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Indian Journal of Biochemistry & Biophysics

 

ISSN : 0301-1208 CODEN : IJBBBQ
VOLUME 40

NUMBER 3

JUNE 2003

 

CONTENTS

 

Minireview

Fluorescence spectroscopy in molecular description of biological processes

147

G Krishnamoorthy

 

 

 

Papers

 

Senescence-induced alterations in the photosystem II functions of Cucumis sativus cotyledons: Probing of senescence driven alterations of photosystem II by chlorophyll a fluorescence induction O-J-I-P transients

160

J S S Prakash, A Srivastava, Reto J Strasser and Prasanna Mohanty*

 

 

 

Delivery in vivo of 14-deoxy-11-oxoandrographolide, an antileishmanial agent, by different drug carriers

169

S Lala, A K Nandy, S B Mahato and M K Basu*

 

 

 

Effects of deuterium oxide on folate metabolism in Lactobacillus casei

175

M S Pote, G Viswanathan and V Kesavan*

 

 

 

Chronic cold stress-induced alterations in brush border membrane composition and enzyme activities in rat intestine

180

Susmita Kaushik, Pawan Kaler and Jyotdeep Kaur*

 

 

 

Characterization of goat plasma vitronectin 

186

S. Suchitra, Vasili Ashok*, Tapas Gupta and Paritosh Joshi

 

 

Dipole moment in TIM a/b fold proteins

194

G Vasanthi and S Krishnaswamy*

 

 

 

QSAR of peripheral benzodiazepine receptor ligand 2-phenylimidazo-[1,2-a]pyridine derivatives with physico-chemical parameters

203

Kunal Roy*, A U De and Chandana Sengupta

 

Notes

 

Analysis of free nucleotide pools of mouse liver tissue by high-pressure liquid chromatography (HPLC)

209

Reza Haji Hosseini Baghdad Abadi

 
 

 

 

Indian Journal of Biochemistry & Biophysics

Vol. 40, June 2003, pp. 147-159

 

 

Minireview

 

Fluorescence spectroscopy in molecular description of biological processes

G Krishnamoorthy

 

Received 20 January 2003; revised & accepted 2 May 2003

 

 

The power of fluorescence spectroscopy in enhancing our ability to obtain molecular description of complex biological processes is discussed. A brief account of sensitivity, selectivity and observable parameters of the various fluorescence methods and some typical applications which are mostly based on work in our laboratory are provided. The applications cover a wide range of areas such as protein dynamics, protein folding dynamics, protein-DNA interactions, membrane dynamics and intracellular dynamics. Interesting information revealed by fluorescence-based methods in each of these applications is highlighted.

 

Keywords: 2-Aminopurine, cell membrane raft, DNA dynamics, Time-resolved fluorescence, fluorescence spectroscopy, fluorescence probes, Maximum entropy method, membrane heterogeneity, protein dynamics, protein folding.

 

 

 

Indian Journal of Biochemistry & Biophysics

Vol. 40, June 2003, pp. 160-168

 

 

Senescence-induced alterations in the photosystem II functions of Cucumis sativus cotyledons: Probing of senescence driven alterations of photosystem II by chlorophyll a fluorescence induction O-J-I-P transients

J S S Prakash, A Srivastava, Reto J Strasser and Prasanna Mohanty*

 

Received 12 July 2002; revised 29 January 2003; rerevised 6 May 2003

 

 

Senescence-induced alterations in photosystem II (PS II) structure and photofunctions were probed in cucumber (Cucumis sativus) cotyledons, using fast O-J-I-P Chlorophyll a (Chl a) fluorescence transients. Analysis of measured and derived parameters of the fast fluorescence O-J-I-P transient revealed senescence-induced alterations in (i), PS II acceptor side electron transfer equilibrium between QA and QB, the primary stable and secondary acceptors of PS II; (ii), intersystem PQ pool size and (iii), affected electron transfer from PS II to PS I. Also, senescence of cotyledons triggered conversion of QA–reducing (fully active) to non- QA-reducing PS II (heat sink) centres. Further, some of the remaining active PS II centres showed a high apparent trapping efficiency due to clustering and energetic connectivity (grouping) between the antennae of active and inactive centers. The overall density of active PS II reaction centers showed a temporal decrease due to the onset of foliar senescence. Thus, the fast Chl a fluorescence transients, with a time resolution of at least 50 μs and use of the equations of JIP-test, provide a valuable, non-invasive rapid biophysical probe to study the ageing in plants in terms of detecting photosynthetic activities and the heterogeneity of different types of photosynthetic units. Further, these results were found to be in agreement with the earlier in vitro studies using thylakoids isolated from senescing cotyledons where it was shown that senescence induced heterogeneity in PS II centers affected acceptor side QA↔QB equilibrium.

 

Keywords: Chl a fluorescence, cotyledons, Cucumis sativus, senescence, JIP-test, photosystem II, O-J-I-P transients

 

 

 

Indian Journal of Biochemistry & Biophysics

Vol. 40, June 2003, pp. 169-174

 

 

Delivery in vivo of 14-deoxy-11-oxoandrographolide, an antileishmanial agent, by different drug carriers

S Lala, A K Nandy, S B Mahato and M K Basu*

 

Received 5 November 2002; revised 7 February 2003; accepted 25 March 2003

 

 

An antileishmanial compound, 14-deoxy-11-oxo-andrographolide, a derivative of andrographlide, isolated from the Indian medicinal plant Andrographis paniculata was evaluated for efficacy in free form and in different vesicular delivery modes on hamster model of Leishmaniasis. The subcutaneous injection of free drug reduced the spleen parasite load by 39%, whereas for drug incorporated in liposomes, niosomes and microspheres, reductions in the parasite load were 78%, 91% and 59%, respectively. Moreover, the drug in various delivery modes, particularly in liposomal and niosomal forms, showed no apparent immediate toxicity. Although an inverse linear relationship between the size of carriers and per cent efficacy in reduction of spleen parasite load was established, involvement of other factors such as drug release profiles or rates remains an open question. Because of greater efficacy and lesser toxicity, liposomal, niosomal and possibly microsphere-incorporated 14-deoxy-11-oxo-andrographolide might have clinical application to combat visceral Leishmaniasis.

 

 

Key words: Andrographis paniculata, antileishmanial compound, 14-deoxy-11-oxoandrographolide, drug carriers, in vivo delivery, liposomes, microspheres, niosomes

 

 

 

Indian Journal of Biochemistry & Biophysics

Vol. 40, June 2003, pp. 175-179

 

 

Effects of deuterium oxide on folate metabolism in Lactobacillus casei

M S Pote, G Viswanathan and V Kesavan*

 

Received 27 August 2002; revised 16 December 2002; accepted 7 February 2003

 

 

Various folate coenzymes and their polyglutamyl derivatives involved in 1-carbon metabolism are modulated as a result of altered physiological states and also vary with respect to growth conditions. We studied the metabolic changes in folic acid and their conjugated polyglutamyl derivatives in Lactobacillus casei cells grown in the presence of D2O. A 40% decrease in methyltetrahydrofolyl polyglutamate derivatives was observed in the cells grown in media prepared with D2O-depleted water (D2O content, 8-10 ppm). Chromatographic analysis of folates showed significant alterations in the formyl- and methyltetrahydrofolate derivatives and their polyglutamylation profiles. Higher amounts of oxidized folates were also present in the cells grown in D2O-depleted conditions. No significant changes were observed in folates and their polyglutamate derivatives when the cells were grown in the presence of 300, 450 and 600 ppm D2O. The altered folate homoestasis is attributed to changes in the metabolic adaptation of cells to D2O-depleted environment.

 

 

 

Indian Journal of Biochemistry & Biophysics

Vol. 40, June 2003, pp. 180-185

 

 

Chronic cold stress-induced alterations in brush border membrane composition and enzyme activities in rat intestine

Susmita Kaushik, Pawan Kaler and Jyotdeep Kaur*

 

Received 20 August 2002; revised 12 December 2002; accepted 17 January 2003

The effect of chronic cold stress on the composition and function of rat intestinal brush border membrane (BBM) was studied. Various lipid fractions from intestinal BBM viz. cholesterol (p<0.01), phospholipids (p<0.01), triglycerides (p<0.05) and gangliosides (p<0.05) were significantly reduced in cold stressed animals, as compared to controls. Analysis of membrane saccharide content revealed a significant increase in sialic acid (25%) and hexosamine (36%) contents and a reduction in fucose (19%) content in cold stressed rats. Determination of various enzyme activities in BBM showed significantly enhanced activities of alkaline phosphatase (p<0.01), lactase (p<0.001) and leucine aminopeptidase (p<0.001), whereas sucrase activity was reduced (p<0.05) under these conditions. The magnitude and site of these alterations across the crypt-villus axis varied from enzyme to enzyme. These findings suggest that chronic cold stress results in profound alterations in intestinal BBM. Altered structure and function of intestinal BBM may play a role in stress-induced derangements in gastrointestinal tract.

 

 

 

Indian Journal of Biochemistry & Biophysics

Vol. 40, June 2003, pp. 186-193

 

 

Characterization of goat plasma vitronectin

S Suchitra, Vasili Ashok, Tapas Gupta and Paritosh Joshi*

 

Received 28 November 2002; revised 10 January 2003; accepted 8 February 2003

 

 

Vitronectin (VN) was isolated and characterized from goat plasma in native and denatured state. Native VN consisted of 160 and >250 kDa polypeptides, whereas denatured VN showed bands of 81 and >250 kDa on SDS-gel. Storage of 81 kDa polypeptide for 3 days at 4°C resulted in formation of 160 and >250 kDa proteins. Hence high molecular weight forms of VN may be dimer and multimeric forms of 81 kDa monomer. Both native as well as denatured VN showed cell adhesive activity. Cells bound to native VN were round, whereas cells adhered to denatured VN were fully spread, a characteristic also observed with 81 kDa polypeptide. The 81 kDa VN bound to Heparin, whereas the 160 kDa preparation did not bind to Heparin in presence of urea. Absence of EDTA resulted in the degradation of goat VN. Similarly, addition of excess Ca2+ caused total degradation of VN polypeptides in buffers with EDTA, suggesting metalloprotease activity in the protein.

 

Key words: Goat, vitronectin

 

 

 

Indian Journal of Biochemistry & Biophysics

Vol. 40, June 2003, pp. 194-202

 

 

Dipole moment in TIM a/b fold proteins

G Vasanthi and S Krishnaswamy*

 

Received 16 January 2003; revised 24 February 2003; accepted 8 April 2003

 

 

TIM proteins of a/b barrel fold from a/b class as given in SCOP database were taken for dipole moment analysis. In all, 32 structures were analyzed for their dipole moment contributions. Representative structures from 20 super families in the a/b fold, with different enzyme functions and 12 protein domains of TIM family in TIM super family were considered. The active sites of these proteins are located on the C-terminal side of the b-strands. The molecules of same a/b fold, but differing in their functionality also showed a common electrostatic field pattern along the barrel axis and had the dipole moment along the barrel axis and towards C-terminal end of the b-strands. However, it is observed from our calculations that the dipole moment direction is possibly a consequence of the structural fold, with distribution of charges playing a modulatory role, and does not contribute to the location of active site. We show here that apart from the commonly held view as proposed by Hol et al [Hol W G L, van Duijnen P T and Berendsen H J C (1978) Nature (London), 273, 443-446] of the role of the a helical dipole moment, the b-sheets in the barrel can also have a considerable dipole moment contribution. Taken together with our dipole moment analysis on integral membrane proteins [Vasanthi G and Krishnaswamy S (2002) Indian J Biochem Biophys 39, 93-100], this suggests the need to examine the role of dipole moment in the case of especially b sheets forming barrels.

 

Keywords: TIM proteins, a/b barrel fold, dipole moment

 

 

 

Indian Journal of Biochemistry & Biophysics

Vol. 40, June 2003, pp. 203-208

 

 

QSAR of peripheral benzodiazepine receptor ligand 2-phenylimidazo- [1,2-a]pyridine derivatives with physico-chemical parameters

Kunal Roy*, A U De and Chandana Sengupta

 

Received 27 August 2002; revised 2 January 2003; accepted 27 January 2003

 

 

QSAR of the binding affinities of [2-phenylimidazo[1,2-a]pyridin-3-yl]acetamide derivatives (Fig. 1) with central and peripheral (from cortex and ovary) benzodiazepine receptors has been explored using physico-chemical parameters. Attempt has been made to explore the structural and/or physico-chemical requirements of the compounds that are responsible for the selective action against peripheral benzodiazepine receptors over central ones. The results indicate that the presence of bi-substitution on the carboxamido nitrogen, presence of substitutions at X and Y positions, especially, chloro substitution at X position, and presence of chloro substitution at Z position in presence of lipophilic X and/or Y substitutions increase selectivity for binding affinity with peripheral benzodiazepine receptors over central ones.

 

 

 

Indian Journal of Biochemistry & Biophysics

Vol. 40, June 2003, pp. 209-212

 

 

Note

 

Analysis of free nucleotide pools of mouse liver tissue by high-pressure liquid chromatography (HPLC)

Reza Haji Hosseini Baghdad Abadi

Department of Biology, Faculty of Science, Payame Noor University, Nakhel St. Lashkarak Rd, 

Tehran, 19569, Iran

Received 28 October 2002; revised 23 April 2003

 

In this paper, analysis of free nucleotides from mouse liver tissue during different day times has been described. Perchloric acid extract of mouse liver tissue was neutralized with tri-N-octylamine in trichlorotriflouroethane and after removal of ClO-4, subjected to preliminary purification on a Cu2+-loaded column of Chelex 100. A high-pressure liquid chromatographic (HPLC) anion-exchange procedure used in the study gave a good resolution of free nucleotides on a single column.