Volume 15, Issue 4
December 1990, pages 239-456
pp 239-248 December 1990
The genomic organization and chromosomal location of theβ-tubulin isogenes inLeishmania donovani promastigotes has been studied by nucleic acid hybridization techniques using a cloned β-tubulin gene. We have cloned aβ-tubulin gene fragment, 3.3 kbp long, from genomic DNA ofLeishmania donovani using a heterologousβ-tubulin DNA as probe. Restriction maps of this clone have been prepared. It has been estimated that there are approximately 11–15 copies of theβ-tubulin genes per haploid genome. The majority of these isogenes are arranged in a tandem repeat with a length of 3.5 kbp on a single chromosome. In addition a few dispersed gene copies at different chromosomal loci were detected by pulse field gradient gel electrophoresis. Part of the internal coding region of the gene has been sequenced to confirm the identity of theβ-tubulin clone and is found to be nearly identical to that ofLeishmania mexicana amazonensis.
pp 249-259 December 1990
Transcription of the multicopyβ-tubulin locus inLeishmania donovani promastigotes was examined by nucleic acid hybridization techniques. By northern analysis of promastigote RNA multipleβ-tubulin mRNAs were detected. The major species of 2.2 kb RNA is derived from the tandem repeat cluster ofβ-tubulin genes, the other two (2.4 and 2.6 kb) are presumably derived from dispersed genomic loci. Combined S1-nuclease and primer extension mapping experiments demonstrated the presence of a single 5′-terminus with a 35 nucleotide spliced-leader sequence. The 3′-termini are heterogeneous. The development of a nuclear run-on system inLeishmania for studying transcription of individual genes is reported. Active but transient RNA polymerase II activity was observed in this system. Using specific DNA probes for labelled run-on RNA it was shown thatβ-tubulin transcription occurs asymmetrically (i.e., on one strand of the DNA template) in anα-amanitin sensitive manner. The significance of these results for the life cycle of the parasite is discussed.
pp 261-269 December 1990
Rice long repetitive DNA (9–20 kbp) reassociating at Cot 50 M.s was cloned in pBR325. Out of several recombinants (Camr Ampr Tets), only a few were selected randomly for further characterization. The insert size in all these clones was 3–4 kbp. Restriction enzyme analysis showed the absence ofEcoRI andBclI sites, presence of a singlePstI andPvuII site and multiple sites forAluI in 3 clones namely pRLl, pRL7 and pRL10.
TheBamHI-PstI fragment of about 0.4 kbp in the pRL7 insert DNA (pRL7-0.4 kbp) was subcloned in M13mpl8 and partially sequenced using Sanger’s dideoxynucleotide chain termination method. Dot matrix comparison of this sequence with rice rDNA sequences revealed low homology with the 25 S rDNA sequence of rice, however, hybridisation did not indicate any homology.
pp 271-279 December 1990
Lead and mercury inhibited porphyrin biosynthesis significantly in the germinating seeds of bajra (Pennisetum typhoideum). Both 5-aminolevulinic acid dehydratase and porphobilinogen deaminase activities were inhibited by these metals. A comparative study of the inhibition of these two enzymes under invivo andin vitro conditions showed that 5-aminolevulinic acid dehydratase is the major site of action of heavy metals in porphyrin biosynthesis. Further, over-all production of porphyrinsviz., protoporphyrin-IX, Mg-protoporphyrin (ester) and protochlorophyllide was repressed by lead and mercury in both light and dark grown seedlings. Similarly, chlorophylla and chlorophyllb and total chlorophyll contents in dark-grown seedlings were also significantly decreased, suggesting the impairment of chlorophyll biosynthesis by lead and mercury in germinating seedlings.
pp 281-288 December 1990
Spinach chloroplast membranes and aqueous dispersions of their extracted lipids have been studied by spin label (stearic acid) electron spin resonance and carbon-13 nuclear magnetic resonance techniques. Combined with electron microscope studies, first systematic evidence is found for the existence of a dynamic lipid-bilayer structure in the chloroplast membranes.
pp 289-295 December 1990
To elucidate the biochemical basis of impaired skin collagen maturity in pyridoxine-or riboflavin-deficient rats the following two mechanistic possibilities were tested: (i) Reduction in the activity of skin lysyl oxidase (EC 1·4·3·13) which initiates the cross-linking of collagen and (ii) putative rise in homocysteine level leading to neutralization of allysine (α-aminoadipic acid δ-5-semialdehyde)or hydroxyallysine (hydroxy α-aminoadepic acid (δ-semialdehyde) in collagen by the formation of thiazine complexes.
Skin lysyl oxidase activity was not affected in pyridoxine deficiency suggesting that pyridoxal phosphate may not be its cofactor. In riboflavin deficiency, lysyl oxidase activity was not altered in the newly regenerated rat skin but a slight reduction was observed in the skin of 18-day-old rat pups. This could be related to the body weight deficit rather than deficiencyper se. Aldehyde content of purified salt soluble collagen of regenerated skin was significantly reduced in both the deficiencies. A 2 to 4-fold increase in the concentration of skin homocysteine was observed in both the deficiencies. The results suggest that increase in skin homocysteine level may be responsible for the impaired skin collagen maturity in riboflavin or pyridoxine deficiency.
pp 297-303 December 1990
The pectin isolated from the juice of the inflorescence stalk of plantain (Musa sapientum) has been found to show significant hypoglycemic effect both in normoglycemic and alloxan diabetic rats. After its administration at a dose of 20mg/100g body weight, there was increase in the concentration of hepatic glycogen, increased glycogenesis as evident from the increased activity of glycogen synthetase and in normoglycemic rats increased incorporation of labelled glucose into hepatic glycogen. Glycogenolysis and glyconeogenesis were lower as was evident from the decreased activity of glycogen phosphorylase and gluconeogenic enzymes.
pp 305-311 December 1990
The effect of alteration of lysine: arginine ratio of the protein on the aortic glycosaminoglycans and glycoproteins was studied in rats fed cholesterol free and atherogenic diet. The concentration of total glycosaminoglycans and of individual fractions was significantly lower in the aorta in the case of diet with lysine: arginine ratio of 1.0, than the diet with a ratio of 2.0. Rats fed globulin fraction isolated from sesame seeds, which has a lysine: arginine ratio of 0.67 also showed significantly lower concentration of total and individual glycosaminoglycan fractions in the aorta than those fed casein (lysine:arginine ratio 2.0). Concentration of total hexose and fucose in the glycoproteins was also lower in the aorta in the case of lysine: arginine ratio 1.0. These results in the light of previous reports of increase in the aortic glycosaminoglycans in the early stages of atherosclerosis and increase in the total hexose and fucose in the glycoproteins in the atherosclerotic aorta indicate that the antiatherogenic effect of a low lysine: arginine ratio in the protein involves alteration in the aortic glycosaminoglycans and glycoproteins.
pp 313-322 December 1990
Using a bacterial speciesPseudomonas PG-1, evidence has been obtained which indicates that uptake ofn-pentane ton-octane by microbial cells takes place primarily from the gas phase either directly orvia the aqueous phase. Specific growth rate increased along with the increase in substrate concentration but above the alkane concentration of 0.3% by volume, specific growth rate decreased indicating substrate inhibition of growth. In the case of less volatile alkanes,n-nonane andn-decane, substrate transfer is predominantly through substrate solubilization system elaborated by the cells. EDTA, a strong inhibitor of hydrocarbon solubilization by the cells, inhibited growth on these two alkanes but had negligible effect on growth onn-pentane ton-octane.
pp 323-328 December 1990
Polyacrylamide gel electrophoresis of the Japanese quail (Coturnix cotunix japonica) muscle extracts revealed a single lactate dehydrogenase isozyme. A month after surgical unilateral brachiotectomy (denervation) there was significant atrophy of the triceps, biceps and radius ulnar muscles accompanied by the appearance of an additional lactate dehydrogenase isozyme band. This extra band may be the result of the synthesis of a new lactate dehydrogenase isozyme. This new isozyme exhibited a lower affinity for lactate, less sensitivity to urea denaturation and was more thermostable than the lactate dehydrogenase of normal (innervated) quail muscles. Based on these properties, it is suggested that the newly synthesised isozyme of the denervated muscles is LDH-1, (or B4/H4) type. Brachiotectomy also resulted in significant quantitative changes in the total lactate dehydrogenase activity of innervated muscles of the same animal.
pp 329-339 December 1990
Role of heme in mitochondrial biogenesis: Transcriptional and post-transcriptional regulation of the expression of Iso-I-cytochrome C gene during glucose repression-derepression in cells ofSaccharomyces cerevisiae
Exogenous addition of hemin to glucose-repressed cells ofSaccharomyces cerevisiae restores the level of Iso-1-cytochrome C messengers to that observed in derepressed cells.In vitro transcription in isolated nuclei has shown a 4-fold stimulation in the synthesis of Iso-1-cytochrome C messengers in repressed but hemin-treated and derepressed cells compared to the repressed cells. Studies onin vitro transport of RNA from isolated nuclei have revealed that there is a 50% drop in the transport of total RNA from nuclei isolated from repressed but hemin-treated and derepressed cells when compared with the nuclei from repressed cells. However, under these conditions, there is an enhanced transport of translatable RNA. Hybridization analysis of the transported RNA using Iso-1-cytochrome C gene-specific probe has shown that there is preferential transport of Iso-1-cytochrome C messengers in repressed but hemin treated and derepressed cells.
pp 341-350 December 1990
The antigenecity of tryptic fragments of reduced and carboxymethylated chicken riboflavin carrier protein were studied. The tryptic sites of the native riboflavin carrier protein bound to riboflavin were inaccessible. The molecular weight and the elution profile on high performance liquid chromatography (TSK 545 DEAE) were unaltered at an enzyme to substrate ratio of 1:31. However, carboxymethylated riboflavin carrier protein could be cleaved into 3 or 4 fragments at an enzyme to substrate ratio of 1:250 or 1:125. Chromatographic separation of the tryptic fragments on high pressure liquid chromatography (TSK 545 DEAE) revealed the presence of two fragments with different elution profiles but similar molecular weight 26 ±2 kDa. Only one fragment (associated with peak 2) had the ability to displace chicken riboflavin carrier protein in an homologous chicken riboflavin carrier protein radioimmunoassay. Thus, carboxymethylated ribotlavin carrier protein which does not compete with chicken riboflavin carrier protein in the radioimmunoassay, on mild trypsinization generates a fragment which interacts with chicken riboflavin carrier protein in radioimmunoassay.
pp 351-359 December 1990
Serum-stimulated mouse embryo fibroblasts specifically secrete two proteins of molecular weights 48,000 and 26,000. The 48 kDa protein showed affinity to concanavalin A and was precipitated by antibody to plasminogen activator inhibitor. Immunoflowcytometry using anti plasminogen activator inhibitor-1 serum indicate the presence of the 48 kDa protein in quiescent cells; this protein was virtually absent in serum-stimulated cells. The presence of the plasminogen activator inhibitor-1 related protein in quiescent cells and its absence in serum-stimulated cells in combination with the observation on the absence of this protein, in the medium of quiescent cells and its presence in the medium of stimulated cells indicate that the 48 kDa protein was transferred from the cells into the medium upon serum-stimulation. The serum-mediated transfer of plasminogen activator inhibitor-1 from the cells into the medium was inhibited by actinomycin-D suggesting that the transfer process required actinomycin-D sensitive events. Treatment of pre-labelled quiescent cells with medium containing 20% fetal calf serum resulted in the gradual transfer of the labelled 48 kDa protein to the extra cellular matrix. These studies indicate that exposure of quiescent cells to fetal calf serum results in the transfer of plasminogen activator inhibitor-1 from the cells to the growth mediumvia extracellular matrix. The translocation of the protease inhibitor from the cells to the matrix and medium may enable the cellular and possibly the membrane proteases to act on growth factors or their receptors thereby initiating the mitogenic response.
pp 361-376 December 1990
Three fragments,viz., BSA-CNBr1–183, BSA-CNBr184–582, and BSA-T377-582 representing domains I, II + III and III of bovine serum albumin have been isolated and purified. The physicochemical properties have been investigated and compared with their parent albumin molecule. The values of Stokes radii (nm) and intrinsic viscosities (ml/g) have been determined to be 2.36, 3.30; 3.43, 4.36; and 2.40, 3.13 for the fragments BSA-CNBr1-183 BSA-CNBr184-582 and BSA-T377-582 respectively. The acid induced unfolding-refolding transitions of intact albumin and the fragment BSA-T377-582 have been shown to occur in two steps while the fragments BSA-CNBr1-183 and BSA-CNBr184-582 underwent single step transitions. The formation of the acid denatured states of intact albumin, BSA-CNBr1–183 and BSA-CNBr184-582 was accompanied by an increase of about 86, 56 and 44% in the values of intrinsic viscosities respectively. Since all the transitions were reversible, the values of equilibrium constants,KD, were calculated. The analysis of the dependence ofKD on pH indicated that the first transition (N-X) of albumin was caused due to the uptake of about 3 protons by the native albumin. The intermediate state,X, is converted to acid unfolded state,D, by taking up another two protons. A comparision of the results on intact albumin with that of its fragments revealed that the second transition of the fragment BSA-T377–582 and the two single step transitions of the fragment BSA-CNBr1-183 and BSA-CNBr184-582 were much closer to the second transition (X-D) of the intact albumin. The first transition of albumin has been attributed to its domain III represented by the fragment BSA-T377-582.
pp 377-388 December 1990
Trehalase found to be associated with the brush border membrane vesicles and the Ca2+ aggregated basolateral membrane vesicles were purified to homogeneity. They were found to differ in their molecular weight, subunit structure, heal stability, N-terminal residues, amino acid composition and also the active site residues. Chemical modification showed the presence of a histidine and tyrosine at the active site of brush border membrane vesicle trehalase and two histidines at the active site of basolateral membrane vesicle.
pp 389-396 December 1990
Modulations of initial rate kinetics of ADP-induced aggregation of citrated calf platelet-rich plasma by adenosine and ATP were investigated employing a spectrophotometric platelet aggregation assay. The data were analysed according to the tenets of sequential shape-change and interaction model of aggregation. Adenosine and ATP increased the slopes and intercepts of double-reciprocal plots of ADP-aggregation kinetics. Examination of their slope and intercept effects together with their effects individually and in combination, on aggregation rates, suggested that adenosine and ATP acted at multiple, nonoverlapping, sites.
pp 397-408 December 1990
Cathepsin B was purified to an apparent homogeneity from goat brain utilizing the techniques of homogenization, autolysis at pH 4, 30–70% (NH4)2SO4 fractionation, Sephadex G-100 column chromatography, organomercurial afinity chromatography and ion-exchange chromatography on CM-Sephadex C-50. The enzyme had a pH optima of 6 with α-N-benzoyl-D, L-arginine-β-naphthIylamide, benzyloxycarbonyl-arginine-arginme-4-methoxy -β-naphthylamide and azocasein as substrates. TheKm values for the hydrolysis of α-N-benzoyl-D, L-arginine-β-naphthylamide and benzyloxycarbonyl-arginine-arginine-4-methoxy -β-naphthylamide were 2.36 and 0.29 mM respectively in 2.5% dimethylsulphoxide. However, the correspondingKm values for these substrates in 1 % dimethylsulphoxide were 0.51 and 0.09 mM. The enzyme was strongly inhibited by thiol inhibitors and tetrapeptidyl chloromethylketones. Leupeptin inhibited the enzyme competitively withKi value of 12.5 × l0−9M. Dithioerythritol was found to be the most potent activator of this sulfhydryl protease. Molecular weight estimations on sodium dodecyl sulphate-polyacrylamide gel electrophoresis and on analytical Sephadex G-75 column were around 27,000 and 29,000 daltons respectively. Cathepsin B was found to reside in the lysosomes of goat brain. The highest percentage of cathepsin B was in cerebrum. However, the specific activity of the enzyme was maximum in pituitary gland.
pp 409-416 December 1990
The present study was undertaken to investigate the role of calcium ions (Ca2+) in the induction and secretion of the dengue type 2 virus induced cytotoxic factor and the cytotoxin. This was done by using calcium channel blocking drugs such as verapamil, nifedipine or diltiazem hydrochloride. The production of cytotoxic factor was significantly reduced by treatment of dengue type 2 virus infected mice with verapamil. Similarly, a dosedependent inhibition of the secretion of cytotoxic factor was observed, when spleen cells of the virus-primed mice were treatedin vitro with the 3 calcium channel blockers. The production of cytotoxin by macrophages was abrogated by pretreatment with calcium channel blockers but had little effect on its secretion as shown by treatment of macrophages with verapamil at 1 h after the induction to later periods up to 18 h. The findings thus show that in the induction of both the cytokines Ca2+ plays a critical role; on the other hand it is required for the secretion of the cytotoxic factor but not for that of the cytotoxin.
pp 417-425 December 1990
Puromycin is known to be an anti-tumor agent. Evaluation of interaction energy of this molecule with nucleic acid bases and base pairs has been performed using quantum-mechanical perturbation technique. Both in-plane and stacking energies have been evaluated. These energy values along with their sites of association have been compared with the standard values during transcription process. The results have been examined in the light of their biological significance.
pp 427-434 December 1990
The subunits of human placental milli calcium activated neutral proteinase and micro calcium activated neutral proteinase have been separated by partial denaturation with urea followed by molecular sieving, with a recovery of 82–91% of activity. The separated subunits were homogeneous, as judged by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Their molecular sizes, catalytic activities and sulphydryl contents suggest that both the subunits of these two calcium activated neutral proteinases are distinct. The subunits were highly specific and could not be interchanged. Both the subunits of micro calcium activated neutral proteinase were catalytically active whereas only the 80 k subunit of milli calcium activated neutral proteinase was active. 30 k subunit of milli calcium activated neutral proteinase has a regulatory role since maximum activity of the 80 k subunit was elicited only in its presence. Activity of the reassociated subunits indicated that interaction is essential for the expression of optimum activity. Interaction of subunits rendered the enzymes less susceptible to inhibition by endogenous calcium activated neutral proteinase inhibitor.
pp 435-442 December 1990
An endogenous inhibitor of calcium activated neutral proteinase has been purified from human placenta. The procedure included chromatography on DEAE cellulose, Ultrogel AcA 22 and milli calcium activated neutral proteinase-sepharose in succession. Endogenous calcium activated neutral proteinase inhibitor was a tetramer with identical subunits of molecular weight 68 kDa. It was specific for milli calcium activated neutral proteinase (Calpain II) which is inhibited by the formation of an inactive enzyme-inhibitor complex and not by sequestering Ca2+ from the medium. Although micro calcium activated neutral proteinase (Calpain I) was not inhibited by endogenous calcium activated neutral proteinase inhibitor, it was protected from autolysis in the presence of the inhibitor. The placental endogenous calcium activated neutral proteinase inhibitor thus regulates Ca2+ activated proteolysis by ensuring micro calcium activated neutral proteinase activity, while inhibiting milli calcium activated neutral proteinase.
pp 455-456 December 1990