Biochemistry and molecular biology international最新文献

The effects of persimmon extract (Diospyros kaki) and related polyphenols on eukaryotic DNA polymerase alpha were examined. It was found that persimmon extract, epigallocatechin gallate, and epicatechin gallate strongly inhibited the activity of DNA polymerase alpha purified from calf thymus. Among these polyphenols, persimmon extract had the most potent effect on DNA polymerase alpha activity and the concentration of persimmon extract producing 50% inhibition of the activity was 0.191 microM. Persimmon extract showed a weaker effect on DNA polymerase beta and slightly inhibited primase and DNA polymerase I. The inhibition of DNA polymerase alpha by persimmon extract was competitive with the template-primer and noncompetitive with dTTP substrate. The Ki value of DNA polymerase alpha for persimmon extract was estimated to be 70 nM. Moreover, persimmon extract inhibited [3H]thymidine incorporation of human peripheral lymphocyte cells stimulated by PHA.

Seeds of Dolichos lablab var. lignosus (field beans) and variety typicus (lablab beans) contain glucose/mannose specific lectins that have been affinity purified and well characterised (Siva Kumar N., and Rajagopal Rao, D., J.Biosci., 1986, 10, 95-109, (1) Rajasekhar et al., (Biochem.Archives. 1997, 13, 233-240) (2). Purified lectins are glycoproteins with a native molecular mass of 60 kDa and are made of two types of subunits (Gowda et al., 1994, J.Biol.Chem. 269, 18789-18793) (3). Chemical modifications of various groups in purified lectins (using group specific reagents) such as lysine (citraconic anhydride), carboxyl groups (water soluble carbodiimide) tyrosine (N-acetyl imidazole) and tryptophan (2-hydroxy 5-nitro benzylbromide) revealed that 14 out of 21 residues of lysines 7 out of 92 residues of carboxyl groups, 16 out of 24 tyrosine residues and 2 out of 10 tryptophan residues were modified. Lysine and carboxyl group modification led to 95% loss in haemaglutinating activity compared to control while tyrosine and tryptophan modifications led to complete loss of lectin activity. Arginine and histidine modifications led to only 50% loss in activity. The extent of modification and loss in activity was same when the lysine and carboxyl groups were modified in the presence and absence of the inhibitory sugar methyl alpha-D-glucopyranoside at 0.1 M concentration. However protection of modification and lectin activity was observed when the tyrosine and tryptophan residues were modified in the presence of the inhibitory sugar. Earlier CD studies carried out (1) and extensive chemical modification studies reported here substantiate the involvement of tyrosine and tryptophan residues in the sugar binding site of these lectins.

Basic proteins were isolated from purified pea chloroplast nucleoids by acid extraction. Using RP-HPLC, the component composition of the basic proteins was studied. SDS-PAGE of major HPLC-fractions showed that the basic nucleoid proteins are heterogeneous with mol. masses of components from 17 to 30 kDa. One polypeptide with mol. mass of 28 kDa (P28) was obtained by RP-HPLC. The sequencing of three tryptic peptides of P28 (T6, T17, and T19) showed that they are homologous to the ribosomal protein L19 of Saccharomyces cerevisiae. The possible functional role of ribosomal proteins in chloroplast nucleoids is discussed.

Human thrombopoietin (hTPO) variant cDNAs truncated in the C-terminal regions of wild-type hTPO (332 amino acids) were constructed by PCR and expressed in Trichoplusia ni (Tn5) insect cells using a baculovirus expression system. Each variant, hTPO163 (amino acids 1-163), hTPO198 (1-198) and hTPO245 (1-245), was produced in insect cells with very low efficiency in comparison with wild-type hTPO. Immunoblot analysis showed that the predicted 20, 25 and 34 kDa molecular sizes corresponding to hTPO163, hTPO198 and hTPO245, respectively, were barely detected in culture medium and most of the proteins remained within the cell. These results suggest that C-terminal regions containing potential N-glycosylation sites of hTPO are required for the secretion of hTPO into culture medium as well as expression in insect cells.

The sodA gene coding for manganese superoxide dismutase from the marine microorganism Vibrio alginolyticus was cloned, sequenced and over-expressed in Escherichia coli using the pET20b (+) expression vector. The full-length gene was consisted of 603bp open reading frame, which encoded a polypeptide of 201 amino acid residues, with a calculated molecular weight of 22672Da. The deduced amino acid sequence of the sodA showed considerable homology to other Mn-SODs. The recombinant enzyme was efficiently purified from crude E. coli cell lysate by the metal ion affinity chromatography. The recombinant VAMn-SOD resisted thermo-denaturation up to 60 degrees C and was insensitive to inhibitors such as H2O2, NaN3 and diethyldithiocarbamic acid.

The structure of bacteriorhodopsin (bR) has been probed by a large number of experimental methods. In earlier work distance constraints measured from the 1BRD Brookhaven structure (1, 2) were used to guide site-directed mutagenesis/affinity labeling experiments (3-5). In the present study we report on the use of limited molecular dynamics (MD) investigations of the same bR/affinity label system. We show here that the chiral center introduced when 4-bromo-all-trans retinal is synthesized produces variable impact on potential crosslinking. Our MD analysis suggests the following ranking of binding site mutants in order of reactivity: R118C > S118C >> S121C > R141C >> S141C >>> R121C, R138C, S138C. Chirality appears to have limited effect for the M118C mutants but shows more dramatic impact for the T121C and S141C mutants. These results are in excellent agreement with the experimental observations and offer encouragement that MD can be a useful component of experimental design with considerable predictive power.

HeLa cells undergo apoptosis after exposure to cisplatin. Since mitochondria have recently been proposed as a probable effector of this type of cell death, we performed an analysis using the fluorescent cation rhodamine 123, which is transported actively by this organelle. Cisplatin induces a decrease in the mitochondrial staining, as assessed by cytofluorometric analysis. Microscopic analysis demonstrated that this effect was accompanied by damage of the mitochondria. These features were not exclusive of cisplatin, as other antineoplasic agents (taxol, etoposide) elicited similar effects. These results point toward the notion of a general effect of antineoplasic drugs over the mitochondria during induction of apoptotic cell death.

Hypertension is dominantly inherited in cross hybrids between hypertensive SHR/Mol and normotensive BB/OK rats. We used these cross hybrids for repeated backcrossing of selected hypertensive animals onto normotensive BB/OK rats to fix high blood pressure and to generate a hypertensive and diabetic BB/OK rat subline. After 8 backcrosses, the backcross parents were genetically analysed with the aid of 259 microsatellite markers to identify SHR genes causing blood pressure of 177 +/- 10 mmHg in this BB/OK rat subline. Loci on chromosomes 1, 14 and 18 showed longest heterozygosity. These loci might contain major genes of the SHR rat causing hypertension in this BB/OK rat subline. This classical strategy seems to be most suitable to fix major genes of hypertension in particular and complex traits in general and therefore to generate new animal models.

Grapefruit juice sac ATP-PFK was studied kinetically for its substrates ATP and Fru-6-P at pH = 7.5. The Km for ATP is equal to 39.8 +/- 4.6 microM. ATP becomes inhibitory at concentrations above 80 microM. The Km for ATP is not affected by the addition of citrate (10 mM). For Fru-6-P, the saturation curve is sigmoidal, with an S0.5 equal to 0.17 +/- 0.03 mM, in the presence of Mg++ (2.5 mM) and ATP (1 mM). ATP-PFK shows a negative cooperativity at lower concentrations of Fru-6-P (h = 0.5), while higher concentrations of the substrate induce a positive cooperation (h = 1.5). The presence of citrate affects the S0.5 affinity value, but not the Vmax. The presence of citrate (10 mM) removes the cooperative effect at higher concentrations of the substrate, as h = 1.0. A theoretical Ki for citrate was calculated and equals 1.30 mM.

Effects of selenite and selenodiglutathione, an initial metabolite of selenite, on the induction of apoptosis and cytotoxicity were investigated in human promyelocytic leukemia HL-60 cells. Treatment of selenite or selenodiglutathione resulted in concentration-dependent cytotoxicity, measured by lactate dehydrogenase leakage assay, and by tetrazolium salt reduction assay. Selenodiglutathione has been shown to exert more cytotoxic effect than selenite in both assay systems. Time-course study of cellular selenium uptake suggests that the higher cytotoxicity of selenodiglutathione be largely due to faster and greater selenium uptake rate. Treatment with selenite or selenodiglutathione also induced apoptosis in a dose-dependent manner, as detected by enzyme-linked immunosorbent assay and by DNA fragmentation assay. The dose-response data of apoptosis induced by selenite or selenodiglutathione were similar to those of cytotoxicity, implicating a relationship between the induction of apoptosis and cytotoxicity. Zn, which is a well-known inhibitor of apoptosis, dose-dependently blocked not only the induction of apoptosis, but also the membrane damage induced by selenium, corroborating this hypothesis. It was noted that the inhibition of apoptosis by Zn exerted little protective effect on cytotoxicity at higher concentrations of selenium, compared with a perfect protective effect at low concentration of selenium. These results suggest that cytotoxicity induced by selenium may be partially correlated with apoptosis.