Indian Journal of Biochemistry

Protein tyrosine kinases (PTKs) are critical in regulating cell growth and differentiation and are deeply involved in several cancers. PTK-inhibitors are mainly ATP-site directed and are finding use in the treatment of several cancers, and more than 30 such agents are now in phase I-III clinical trials. The present review focuses mainly on the development of PTK inhibitors in clinical trials, with special emphasis on imatinib mesylate, a rationally designed, potent oral anticancer agent and selective inhibitor for Abl tyrosine kinase, including Bcr-Abl, C-kit and platelet-derived growth factor-receptor tyrosine kinases, which has been implicated in several malignancies, including chronic myeloid leukemia and gastro-intestinal stromal tumour.

Keywords: Protein tyrosine kinase, tyrosine kinase inhibitors, imatinib mesylate, chronic myeloid leukemia, acute myeloid leukemia, anticancer agent, gastrointestinal stromal tumor, epidermal growth factor receptor, vascular endothelial growth factor

Papers

Immunocontraceptive potential of recombinantly expressed minimized chicken riboflavin carrier protein (mini-RCP) in rodents

Chicken riboflavin carrier protein (RCP; 219 AA) harbours four linear epitopes, constituted by the peptide residues 3-23, 64-83, 130-147 and 200-219. Antibodies to these sequences bioneutralize maternal RCP and provide protection from pregnancy in rodents. In order to overcome the major histocompatibility complex-dependent variability in immune response often encountered with use of single peptides for vaccination in genetically outbred populations, we have assembled a novel synthetic gene, incorporating in tandem the nucleotide sequences coding for all the four neutralizing epitopes of chicken RCP and expressed in Escherichia coli. The gene product, mini-RCP has been characterized for its immunogenic properties and contraceptive potential in rodents. Immunization of rabbits and rats led to generation of antibodies against individual peptide components, as determined by enzyme-linked-immunosorbent assay (ELISA). However, immunized rats carried pregnancy to term and delivered healthy offsprings. Antisera from these rats exhibited decreased affinity of binding to the native protein. These findings suggest that the prospects of covalently-linked epitope peptides need to be cautiously evaluated during the design and development of peptide-based vaccines.

Keywords: Immunocontraception, immunogenicity, chicken riboflavin carrier protein, major histocompatibility complex, minimized riboflavin carrier protein, synthetic gene, rat, rabbit

Partial purification, characterization and properties of two isoforms of glutamine synthetase from Pennisetum glaucum L. leaves

Two isozymes of glutamine synthetase GS₁ and GS₂ were partially purified from Pennisetum glaucum leaves by ion-exchange and gel filtration chromatography and their kinetic and regulatory properties were studied using semisynthetase assay of GS. Mg²⁺was the most effective cation for activity of both the isozymes; however, it could be efficiently replaced by Co²⁺. The pH optima for GS₁ and GS₂were 7.0 and 8.0, respectively. GS₁exhibited maximum activity at 42°C, with activation energy of 18 KJ mol^-1 and a Q₁₀ of 3.0, whereas GS₂ showed maximum activity at 50°C, with activation energy of 40 KJ mol^-1 and Q₁₀ of 2.25. GS₁ was more thermostable than GS₂. The K_m value for Mg²⁺ of GS₁ was 2-fold higher than GS₂; however, these isozymes did not differ much in their affinity for other substrates. Alanine, serine and glycine lowered GS₁ and GS₂ activities, whereas cysteine enhanced their activities with a more pronounced effect on GS₂. Serine inhibited the activity of both the isoforms in a competitive-manner, whereas alanine was a non-competitive inhibitor, with respect to glutamate. AMP and ADP were competitive inhibitor with respect to ATP for both the isozymes.

Keywords: Pennisetum glaucum leaves, glutamine synthetase, chloroplastic, cytosolic, isozymes.

cAMP-mediated upregulation of gelatinases in primary cultures of isolated rat hepatocytes

Matrix metalloproteinases (MMPs) play a major role in tissue remodelling and repair in pathophysiological conditions, such as liver fibrosis and regeneration. Regulation of the MMPs produced by liver cells is important in maintaining cell-matrix ratio in liver, which is a major target site for hormones that mediate their intracellular effects through cAMP. The possibility of cAMP affecting the activity of MMPs and their endogenous inhibitors, tissue inhibitor of MMPs (TIMPs) was studied using isolated rat hepatocytes in culture. Zymographic analysis showed that treatment with hormones like epinephrine, thyroxine and dexamethasone and Bt₂ cAMP increased 92 kDa MMP-9 activity. Bt₂ cAMP caused upregulation of MMP-9 in a dose-dependent manner. The effect of hormones was less on MMP-2. ELISA using specific antibodies showed increase in levels of MMP-9 and TIMP-1 protein. Kinetic analysis of production of MMPs and TIMPs showed that the response to Bt₂cAMP was a delayed one, indicating its effect on de novo protein synthesis. These results suggest the possibility of cAMP dependent regulation of MMP-9 in the hepatocytes.

Keywords: Rat hepatocytes, matrix metalloproteinases (MMPs), tissue inhibitor of MMP (TIMP), cAMP-mediated upregulation, extracellular matrix (ECM), horse radish peroxidase.

Physico-chemical and antigenic characterization of unconventional heavy chain antibodies of Indian desert camel (Camelus dromedarius L.)

Heavy chain antibodies (HCAbs) of IgG2 and IgG3 subtypes were purified from the sera of Indian desert camel (Camelus dromedarius L.) by ammonium sulphate precipitation, followed by ion-exchange chromatography on DEAE-cellulose and affinity chromatography on protein A-sepharose and protein G-sepharose, and characterized by SDS-polyacrylamide gel electrophoresis, agar gel immunodiffusion (AGID), counter-immunoelectrophoresis (CIEP), immunoelectrophoresis (IEP), ELISA and immunoblotting. IgG2 and IgG3 were found to have molecular mass 46.77 kDa and 43.65 kDa, respectively by SDS-PAGE under reducing conditions. They migrated in β-region in IEP and could be detected in CIEP, because of being more negatively charged and smaller size. Anti-camel IgG3 cross-reacted in AGID, ELISA and immunoblotting with IgGs of pig and ruminants (cattle, buffalo, sheep and goat), but not with immunoglobulins from horse, dog, guinea pigs, mice, fish, poultry and human. The present findings suggest close antigenic relationship of camels with pigs and ruminants.

Keywords: Indian camel, heavy chain antibodies, purification, electrophoresis, antigenic relationship, immunoblots, Camelus dromedarius L.

A theoretical study of the stability of DNA binding with cis/trans platin

Seema Srivastava, Irfan Ali Khan, Shinoo Srivastava and Vishwambhar Dayal Gupta*

Both cis- and trans-platins are known to form intra- and interstrand cross-linking with DNA. Since the nature and strength of binding is different, it makes their efficacy as anti-tumour drug different. In the present communication, we report theoretical analysis by using an amended Zimm and Bragg theory, to explain the melting behaviour and heat capacity of DNA with and without platin binding. The sharpness of transition has been examined in terms of half width and sensitivity parameter (DH/s). The experimental measurements of Pilch et al (J Mol Biol 2000, 296, 803) and Ctirad and Brabec (J Biol Chem 2001, 276, 9655) have been used.

Keywords: cis/trans Platin, nucleation parameter, co-operativity, l-point anomaly, transition profile, DNA binding, heat capacity

Phonon dispersion in polyinosinic acid

A study of the normal modes of vibration and their dispersion in polyinosinic acid [poly (I)] along the helix axis based on Urey-Bradley force field is reported. It leads to a better interpretation of Raman and FTIR spectra. A comparison of dispersion curves of poly (I) with poly (G) has been made. Characteristic features of dispersion curves, such as regions of high density-of-states, repulsion and character mixing are discussed. Predictive value of heat capacity as a function of temperature is reported.

Keywords: Normal modes, dispersion curves, Fourier transform, density-of-states, polyinosinic acid

Notes

Automated derivatization with o-phthalaldehyde for the estimation of amino acids in plasma using reversed-phase high performance liquid chromatography

A fully automated method for quantitative estimation of plasma amino acids using fluorescence detection of o-phthaladehyde/2-mercaptoethanol derivatives of the analytes and their separation by gradient elution reversed-phase HPLC has been described. The method is simple and the three-step gradient elution is suitable for routine analysis of a large number of biological samples due to clear resolution, high degree of precision, accuracy, cost-effectiveness and lack of interference from chemical contaminations. Using this method, 19 amino acids were completely resolved and the within-run coefficients of variation ranged from 2.53 to 10.7% with a mean variation of 5.68%.

Keywords: o-Phthaladehyde derivatization, reversed-phase high performance liquid chromatography, plasma amino acid analysis.

Determination of serum triglycerides using lipase, glycerol kinase, glycerol-3-phosphate oxidase and peroxidase co-immobilized onto alkylamine glass beads

A method for determination of serum triglycerides (Tgs) using lipase, glycerol kinase, glycerol-3-phosphate oxidase and peroxidase co-immobilized onto alkylamine glass beads (pore diameter 55 nm) through glutaraldehyde coupling was developed and evaluated. The minimum detection limit of the method was 0.54 mM. The analytical recovery of added triolein in the serum was 97.55±1.5% (mean ± S.D.). The mean value of serum Tgs, determined by the present method showed a good correlation (r=0.984) with the Bayer’s kit method, employing free enzymes. The within and between batch coefficients of variation (CV) were <2.25% and <1.35% respectively. No significant loss of activity was observed, when co-immobilized enzymes were reused for about 200 times and stored at 4°C in distilled water. The cost of Tg determination for 200 serum samples was less, as compared with Bayer’s kit method.

Keywords: Triglyceride, lipase, glycerol kinase, glycerol-3-phosphate oxidase, peroxidase, co-immobilization, alkylamine glass, serum.

Inhibition of the hexokinase/hexose transporter region in the glycosomal membrane of bloodstream Trypanosoma brucei by oligomycin and digitonin

Glycolysis in bloodstream T. brucei is the sole source of energy and remains a favourable chemotherapeutic target. In furtherance of this, an attempt has been made to understand better the contribution of glucose, fructose, mannose and glycerol to the energy charge of these parasites incubated in the presence of oligomycin, salicyhydroxamic acid (SHAM) and digitonin. Their cellular energy charge, when catabolizing glucose was 0.860, and under inhibition by oligomycin (10 mg), SHAM (2 mM) or oligomycin plus SHAM, 0.800, 0.444 and 0.405, respectively. Oligomycin inhibited the rate of catabolism of glucose, mannose and fructose up to 80%. The inhibition could not be alleviated by uncouplers, such as 2,4-dinitrophenol or permeabilization of the membranes by digitonin. Glucose-6-phosphate and other phosphorylated glycolytic intermediates, such as fructose-6-phosphate were catabolized by the permeabilized parasites in the presence of oligomycin, implying that except hexokinase, all the other glycolytic enzymes were active. Glucose oxidation was stimulated by low concentrations of digitonin (up to 4 mg), but at higher concentrations, it was significantly inhibited (up to 90% inhibition at 10 mg). Apparently, the inhibitory effects of oligomycin and digitonin were confined to glucose uptake and hexokinase catalysis. The above observations suggest that the hexose transporter and the enzyme hexokinase might be functionally-linked in the glycosomal membrane and oligomycin inhibits the linkage, by using a mechanism not linked to the energy charge of the cell. Digitonin at concentrations higher than 4 mg disrupted the membrane, rendering the complex in-operative. A hexokinase/hexose transporter complex in the glycosomal membrane is envisaged.

Keywords: Energy charge, glycosome, glycosomal membrane, hexokinase, hexose-transporter, oligomycin, salicyhydroxamic acid, digitonin, Trypanosoma brucei

Author Index

Annual Author Index

Adiga P R	281	Jang Moon-Sun	141	Pawar R	254
Ahmad R	148	Juneja M	53	Phale P S	227
Akdogan M	57			Ponnuswamy M N	184
Akgul C	120	Kalia V	326	Porwal V	34
Ali A M	167	Kamat B P	173	Prabhu Y	227
Altintas L	57	Kapoor H C	29	Prasanna S	179
An Sun-Young	141	Karande A A	281	Pundir C S	102, 326
Annamalai P T	40	Kaur K	116, 221	Punekar N S	205
Arif S H	148	Kaya I	120
Arunkumar N S	96	Khan I A	305, 311	Rajasekar P	188
		Khan M M	148	Ranhotra H S	246
Bahadur A	107	Khar A	167	Rao J A	241
Bahadur P	107	Kiaira J K	329	Rath L S	258
Banerjee S	81	Kim Cheorl-Ho	141	Raval M K	258
Bansal D D	20	Kim Ji-Youn	141	Ray M	7
Bansal M P	14	Kole P L	48	Ray S	7
Basu A	162	Krishnan S	227	Reddy M	167
Bhalla S	116, 221	Kumari A L	167
Bhatt P N	123	Kumari M	102	Sahindokuyucu F	57
Bhattacharya I	89	Kurpad A V	322	Saja K	294
Bhattacharyya D	162			Saraboji K	184
Bhattacharyya Dhananjay	233	Lakshmi V P	205	Sawant M S	216
Bhise S B	273	Lee Young-Choon	141	Seetharamappa J	173
Bhowal J	81	Lodha M L	29	Sehrawat S	299
Bindu M P	40			Sen P C	162
Biswas S	89	Mahmood A	116, 221	Shalini S	14
		Mahmood S	116, 221	Shanmukha I	48
Cetinkaya O	45	Majumdar R	233	Sharma A	154
Chandra A	191	Manivannan E	179	Sharma R	246
Chatterjee M	162	Manivasagam T	188	Sharma S	113
Chatterjee B P	81	Mehra R	53	Shi X	216
Chaturvedi S C	179	Mehta J P	123	Sikdar S	81
Cho Young-Su	141	Melwanki M B	173	Silig Y	45
Choi Yong-Lark	141	Mishra S	254	Singh A	299
		Misra S N	123	Sonawane S	254
Das Hasi R	89	Misra R M	34	Sreedhar B	250
Das R H	89	Mitra A	81	Srinivas S K	322
Dasgupta A K	233	Mohankumar C	96	Srivastava Seema	305, 311
Demirezer L O	45	Murugan K	96	Srivastava Shinoo	305, 311
Dutt, S	23	Mwangi D W	20	Subramanian P	188
				Subramanian S	281
Eraslan G	57	Nagappa A N	48	Sudhakaran P R	294
Essiz D	57	Nair K M	250	Sun Y	216
		Nalawade A D	273
Ghosh P K	254	Nallini A	184	Tandon P	34
Ghosh S	7	Neekhra N	69	Tyagi A	191
Ghosh Shilpi	288	Njogu M R	329
Ghosh Subho	162			Varade D	107
Gnanou J V	322	Ok Min	141	Venkateswarlu K	241
Guha A K	81			Vethamuthu M S	107
Gupta M N	113	Padh H	69
Gupta V D	34, 305, 311	Pandey M K	311	Wadhawa H	273
		Pandi P V	48
Hasnain A	148	Pang Q	216	Yadav O P	29
		Pardhasaradhi B V V	167	Yarsan E	57
Jabeen M	148	Patel A	254
Jain S	113	Patel V	107	Zeeyauddin K	48
Janave M T	154	Pathak T V	123	Zhang S	216
		Patil R T	48

Annual Subject Index

absorption, infrared	34	Dioxygenase		Operativity, Co-	305
acetaminophen	277	1-hydroxy-2-naphthoic acid	227	Orbitals, Pseudo p	184
Acetogenins	167	ring-cleaving	228	Oscillations, Circadian	188
acetone	328	dipole(s)	179	osteoblastic	57
acetosyringone	206	atomic	35	Over-expression	102, 141, 211
acid(s)		ion	255	oxalate oxidase	102
abscisic	191	Dipyridyl, α,α-	228	oxidants	294
amino	188	disease(s)		Oxidase, Glycerol-3-phosphate	326, 329
8-anilino-1-naphthalene sulphonic	173	Hodgkins	274	oxidative damage	14
arachidonic	246	Parkinson’s	123	oxygenase, pheophorbide a	154, 159
aromatic amino	184	inflammatory	20	oxyradicals	43
ascorbic	328	Dismutase, Superoxide	20, 40	oxysterol	14, 18
cis-4-(1-hydroxynaphth-2-yl)-2-oxobut-3-enoic	231	disorders		paclitaxel	167
colominic	81	gastro-intestinal	53	paracetamol	173
deoxyoctulosonic	89	respiratory	53	Parameter, Nucleation	305
diaminetetraacetic	143	Dispersion, Phonon	34, 311	parasites	329
5,5-dithiobis-2-nitrobenzoic	20	Distribution, Potential energy	37	pathway(s)
ethylene diamine tetraacetic	20, 162	5,5′-dithiobis-(2-nitrobenzoic)	7	biosynthetic-secretory	70
ethylene glycol bis (b-amino		dithiothreitol	7	chlorophyll oxidase	160
ethyl ether) N, N, N’, N’-tetraacetic	162	DNA		chlorophyllase	158, 160
5-fluoroorotic	205, 207	k	305	clathrin-mediated	73
gallic	21	melting behaviour	305	1, 2-dihydroxynaphthalene	227
gallotannic	20	transition	305	endocytic	70, 74
1-hydroxy-2-naphthoic	227	Docking	76	endolysosomal	71
lyso bis-phosphatidic	69, 75	donor		endosomal	282
N-acetyl-neuraminic	20	electronegative	184	gentisate	227
nalidixic	48	proton	184, 258	hydrophobic	48
neuraminic	20, 81	drug(s)		lysosomal	282
N-glycolylneuraminic	20	anti-tumour	305	non-clathrin-mediated	73
nucleic	311	anti-inflammatory	179	o-phthalic acid	227
oleic	246	design	275	phagocytic	71
o-phthalic	227	quinone	45	signal transduction	274
palmitic	246	surface-active	48	stimulicaspase-3-related	167
perchloric	330	D-tagatose	81	Patients, Hypercholesterolemic	15
polycytidilic	311	D-talose	81	Pennisetum glaucum	288
polyinosinic	311	Dye, Quinoneimine	327	pepstatin A	143
polyunsaturated fatty	246	Dynamics, Vibrational	311	Peptide groups	233
salicyhydroxamic	329	Dysmorphology, Craniofacial	221	permeability	48
saturated fatty	246	Edema, Peripheral	277	permeabilization	329
sialic	81	eel	148, 219	permeants	48
tannic	26	Effect(s)		peroxidase
thiobarbituric	20	alcohol	40	glutathione	40
tricarboxylic	14	antiulcerogenic	45	horse radish	294, 326
trichloroacetic	89, 91, 162	circular dichroism cotton	148	phenylalanine ammonia	96
uric	57, 328	pro-atherogenic	14	peroxidation, lipid	40
uronic	89	Ehrlich ascites carcinoma	7, 81	Peroxides, Lipid	21
Acidogenesis	212	electroendosmosis	299	Phagocytosis	69
aconitase		Electrophoresis		phagosome	69
duodenal cytosolic	250	2-D polyacrylamide gel	192	Phase, Hexadecane	92
inhibition of duodenal cytosolic	250	polyacrylamide gel	29, 149, 216	phenanthrene	227
Acrophase	188	sodium dodecyl sulphate-poly		1,10-phenanthroline	227
actin	217	acrylamide gel	20, 81, 143, 299	Phenol, p-nitro	164
activation		electroporation	206	Phenyl, Methyl sulfonyl fluoride	14
hepatic GR	246	Elements, Trace/toxic	53	Phenylalanine, 3, 4-dihydroxy	123
in vitro heat	246	emodin	46	phenylisothiocyanate	322
inhibitors of GR heat	246	Endocytosis	69	phenylketonuria	322
receptor	246	Endoplasmic, Reticulum	26, 69	pheophorbide	154
activator, plasminogen	211	Endosomes	69	pheophytin	154
Activit(y)ies		tubular	74	Phosphatase(s)	288
antimicrobial	48,167	Endothapepsin	237	alkaline	221
alkaline phosphatase	45, 57, 190	Energy, Activation	255, 291	phosphate,
anti-angiogenic	276	Enterococcus faecalis	120	cytidine mono	20
antibacterial	120	enterocytes	119, 221	D-glyceraldehyde-3-	7
antigen-degrading	26	Enzyme		glucose-6-	329
anti-inflammatory	45	antioxidant	14, 20, 26, 40	orotidine-5’-mono	205
anti-metabolite	120	ATPase inhibitor of Na⁺, K⁺-	162	p-nitrophenyl	162
anti-microbial	167	chlorophyll degrading	154	Phosphokinase, tyrosine	276
anti-neoplastic	167	chloroplastic	288	phospholipid	49, 77
antipyretic	45	cyclooxygenase-2	179	phosphoprotein	288
anti-tumor	167, 275, 305	digestive	69	phosphorylase B	217
caspase-3	167	drug-metabolizing	45	phosphorylation	162, 329
catalase	22, 46	glycolytic	329	reversible	288
chlorophyll-A degrading	156	immobilized	327	Photo-oxidation	12
chlorophylloxidase	158	inhibition of intracellular	48	photorespiration	288
cinnamyl alcohol-NADPH-dehydrogenase	96	intestinal brush border	221	photosynthesis	159, 254, 292
cytosolic aconitase	251	marker	96	photosystem II	159, 254, 258
cytotoxic	167, 306	mycolytic	206	Phthaladehyde, o-	15, 168, 322
1-hydroxy-2-naphthoic acid dioxygenase	227	proteolytic	141	phycobilisomes	254
DNase	29	recombinant	141	Phycocyanin, C-	254
fucosyl transferase	119	recovery	102	phycoerythrin	254
gelatinase	295	ring-cleaving	227	Phytohaemagglutinin	283
glutathione peroxidase	41	epinephrine	296	Pinocytosis	69
glutathione reductase	22	erythrocytes	241	Plaques, Atherosclerotic	15
glutathione-S-transferase	45	Estimation		Plasmid (s)	142, 206, 282
glycosyl transferases	119	lipid peroxides	21	platin cis/trans	305
hemagglutinating	81	protein	8	Plot, Sctachard	85
hydroxylase	20, 26	reduced glutathione	22	pokeweed	29
intracellular serine protease	141	sialic acids	22	Polyethylene	37
lactase	221	total thiols	22	polyglycine I	34
Mg-dechelatase	157, 158	estrogen	216	polymer	312
microbicidal	14, 26	Ethanol, b-Mercapto	162	Polyporus squamosus	81
N-glycosidase	29	Ether, Octaethylene glycol mono-n-dodecyl	162	polythymidilic,	311
parasiticidal	167	ethylene diaminetetracetate	322	polyvinyl polypyrrolidone	29
pesticidal	167	exocytosis	70, 77	Porins	48
photosynthetic	292	exopolysaccharide	89	Position, Planar	233
p-nitrophenyl phosphatase	163	exposure		Powder, Acetone	155
protocatechate-3, 4-dioxygenase	231	cadmium	53	Precipitation, Ammonium sulphate	299
radical scavenging	254	electromagnetic field	57	pregnancy	221, 281
RNase	29	ethanol	116	primers	142
semisynthetase	293	lead	53	hexamer	169
sialyl transferase	119	prenatal ethanol	221	probe oxidation-sensitive fluorescent	168
SOD	22, 46	expression		Probe(s)
tumouricidal	14	anachronistic	209	hydrophobic	173
Adaptins	72	gene	170, 191	cDNA	169
adipocytes	246	high-level	141	Products, Phytylated	160
adjuvant, Freund’s	281	mini-RCP gene	282	profile, transition	305, 309
agent (s)		over-	209, 274	Profile, Protein	191
antibacterial	48, 120	Extract, Seed	167	Promoter(s)
anticancer	273	Factor(s)		enolase	211
antiseptic	122	ADP ribosylation	69	fungal	209
bacterostatic	48	epidermal growth	69	properties
reducing	50	guanine nucleotide-exchange	73	anti-bacterial	48
sulfhydryl	16	N-ethylmaleimide sensitive	69	anticancer	254
agglutinin (s)	81	sexual agglutination	85	anti-inflammatory	254
dermatophyte	86	steric	238	antioxidant	254
fungal

ISSN : 0301-1208		CODEN : IJBBBQ
VOLUME 41	NUMBER 6	DECEMBER 2004