Indian Journal of Biochemistry & Biophysics

CONTENTS

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*Author for correspondence

Indian Journal of Biochemistry & Biophysics

Vol. 40, December 2003, pp. 377-391

Minireview

Recent advances in oligonucleotide synthesis and

their applications

B Vaijayanthi, P Kumar, P K Ghosh^‡ and K C Gupta*

Short synthetic oligonucleotides are finding wide variety of applications in area of genomics and medicinal chemistry. Since the isolation of nucleic acids to the mapping of human genome, chemical synthesis of nucleic acids has undergone tremendous advancements. Further improvements in this area such as, introduction of high throughput synthesizers, better coupling reagents, improved polymer supports, newer sets of protecting groups for exocyclic amino groups of nucleic bases and introduction of universal polymer supports have completely revolutionized the entire field of nucleic acids chemistry. Most of these developments have been targeted to assemble these molecules more efficiently in a cost-effective manner and rapidly. Preparation of oligonucleotide conjugates has further helped in identifying the newer areas of their applications. A number of conjugates with biological and abiological ligands have been discussed in this article along with their possible wide spectrum of applications. Recently developed microarray technology, which refers to attachment of short oligonucleotides on a solid/polymeric surface, has proved to be useful for screening of genetic mutations, study of polymorphism, as diagnostics, etc. The major developments in these areas are presented in the review.

Keywords: Oligonucleotides, universal polymer support, reusable support, modifications, conjugates, immobilization, microarray.

Indian Journal of Biochemistry & Biophysics

Vol. 40, December 2003, pp. 392-399

Enzymatic transesterification for biodiesel production

Shweta Shah, Shweta Sharma and M N Gupta*

Biodiesel consists monoalkyl esters of long chain fatty acids. It is produced from vegetable oils or fats either by chemical transesterification or by lipase-catalyzed transesterification with methanol or ethanol. Biodiesel is a green fuel and can be used as a blend with diesel or alone. Either way, it does not require any modification in engine design or storage facilities. The enzymatic process offers several advantages over the chemical routes. The handicap of increase in process cost because of the cost of the enzyme can be overcome by using efficient production process for enzyme and using reusable derivatives of enzymes, such as immobilized enzyme. Numerous strategies available in the area of non-aqueous enzymology can be exploited during the enzymatic alcoholysis for biodiesel production. Some of the technical challenges and their possible solutions are also discussed.

Keywords: Biodiesel, transesterification reactions, lipases, oil-extraction, enzymes in organic solvents

Indian Journal of Biochemistry & Biophysics

Vol. 40, December 2003, pp. 400-408

Microheterogeneity of molecular forms of arginase in mammalian tissues

Gita Venkatakrishnan, V Shankar and S R R Reddy^*

Two isoforms of arginase, A₁ and A₂, were found in rat liver, submaxillary gland and kidney as well as beef kidney. In beef liver, however, A₂ was the only detectable form. Two additional forms, A₃ and A₄, found only in rat kidney were probably artifactitious. A₁ and A₂ exhibited chromatographic and immunological microheterogeneity. While A₁ in rat liver and submaxillary gland was excluded by DEAE-cellulose (pH 8.3) and retained on CM-cellulose (pH 7.5), that (A’₁) in beef and rat kidneys was excluded by both ion-exchangers. A₂ in all tissues was retained on DEAE-cellulose, but not on CM-cellulose. Both A₁ and A₂ in rat liver and beef kidney, A₁ from rat submaxillary gland and A₂ from beef liver were precipitated by antibodies to rat and beef liver arginases. None of the forms in rat kidney (A₁, A₂, A₃ and A₄) showed any cross-reactivity to either antibody. Rat submaxillary gland A₂ was precipitated by anti-rat liver arginase, but activated by anti-beef liver arginase. While the major molecular forms were A₁ in rat liver and submaxillary gland and A₂ in beef liver and rat kidney, the two forms occurred in equal proportions in beef kidney. It appears that different isoforms might function as components of the urea cycle in the liver of different mammals and of the arginine catabolic pathway in different extrahepatic tissues.

Keywords: Arginase isoforms; mammalian tissues, chromatographic behaviour; immunological characterization, kineticand physical properties, activation by antibodies, microheterogeneity

Indian Journal of Biochemistry & Biophysics

Vol. 40, December 2003, pp. 409-415

Fibronectin dependent upregulation of matrix metalloproteinases in hepatic stellate cells

P Pranitha and P R Sudhakaran*

Activation and transition of hepatic stellate cells (HSCs) to myofibroblast (MFB)-like cells is influenced by growth factors, cytokines and matrix proteins like fibronectin (FN). To examine whether the FN-dependent transition of HSCs is mediated through FN receptor, a marker function, such as matrix metallo-proteinase (MMP) production by HSCs in primary culture was studied. An upregulation of MMP production by HSCs maintained on FN was observed. FN-dependent upregulation of MMPs was significantly reduced when cells were pre-treated with antibodies to a5b1 integrin. Treatment of cells with genistein, a protein kinase C inhibitor completely blocked the gelatinase production by HSCs, indicating that the FN-dependent upregulation of MMPs is mediated through integrins and it involves tyrosine phosphorylation dependent signaling pathways.

Keywords: Hepatic stellate cells, fibronectin-dependent upregulation, matrix metalloproteinase

Indian Journal of Biochemistry & Biophysics

Vol. 40, December 2003, pp. 416-422

Stable free radical scavenging and antiperoxidative properties of resveratrol compared in vitro with some other bioflavonoids

K L Khanduja* and Anjana Bhardwaj

Stable free radical scavenging and antiperoxidative activities of resveratrol, a component of grapes and red wine, were evaluated and compared with some other known bioflavonoids (quercetin, catechin, kaempferol, myricetin, fisetin, ellagic acid and naringenin) widely present in the plant kingdom. Free radical scavenging activity was measured in an in vitro chemical system (DPPH assay), while for antiperoxidative activity, biological system comprising of hepatic and pulmonary homogenates was employed. Antiradical activity assay showed quercetin and myricetin to be stronger antiradical agents than resveratrol. Structure-activity study revealed that O-dihydroxy group on ring B of flavonoid plays a crucial role. A double bond at 2-3 position conjugated with a 4-oxo function and hydroxy groups at positions 3 and 5 also contribute towards antiradical activity of flavonoids. Resveratrol exhibited stronger antiradical activity than kaempferol and naringenin and was also more efficient than a-tocopherol, a known strong endogenous non-flavonoid antioxidant, used for comparison. In vitro antiperoxidative assay showed fisetin as the strongest and kaempferol as the weakest antioxidant. Resveratrol was found to be stronger antioxidant than catechin, myricetin, kaempferol and naringenin, but was weaker than quercetin, fisetin and a-tocopherol. Antiradical and antiperoxidative activities of resveratrol may explain its beneficial effects in disease states. Assays exhibited no direct correlation between antiradical and antiperoxidative activities of the phenolics.

Keywords: antiradical, antioxidant, DPPH, resveratrol, lipid peroxidation, flavonoids, polyphenols, α-tocopherol

Indian Journal of Biochemistry & Biophysics

Vol. 40, December 2003, pp. 423-428

Effect of chlorpyrifos on thiobarbituric acid reactive substances, scavenging enzymes and glutathione in rat tissues

Radhey S Verma and Nalini Srivastava*

The present study showed that exposure of chlorpyrifos, O,O¢-diethyl-O-3,5,6-trichloro-2-pyridyl phosphorothionate (CPF), a widely used pesticide in rats caused significant inhibition of acetylcholinesterase (AChE) activity in different tissues viz., liver, kidney and spleen. CPF exposure also generated oxidative stress in the body, as evidenced by increase in thiobarbituric acid reactive substances (TBARS), decrease in the levels of superoxide scavenging enzymes viz., superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) in liver, kidney and spleen at all doses. Malondialdehyde levels were increased by 14%, 31% and 76% in liver, 11%, 31% and 64% in kidney and 32%, 75% and 99.9% in spleen when 50 mg, 100 mg and 200 mg/kg body wt. CPF was administered for three days. SOD and CAT activities were decreased in liver, kidney and spleen, while GPx activity showed slight increase in kidney at 50 mg and 100 mg dose, and decreased on further increase in dose of CPF. Liver and spleen showed dose-dependent decrease in GPx activity. The levels of reduced glutathione (GSH) was decreased, while oxidized glutathione (GSSG) was increased, thus a marked fall in GSH/GSSG ratio was observed in all tissues. A maximum decrease of 83% was observed in liver, followed by kidney and spleen, which showed 78% and 57% decrease, respectively in group given 200 mg/kg CPF. The levels of glucose-6-phosphate dehydrogenase (G6PDH) and glutathione reductase (GR) were also decreased in liver and kidney, while spleen showed increase at lower doses, but decrease at high dose of CPF. The data provide evidence for induction of oxidative stress on CPF exposure.

Key words: Chlorpyrifos, oxidative stress, lipid peroxidation, scavenging enzymes,
organophosphate pesticides

Indian Journal of Biochemistry & Biophysics

Vol. 40, December 2003, pp. 429-438

Effect of side chain length on the aggregation of amphiphilic 5,10,15-tris (1-methylpyridinium-4-yl)-20-[4-(alkoxy) phenyl]
21H, 23H porphyrin tritosylates

S M S Chauhan*, Anil Kumar, K A Srinivas and M K Mishra

Self-aggregation of cationic amphiphilic 5, 10, 15-tris-(1-methylpyridinium-4-yl)-20-[4-(alkoxy)phenyl]-21H, 23H porphyrin tritosylates (2a-e) with different alkoxy chain length in aqueous and binary solvent systems has been studied by UV-visible, fluorescence and 1H NMR spectroscopy. Binary solvent, concentration, ionic strength, presence of surfactants, and temperature govern the aggregations of 2a-e. Porphyrins having side chain length more than ten carbon atoms (2c-e) form higher aggregates, such as vesicles by sonication of dimers formed initially, whereas porphyrins with lesser side chain length (2a & b) form lower aggregates only. Further, the size and the formation of vesicles have been confirmed by transmission electron microscopy (TEM) and dye entrapment experiments for 2e.

Key words: Aggregation, binary solvent, amphiphilic porphyrins, vesicles, higher aggregates.

Indian Journal of Biochemistry & Biophysics

Vol. 40, December 2003, pp. 439-441

Mutation analysis in spinal muscular atrophy using allele-specific polymerase chain reaction

Akanchha Kesari, Monisha Mukherjee and Balraj Mittal*

Polymerase chain reaction (PCR), followed by restriction digestion is universally used for molecular diagnosis of spinal muscular atrophy (SMA). In the present study, we have used a modified strategy based on amplification refractory mutation system-polymerase chain reaction (ARMS-PCR) to develop a rapid and reliable method for mutation detection and prenatal diagnosis in SMA patients. The telomeric (SMN1) and centromeric (SMN2) copies of exon 7 of the survival motor neuron (SMN) gene were amplified by ARMS-PCR, using primers specific to SMN1 and SMN2 nucleotide sequence with the exonic mismatch G (for SMN1) and A (for SMN2) at the 3’ end. The PCR products were analyzed on agarose gels. All the patients who had homozygous deletion of exon 7 of SMN1 gene by conventional PCR-restriction fragment length polymorphism (PCR-RFLP) method showed the same deletion status by ARMS-PCR. This procedure showed a 100% concordance between PCR-RFLP and ARMS-PCR methods for the detection of SMN1/SMN2 status in patients with SMA. An artifact due to incomplete digestion is not a problem while using ARMS-PCR. The modified protocol is specific, rapid and highly reliable for use in prenatal diagnosis as well.

Keywords: spinal muscular atrophy, allele-specific amplification, DNA diagnosis, prenatal diagnosis, SMN gene, mutation analysis, polymerase chain reaction

Indian Journal of Biochemistry & Biophysics

Vol. 40, December 2003, pp. 442-446

Purification of catecholase from Solanum melangena (brinjal)

Anita P Goswami and Sudha V Amarapurkar

Catecholase was purified from cortex of Solanum melangena (brinjal) on natural affiant, lignin. The elution profile showed seven peaks with the 6^th peak having 4616-fold purity. The 6^th pure fraction loaded on PAGE showed two protein bands on staining with Coomassie brilliant blue, one at the point of application and other near the dye front. These bands exhibited catecholase activity, when stained with 4-methyl catechol and proline, Basic fuschin and ethidium bromide showed positive tests, indicating that catecholase is a ribonucleoglycoprotein.

Keywords: brinjal, Solanum melangena, polyphenol oxidase, 4-methyl catecholase, o-diphenol oxidase

Indian Journal of Biochemistry & Biophysics

Vol. 40, December 2003, pp. 447-450

Lipid peroxidation and scavenging enzyme levels in the liver of streptozotocin-induced diabetic rats

Neslihan Bukan*, Banu Sancak, Özlem Yavuz, Cemile Koca, Funda Tutkun,
A Tanju Özçelikay  and Nilgün Altan

In this study, alterations in the liver antioxidant enzymes status and lipid peroxidation in short-term (8-weeks) and long-term (24-weeks) diabetic rats were examined. Glutathione peroxidase (GSH-Px) activity and malondialdehyde (MDA) levels were significantly increased, but superoxide dismutase (SOD) activity was significantly reduced in 8-weeks diabetic rats, compared to control. Catalase (CAT) activity, however, was found unchanged. In 24-weeks diabetic rats, while GSH-Px activity was unchanged, but SOD and CAT activities and MDA levels were significantly increased, compared to control. These results suggest that diabetes-induced alterations in tissue antioxidant system may reflect a generalized increase in tissue oxidative stress. It can be concluded that lipid peroxidation and antioxidant enzyme levels are elevated in diabetic condition. Hence, diabetes mellitus, if left untreated, may increase degenerative processes due to accumulation of oxidative free radicals.

Key words: lipid peroxidation, antioxidant enzymes, diabetic rats

Indian Journal of Biochemistry & Biophysics

Vol. 40, December 2003, pp. 451-454

Hydroxyproline concentrations in ocular tissues of Arabian camel  (Camelus dromedarius Linn.)

N J Siddiqi and A S Alhomida*

Hydroxyproline (Hyp) concentrations (total, free, peptide-bound and protein-bound) in camel eye tissues were determined. Total Hyp concentration was highest in iris, followed by ciliary body, sclera, cornea, lens and retina; the difference between total Hyp concentration of iris and sclera (P < 0.05) and cornea, lens and retina (P < 0.001) was statistically significant. Cornea had the highest concentration of free Hyp, followed by ciliary body, retina, iris, sclera and lens (P < 0.001). Peptide-bound Hyp concentration was highest in iris, followed by lens, cornea, ciliary body, retina and sclera (P < 0.001). Iris also had the highest concentration of protein-bound Hyp, followed by ciliary body, sclera, cornea, retina and lens; the difference in the protein-bound Hyp concentration between iris and sclera (P < 0.05) and cornea, retina and lens (P < 0.001) was statistically significant. Iris was also found to have the highest concentration of collagen, followed by ciliary body, sclera, cornea, lens and retina; the difference between the collagen concentration of iris and sclera (P < 0.05) and cornea, lens and retina (P < 0.001) was statistically significant. These variations may result from differences in the collagen structure and/or composition in these tissues.

Keywords: Hydroxyproline, collagen, ocular tissues, camel

Indian Journal of Biochemistry & Biophysics

Vol. 40, December 2003, pp. 455-467

Author Index