Biochemistry and molecular biology international (Biochem Mol Biol Int)

Publications in this journal

• Article: Overexpression and Purification of PreS Region of Hepatitis

  Naaz Abbas, aftab ahmad, A.R. Shakoori
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  ABSTRACT: PreS domain of Hepatitis B virus (HBV) surface antigen is a good candidate for an effective vaccine as it activates both B and T cells besides binding to hepatocytes. This report deals with overexpression and purification of adr subtype of surface antigen that is more prevalent in Pakistan. PreS region, comprising 119 aa preS1 region plus a 55 aa preS2 region plus 11 aa from the N-terminal S region, was inserted in pET21a+ vector, cloned in E. coli DH5α cells and expressed in E. coli BL21 codon+ cells. The conditions for over expression were optimized using different concentrations of IPTG (0.01-5 mM), and incubating the cells at different temperatures (23-41°C) for different durations (0-6 h). The cells were grown under the given optimized conditions (0.5 mM IPTG concentration at 37°C for 4 h), lysed by sonication and the protein was purified by ion exchange chromatography. On the average, 24.5 mg of recombinant protein was purified per liter of culture. The purified protein was later lyophilized and stored at -80°C.
  Biochemistry and molecular biology international 01/2007;
• Article: Total reactive antioxidant potential in human saliva of smokers and non-smokers.

  I Kondakova, E A Lissi, M Pizarro
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  ABSTRACT: Uric acid is the most important non-enzymatic antioxidant present in human saliva. There is a great variability among individuals, both in salivary uric acid content and saliva total reactive antioxidant potential (TRAP). The uric acid present in saliva correlates with plasma uric acid, suggesting that the former is imported from plasma. There are not statistical differences between uric acid or TRAP values in saliva of smokers and non-smokers. Also, smoking a cigarette does not modify the levels of antioxidants present in saliva.
  Biochemistry and molecular biology international 07/1999; 47(6):911-20.
• Article: The lysine and methionine rich basic subunit of buckwheat grain legumin: some results of a structural study.

  M K Rout, N K Chrungoo
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  ABSTRACT: The 26 kD basic subunit of 280 kD buckwheat grain legumin has been partially characterized by measurement of its fluorescence and CD spectra. The protein has 22% alpha-helix, 36% beta-sheet, 12% beta-turn and 30% random coil secondary structure. In comparison with the basic subunits of other legumin-type proteins, the buckwheat legumin subunit has a high content of lysine and methionine. The protein also has higher ratios of lysine to arginine and methionine to arginine.
  Biochemistry and molecular biology international 07/1999; 47(6):921-6.
• Article: Preparation and characterization of beta 1-bungarotoxin bispecific monoclonal antibody.

  C C Yang, H L Chan
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  ABSTRACT: A hybrid hybridoma (tetradoma) that produces bispecific monoclonal antibodies (mAbs)2, which recognize two different epitopes on the A chain of beta 1-bungarotoxin (beta 1-bgt) at peptide sequences 46-51 and 100-106, has been obtained by fusing two hybridoma cell lines. The bispecific mAb were observed to inhibit 98% of the enzymatic activity of beta 1-bgt and neutralize its lethal toxicity completely. The avidity between the bispecific mAb and beta 1-bgt was noted to be 4.5 x 10(10) (liter/nmol), which is about 45-150 folds higher than the avidity of its two parental mAbs. All the soluble complexes, obtained from bispecific mAb and beta 1-bgt with different molar ratios, emerged in the void volume of size exclusion chromatography column, indicating multiple complexes of beta 1-bgt and bispecific mAb were formed. Based on these results, it indicated that the binding of bispecific mAb with its two epitopes on beta 1-bgt, which facilitates the immuno-complex formation and enhances the avidity, also highly neutralizes the biological activity of beta 1-bgt.
  Biochemistry and molecular biology international 07/1999; 47(6):1039-48.
• Article: Denitration of peroxynitrite-treated proteins by "protein nitratases" from dog prostate.

  W N Kuo, R N Kanadia, V P Shanbhag
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  ABSTRACT: Putative "protein nitratases," which catalyze denitration of peroxynitrite (PN)-treated, proteins, were detected in the crude extract of dog prostate. Nitratase activity was monitored by the decreased intensity of nitrotyrosine immunoreactive-bands in Western blot and increased nitrate level in dialysate of incubation mixture, which contained prostate crude extract, protease inhibitors and a PN-treated substrate, such as treated histone (III-S), BSA, invertase, or polylysine. Nitratases were activated by preincubation with m-calpain/Ca2+. Furthermore, after denitration, the activity of PN/DTT-treated invertase decreased to the similar activity level of DTT-treated invertase. At least two different types of nitratases may occur: type I, reductant-dependent, and type II, reductant-independent.
  Biochemistry and molecular biology international 07/1999; 47(6):1061-7.
• Article: Synthetic insulin fragment with insulin-like biological activity.

  V N Prozorovskiy, E M Maksimova, A E Alekseeva, O G Grebenschikova, Abakumova OYu, N G Kutsenko, A S Ivanov, V N Kniazhev, A I Archakov
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  ABSTRACT: An insulin fragment, representing the C-terminal functionally important site of its molecule and responsible for receptor binding, was synthesized. The fragment consists of two peptides: a dipeptide (A 20-21) and an octapeptide (B 19-26), linked with a disulfide bond (A20-B19). The biological activity of the newly synthesized fragment relative to insulin was assayed for the influence on glycogenesis and for the ability to stimulate glucose uptake. Comparative tests for the biological activity of the synthesized fragment and of the intact hormone allowed us to conclude that the fragment has insulin-like properties.
  Biochemistry and molecular biology international 07/1999; 47(6):957-63.
• Article: RAPD profile based genetic characterization of chemotypic variants of Artemisia annua L.

  R S Sangwan, N S Sangwan, D C Jain, S Kumar, S A Ranade
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  ABSTRACT: The annual herbaceous plant, Artemisia annua L., belonging to family Asteraceae, is the natural source of the highly potent antimalarial compound, artemisinin, besides producing valuable essential oil. The plant is at present the sole commercial source for artemisinin production since all the chemical syntheses are non-viable. Therefore, economic and practical considerations dictate that plants with maximum content of artemisinin be found and/or ways to increase their artemisinin content be sought. The key to this selection and breeding is a comprehension of chemical and genetic variability and suitable selection(s) of elites from within the available population. In the present study, RAPD analyses of selected chemotypes from a decade old introduced population in India were carried out using arbitrary primers. The RAPD data clearly indicate the distinction amongst these plants. Further, the detection of highly polymorphic profiles (97 polymorphic markers out of a total of 101 markers) suggests the existence of very high levels of genetic variation in the Indian population despite geographical isolation and opens out a strong possibility of further genetic improvement for superior artemisinin content. UPGMA analyses of RAPD and phytochemical trait data indicate that the wide phytochemical diversity is included within the genetic diversity. These results further support the prospects for selection and breeding of superior artemisinin containing lines.
  Biochemistry and molecular biology international 07/1999; 47(6):935-44.
• Article: Superoxide anion and hydroxyl radical scavenging activities of vegetable extracts measured using electron spin resonance.

  T Kaneyuki, Y Noda, M G Traber, A Mori, L Packer
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  ABSTRACT: Radical scavenging by reconstituted lyophilized powders of water extracts from 16 common vegetables was measured using electron spin resonance (ESR) with 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), hydroxyl radicals, (.OH) or superoxide anion radicals (O2.-), as DMPO-OH or DMPO-OOH spin adducts. On a dry weight basis, eggplant, and red, yellow and green bell pepper extracts showed potent superoxide anion radical scavenging activities (SOD-like activities). Ascorbate oxidase- or heat-treatments, decreased SOD-like activities in bell pepper extracts suggesting that ascorbate accounts for much of their free radical scavenging activity. Eggplant epidermis extract exhibited the most potent hydroxyl radical scavenging and SOD-like activities. Eggplant SOD-like activity did not decrease after ascorbate oxidase treatment, but decreased following ultrafiltration demonstrating that SOD-like activity is partially due to high molecular weight substances. Nasunin, an anthocyanin in eggplant epidermis, showed markedly potent superoxide anion radical scavenging activity, while it inhibited hydroxyl radical generation probably by chelating ferrous ion.
  Biochemistry and molecular biology international 07/1999; 47(6):979-89.
• Article: Purification and partial characterization of a new proteolytic enzyme from the venom of Bothrops moojeni (CAISSACA).

  F Oliveira, V M Rodrigues, M H Borges, A M Soares, A Hamaguchi, J R Giglio, M I Homsi-Brandeburgo
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  ABSTRACT: A basic serine protease which is active on casein and fibrinogen was purified from Bothrops moojeni venom using a single step chromatography on a CM-Sepharose fast flow column. The enzyme, MOO3, was not hemorrhagic and presented only a trace of blood-clotting activity. Synthetic chromogenic substrates (azoacasein and azoalbumin) where not hydrolyzed by MOO3. Using polyacrylamide gel electrophoresis at pH 4.3, MOO3 showed as a single protein band. Using sodium dodecyl sulfate-polyacrylamide electrophoresis, MOO3 behaved as a single-chain protein with an approximate mol. weight of 27,000, both in the presence and absence of beta-mercaptoethanol. Its pI was 7.8 by electrofocusing. The enzyme did not contain neutral carbohydrates and its N-terminal amino acid was alanine. The amino acid composition showed 249 residues/mole, a high content of hydrophilic amino acids and 14 half-cystine residues, which should account for 7 disulfide bonds. The protease cleaved the A-alpha chain faster than the B-beta of bovine fibrinogen and showed no effect on the delta-chain. Specific esterolytic activity of MOO3 on alpha-N-tosyl-l-arginine methyl ester was 29.64 mumol min-1 x mg-1. MOO3 represented 1.42% (w/w) of the initial desiccated venom. Its proteolytic activity was inhibited by beta-mercaptoethanol, leupeptin, phenylmethylsulphonyl fluoride and ethylenediamine tetraacetate.
  Biochemistry and molecular biology international 07/1999; 47(6):1069-77.
• Article: Changes of nuclear membrane fluidity during rat liver regeneration.

  M L Tomassoni, E Albi, M V Magni
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  ABSTRACT: We have previously shown that the nuclear membrane fluidity is affected by lipid composition changes and that is very high, particularly in the hydrophobic core. The aim of this work is to study the modifications of nuclear membrane fluidity in relation to the cell cycle. Since compensatory hepatic growth is an informative and well characterised model for natural cell proliferation, the nuclear membrane fluidity, detected by two fluorescent probes, was studied at various regenerating times, ranging from 0 to 30 hours after partial hepatectomy. At 18 hours after partial hepatectomy the nuclear membrane fluidity increased and at 30 hours the higher values of hydrophobic core fluidity were observed. The behaviour of fluidity was related to the nuclear membrane neutral-sphingomyelinase activity and, then, to the content of sphingomyelin. Therefore, the significant changes of the nuclear membrane fluidity and of the neutral-sphingomyelinase activity found during rat liver regeneration suggested a their likely role in signal transduction pathways implying cell regeneration.
  Biochemistry and molecular biology international 07/1999; 47(6):1049-59.
• Article: The amino acid sequence of ovocleidin 17, a major protein of the avian eggshell calcified layer.

  K Mann, F Siedler
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  ABSTRACT: The amino acid sequence of ovocleidin 17, a major protein of the chicken eggshell calcified layer, contains 142 amino acids including 2 phosphorylated serines. Data base searches show that ovocleidin belongs to a heterogeneous group of proteins consisting of a single C-type lectin domain (CTL). The most similar sequences with an average of 30% identical amino acids were those of pancreatic stone protein (lithostathine) and lectins and anticoagulant proteins from snake venom.
  Biochemistry and molecular biology international 07/1999; 47(6):997-1007.
• Article: Overexpression of human testis antigens in Escherichia coli host cells is influenced by site of expression and the induction temperature.

  D N Mburu, T K Roberts, B Boettcher
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  ABSTRACT: A panel of twenty human testis cDNA clones were expressed in an Escherichia coli expression system and six clones were found to express identifiable fusion polypeptides. Expression was found to be influenced not only by the site of localization of the polypeptide in the host cells, but also by the temperature used for induction. This emphasized the need for cytoplasmic and periplasmic expression of new antigens of unknown properties, as well as the use of temperatures of 30 degrees C or lower. A majority of the expressed polypeptides were mainly in an insoluble form. By reducing the induction temperature to 30 degrees C production of the soluble fraction was further improved.
  Biochemistry and molecular biology international 07/1999; 47(6):1009-18.
• Article: Evidence of carbamoylphosphate induced conformational changes upon binding to human ornithine carbamoyltransferase.

  A De Gregorio, A Risitano, C Capo, C Criniò, R Petruzzelli, A Desideri
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  ABSTRACT: Human liver ornithine carbamoyltransferase undergoes absorbance changes in the UV region upon formation of the carbamoylphosphate-norvaline-enzyme ternary complex. The UV changes are similar in the presence of carbamoylphosphate alone, whilst they are lower in the presence of ornithine or norvaline alone. The extent of the UV changes correlates with the enzyme susceptibility to proteolytic degradation. The free native enzyme is completely and rapidly hydrolyzed by trypsin, whilst it is partially protected upon carbamoylphosphate binding. The extent of protection increases for the carbamoylphosphate-norvaline-enzyme ternary complex. These results strongly suggest that the binding of the first substrate, i.e. carbamoylphosphate, to human ornithine carbamoyltransferase induces a large protein isomerization, which regards the polar domain plus a part of equatorial domain of each subunit.
  Biochemistry and molecular biology international 07/1999; 47(6):965-70.
• Article: Activation of bradykinin B2 receptors increases calcium entry and intracellular mobilization in C9 liver cells.

  J A García-Sáinz, S E Avendaño-Vázquez
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  ABSTRACT: In C9 rat liver cells bradykinin and kallidin increased (approximately 2-fold) the intracellular concentration of calcium, but the B1 agonist, des-Arg9-bradykinin did not. The effect of bradykinin was inhibited by the B2 antagonists, Hoe 140 and N-alpha-adamantaneacetyl-D-Arg-[Hyp3, Thi5,8, D-Phe7]-bradykinin, but not by the B1 antagonist, des-Arg9-[Leu8]-bradykinin. The action of bradykinin was diminished, but not abolished, in medium without calcium. The peptide was able to increase intracellular calcium concentration in cells treated with thapsigargin. Bradykinin action was not observed in cells previously stimulated with this local mediator: however, under the same conditions, angiotensin II induced a clear increase in intracellular calcium concentration. Our data indicate that activation of bradykinin B2 receptors increase intracellular calcium concentrations by inducing both gating of the cation and intracellular mobilization in C9 liver cells. In addition, homologous desensitization was observed.
  Biochemistry and molecular biology international 07/1999; 47(6):927-33.
• Article: Porphyria-induced hepatic porphyrinogen carboxy-lyase inhibitor and its interaction with the active site(s) of the enzyme.

  S B de Catabbi, R W de Calmanovici, C Minutolo, C Aldonatti, L C San Martin de Viale
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  ABSTRACT: Porphyrinogen carboxy-lyase is an enzyme that sequentially decarboxylates uroporphyrinogen III (8-COOH) to yield coproporphyrinogen III (4-COOH). In mammals this enzyme activity is impaired by hexachlorobenzene treatment, through generation of an enzyme inhibitor. The interaction of porphyrinogen carboxy-lyase inhibitor, extracted from the liver of hexachlorobenzene-treated rats, with substrate decarboxylation sites on the enzyme, was studied using four different carboxylated substrates belonging to the isomeric III series of naturally-formed porphyrinogens containing 8-,7-,6- and 5-COOH. Similar inhibitor effects were elicited against all the substrates assayed, with the exception of pentacarboxyporphyrinogen III in which decarboxylation was not inhibited to same extent. Enzyme protection assays in the presence of the different substrates, indicated that each porphyrinogen protects its own decarboxylation from inhibitor action. Preincubation of the inhibitor with normal enzyme increased its inhibitory effect. On the other hand, preincubation of both enzyme and inhibitor with superoxide dismutase or mannitol, did not alter inhibitory activity. Preincubation of the inhibitor with a number of amino acids showed that only arginine and its derivative N alpha-Benzoyl-L-Arginine ethyl ester interact with the inhibitor, noticeably reducing its ability to inhibit porphyrinogen carboxy-lyase. Albumin, histidine, serine, cysteine and imidazol, were unable to quench inhibitor activity. The present results indicate that the inhibitor acts at the binding site of each porphyrinogen. Taking into account that arginine is related to enzyme activity, and that histidine is found at the binding site of the substrates, the results suggest that the inhibitor could bind to arginine residues, blocking the access of substrates to histidine and altering the adequate orientation for decarboxylation by masking the positively charged active site necessary for porphyrinogen binding to the enzyme. In addition an indirect effect of the inhibitor mediated through free radicals could be discarded.
  Biochemistry and molecular biology international 07/1999; 47(6):945-56.
• Article: Stabilization of the T-state of human hemoglobin by proflavine, an antiseptic drug.

  P Ascenzi, M Colasanti, M Fasano, A Bertollini
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  ABSTRACT: The effect of proflavine (3,6-diaminoacridine), an antiseptic drug, on the spectroscopic and oxygen binding properties of ferrous human adult hemoglobin (Hb) has been investigated. Upon binding of proflavine to the nitric oxide derivative of ferrous human adult hemoglobin (HbNO), the X-band EPR spectrum displays the characteristics which have been attributed to the T-state of the ligated tetramer. In parallel, oxygen affinity for the deoxygenated derivative of ferrous human adult Hb decreases in the presence of proflavine. The effect of proflavine on the spectroscopic and ligand binding properties of ferrous human adult Hb is reminiscent that of 2,3-D-glycerate bisphosphate, the physiological modulator of Hb action.
  Biochemistry and molecular biology international 07/1999; 47(6):991-5.
• Article: Expression of the divergent transcription unit containing the yeast PET122 and OXA1 genes.

  S V Marathe, J E McEwen
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  ABSTRACT: The nuclear PET122 gene of S. cerevisiae encodes a mitochondrial-localized protein that activates initiation of translation of the mitochondrial mRNA from the COX3 gene, which encodes subunit III of cytochrome c oxidase. The PET122 locus contains two divergent transcription units: one is involved in expression of PET122 mRNA and the mRNA for an adjacent gene OXA1, which is also required for cytochrome c oxidase biogenesis, and the other is involved in expression of an antisense RNA that is complementary to about two thirds of the PET122 mRNA and an adjacent gene YER152C of unknown function. Steady state levels of OXA1, PET122 sense and PET122 antisense RNAs were measured after growth of yeast cells under catabolite repressing or derepressing conditions, or after deletion of portions of the 5' flanking DNA of the genes. The results reported here indicate that the PET122 and OXA1 genes are unconventional in terms of the control of their transcription. Neither possesses a canonical TATA element and they exhibit no apparent need for native upstream DNA. These results raise the interesting possibility that PET122 and OXA1 transcription is controlled by downstream DNA, perhaps located within the coding regions of the respective genes.
  Biochemistry and molecular biology international 07/1999; 47(6):971-7.
• Article: Purification and characterization of tripeptidyl peptidase I from Dictyostelium discoideum.

  R P Krimper, T H Jones
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  ABSTRACT: A tripeptidyl peptidase I from Dictyostelium discoideum was purified 744-fold to near homogeneity. The enzyme is 214 kDa in size and is composed of two monomers with a M(r) of 107 kDa. It has two pH optima at pH 4.5 and 5.9 and is a serine peptidase with no aminopeptidase or dipeptidyl peptidase activity. The enzyme was relatively specific showing activity on ala-ala-phe-p-nitroaniline but also acted on substrates with proline in the P1 position in contrast to mammalian TPP I. The K(m) values of the enzyme at pH 4.5 for ala-ala-phe-, ala-phe-pro- and ala-ala-pro-p-nitroanilines were 27 microM, 437 microM and 888 microM, respectively. The enzyme is most abundant during the amoeba stage of the life cycle but is present in the early stages of development and may therefore have a dual role in the organism in mobilizing amino acids or in processing specific peptides or proteins.
  Biochemistry and molecular biology international 07/1999; 47(6):1079-88.
• Article: Influence of atropine on carbachol dual effect on Ca2+ mobilization in SH-SY5Y neuroblastoma cells.

  A Oras, J Järv, K E Akerman
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  ABSTRACT: The muscarinic receptor stimulated mobilisation of calcium ions in SH-SY5Y neuroblastoma cells was measured as a function on carbachol and atropine concentrations. The combined application of this pair of muscarinic agonist and antagonist yielded a set of bell-shaped dose-response curves. In the presence of atropine the cell responses were smaller and the up-going phase of these relationships was shifted towards higher agonist concentration, while the down-going phase of these curves was not influenced by the antagonist. These results pointed to a similar mechanism of the receptor inhibition at high carbachol (agonist) concentrations and by atropine (antagonist).
  Biochemistry and molecular biology international 06/1999; 47(5):743-7.
• Article: Noncompetitive, Ca(2+)-independent inhibition of pyruvate dehydrogenase phosphatase by fluphenazine.

  C I Bak, J W Huh, S Hong, B J Song
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  ABSTRACT: The effects of two different classes of calmodulin antagonists on the catalytic activities of purified pyruvate dehydrogenase (PDH) phosphatase and PDH complex (PDC) were studied. In general, PDH phosphatase was more strongly inhibited than PDC by the calmodulin antagonists with the following potency order: fluphenazine > chlorpromazine > thioridazine > triflupromazine. Promazine and two sulfonamides (W-5 and W-7) did not suppress PDH phosphatase activity at 1 mM concentrations, while about 20% of PDC activity was inhibited by these antagonists. Fluphenazine-mediated inhibition of PDH phosphatase was observed with the purified PDC as well as intact mitochondria. Although Ca2+ stimulates PDH phosphatase activity, the addition of exogenous Ca2+ did not overcome the inhibition by calmodulin antagonists. These results suggest that the suppression of PDH phosphatase activity is dependent upon the structure of the individual calmodulin antagonist and appears to be Ca(2+)-independent. Kinetic analysis showed a noncompetitive inhibition of PDH phosphatase by fluphenazine, indicating that it binds to different site(s) from the catalytic site of the enzyme.
  Biochemistry and molecular biology international 06/1999; 47(6):1029-37.