• Volume 6, Issue 5

      December 1984,   pages  585-794

    • An overview

      L K Ramachandran

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    • Thermodynamics of binding water and solute to myosin

      Madhusweta Das D K Chattoraj

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      From the isopiestic measurements of the extents of adsorption of water vapour by fish myosin at various values of water activities at three different temperatures, the changes in free energy, enthalpy and entropy of dehydration of the protein have been calculated. Extents of excess binding of solvent and solute to myosin have also been determined from isopiestic experiments in the presence of different inorganic salts, sucrose and urea respectively. Mols of water and solute respectively bound in absolute amounts to myosin have been evaluated from these data in limited range of solute concentrations. Free energy changes at different concentrations of these solutes have also been evaluated and their relations with ‘salting-in’ and ‘salting-out’ phenomena have been discussed. The order of the values of the standard free energy change for excess binding calculated with respect to an unified thermodynamic scale are found to be consistent with relative reactivity of binding water to myosin in the presence of inorganic salts, sucrose and urea.

    • Structure and stability of glucoamylase II fromAspergillus niger: A circular dichroism study

      B C Shenoy A G Appu Rao M R Raghavendra Rao

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      Glucoamylase II (EC 3.2.1.3) fromAspergillus niger has 31 % α-helix, 36 %Β- structure and rest aperiodic structure at pH 4.8 as analysed by the method of Provencher and Glockner (1981,Biochemistry, 20,33). In the near ultra-violet circular dichroism spectrum the enzyme exhibits peaks at 304, 289, 282 and 257 nm and troughs at 285, 277 and 265 nm respectively. The enzyme activity and structure showed greater stability at pH 4.8 than at pH 7.0, were highly sensitive to alkaline pH but less sensitive to acid pH values. The enzyme retained most of its catalytic activity and structure even on partial removal of carbohydrate moieties by periodate treatment but was less stable at higher temperatures and storage at 30‡C. Reduction of the periodate treated enzyme did not reverse the loss of stability. Binding of the synthetic substrate,p-nitrophenyl-α-D-glucoside, perturbed the environment around aromatic amino acids and caused a decrease in the ordered structure.

    • Interaction of rose bengal with mung bean aspartate transcarbamylase

      P V Prasad N Appaji Rao

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      The fluorescein dye, rose bengal in the dark: (i) inhibited the activity of mung bean aspartate transcarbamylase (EC 2.1.3.2) in a non-competitive manner, when aspartate was the varied substrate; (ii) induced a lag in the time course of reaction and this hysteresis was abolished upon preincubation with carbamyl phosphate; and (iii) converted the multiple bands observed on polyacrylamide gel electrophoresis of enzyme into a single band. The binding of the dye to the enzyme induced a red shift in the visible spectrum of dye suggesting that it was probably interacting at a hydrophobic region in the enzyme. The dye, in the presence of light, inactivated the enzyme and the inactivation was not dependent on pH. All the effects of the dye could be reversed by UMP, an allosteric inhibitor of the enzyme. The loss of enzyme activity on photoinactivation and the partial protection afforded by N-phosphonoacetyl-L-aspartate, a transition state analog and carbamyl phosphate plus succinate, a competitive inhibitor for aspartate, as well as the reversal of the dye difference spectrum by N-phosphonoacetyl-L-aspartate suggested that in the mung bean aspartate transcarbamylase, unlike in the case ofEscherichia coli enzyme, the active and allosteric sites may be located close to each other.

    • Studies on the conformations of sialyloligosaccharides and implications

      K Veluraja V S R Rao

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      Theoretical investigations, using semi-empirical potential functions have been carried out to predict the favoured conformations of the terminal dissaccharide fragments of various sialyloligosaccharides. The proposed conformational similarity for these fragments has been correlated to the binding specificity of neuraminidases. These calculations predict that bacterial neuraminidases have a binding site which can accommodate only two sugar residues and virus neuraminidases have a binding site which can accommodate more than two sugar residues.

    • Location of valinomycin in lipid vesicles

      M B Sankaram K R K Easwaran

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      The location of the cyclododecadepsipeptide, valinomycin in vesicles formed from two synthetic lipids is studied by differential scanning calorimetry, spin-label partitioning electron paramagnetic resonance and [1H]-nuclear magnetic resonance. The results show that valinomycin is located near the head group region of dipalmitoyl phosphatidyl choline vesicles and in the hydrophobic core of the dimyristoyl phosphatidyl choline vesicles in the liquid crystalline phase.

    • Purification of rabbit liver phosphofructokinase and its properties under simulatingin vivo conditions

      Ganta Roman Reddy Abburi Ramaiah

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      Phosphofructokinase (EC 2.7.1.11) from rabbit liver was purified to homogeneity. Preincubation of enzyme results in nonlinearity of enzyme activity with enzyme concentration. Therefore K0.5 of enzyme for fructose 6 phosphate in the absence or presence of fructose 2,6 bisphosphate or polyethylene glycol or in the presence of both was determined at physiological concentrations of its various effectors by taking the initial rate obtained by adding the enzyme last. They decrease the K0.5 value from 4.1 mM to about 0.2mM. The K0.5 of enzyme for fructose 2,6 bisphosphate was also determined under the above conditions. It is about 4.3ΜM. Transient kinetics of phosphofructokinase at varying concentrations of enzyme in the presence of fructose 2,6 bisphosphate or polyethylene glycol or in the presence of both were studied. It was found that although they decrease t1/2i.e. the time to reach half the maximal steady rate by about 5–8 fold, it was about constant at varying concentrations of the enzyme. These results indicate that fructose 2,6 bisphosphate and polyethylene glycol decrease K0.5 of the enzyme for fructose 6 phosphate not by associating the enzyme to higher aggregates, but by a different mechanism.

    • Relationship between relative protein value and somein vitro indices of protein quality

      M Raghunath B S Narasinga Rao

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      Besides amino acid composition of a protein, their bioavailability is an important determinant of the protein quality. In view of the observations over the last decade or two, implicating the small peptide uptake by the mammalian intestine as a major route of protein absorption, a few animal and plant proteins were subjected to sequential enzymatic digestionin vitro with pepsin, pancreatin + trypsin and erepsin and the release of amino acids as small (including dipeptides) and large peptides and free amino acids, was determined. The relative protein values of α-lactalbumin, egg whites, casein, gluten, zein and protein isolates of soyabeans and groundnuts was determined using rat growth method. It was observed that relative protein value were positively correlated with the essential amino acid index of protein, quantity of essential amino acids released as small peptides and the dipeptide content of enzymatic digests, while there was a negative correlation between relative protein value and essential amino acid content of large peptide fraction.

    • An overview

      P R Mahadevan

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    • Immunological unresponsiveness in leprosy and its relevance to immunoregulation in man

      Barry R Bloom Vijay Mehra

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      The varied forms of leprosy form a clinical and immunological spectrum which offers extraordinary possibilities for insight into immunoregulatory mechanisms in man. At one pole, tuberculoid leprosy, patients develop high levels of cell-mediated immunity which ultimately results in killing of bacilli in the tissues, albeit often with damage to nerves. At the lepromatous pole, patients exhibit selective immunological unresponsiveness to antigens ofMycobacterium leprae. Even though all currently known protein species ofMycobacterium leprae and BCG are cross-reactive, lepromatous patients unreactive toMycobacterium leprae antigens frequently respond strongly to tuberculin.In vitro experiments suggest the existence of lepromin-induced suppressor activity, mediated by both monocytes andT cells. TheT suppressor cells have the T8 phenotype of which 50% express the activation markers,Ia and FcR. The one unique species of antigen of the leprosy bacillus is a phenolic glycolipid, and it appears that theTs cells largely recognize the terminal trisaccharide of this unique antigen. Depletion ofTs cells restoresin vitro reactivity of lymphocytes to lepromin in a portion of lepromatous patients, and addition of IL-2 containing supernatants partially restores responsiveness toMycobacterium leprae antigens. Vaccination of lepromatous patients with a mixture ofMycobacterium leprae and live BCG restores cell-mediated immunity in the majority of lepromatous patients, and concomitantly reduces thein vitro suppressor activity and number of activated T8 cells.

      These experiments suggest the existence of stage-of-disease related suppressor cells in leprosy which appear to block the responsiveness ofTH capable of responding to either specific or cross-reactive mycobacterial antigens. The mode of action of theseTs appears to be the inhibition of production of IL-2 and other lymphokines. Successful immunotherapeutic vaccination appears to overcome this block in the majority of patients.

    • Mycobacterium leprae — The outer lipoidal surface

      Patrick J Brennan

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      There is now a considerable body of evidence to suggest that the phthiocerol-containing lipids, including the phenolic glycolipids, comprise the so-called “peribacillary substance”, “spherical droplets”, “foamy structures” and “capsular materials” ofMycobacterium leprae. Thus, the phthiocerol-containing lipid capsule may be directly responsible for the intracellular survival ofMycobacterium leprae.

    • Immunodiagnosis of bancroftian filariasis—Problems and progress

      Bhaskar C Harinath

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      The immunodiagnosis of bancroftian filariasis is a major challenge to the immunoparasitologist. Significant progress is yet to be made in developing convenient laboratory animal model and inin vitro cultivation of filarial parasites making it very difficult to obtain required amount of parasite material for research. Parasitological examination techniques are not useful in low microfilaraemia, occult or chronic.filarial infections. A precise and accurate immunodiagnostic technique is very much needed for successful filaria control programmes. Such a test will also avoid the need for laborious night blood examination in bancroftian filariasis.

      Due to comparatively easy availability, a good amount of work has been done to explore immunodiagnostic potential of heterologous filarial antigens isolated fromLitomosoide carinii, Dirofilaria immitis, Brugia malayi, Setaria digitata, Setaria cervi and number of other filarial species. However, there has been limited or no significant success due to number of false negative and false positive reactions.

      Extensive study has also been made with antigens isolated fromWuchereria bancrofti microfilariae. Soluble antigens of microfilariae have been used in different immunological techniques such as skin test, counter immuno electrophoresis, indirect haemagglutination test, indirect fluorescent antibody test and enzyme linked immunosorbent assay. Fractionation of

      Wuchereria bancrofti microfilarial soluble antigens yielded mfS3e antigen fraction which was found to be highly reactive in microfilaraemia by enzyme linked immunosorbent assay, but the yield of the purified antigen was not sufficient enough to make it a practical proposition for large scale isolation of antigen.

      Wuchereria bancrofti microfilarial excretory-secretory antigens were found to be specific and highly sensitive requiring as little as 0.35 ng antigen protein per well in penicillinase enzyme linked immunosorbent assay for detection of filarial antibody. One ml of culture fluid was found to be sufficient for 400,000 tests. Field evaluation of this test showed that it can replace laborious night blood examination.

      Assay systems have been developed for detection of filarial antigen in serum, urine, hydrocele fluid and immune complexes using immunoglobulins from chronic filarial sera and antisera to excretory filarial antigens. Further purification of excretory-secretory antigens by affinity chromatography and production of monoclonal antibodies should hopefully give suitable reagents for use in sensitive assays such as enzyme immuno assay and immuno radiometric assay, providing an ideal assay system for detection of active filarial infection in the not too distant future.

    • Role of infection in the pathogenesis of rheumatic diseases

      A N Chandrasekaran Radha Madhavan

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      Advanced immunological technology has revealed immunological abnormalities not only in some chronic and autoimmune connective tissue disorders but also in conditions like infective arthritis where infection apparently seems to play the only role. On the other hand role of infection in the pathogenesis of some connective tissue disorders has recently gained much importance from the observation of clinical, pathological and immunological similarities between these diseases and certain infectious diseases occurring in animal models. Meanwhile, knowledge gained into human leucocyte-A system and its association with certain diseases opens another angle in etiopathogenesis of certain rheumatic diseases. It has been postulated that adaptive mechanism of a microbe or the binding between the human leucocyte-A molecule and carbohydrate moiety of a microbe may set up an autoimmune reaction and in the presence of some triggering factors in the environment may lead on to disease manifestations. An attempt has been made to discuss the role of infection in the outcome of rheumatic diseases such as septic arthritis, polyarteritis nodosa, rheumatic fever, enteropathic arthritis, ankylosing spondylitis, rheumatoid arthritis and systemic lupus erythematoses in genetically susceptible individuals producing immunological abnormalities.

    • In vitro drug screening system using membrane alteration in macrophages byMycobacterium leprae

      M V Mankar R Jagannathan P R Mahadevan

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      The observation that liveMycobacterium leprae on entry into macrophages from lepromatous leprosy patients reduced the number of EA rosetting macrophages, was extended to macrophages from Swiss white mice also. Further, the fact that deadMycobacterium leprae do not bring about such a change in macrophages from mice, allowed us to develop this into a bacterial viability testing system. Thus drug treated macrophages in the presence ofMycobacterium leprae showed normal rosetting ability ifMycobacterium leprae are inactivated by the drug, but showed reduced level of rosetting when bacteria were not susceptible to the drug. It was shown that a drug like dapsone, does act onMycobacterium leprae based on its permeability, quantity available inside the macrophages and inhibition of its action by Para amino benzoic acid. The inactivation ofMycobacterium leprae by sulphone and rifampicin was also proved by the flourescence diacetate method, which showed poorly viable bacteria after exposure to drugs. Thus it has been possible to develop a rapid drug screening method for testing the activity of unknown compound againstMycobacterium leprae.

    • Monoclonal antibodies againstWuchereria bancrofti microfilarial excretory-secretory antigens

      M V R Reddy W F Piessens B C Harinath

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      A battery of monoclonal antibodies were produced againstWuchereria bancrofti microfilarial excretory-secretory antigens and their specificity was studied using different filarial antigens. Among the 1116 wells plated out, 42 % of the wells developed hybrids and 5 % of the hybrids showed antiWuchereria bancrofti microfilarial excretory-secretory antigens. Specificity studies on the antibodies produced from 63 cloned and expanded hybrids showed 10 clones which were specifically positive only toWuchereria bancrofti microfilarial excretory-secretory antigens.

    • Humoral immune response to filarial antigens in chyluria

      Alamelu Raja Rabia Hussain R Prabhakar P R Narayanan

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      Humoral immune parameters like total immunoglobulins and specific antibody levels in serum were studied in filarial chyluria patients. Mean serum IgG was significantly reduced in this group compared to normal controls, while IgA and IgM levels remained comparable to controls. Anti-filarial antibody titre as measured by enzyme-linked immunosorbent assay also was significantly reduced. However, the total and specific IgE antibody titre was similar to that of controls. Specific IgE contents of the patients’ sera could be related to their microfilaraemic status.

    • Characterization of a toxin fromParthenium hysterophorus and its mode of excretion in animals

      T R Narasimhan B S Keshava Murthy N Harindranath P V Subba Rao

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      Fractionation of methanolic extracts of air dried aerial parts ofParthenium resulted in the isolation of a toxic constituent which was identified as parthenin, the major sesquiterpene lactone from the weed. The LD50 (minimal lethal dose required to cause 50% mortality) for parthenin in rats was 42 mg/kg body weight. When [3H]-parthenin was given orally or by intravenous administration, radioactivity appeared in the milk of lactating laboratory and dairy animals. Tissue distribution of radioactivity revealed that maximum label was detectable in kidneys.

    • Chemical synthesis, cloning and expression of human preproinsulin gene

      Saran A Narang

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      A 355 base pair DNA sequence coding for human preproinsulin has been assembled by joining 55 synthetic deoxyoligonucleotide fragments prepared by the modified phosphotriester methodology. Proinsulin was expressed underlac promoter control and truncatedΒ-galactosidase 590 amino acid long sequence. The fusedΒ-galactosidase proinsulin protein was produced in amount to 30 % of the totalEscherichia coli proteins. It was also expressed in M13 bacteriophage and yeast system.

    • Modified bases in transfer RNA

      Vani Brahmachari T Ramakrishnan

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      Transfer RNA is uniquely enriched with modified bases. A large body of information has accumulated about the occurrence, nature and distribution of modified bases in tRNA. But similar investigations on the enzymes involved in this post-transcriptional modification have been hampered by the instability of the enzymes and lack of suitable substrates. The present review summarises briefly, the occurrence and methods of detection of modified bases, the enzymes involved in their formation and also certain suggestive evidence for the role of modification in cellular metabolism.

    • Sodium requirement and metabolism in nitrogen-fixing cyanobacteria

      Joseph Thomas Shree Kumar Apte

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      Sodium affects the metabolism of eukaryotes and prokaryotes in several ways. This review collates information on the effects of Na+ on the metabolism of cyanobacteria with emphasis on the N2,fixing filamentous species. Na+ is required for nitrogenase activity inAnabaena torulosa, Anabaena L-31 andPlectonema boryanum. The features of this requirement have been mainly studied inAnabaena torulosa. The need for Na+ is specific and cannot be replaced by K+, Li+, Ca 2 + or Mg2+. Processes crucial for expression of nitrogenase such as molybdenum uptake, protection of the enzyme from oxygen inactivation and conformational activation of the enzyme are not affected by Na+. Mo-Fe protein and Fe protein, the two components of nitrogenase are synthesized in the absence of Na+ but the enzyme complex is catalytically inactive. Photoevolution of O2 and CO2 fixation, which are severely inhibited in the absence of Na+, are quickly restored by glutamine or glutamate indicating that Na+ deprivation affects photosynthesis indirectly due to deficiency in the products of N2 fixation. Na+ deprivation decreases phosphate uptake, nucleoside phosphate pool and nitrogenase activity. These effects are reversed by the addition of Na+ suggesting that a limitation of available ATP caused by reduced phosphate uptake results in loss of nitrogenase activity during Na+ starvation.

      Na+ influx inAnabaena torulosa andAnabaena L-31 is unaffected by low K+ concentration, is carrier mediated, follows Michaelis-Menten kinetics and is modulated mainly by membrane potential. Treatments which cause membrane depolarisation and hyperpolarisation inhibit and enhance Na+ influx respectively. These cyanobacteria exhibit rapid active efflux of Na+, in a manner different from the Na+/H+ antiporter mechanism found inAnacystis nidulans.

      Na+ requirement in nitrogen metabolism including nitrate assimilation, synthesis of amino acids and proteins, in respiration and oxidative phosphorylation, in transport of sugars and amino acids, cellular distribution of absorbed sodium, physiological basis of salt tolerance and prospects of reclamation of saline soils by cyanobacteria are the other aspects discussed in this review.

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