Indian Journal of Biochemistry & Biophysics 

CONTENTS

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*Author for correspondence

Indian Journal of Biochemistry & Biophysics

Vol. 40, October 2003, pp. 291-299

Minireview

Activating and inhibitory receptors and their role in Natural Killer cell function

Raghvendra M Srivastava, B Savithri and Ashok Khar*

Last decade has seen a significant advancement in our understanding of the Natural Killer (NK) cell biology and function. Several receptors present on NK cells have been identified, which are involved in either their activation or inhibition. Similarly, a large number of interacting ligands have been identified on the target cells that upon interaction transmit either activating or inhibitory signals. This review is oriented towards understanding the role of these receptors on the NK cells, which are considered as the first line of defense.

Keywords: NK cells, receptors, ligands, function, inhibition, activation, cytotoxicity

Indian Journal of Biochemistry & Biophysics

Vol. 40, October 2003, pp. 300-308

Minireview

  (Methods)

Methods for estimating lipid peroxidation: An analysis of merits and demerits

 T P A Devasagayam, K K Boloor and T Ramasarma^2*

Among the cellular molecules, lipids that contain unsaturated fatty acids with more than one double bond are particularly susceptible to action of free radicals. The resulting reaction, known as lipid peroxidation, disrupts biological membranes and is thereby highly deleterious to their structure and function. Lipid peroxidation is being studied extensively in relation to disease, modulation by antioxidants and other contexts. A large number of by-products are formed during this process. These can be measured by different assays. The most common method used is the estimation of aldehydic products by their ability to react with thiobarbituric acid (TBA) that yield ‘thiobarbituric acid reactive substances’ (TBARS), which can be easily measured by spectrophotometry. Though this assay is sensitive and widely used, it is not specific and TBA reacts with a number of components present in biological samples. Hence caution should be used while employing this method. Wherever possible this assay should be combined with other assays for lipid peroxidation. Such methods are measurement of conjugated dienes, lipid hydroperoxides, individual aldehydes, exhaled gases like pentane, isoprostanes, etc. The modern methods also involve newer techniques involving HPLC, spectrofluorimetry, mass spectrometry, chemiluminescence etc. These and other modern methods are more specific and can be applied to measure lipid peroxidation. There are certain restraints, in terms of high cost and certain artifacts, and these should be considered while selecting the method for estimation. This review analyses the merits and demerits of various assays to measure lipid peroxidation.

Keywords: Lipid peroxidation, thiobarbituric acid reactive substances, malondialdehyde, polyunsaturated fatty acid, conjugated dienes, lipid hydroperoxides, estimation

Indian Journal of Biochemistry & Biophysics

Vol. 40, October 2003, pp. 309-314

Cytochrome P450-mediated oxidative damage of nuclear membrane proteins and its prevention by vitamin C

 M K Ghosh, R Arun, D J Chattopadhyay and I B Chatterjee*

In this report, we present data to indicate that NADPH-cytochrome P450 reductase/cytochrome P450 system is present in the nuclear membrane. The reactive oxygen species generated in this free metal ion–independent P450 system oxidatively modifies and degrades the membrane proteins. The oxidative modification is evidenced by the formation of carbonyl, bityrosine and tryptophan loss. The degradation of membrane proteins is manifested using fluorescamine reactivity and SDS-PAGE. Ascorbic acid exclusively prevents the oxidative modification and degradation of the membrane proteins. Other antioxidants, such as superoxide dismutase, catalase, glutathione, a-tocopherol, probucol, b-carotene, mannitol, histidine and thiourea are found to be ineffective. The observation assumes significance, particularly in subclinical ascorbic acid deficiency, where oxidative damage of the nuclear membrane would occur. This, in turn, would affect the traffic of cytoplasmic enzymes and proteins required for DNA replication and repair, transcription and RNA processing, ultimately leading to disruption of gene regulation of the cell.

Keywords: nuclear membrane, cytochrome P450, NADPH-cytochrome P450 reductase, oxidative damage, ascorbic acid, antioxidants

Indian Journal of Biochemistry & Biophysics

Vol. 40, October 2003, pp. 315-323

Purification and characterization of cathepsin l-like proteinase from goat brain

Rachna Sadana, Ashwani Mittal, Shiwani Khurana, Hari Singh and Ramesh C Kamboj^*

Cathepsin l-like proteinase was purified ~1708-fold with 40% activity yield to an apparent electrophoretic homogeneity from goat brain by homogenization, acid-autolysis at pH 4.2, 30-80% (NH₄)₂SO₄ fractionation, Sephadex G-100 column chromatography and ion-exchange chromatography on CM-Sephadex C-50 at pH 5.0 and 5.6. The molecular weight of proteinase was found to be ~65,000 Da, by gel-filtration chromatography. The pH optima were 5.9 and 4.5 for the hydrolysis of Z-Phe-Arg-4mbNA (benzyloxycarbonyl-l-phenylalanine-l-arginine-4-methoxy-b-naphthylamide) and azocasein, respectively. Of the synthetic chromogenic substrates tested, Z-Phe-Arg-4mbNA was hydrolyzed maximally by the enzyme (K_m value for hydrolysis was 0.06 mM), followed by Z-Val-Lys-Lys-Arg-4mbNA, Z-Phe-Val-Arg-4mbNA, Z-Arg-Arg-4mbNA and Z-Ala-Arg-Arg-4mbNA. The proteinase was activated maximally by glutathione in conjunction with EDTA, followed by cysteine, dithioerythritol, thioglycolic acid, dithiothreitol and b-mercaptoethanol. It was strongly inhibited by p-hydroxymercuribenzenesulphonic acid, iodoacetic acid, iodoacetamide and microbial peptide inhibitors, leupeptin and antipain. Leupeptin inhibited the enzyme competitively with K_i value 44 ´ 10^-9 M. The enzyme was strongly inhibited by 4 M urea. Metal ions, Hg²⁺, Ca²⁺, Cu²⁺, Li²⁺, K⁺, Cd²⁺, Ni²⁺, Ba²⁺, Mn²⁺, Co²⁺ and Sn²⁺ also inhibited the activity of the enzyme. The enzyme was stable between pH 4.0-6.0 and up to 40ºC. The optimum temperature for the hydrolysis of Z-Phe-Arg-4mbNA was ~50-55ºC with an activation energy E_a of ~6.34 KCal mole^-1.

Keywords: cathepsin l-like proteinase, purification, characterization, goat brain

Indian Journal of Biochemistry & Biophysics

Vol. 40, October 2003, pp. 324-329

Changes in the activity of matrix metalloproteinases in regenerating rat liver after CCl₄-induced injury

 Shiju Jacob and P R Sudhakaran*

Molecular mechanisms involved in mediating alteration in cell matrix interaction have been examined by studying the changes in the activity of matrix metalloproteinases (MMPs) in CCl₄-induced regenerating liver, using zymography and ELISA. Activity of MMPs (72 kD, 92 kD and 130 kD gelatinases) in the rat liver increased progressively during acute injury till the 4th day and then decreased to near normal level after CCl₄ administration (0.5 ml/100 g body wt.) on the 6^th day. Hepatocyte lysate of injured liver on the 4^th day showed significantly higher levels of MMP2 and MMP9 compared to the control. In the culture medium of hepatocytes, the levels of MMP2 and MMP9 increased progressively with the duration of culture, indicating that hepatocytes are the major source of these MMPs in regenerating liver. These results suggest an involvement of MMPs in matrix degradation and remodeling during regeneration after acute liver injury induced by CCl₄.

Keywords: hepatic regeneration, matrix metalloproteinases (MMPs), extracelluar matrix (ECM), hepatocytes, CCl₄

Indian Journal of Biochemistry & Biophysics

Vol. 40, October 2003, pp. 330-339

Aluminium-induced enhancement of ageing-related biochemical and electrophysiological parameters in rat brain regions

Jaspreet Kaur, Sangeeta Singh, Deepak Sharma and Rameshwar Singh^*

We investigated alterations in the ageing-related parameters: multiple-unit action potentials, Na⁺, K⁺-ATPase activity, glutathione-s-transferase (GST) activity, glutathione peroxidase (GPx) activity, lipid peroxidation and lipofuscin contents in the brain regions cerebral cortex, striatum, hippocampus and thalamus, resulting from the chronic administration of aluminium chloride (AlCl₃) in drinking water to rats of 6 and 12 months of age. Aluminium treatment significantly depressed Na⁺, K⁺-ATPase, GST and GPx activities, elevated lipid peroxidation and lipofuscin contents, and produced intense epileptiform activity in the electroencephalograms of the studied brain regions together with a concomitant increase in the multiple-unit action potentials (MUA) indicating a vigorous neuronal epileptic hyperactivity. Taken together the aluminium-induced alterations in these parameters are indicative of an accelerated ageing process.

Keywords:  Aluminium, brain ageing, Na⁺, K⁺-ATPase, multiple unit action potentials, antioxidant enzymes, lipofuscin, lipid peroxidation, epileptic activity, acceleration of ageing

Indian Journal of Biochemistry & Biophysics

Vol. 40, October 2003, pp. 340-349

Kinetics in microemulsion V. Glucose oxidase catalyzed oxidation of b-D-glucose in aqueous, micellar and water-in-oil microemulsion media

Syamasri Gupta*, Lana Mukhopadhyay and S P Moulik

The oxidation of b-D-glucose by the enzyme glucose oxidase was studied in aqueous medium, in solutions of surfactants AOT (2-ethylhexylsulfosuccinate, sodium salt) TX-100 (polyethylene glycol p-tert octyl phenyl ether) and in w/o microemulsion medium (water/AOT/decane) at different water/AOT mole ratio (w), pH, temperature and in presence of additives. The time-dependent activities of the enzyme in aqueous and microemulsion media were determined. The catalytic process was retarded in the presence of TX-100 and AOT. In microemulsion medium, k_cat values exhibited a deformed W-shaped profile with w. At pH 7, a maximum value of k_cat was observed at w = 10.6. The k_cat values were found to be higher in microemulsion medium than in aqueous medium at both pH’s 7 and 8. Activation parameters for the kinetic process were evaluated together with the thermodynamics of the enzyme-substrate Michaelis complex. The DG* was lower, whereas DH* and DS* were higher in microemulsion than in water. The Michaelis constant, K_M was also lower in microemulsion. The inhibition effects of the additives, NaNO₃ and NaC were studied in both aqueous and microemulsion media by examining their influences on catalytic constant, k_cat and Michaelis constant K_M. In microemulsion, both the additives NaNO₃ and NaC produced non-competitive inhibition.

Keywords: microemulsion, enzyme activity, inhibitor, anisotropy

Indian Journal of Biochemistry & Biophysics

Vol. 40, October 2003, pp. 350-353

Activation of tobacco leaf polyphenol oxidase by

sodium dodecyl sulfate

Hui Jiang, Chunhua Shi, Yongshu Xie, Xiaolong Xu and Qingliang Liu*

The effect of sodium dodecyl sulfate (SDS) on purified tobacco leaf PPO (PPO II) was investigated at various pHs and temperatures. SDS increased the activity of PPO II due to the formation of SDS-PPO II complex, leading to conformational changes, thus making access to active center easier. The relationship between the activity and the molar ratio of SDS-PPO II to PPO II showed that the critical point reached a plateau of activity at the molar ratio of about 1.2. The pH had a significant effect on interaction between SDS and PPO II, as compared to PPO II. The optimum catalytic temperature of the complex rose by 10°C, suggesting that stabilization of the structure had been improved by the formation of complex.

Keywords: Sodium dodecyl sulfate (SDS), polyphenol oxidase, tobacco leaf, pH effect, temperature effect

Indian Journal of Biochemistry & Biophysics

Vol. 40, October 2003, pp. 354-357

Hepatoprotective and antioxidant effect of tender coconut water on carbon tetrachloride induced liver injury in rats

Anthony Loperito Loki and T Rajamohan*

Hepatoprotective and antioxidant effects of tender coconut water (TCW) were investigated in carbon tetrachloride (CCl₄)-intoxicated female rats. Liver damage was evidenced by the increased levels of serum glutamate oxaloacetate transaminase (SGOT), serum glutamate pyruvate transaminase (SGPT) and decreased levels of serum proteins and by histopathological studies in CCl₄-intoxicated rats. Increased lipid peroxidation was evidenced by elevated levels of thiobarbituric acid reactive substance (TBARS) viz, malondialdehyde (MDA), hydroperoxides (HP) and conjugated dienes (CD), and also by significant decrease in antioxidant enzymes activities, such as superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (Gpx) and glutathione reductase (GR) and also reduced glutathione (GSH) content in liver. On the other hand, CCl₄^-intoxicated rats treated with TCW retained almost normal levels of these constituents. Decreased activities of antioxidant enzymes in CCl₄-intoxicated rats and their reversal of antioxidant enzyme activities in TCW treated rats, shows the effectiveness of TCW in combating CCl₄-induced oxidative stress. Hepatoprotective effect of TCW is also evidenced from the histopathological studies of liver, which did not show any fatty infiltration or necrosis, as observed in CCl₄-intoxicated rats.

Keywords:  Hepatoprotective effect, antioxidant effect, tender coconut water, carbon tetrachloride-intoxication, rats

Indian Journal of Biochemistry & Biophysics

Vol. 40, October 2003, pp. 358-361

Effects of some flavonoids on the susceptibility of low-density lipoprotein to oxidative modification

Mohammad-Reza Safari^*, Mohsen Rezaie and Nasrin Sheikh

The effects of six flavonoids viz., apigenin, genistein, morin, naringin, pelargonidin and quercetin on the susceptibility of low-density lipoprotein (LDL) to oxidative modification were investigated. Flavonoids were added to plasma and incubated for 3 hr at 37°C, and the LDL fraction was separated by ultracentrifugation. Oxidizability of LDL was estimated by measuring conjugated diene (CD), lipid peroxides and thiobarbituric acid-reactive substances (TBARS), after cupric sulfate solution was added. Quercetin and morin significantly (P<0.01 by ANOVA) prolonged the lag time before initiation of oxidation reaction in dose-dependent manner. They also suppressed the formation of lipid peroxides and TBARS more markedly than other flavonoids. The ability to prolong lag time and suppression of lipid peroxides and TBARS formation was in the following order: quercetin >morin >pelargonidin >genistein >naringin >apigenin. LDL exposed to flavonoids reduced oxidizability. These findings suggest that flavonoids may have a role in ameliorating atherosclerosis.

Keywords: Flavonoids, oxidative modification, low-density   lipoprotein (LDL)

Indian Journal of Biochemistry & Biophysics

Vol. 40, October 2003, pp. 362-366

An assessment of biopotential of three cyanobacterial isolates from Antarctic for carotenoid production

S P Shukla* and A K Kashyap

Specific growth rates and carotenoid contents of three Antarctic and tropical strains of cyanobacteria viz. Anabaena sp., Phormidium sp. and Nostoc sp. were compared in batch and mass cultures to assess bio-potential of Antarctic strains for cost-effective carotenoid production. Antarctic strains though exhibited slightly lower specific growth rates, but contained higher carotenoid contents (per unit dry wt.), than tropical strains. Modification of normal composition of BG-11 culture medium, by altering nitrogen and carbon sources resulted in 25-38% increase in carotenoid content in both types of strains. Mass-culture in indoor and semi-outdoor bio-reactors resulted in 39-113% higher carotenoid content in Antarctic strains, compared to their respective tropical strains. The observations suggest that Antarctic cyanobacteria may have potential as superior strains for maximizing the yield of carotenoids.

Key words: Antarctic, cyanobacteria, carotenoids, mass-culture