Indian Journal of Biochemistry & Biophysics

CONTENTS

Indian Journal of Biochemistry & Biophysics

Vol. 41, February 2004, pp. 7-13

Evidence for the presence of a critical histidine residue at the active site in

glyceraldehyde-3-phosphate dehydrogenase of

Ehrlich ascites carcinoma cells

Swapna Ghosh, Manju Ray* and Subhankar Ray

Ehrlich ascites carcinoma (EAC) cell glyceraldehyde-3-phosphate dehydrogenase (GA3PD) (EC. 1.2.1.12) was completely inactivated by diethyl pyrocarbonate (DEPC), a fairly specific reagent for histidine residues in the pH range of 6.0-7.5. The rate of inactivation was dependent on pH and followed pseudo-first order reaction kinetics. The difference spectrum of the inactivated and native enzymes showed an increase in the absorption maximum at 242 nm, indicating the modification of histidine residues. Statistical analysis of the residual enzyme activity and the extent of modification indicated modification of one essential histidine residue to be responsible for loss of the catalytic activity of EAC cell GA3PD. DEPC inactivation was protected by substrates, D-glyceraldehyde-3-phosphate and NAD, indicating the presence of essential histidine residue at the substrate-binding region of the active site. Double inhibition studies also provide evidence for the presence of histidine residue at the active site.

Keywords:         Glyceraldehyde-3-phosphate dehydrogenase, Ehrlich ascites carcinoma cell, active site histidine, diethyl pyrocarbonate (DEPC), anticancer drug, inactivation

Indian Journal of Biochemistry & Biophysics

Vol. 41, February 2004, pp. 14-19

Effect of cholesterol and 7-b-hydroxycholesterol on glutathione status and expression of hsp70 in

cultured murine peritoneal macrophages

M P Bansal^* and Sonia Shalini

Using cultured murine peritoneal macrophages, the change in redox ratio (oxidized/reduced glutathione) was studied at different incubation intervals (6, 12, 18 and 24 hr) with different concentrations (2.5, 5 and 7.5 μg/ml) of cholesterol and 7b-hydroxycholesterol (7b-OH), using fluorimeter. The changes in the levels of heat shock protein, hsp70 was determined using ELISA. Both cholesterol/7b-OH caused a decrease in hsp70 protein levels at all the incubation intervals in dose dependent manner but the decrease was significantly higher with 7β-OH. Treatment with 7b-OH also resulted in significantly increased levels of oxidized glutathione (GSSG) and decreased reduced glutathione (GSH) while cholesterol showed no effect on GSSG levels. Moreover, GSH levels were lowered only at the highest concentration (7.5 mg/ml) at longer incubation intervals (18 and 24 hr) with cholesterol exposure. This altered the redox status in both cholesterol/7b-OH treated macrophages. These results suggest that cholesterol and more likely 7b-OH may exert their pro-atherogenic effects by lowering hsp70 protein production and inhibiting glutathione synthesis by macrophages present in the arterial wall.

Key words: cholesterol, 7-b hydroxycholesterol, glutathione status, hsp70, murine peritoneal macrophages

Indian Journal of Biochemistry & Biophysics

Vol. 41, February 2004, pp. 20-28

Evidence of free radical participation in

N-glycolylneuraminic acid generation in liver of chicken treated with gallotannic acid

Danny W Mwangi and Devi D Bansal*

The occurrence of N-glycolylneuraminic acid (Neu5Gc) in cancerous tissue and inflammatory diseases, conditions associated with increased oxidative stress suggests the participation of reactive oxygen radicals in Neu5Gc generation, where an oxygen atom is transferred. To study this possibility, we treated two groups of domesticated birds and rabbits with different dosages of gallotannic acid (GTA), a compound known to cause generation of reactive oxygen species (ROS). The antioxidant status and leukocyte capacity, as well as amount and form of sialic acids were assessed in plasma and liver. Results showed that while lipid peroxides were increased, white blood cell (WBC) count was decreased significantly in all treated groups. The increased sialic acids and low protein contents were observed in plasma, possibly as a result of decreased sialic acid cycling crucial for formation of new glycoconjugates in tissues, caused by decreased protein synthesis due to microsomal degranulation. The activities of antioxidant enzymes were also decreased in treated groups, implying increased oxidative stress. The presence of Neu5Gc and apparent absence of Neu5Ac hydroxylase activity in liver of chicken treated with GTA indicate that free radicals might be involved in the non-enzymatic hydroxylation of N-acetylneuraminic acid (Neu5Ac) to Neu5Gc in liver, which normally does not express this sialic acid.

Key words: Gallotannic acid, N-glycolylneuraminic acid, oxidative stress

Indian Journal of Biochemistry & Biophysics

 Vol. 41, February 2004, pp. 29-33

Possible mechanism of action of antiviral proteins from the leaves of Chenopodium album L.¹

Som Dutt, O P Yadav, H C Kapoor and M L Lodha*

Antiviral proteins (AVPs) named CAP-I and CAP-II purified from the leaves of Chenopodium album cv Pusa Bathua-1 induced systemic resistance against tobacco mosaic virus (TMV) and sunnhemp rosette virus (SRV) in both hypersensitive as well as systemic hosts. An increased accumulation of two polypeptides (~17 kDa and ~26 kDa) was observed in untreated upper leaves of Cyamopsis tetragonoloba plants whose basal leaves were treated with CAP-I/CAP-II. Both AVPs exhibited ribosomal RNA N-glycosidase activity on 28S rRNA of tobacco leaves and also caused in vitro degradation of TMV RNA. It is suggested that the CAP-I and -II are multi-functional and may be acting at multiple levels to ensure maximum possible inhibition of viral infection.

Keywords: Chenopodium album, antiviral proteins, mechanism, N-glycosidase activity, systemic response, RNase activity, tobacco mosaic virus, sunnhemp rosette virus

Indian Journal of Biochemistry & Biophysics

Vol. 41, February 2004, pp. 34-39

Vibrational dynamics and heat capacity

of polyglycine I

Vikas Porwal, Radha Mohan Misra, Poonam Tandon* and Vishwambhar Dayal Gupta

Earlier works on polyglycine I suffer from several infirmities, such as the dynamic methylene group being replaced by a mass unit and the use of poorly resolved inelastic neutron spectra, which have resulted in wrong assignments and imprecise profile of dispersion curves. In addition, the density-of-states and heat capacity variation as a function of temperature are being reported for the first time. The heat capacity is in good agreement with the measurements reported earlier by Roles and Wunderlich¹⁹ within a certain range (230-350 K). Deviations set in beyond this could be due to the presence of two crystalline states (I and II) in the sample used for the heat capacity measurements.

Keywords: Polyglycine I, b conformation, density-of-states, phonon dispersion, heat capacity

Indian Journal of Biochemistry & Biophysics

Vol. 41, February 2004, pp. 40-44

Combined effect of alcohol and cigarette smoke on lipid peroxidation and antioxidant status in rats

M P Bindu and P T Annamalai*

The effect of long-term administration of alcohol and cigarette smoke independently and both in combination on lipid peroxidation and antioxidant enzymes, such as superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), and glutathione-S-transferase (GST) was studied in liver, kidney, heart and lungs of albino rats. The levels of peroxidation products viz., malondialdehyde, hydroperoxides and conjugated dienes were increased in all the tissues of alcohol administered and smoke-exposed rats. Activities of SOD and CAT were decreased in alcohol-treated and alcohol and smoke combination groups, but increased in smoke-exposed group. Activities of GPx and GST have shown an increase, while concentration of reduced glutathione was found decreased in all the three groups.

Keywords: Alcohol, cigarette smoke, lipid peroxidation, antioxidant status

Indian Journal of Biochemistry & Biophysics

Vol. 41, February 2004, pp. 45-47

Effects of Rumex patientia L. extract on some drug-metabolizing enzymes in rat liver

Yavuz Silig^*, Oge Cetinkaya and L Omur Demirezer

The effect of aqueous extract from the roots of Rumex patientia L. (Polygonaceae) (D-1), a traditional Turkish medicine used as a laxative and cholagogue, on drug-metabolizing enzymes, such as cytochrome P4502E1, NADPH cytochrome c reductase, NADH cytochrome b₅ reductase and glutathione-S-transferase (GST); and serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were studied in male Wistar albino rat liver. A significant increase was observed in cytochrome P4502E1 and GST activities, but not in NADPH-cytochrome c reductase and NADH-cytochrome b₅ reductase activities. Serum AST and ALT activities were found within the normal laboratory range values. The results demonstrated that the aqueous extract of R. patientia triggers induction of cytochrome P4502E1 in liver and cytosolic GST activity.

Keywords: Rumex patientia, drug-metabolizing enzymes, cyto-chrome P4502E1, glutathione-S-transfrase, NADPH-cytochrome c reductase, NADH-cytochrome b₅ reductase

Indian Journal of Biochemistry & Biophysics

 Vol. 41, February 2004, pp. 48-52

Transport studies through liquid membranes of ciprofloxacin and norfloxacin

A N Nagappa*, P L Kole, P V Pandi, R T Patil, K Zeeyauddin and I Shanmukha

Ciprofloxacin and norfloxacin, the widely used drugs have been shown to generate liquid membranes in series with a supporting membrane (Sartorius celluose acetate microfiltration membrane). Transport of dextrose and ions, such as NH₄⁺_, Mg²⁺, Ca²⁺, K⁺ and PO₄^3- has been studied in the presence of liquid membranes generated by these drugs. The data obtained on the modification in the permeability of dextrose and ions in the presence of liquid membrane indicate the significance of liquid membranes in passive transport.

Keywords : Ciprofloxacin, norfloxacin, liquid membrane, fluoro-quinolones antibiotics, permeants, permeability, transport study

Indian Journal of Biochemistry & Biophysics

Vol. 41, February 2004, pp. 53-56

Biological monitoring of lead and cadmium in

human hair and nail and their correlations with biopsy materials, age and exposure

Rita Mehra* and Meenu Juneja

Hair and fingernails of exposed and unexposed subjects were analyzed for their lead (Pb) and cadmium (Cd) contents by atomic absorption spectrophotometer with graphite furnace and air-acetylene flame. Hair and nail Pb concentrations in occupationally exposed subjects ranged between 1.020-409.726 and 8.130-765.306 µg/g and in environmentally unexposed subjects 0.123-25.160 and 1.076-65.613 µg/g, respectively. Similarly, hair and nail Cd concentrations in occupationally exposed subjects ranged between 0.014-22.086 and 0.214-35.714 µg/g and in environmentally unexposed subjects 0.113-1.627 and 0.028-8.108 µg/g, respectively. A significant correlation was observed between Pb hair and nail concentrations in exposed subjects at P<0.05, as compared to unexposed subjects and Cd hair and nail in exposed, as well as unexposed subjects. With respect to exposure, levels of Pb in hair and nails were found to be significant in exposed subjects, compared to unexposed ones and levels of Cd were significant only in nails of exposed ones. With respect to age, no significant correlation was found between hair and nail Pb and Cd concentrations in both exposed and unexposed subjects.

Keywords:   Metal body burden, trace/toxic elements, Pb and Cd in hair/nail, biomonitoring

Indian Journal of Biochemistry & Biophysics

Vol. 41, February 2004, pp. 57-59

Effects of short-term exposure to pulsed electromagnetic field on some biochemical parameters in mice

Gokhan Eraslan*, Mehmet Akdogan, Ender Yarsan, Dinc Essiz,

Fatma Sahindokuyucu and Levent Altintas

Five-months-old male albino mice were subjected to an electromagnetic field (EMF) of 5 mT of magni-tude with a frequency of 60 Hz for 8hr of single application. Analysis of blood sampled on hourly basis (up to 8 hr) for levels/activities of total protein, albumin, globulin, uric acid, creatinine, cholesterol, and alkaline phosphatase indicated no significant differences (p>0.05) from that of the control group.

Keywords: Pulsed EMF, biochemical parameters, short-term exposure

Author Index