• Volume 13, Issue 1

      March 1988,   pages  1-104

    • Effect of hydroxyurea on subcellular activities of thymidine kinase in developing and aging rat brain

      V Prabhakar Kalluri Subba Rao

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      Hydroxyurea, when injected intraperitoneally at a dose of 1 mg/g body weight, inhibited thymidine kinase activity in developing rat cerebrum (16-day-embryonic) and cerebellum (7-day-postnatal) within a few hours of administration. The inhibition was time-dependent and both cytosolic and mitochondrial thymidine kinases were affected. Under the same conditions, the activities of certain other enzymes concerned with DNA metabolism,viz., DNA polymerase, and acid and alkaline DNases were not inhibited. Further, the addition of hydroxyureain vitro had no effect on the activity of any of the enzymes studied. However, similar treatment given to 2-year-old rat failed to exert any inhibition on either the mitochondrial or soluble thymidine kinase activities in grey and white matter regions of cerebrum and cerebellum. It is inferred that hydroxyurea, apart from its already known effect on ribonucleotide reductase of replicating cells, also affects thymidine kinase.

    • Comparative study of conformational behaviour of leucine and methionine enkephalinamides by1H-nuclear magnetic resonance spectroscopy

      M M Dhingra Anil Saran

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      The conformational proclivity of leucine and methionine enkephalinamides in deuterated dimethyl sulphoxide has been investigated using proton magnetic resonance at 500 MHz. The resonances from the spin system of the various amino acid residues have been assigned from the 2-dimensional correlated spectroscopy spectra. The temperature variation of the amide proton shifts indicates that none of the amide proton is intramolecularly hydrogen-bonded or solvent-shielded. The analysis of vicinal coupling constants,3JHN.C2H,along with temperature coefficients and the absence of characteristic nuclear Overhauser effect cross peaks between the NH protons reveal that there is no evidence of the chain folding in these molecules. However, the observation of nuclear Overhauser effect cross peaks between the NH and the CαH of the preceding residue indicates preference for extended backbone conformation with preferred side chain orientations particularly of Tyr and Phe in both [Leu5]- and [Met5]-enkephalinamides.

    • Synthesis of ribulose 1,5-bisphosphate carboxylase by isolatedSorghum mesophyll chloroplasts

      T Valliammai S Krishnasamy A Gnanam

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      Chloroplasts isolated fromSorghum vulgare are active in light-dependent, organelle protein synthesis. Intact chloroplasts can use light as an energy source; photosynthetically inactive chloroplasts require the addition of ATP for this protein synthesis. Preincubation of chloroplasts in light at 25°C for 1 h depleted the endogenous templates completely; such preincubated chloroplasts translated exogenously added heterologous templates efficiently. When total cellular RNA fromChlorella protothecoides, a C3 plant, was used as template for translation in a cell-free light-dependent system of isolated mesophyll chloroplasts fromSorghum vulgare, a C4 type plant, polypeptides of 55 kDa (large subunit) and 15 kDa (small subunit) were detectable in the fluorographic profile of the newly synthesized proteins; these polypeptides were absent in the products obtained with endogenous RNA. Evidence for the fidelity of the system was obtained by immunological analysis of ribulose 1, 5-bisphosphate carboxylase obtained by the translation ofChlorella cellular RNAs.

    • Lipid profile of cultured cells of apple (Malus sylvestris) and apple tissue

      T N Prabha P L Raina M V Patwardhan

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      The potentiality of apple cell cultures to synthesize not only higher quantities of lipids than apple fruit but also different classes of lipids is noted. Total lipid was 15-fold higher in apple callus than in the original tissue. On callusing, linoleic acid decreased from 66% to 14%, while linolenic acid showed a very large increase from 0.9% to 44%. Stearic and oleic acids also increased in callus. The relative amounts of sterol/hydrocarbon and diglyceride fractions were higher in callus cultures, while apple tissue showed higher levels of triglycerides and sterol. Phosphatidylethanolamine and phosphatidylglycerol seemed to be newly synthesized during callusing while other phospholipids such as lysophosphatidylcholine, lysophosphatidylethanolamine, phosphatidylinositol and phosphatidic acid decreased. There was much higher glycolipid in apple callus than in the original tissue. The ratio of neutral lipid to polar lipid was higher in apple than in apple callus.

    • Characterization of foot-and-mouth disease virus types O and Asia 1 RNA

      S Vasantha S M Lal A Antony

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      Poly (A) RNA was isolated from foot-and-mouth disease virus-infected cells by oligo (dT)-cellulose chromatography. One-dimensional oligonucleotide mapping of virus-induced poly (A) RNA indicated major differences between virus types O and Asia 1. Base composition analysis of virus-induced RNA showed no significant differences between types O and Asia 1.

    • Device for miniscale isoelectric focusing of proteins

      H S Pawar A M Bodhe M V Rele H G Vartak

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      A simple device is developed for mini-scale electrofocusing of proteins. The main apparatus consists of only two glass tubes joined by a small tubing. No special cooling system, stopcocks, stands, etc., are needed. Even the need for a peristaltic pump for fractionation is eliminated. The apparatus does not require very high voltages and the amount of Ampholines is drastically reduced. The model can be used for analytical as well as semi-quantitative purposes.

    • Preparation of Concanavalin A-β-galactosidase conjugate and its application in lactose hydrolysis

      Sunil Kumar khare Munishwar Nath Gupta

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      A Concanavalin A-β-galactosidase conjugate was prepared using glutaraldehyde as the crosslmking reagent. The conjugate bound to Sephadex G-50 beads was more thermostable and hydrolyzed lactose faster than the free enzyme. The immobilized enzyme may prove useful in the preparation of low lactose milk which is required by persons suffering from lactose intolerance.

    • Bioorganic chemistry of the purple membrane ofHalobacterium halobium — Chromophore and apoprotein modified bacteriorhodopsins

      Anil K Singh M Roy S Sonar M Kapil

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      Iodophenyl and anthryl retinal analogues have been synthesized. Thetrans-isomers have been isolated and purified by high pressure liquid chromatography. The purified isomers have been further characterized by nuclear magnetic resonance and ultraviolet-visible spectroscopy. Incubation of these retinal analogues with apoprotein (bacterioopsin), isolated from the purple membrane ofHalobacterium halobium gave new bacteriorhodopsin analogues. These analogues have been investigated for their absorption properties and stability. The iodophenyl analogue has been found to bind to bacterioopsin rapidly. The pigment obtained from this analogue showed a dramatically altered opsin shift of 1343 cm-1. The anthryl analogue based bacteriorhodopsin, however, showed an opsin shift of 3849 cm-1. It has been found that bacteriorhodopsin is quite unrestrictive in the ionone ring site. The apoprotein seems to prefer chromophores that have the ring portion co-planar with the polyene side chain.

      The purple membrane has also been modified by treatment with fluorescamine, a surface active reagent specific for amino groups. Reaction under controlled stoichiometric conditions resulted in the formation of a modified pigment. The new pigment showed a band at 390 nm—indicative of fluorescamine reaction with amino group (s) of apoprotein-besides retaining its original absorption band at 560 nm. Analysis of the fluorescamine modified bacteriorhodopsin resulted in the identification of lysine 129 as the modified amino acid residue. Fluorescamine-modified-bacteriorhodopsin suspension did not release protons under photolytic conditions. However, proteoliposomes of fluorescamine-modified-bacteriorhodopsin were found to show proton uptake, though at a reduced rate.

    • Refined procedures for accurate determination of solution structures of nucleic acids by two dimensional nuclear magnetic resonance spectroscopy

      R V Hosur K V R Chary A Sheth Girjesh Govil H T Miles

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      New procedures have been described for accurate determination of solution structures of nucleic acids. These are two fold; new two dimensional nuclear magnetic resonance techniques and better approaches for interpretation of nuclear magnetic resonance data for structure determination purposes. The significant development in two dimensional nuclear magnetic resonance techniques for this purpose areω1 -scaling and recording of pure phase spectra. Use ofω1-scaled correlated and nuclear Overhauser effect spectra for estimation of interproton distances and 1H-1H coupling constants has been described. Computer simulation procedures for exact determination of structure have been described. Experimental spectra demonstrating the application of new procedures have been presented.

    • Biochemical and immunological aspects of riboflavin carrier protein

      P R Adiga S S Visweswariah A Karande N Kuzhandhaivelu

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      Riboflavin carrier protein which is obligatorily involved in yolk deposition of the vitamin in the chicken egg, is a unique glycophosphoprotein present in both the yolk and white compartments. The yolk and egg white proteins are products of a single estrogen-inducible gene expressed in the liver and the oviduct respectively of egg laying birds. Despite the fact that the carbohydrate composition of the yolk and white riboflavin carrier proteins differ presumably due to differential post-translational modification, the proteins are immunologically similar and have identical amino acid sequence (including a cluster of 8 phosphoser residues towards the C-terminus) except at the carboxy terminus where the yolk riboflavin carrier protein lacks 13 amino acids as a consequence of proteolytic cleavage during uptake by oocytes. The protein is highly conserved throughout evolution all the way to humans in terms of gross molecular characteristics such as molecular weight and isoelectric point, and in immunological properties, preferential affinity for free riboflavin and estrogen inducibility at the biosynthetic locusviz., liver. Obligatory involvement of the mammalian riboflavin carrier protein in transplacental flavin transport to subserve fetal vitamin nutrition during gestation is revealed by experiments using pregnant rodent or subhuman primate models wherein immunoneutralisation of endogenous maternal riboflavin carrier protein results in fetal wastage followed by pregnancy termination due to selective yet drastic curtailment of vitamin efflux into the fetoplacental unit. Using monoclonal antibodies to chicken riboflavin carrier protein, it could be shown that all the major epitopes of the avian riboflavin carrier protein are highly conserved throughout evolution although the relative affinities of some of the epitopes for different monoclonal antibodies have undergone progressive changes during evolution. Using these monoclonal antibodies, an attempt is being made to map the different epitopes on the riboflavin carrier protein molecule with a view to delineate the immunodominant regions of the vitamin carrier to understand its structure-immunogenicity relationship.

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