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Indian Journal of Biochemistry & Biophysics

 

 

ISSN : 0301-1208

 

CODEN : IJBBBQ  

VOLUME 41

NUMBER 2&3

APRIL-JUNE 2004

 

CONTENTS

 

Minireview

 

An insight into molecular mechanism of endocytosis

69

Nidhi Neekhra and Harish Padh*

 

 

 

Papers

 

Purification and characterization of an extracellular agglutinin from Tricophyton rubrum with specificity towards sialic acid containing glycoconjugates

81

J Bhowal, A Mitra, S Banerjee, S Sikdar, A K Guha and B P Chatterjee*

 

 

 

Rhizobial lipopolysaccharide as the receptor in lectin-Rhizobium interaction

89

Indranil Bhattacharya, Sagarika Biswas, Rakha H Das and Hasi R Das*

 

 

 

Purification and characterization of cinnamyl alcohol-NADPH-dehydrogenase from the leaf tissues of a basin mangrove Lumnitzera racemosa Willd.

96

K Murugan, N S Arunkumar and C Mohankumar*

 

 

 

Measurement of urinary oxalate by grain sorghum leaf oxalate oxidase immobilized to affixed alkylamine glass beads

 

102

M Kumari and C S Pundir*

 

 

 

Mixed micelles of cationic surfactants and sodium cholate in water

107

Dharmesh Varade, Vijay Patel, Anita Bahadur, Pratap Bahadur* and Martin Swanson Vethamuthu

 

 

 

Notes

 

Microwave-assisted method for microassay of proteins using bicinchoninic acid

113

Sulakshana Jain, Shweta Sharma and Munishwar N Gupta*

 

 

 

Effect of prenatal ethanol administration on microvillus membrane glycosylation in developing rat intestine

116

Sonali Bhalla, Kamaljit Kaur, Akhtar Mahmood and Safrun Mahmood*

 

 

 

Potent antibacterial activity of oligo-3-aminopyridine against Staphylococcus aureus and Enterococcus faecalis

120

Cahit Akgul* and Ismet Kaya

 

 

 

Absorption spectral study of 3, 4-dihydroxy phenyl alanine (DOPA) with Nd (III) in aqueous medium

 

123

Prashant N Bhatt*, Trupti V Pathak, Jignasu P Mehta and Sudhindra N Misra

 

 

 

Meeting Report (Trendys)

128

 

 

Book Review
131

 

 
Instructions to Authors
133

 

 
Announcement
135
   
AUTHOR INDEX  

 

 

*Author for correspondence

 


 

Indian Journal of Biochemistry & Biophysics

Vol. 41, .April-June 2004, pp. 69-80

 

An insight into molecular mechanism of endocytosis

Nidhi Neekhra and Harish Padh*

 

Endocytosis involving pinocytosis, phagocytosis and receptor-mediated pathway is a central process involved in numerous cellular events: receptor recycling, nutrient uptake, transcytosis, antigen processing and presentation, pathogen uptake etc. Traditionally, the process has been studied using uptake kinetics and immunocytochemistry. In last few decades, additional tools, like mutant analysis, density shift, semi-intact and cell-free systems and electromagnetic separation have helped us to obtain molecular insight into many of the steps involved in endocytosis. New chemical entities, like clathrin and coatomer proteins involved in internalization have been fully characterized. Biomolecules involved in vesicle budding and fusion reactions, like ARF (ADP ribosylation factors), COPs (coat proteins), and SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) have been identified and characterized. Compartment specific molecules, like Rab have also added to our understanding of complex process of endocytosis. Collectively these have led us to an era of molecular endocytosis. The present review gives an overview of the process and describes some of the molecular events of the endocytic process. It also describes methods and approaches used in deciphering the events at cellular and molecular levels.

 

Keywords: Endocytosis, receptors, pinocytosis, phagocytosis, endosomes, clathrin, adaptor proteins, coatomer proteins.

 

 

 

Indian Journal of Biochemistry & Biophysics

 Vol. 41, .April-June 2004, pp. 81-88

 

Purification and characterization of an extracellular agglutinin from  Tricophyton rubrum with specificity towards sialic acid containing glycoconjugates

J Bhowal, A Mitra, S Banerjee, S Sikdar, A K Guha and B P Chatterjee*

 

An agglutinin, a monomeric glycoprotein with a molecular mass of about 6.5 kDa and containing 18% sugar has been purified to an apparent homogeneity from a 21 days old culture filtrate of an anthropophilic dermatophyte Tricophyton rubrum. It is a human blood group non-specific agglutinin which also agglutinates animal erythrocytes and Ehrlich ascites carcinoma and Sarcoma-180 cells. It is thermally stable and exhibits maximum activity at pH 8. Amino acid analysis shows a significant amount of glycine, with no cysteine. Glycoproteins inhibited the hemagglutination of the agglutinin, but not the simple sugars, including sialic acid. Fetuin is the most potent inhibitor among the glycoproteins tested. This inhibition gives a hint to binding with Galb1-3GalNAc or Galb1-4GlcNAc residue containing sialic acid at the terminal position with α 2-6 or α 2-3 linkage.

 

Keywords: Tricophyton rubrum, dermatophyte, agglutinin, glycoproteins, hemagglutination

 

 

 

Indian Journal of Biochemistry & Biophysics

 Vol. 41, .April-June 2004, pp. 89-95

 

Rhizobial lipopolysaccharide as the receptor in lectin-Rhizobium interaction

Indranil Bhattacharya, Sagarika Biswas, Rakha H Das and Hasi R Das*

 

Rhizobial specificity was examined on the basis of interaction between legume lectins (peanut, pea and soybean) and different rhizobial species (various bradyrhizobia specific for peanut, P 14-93 and SB16). Legume lectins showed higher affinity towards host-specific Rhizobium and lipopolysaccharides (LPS) isolated from those particular rhizobia. Two LPS mutants of peanut-specific Bradyrhizobium sp. (Arachis) strain GN17 were isolated by Tn5 mutagenesis. These mutants (GN17M1 and GN17M2) were characterized by their higher hydrophobicity with respect to the parent cells. The hexose content in exopolysaccharides (EPS) and LPS of the mutants was found reduced significantly, whereas 2-keto-3-deoxyoctulosonic acid (Kdo) and uronic acid in LPS were less by 20-times and thrice, respectively in the mutants. Glucose was the major sugar in LPS from all the strains. However, glucosamine appeared only in the mutants. Spectrofluorimetric analysis showed that LPS from GN17M1 mutant interacted most significantly with peanut root agglutinin or lectin (PRA II). The results indicate that LPS on the surface of rhizobial cells is the possible receptor for lectin.

 

Keywords: Lipopolysaccharide, peanut root lectin, exopolysaccharide, receptor, lectin-Rhizobium interaction

 

 

 

Indian Journal of Biochemistry & Biophysics

 Vol. 41, .April-June 2004, pp. 96-101

 

Purification and characterization of cinnamyl alcohol-NADPH-dehydrogenase from
the leaf tissues of a basin mangrove Lumnitzera racemosa Willd

K Murugan, N S Arunkumar and C Mohankumar*

 

Cinnamyl alcohol-NADPH-dehydrogenase (CAD), the marker enzyme of lignin biosynthesis was purified from the leaf tissues of a basin mangrove Lumnitzera racemosa by ammonium sulphate precipitation, followed by anion-exchange, gel filtration and affinity chromatography. The molecular mass of the CAD enzyme was determined as 89 kDa, by size elution chromatography. SDS-PAGE of CAD revealed two closely associated bands of 45 kDa and 42 kDa as heterogenous subunits. The optimum pH of CAD was found to be 4.0. Km for the substrates cinnamaldehyde, coniferaldehyde and sinapaldehyde was determined. Cinnamaldehyde showed higher Km value than sinapaldehyde and coniferaldehyde. The correlation of activity of CAD with the amount of lignin was found less significant in L. racemosa, compared to plant species of other habitats viz., mesophytes, xerophytes and hydrophytes, suggesting that CAD possibly exhibits physiological suppression due to the saline habitat of the plant.

 

Keywords: Lumnitzera racemosa, cinnamyl alcohol-NADPH-dehydrogenase, basin mangrove.

 

 

 

Indian Journal of Biochemistry & Biophysics

 Vol. 41, .April-June 2004, pp. 102-106

 

Measurement of urinary oxalate by grain sorghum leaf oxalate oxidase immobilized to affixed alkylamine glass beads

M Kumari and C S Pundir*

 

Oxalate in urine was measured by grain Sorghum leaf oxalate oxidase conjugated to alkyl amine glass beads affixed in a beaker. The minimum detection limit was 0.05 mM/L in urine. Recovery of added oxalate in urine was 80.5% and within and between assay, coefficients of variation (CV) were <4% and <5.5%, respectively. Urinary oxalate values obtained by the present method showed a good correlation (r = 0.947) with those by Sigma kit method. The method is not only free from tedious handling of free glass beads and Cl- interference, but also has longer stability and reusability of immobilized enzyme compared to that of barley root and forage Sorghum leaf.

 

Keywords: Oxalate, oxalate oxidase, urine, grain Sorghum, alkylamine glass, immobilized enzyme

 

 

 

Indian Journal of Biochemistry & Biophysics

 Vol. 41, .April-June 2004, pp. 107-112

 

Mixed micelles of cationic surfactants and sodium cholate in water

Dharmesh Varade, Vijay Patel, Anita Bahadur, Pratap Bahadur* and Martin Swanson Vethamuthu

 

Critical micelle concentrations (CMCs) of cationic surfactant (alkyltrimethylammonium bromides, CnTABr, where n = 10, 12, 14, 16 and 18), and a bile salt sodium cholate (NaC) were determined from surface tension, conductance and dye solubilization methods, while of their equimolar mixtures from surface tension and dye solubilization methods. The interaction parameter (β) obtained from analysis of data, using Rubingh’s theory showed strong interaction between NaC and cationic surfactant. Time-resolved fluorescence-quenching results revealed small-sized mixed spherical micelle with aggregation number much less than micelles of cationic surfactant.

 

Keywords: Bile salts, cationic surfactants, mixed micelles, interaction parameter

 

 

 

Indian Journal of Biochemistry & Biophysics

 Vol. 41, .April-June 2004, pp. 113-115

 

Microwave-assisted method for microassay of proteins using bicinchoninic acid

Sulakshana Jain, Shweta Sharma and Munishwar N Gupta*

 

A microwave-assisted microassay has been developed for estimation of proteins, using bicinchoninic acid (BCA). The protocol described here is rapid with a reaction time of merely 95 sec, requires lesser amounts of BCA and other reagents, and tolerates larger or comparable concentration of various interfering substances.

 

Keywords: Bicinchoninic acid assay, microwave-assisted microassay, protein estimation.

 

 

 

Indian Journal of Biochemistry & Biophysics

 Vol. 41, .April-June 2004, pp. 116-119

 

Effect of prenatal ethanol administration on microvillus membrane glycosylation in
developing rat intestine

Sonali Bhalla, Kamaljit Kaur, Akhtar Mahmood and Safrun Mahmood*

 

Effect of prenatal ethanol exposure has been studied on microvillus membrane glycosylation in developing rat intestine. In utero ethanol administration did not affect the gestation period but reduced litter size in ethanol-exposed group. Body weight, intestinal length and weight of pups born to ethanol-exposed rats during gestation, aged 4 to 30 days were significantly low compared to the respective controls. Total hexoses (p<0.05) and sialic acid (p<0.05, p<0.01) contents of purified brush border membranes increased in pups exposed to ethanol prenatally, while the fucose content showed significant (p<0.05, p<0.01) decrease. The fucose-sialic acid molar ratio was also low in all age-groups of prenatally ethanol-exposed pups, compared to respective controls. The results indicate that ethanol ingestion during gestation alters the glycosylation pattern of intestine in rats, which may impair its functions during postnatal development.

 

Keywords: Glycosylation, prenatal ethanol exposure, microvillus membrane, brush border membrane, total hexoses, fucose, sialic acidKeywords: Pulsed EMF, biochemical parameters, short-term exposure

 

 

 

Indian Journal of Biochemistry & Biophysics

 Vol. 41, .April-June 2004, pp. 120-122

 

Potent antibacterial activity of oligo-3-aminopyridine against Staphylococcus aureus
and Enterococcus faecalis

Cahit Akgul* and Ismet Kaya

 

Oligo-3-aminopyridine (OAP) was tested for its antibacterial activity against two gram positive (Staphylococcus aureus and Enterococcus faecalis) and two gram negative (Escherchia coli and Pseudomonas aeruginosa) bacterial strains. Antibacterial efficacy was measured by disk diffusion assay. Inhibition zones demonstrated that 50 μg OAP showed strong antibacterial activity against the gram-positive strains; the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were found to be 25 μg/ml and 50 μg/ml, respectively. The results indicate that OAP is a potent antibacterial agent against S. aureus and E. faecalis.

 

Keywords: Antibacterial activity, S. aureus, E. faecalis, oligo-3-aminopyridine.

 

 

 

Indian Journal of Biochemistry & Biophysics

 Vol. 41, .April-June 2004, pp. 123-127

 

Absorption spectral study of 3, 4-dihydroxy phenyl alanine (DOPA) with Nd (III)
in aqueous medium

Prashant N Bhatt*, Trupti V Pathak, Jignasu P Mehta and Sudhindra N Misra

 

Spectroscopic properties of Nd (III)-DOPA in aquated organic solvents are investigated through absorption difference and comparative absorption spectroscopy involving 4f-4f transitions. The change in coordination in different conditions is observed from the absorption intensity analysis when Nd (III) interacts with DOPA. The interaction induced substantial changes in the intensities of 4f-4f bands and their perturbation was reflected through oscillator strength Judd-Ofelt intensity (Tl) parameters. It is suggested that the changes in the oscillator strengths of different 4f-4f bands and Judd-Ofelt intensity (Tl) parameters can be used to predict in vivo intracellular complexation of DOPA with Ca(II) through Nd (III)-DOPA absorption spectral studies in-vitro as both Nd (III) and Ca(II) have unique similarity in their coordination behaviour.

 

Keywords: 4f-4f transitions, Nd (III)-DOPA interaction

 

 

 

Indian Journal of Biochemistry & Biophysics 

Vol. 41, .April-June 2004, pp. 128-130

 

Trendys Meeting 2003

A P Sowjanya and G Ramakrishna

 

TRendys in Biochemistry, started as a small scientific discussion group in IISc Bangalore a decade ago by Prof. T Ramasarma, has now grown to the stature of an important national forum primarily through the nurturing efforts of Prof. K Subba Rao (University of Hyderabad) and other like-minded scientists. TRendys held its 10th meeting at the Centre for DNA Fingerprinting and Diagnostics (CDFD), Hyderabad on 24th and 25th November, 2003. Students and scientists from various universities and institutes in Hyderabad attended the meeting. A series of lectures by eminent scientists, experienced and budding, covering new facets of Biochemistry, Molecular Bio-logy and some recent advances in the Clinical Sciences were indeed impressive. Prof. S E Hasnain (Director, CDFD) presented the welcome address, followed by a brief introduction by Prof. K Subba Rao.

 

Prof. Ramasarma (CDFD, Hyderabad) spoke on the fascinating aspect of nucleic acid catalysis. The discovery of RNA-catalysts (ribozymes) demolished the central dogma that proteins are exclusive cellular catalysts. He revealed the mechanistic aspect of how polynucleotides can gain catalysis and proposed a hypothesis about catalysis involving intramolecular electron transfer over H-bond interlinked by p-electron clouds of nucleotide structures. By elaborating on the extensive secondary structures occurring in ribozymal RNA with canonical and unusual H-bonds, Prof. Ramasarma explained the possible existence of p-H pathways in ribozymes.

 

The next lecture, “Channels - facts and fears… Nobel prizes notwithstanding” by Prof. V Sitaramam (University of Pune, Pune) highlighted the prize winning work of Roderick MacKinnon (Nobel Prize for Chemistry in 2003) on channels in cell membranes. He described how potassium channel with extraordinary selectivity for potassium still maintained diffusion rates nearly matching self-diffusion in water. He also touched up on aquaporins, the membrane water channels, which play critical role in controlling water contents of the cells. Aquaporins, in fact, form tetramers in the cell membrane and facilitate the transport of water. However, water pores are completely impermeable to charged species, such as protons, a remarkable property that is critical for conservation of membrane's electrochemical potential, but paradoxical at the same time, since protons can be transported readily through water molecules.

 

Dr. Shashidhara, Centre for Cellular and Molecular Biology (CCMB), Hyderabad described how membrane trafficking paves the way to morphogen gradient and the mechanisms on how concentration gradient of the morphogen is established to confer different identities to the cells in a multicellular (specific Ex. Drosophila). He covered many aspects on the endocytic pathway in the secretion of signal molecules from the source cells, their movement through the target tissues and transduction of signal from endocytic compartments/vesicles.

 

Dr. Gopal Kundu (NCCS, Pune) chose the signal transducer Nuclear factor-B (NF-B) which plays a crucial role in many of the signaling events to unravel the emerging mind-boggling complexities of various signal transduction pathways. NF-B is a family of transcription factors that has been shown to be involved in regulation of several diseases. The speaker elaborated on how in the nucleus NF-B modulates the expression of a variety of genes including those encoding cytokines, growth factors, cell adhesion molecules and urokinase type plasminogen activator, thereby modulating events such as apoptosis, proliferation, cellular motility, etc.

 

Application of gene chips/microarrays in cancer diagnostics was touched by Prof. P Kondaiah (IISc, Bangalore) in his talk, “Molecular signatures of cancer: Do they improve patient management?”. He discussed how by labeling cancerous cell and its normal counterpart using two different fluorescent dyes and hybridizing them together on a oligo/cDNA gene chip can help identify upregulated or downregulated genes in cancerous cells. By nailing down on few such target genes one can explore these key molecules for cancer therapeutics. He described how difficult it is to predict clinical outcome of such a disease on the basis of histology, but gene expression profiles using microarray can help define a set of genes thus providing quite a strong indicator of long-term survival.

 

The possibility of development of male contraceptives including the use of synthetic androgen like 7-α-methyl-19-nortestosterone which is more potent than testosterone and is more efficacious in controlling fertility was discussed by Prof. A J Rao (IISc, Bangalore). Hormonal approaches to male contraception based on the suppression of luteinizing hormone secretion require androgen replacement treatment to maintain sexual behaviour and secondary sexual characteristics. Androgen supplementation not only involves large and frequent doses of testosterone esters, but also results in undesirable effects on the prostate gland.

 

In a thought-provoking seminar on kinetics and energetics of protein folding, “Say no to what they say how proteins fold” Dr. Abani Bhuyan (University of Hyderabad) discussed newer concepts on protein folding and put forth his views on (i) the folding time correlation with the energy of native state, (ii) the folding transition-state region that appears rather late at a time when a substantial fraction of native contacts are already formed, and (iii) the rate-limiting barrier which is early and low in energy and has shortcomings.

 

Though protein folding is essential for stability and activity of the proteins, its degradation is equally important for basal protein turnover. The major regulatory mechanism controlling protein degradation is by unbiquitin-proteosome mediated pathway. Dr. S Jonnalagadda (Osmania University) discussed in detail, the two main steps in the ubiquitination pathway: (i) covalent attachment of a polyubiquitin chain to the substrate, and (ii) specific recognition of this signal, and degradation of the tagged protein by the proteosome.

 

Dr. Shekhar Mande (CDFD, Hyderabad) highlighted the X-ray crystallography as a major tool in understanding biological phenomena. Rapid advancements in these techniques in recent years have facilitated study of complex problems. He explained the structure and functioning of some of the important protein complexes, such as ATPase, now recognized as tiny molecular motors.

 

Stem cell research in recent years has seen a lot of ‘hype and hope’ among the scientific community. While the hype is on bioethical issues, the hope is the use of stem cells as elixirs of life, supposedly a wonder cure of everything. The 10th TRendys meeting figured two such talks. Prof. D Balasubramanian of L V Prasad Eye Institute (LVPEI), Hyderabad revealed how limbal stem cells in India are cultured for corneal transplants. The discovery that the limbus has a supply of (adult) stem cells led to the possibility of harvesting and cultivating them in order to produce the corneal epithelium. He described how the amniotic membrane and the limbal stem cells taken from the healthy eye are co-cultured together in vitro and few days of culturing results in desired epithelium which is then grafted to the diseased eye. There was a lot of excitement in the audience as the video clippings showed successful transplantation and total recovery of the damaged eyes in the patients. Dr. Shyamala Mani, National Brain Research Centre (NBRC), Gurgaon, elaborated on the neuronal stem cells, their diffrentiation and utility in curing a number of neuronal diseases like Alzheimers, Parkinsons, etc.

 

Dr. Sanjeev Khosla (CDFD, Hyderabad) gave an interesting talk on epigenetic circuitry focusing the phenomenon of imprinting which can be loosely defined as the gamete-of-origin dependent modification of phenotype, that is, the phenotype elicited from a locus is differentially modified by the sex of the parent contributing that particular allele. He described various examples of maternally and paternally imprinted genes. One of the major highlights of this talk was on chromatin remodeling and how methylation of the CpG islands regulates gene expression. He also discussed recent advances in cancer epigentics where it has been shown that tumor suppressors are turned off by hypermethylation.

 

The series of interesting lectures at the TRendys 10th meeting came to an end with the exciting presentation of Dr. Raj Gopal (Tata Consultancy Service, Hyderabad) on the power of in silico approach, a recent trend in “Designing novel protein folds”. He highlighted construction of a novel functional, but unnatural protein using sophisticated computer algorithms. He discussed the paper by Baker and colleagues later published in Science, November 21, 2003 issue (Kuhlman et al, Science, 2003, 302, 1364) on successful understanding of the language proteins, wherein through an integrated approach of protein design algorithms with protein structure-prediction algorithms, researchers predicted a 93-amino acid protein structure called ‘Top7’. They synthesized ‘Top7’ to determine its 3D-structure by NMR spectroscopy and X-ray crystallography and found that the solved crystal structure was almost identical to the predicted structure. The speaker while admitting that designing a specified protein fold is a big achievement, he was keen on encouraging researchers to explore larger questions about how proteins evolved and why nature “chose” certain protein folds over others and a bigger challenge lies ahead in future for designing of proteins with specified functions.

 

The new concepts, hypotheses and views of the speakers lead to active discussions and contemplations on the varied topics. The meeting concluded with views from the speakers and the students regarding outcome of the two-day symposia. It was noted that meetings like TRendys are informative, especially on emerging areas in Biochemistry and Molecular Biology and provide a forum for scientific discussion on thought-provoking ideas nurturing and inculcating the scientific temper.

It was also decided by the ‘Standing Committee’ of TRendys that from next year onwards the first lecture (one hour duration) of the meeting would be given by a person specially invited for the purpose and the lecture will be known as “TRendys Oration”.

 

 

 
AUTHOR INDEX

 

Akgul C

120

Guha A K                         

81

Neekhra N                   

69

Arunkumar S               

96

Gupta M N          

113

 

 

 

 

 

 

Padh H                               

69

Bahadur A                        

107

Jain S                                 

113

Patel V                         

107

Bahadur P                    

107

 

 

Pathak T V                           

123

Banerjee S                            

81

Kaur K                              

116

Pundir C S                           

102

Bhalla S

116

Kaya I                               

120

 

 

Bhatt P N                           

123

Kumari M                         

102

Sharma S                

113

Bhattacharya I               

89

 

 

Sikdar S                    

81

Bhowal J 81

Mahmood A                    

116

   

Biswas S                              

89

Mahmood S                     

116

Varade D                     

107

 

 

Mehta J P                         

123

Vethamuthu M S        

107

Chatterjee* B P                  

81

Misra S N                         

123

 

 

 

 

Mitra A                             

81

 

 

Das Hasi R       

89

Mohankumar C                

96

 

 

Das R H                               

89

Murugan K                      

96