression was analysed simultaneously in testis, epididymis, seminal vesicle, prostate and pituitary utilising three independent tools i.e. immunohistochemistry, Western blotting and RT‐PCR. All the five organs showed higher AR transcriptional activity gradually increasing from 15 to 30 days of oestrogen treatment. However, the AR protein expression either through immunostaining or Western blotting demonstrated a significant decline in all the reproductive organs. In the pituitary, on the other hand, the decline coincided with a distinct breakdown of the AR protein into two bands with increasing duration of treatment. Serum and intra‐testicular testosterone levels were found significantly lowered. Spermatogenesis was adversely affected with concurrent decrease in weights of testis and accessory sex organs. Decrease in testis weight was consistent with the reduction in the number of maturing germ cells per tubule. Despite the decrease in weight, accessory sex organs like epididymis, seminal vesicle and prostate were completely devoid of any apoptotic cells which were characterised only in testis and pituitary. Seminiferous epithelium demonstrated a marked increase in the number of germ cells undergoing apoptosis. However, the rate of cell apoptosis was much higher in the pituitary than in the testis at the end of 30 days treatment. It is therefore concluded that degradation of AR protein expression after oestrogen treatment is probably directly linked to an increase in cell apoptosis both in testis and pituitary.

Middle-redox potential laccase from Ganoderma sp.: its application in improvement of feed for monogastric animals

The variables influencing laccase production by white-rot fungus Ganoderma sp. rckk-02 were optimized employing response surface methodology. Malt extract (6.0% w/v), lignin (0.5% w/v) and pH (5.5) were found to be the most significant factors for enhanced laccase production by 7 fold (226.0 U/ml) as compared to unoptimized growth conditions (32.0 U/ml). The N-terminal sequence of laccase revealed its distinct amino acid profile (S- I- R- N- S- G), which suggested it as a novel enzyme. The Far-UV CD spectrum of the laccase showed single broad negative trough at around 213 nm, a typical signature of all β proteins. The laccase was found to fall in the range of middle redox potential laccases. Purified laccase at dosage of 2.5 Ug−1 body weight when supplemented with pelleted diet of rats, a significant improvement (p < 0.05) in nutrients digestibility without causing any elevation of blood stress enzymes was observed.

N-acetylcysteine counteracts oxidative stress and prevents hCG-induced apoptosis in rat Leydig cells through down regulation of caspase-8 and JNK

We have earlier reported that following persistent stimulation with hCG, oxidative stress‐induced apoptosis in rat Leydig cells was mainly achieved through the extrinsic pathway. In the present study, the role of N‐acetylcysteine (NAC) in counteracting the oxidative stress and the mechanisms of inhibition of apoptosis under such conditions were investigated. NAC (1 mM) intervention with repeated hCG stimulation (50 ng/ml, four times, each with 30 min challenge) prevented the decline in Leydig cell viability and the rise in lipid peroxidation and reactive oxygen species. Simultaneously, the activities of the enzymes glutathione‐S‐transferase, catalase, superoxide dismutase and the intracellular glutathione and antioxidant capacity of the treated cells improved significantly. Apoptotic markers Fas, FasL, and caspase‐8, up‐regulated following repeated hCG exposure, were significantly down‐regulated following NAC co‐incubation. While Bcl‐2 expression was fully restored, Bax and caspase‐9 remained unchanged. NAC treatment induced down‐regulation of upstream JNK/pJNK and down‐stream caspase‐3 in the target cells. Taken together, the above findings indicate that NAC counteracted the oxidative stress in Leydig cells induced as a result of repeated hCG stimulation, and inhibited apoptosis by mainly regulating the extrinsic and JNK pathways of metazoan apoptosis. Mol. Reprod. Dev. 77:900–909, 2010.

A quick and simple biostrip technique for detection of lactose

A quick, simple and economical biostrip technology was developed for estimation of lactose by immobilizing β-galactosidase, galactose oxidase and horseradish peroxidase on to a polymeric support. The biostrip is dipped in milk or milk products and, from the colour that develops from an added chromogen, the concentration of lactose can be estimated from < 20 to 100+g l−1. The biostrips may be used in dairy industries, hospitals and remote areas where expensive instruments are not available.

Immunostimulatory effect of artificial feed supplemented with indigenous plants on Clarias gariepinus against Aeromonas hydrophila

The antibacterial activity of methanol extracts of Ficus benghalensis (prop-root) and Leucaena leucocephala (pod seed) was evaluated by measurement of zone of inhibition against pathogenic bacteria, Escherichia coli and Aeromonas hydrophila. Control artificial feed and artificial feed supplemented with 5% powder of F. benghalensis and L. leucocephala were prepared. Juvenile Clarias gariepinus were divided into four groups, acclimatized to laboratory conditions and fed with respective feeds for 20 days prior to the experiment. Immunomodulatory response of supplementary feed was studied by challenging the fish intraperitoneally at weekly intervals, with A. hydrophila. One set of fish, not challenged with A. hydrophila was used as a negative control, to analyze any detrimental effect of supplementary feed, while positive control, comprised of challenged fish fed with non-supplemented feed. Other two groups of fish were challenged with A. hydrophila and fed with respective supplementary feeds. Blood was collected on weekly intervals for four weeks and serum samples were analyzed to evaluate the damage of fish by A. hydrophila through liver function tests. The increase in the levels of Serum glutamic oxaloacetic transaminase (SGOT) and Serum Glutamic pyruvate transaminase (SGPT) in positive control group indicated the damage of liver & kidney. However the levels did not change significantly in fish fed with supplementary feeds when compared to negative control group. Nitric oxide, SOD, ALP and lipid peroxidase indicated lower stress levels in these fish compared to positive control. Fish fed with supplementary feed showed increased lysozyme activity and phagocytic index indicating an increase in non-specific immune response. The immunoglobulin levels of in serum were analyzed by homologous sandwich ELISA, which showed higher antibody production in fish fed with supplementary feed. The current study suggests conclusively, immunostimulatory role of F. benghalensis (prop-roots) and L. leucocephala (pod seed) in C. gariepinus when supplemented in artificial feed.

Differential modulation of apoptotic gene expression by N-acetyl-L-cysteine in Leydig cells stimulated persistently with hCG in vivo.

The present study was designed to investigate the molecular mechanisms of NAC (150 mg/kg bw twice/week) action in vivo under repeated hCG (100 IU/rat/day) stimulation to adult rats. Leydig cell refractoriness led to a significant decline in serum testosterone and intracellular cAMP by day 30 of chronic hCG intervention which improved significantly following NAC co-administration. It inhibited the rise in lipid peroxidation, improved the activity of antioxidant enzymes along with intracellular glutathione and total antioxidant capacity in the target cells. Leydig cell apoptosis declined significantly (P<0.001) with down-regulation of upstream, Fas, FasL, caspase-8, Bax and caspase-9, JNK/pJNK and downstream caspase-3 and PARP. On the other hand, anti-apoptotic Bcl2, NF-kβ, and Akt were up-regulated. Taken together, the above findings indicate that the specificity of NAC action was not restricted to regulating marker proteins in the extrinsic and JNK pathways as seen in vitro but extended to include intrinsic pathway of metazoan apoptosis as well.

AR versus ER (α) Expression in the Testis and Pituitary Following Chronic Estrogen Administration in Adult Rat

Modulation of the testis-pituitary axis has direct relevance to the expression of androgen and estrogen receptors. Androgen receptor (AR) and estrogen receptor (ERα) expression during hypospermatognesis after chronic estrogen administration to rats was studied in the adult testis and pituitary utilizing immunohistochemistry, western blotting, and RT-PCR. Both organs demonstrated higher AR transcriptional activity gradually increasing from 15 days (d) to 30 d of estrogen treatment. However, the AR protein as measured by either immunostaining or western blotting demonstrated a significant decline. A distinct break down of the AR protein in the pituitary into two specific bands was seen. In contrast, higher ERα transcriptional activity coincided well with the rise in protein and immunoexpression in both organs. FSH and testosterone (serum, intra-testicular testosterone) were found significantly (p < 0.001) lowered compared with raised estradiol levels. Spermatogenesis was adversely affected and was associated with a significant increase in cell apoptosis in both organs. The pituitary demonstrated a higher rate of apoptosis at the end of 30 d of estrogen treatment. Taken together, the above data indicate that chronic estrogenization to adult rats up-regulates ERα but down-regulates AR protein expression in testis and pituitary which probably has a direct association to the marked rise in cell apoptosis in these organs.