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Biomedical subjects

S Hua

Publications and source records attributed to S Hua.

34 records · Page 2Linked to original sources
PubMed1995

Synthesis and interaction of fluorescent thapsigargin derivatives with the sarcoplasmic reticulum ATPase membrane-bound region.

Fluorescent derivatives of thapsigargin (TG) were synthesized by replacing the C8-butanoyl chain with a dansyl (DTG) or eosin (ETG) moiety. DTG and ETG retain the inhibitory effect of TG on the sarcoplasmic reticulum (SR) ATPase, displaying a 2 and 10 microM Ki, respectively. Steady state and lifetime fluorescence measurements are consistent with energy transfer between tryptophanyl residues assigned to the ATPase membrane-bound region and DTG. This phenomenon exhibits saturation behavior, occurs in the presence of DTG concentrations producing ATPase inhibition, and is partially prevented by inhibitory concentrations of TG. Although long range conformational effects of TG binding affect the fluorescence properties of endogenous tryptophans as well as of a fluorescein 5'-isothiocyanate (FITC) label of the ATPase extramembranous region, no significant energy transfer was detected between DTG and the FITC label. It is concluded that the inhibitors partition within the membrane and the binding domain resides within or near the membrane-bound region of the ATPase.

Animals↗
PubMed1995

Hydrophobic surfaces that are hidden in chaperonin Cpn60 can be exposed by formation of assembly-competent monomers or by ionic perturbation of the oligomer.

The oligomeric form (14-mer) of the chaperonin protein, Cpn60 (GroEL) from Eschericia coli, displays restricted hydrophobic surfaces and binds tightly one to two molecules of the fluorescent hydrophobic reporter, 1,1'-bi(4-anilino)naphthalene-5,5'-disulfonic acid (bisANS). The 14-mer is resistant to proteolysis by chymotrypsin, and none of the three sulfhydryl groups/monomer react with 6-iodoacetamidofluorescein. When monomers of Cpn60 that are folded and competent to participate in protein folding are formed by low concentrations of urea (< 2.5 M), the hydrophobic exposure increases to accommodate approximately 14 molecules of bisANS/14-mer, the binding affinity for bisANS decreases, and 1 sulfhydryl group/monomer reacts with 6-iodoacetamidofluorescein. These monomers display limited proteolysis by chymotrypsin at several points within a hydrophobic sequence centered around residue 250 to produce a relatively stable N-terminal fragment (approximately = to 26 kDa) and a partially overlapping C-terminal fragment (approximately = to 44 kDa). The exposure of hydrophobic surfaces is facilitated by ATPMg. Ions increase hydrophobic exposure more effectively than urea without dissociation of Cpn60. For example, subdenaturing concentrations of guanidinium chloride (< or = 0.75 M) or the stabilizing salt, guanidinium sulfate, as well as NaCl or KCl are effective. The trivalent cation, spermidine, induces maximum exposure at 5 mM. The results suggest that hydrophobic surfaces can be involved in stabilizing the oligomer and/or in binding proteins to be folded, and they are consistent with suggestions that amphiphilic structures, presenting hydrophobic surfaces within a charged context, would be particularly effective in binding to Cpn60.

Amino Acid Sequence↗
PubMed1995

Assay and purity control of minocycline by thin-layer chromatography using UV and fluorescence densitometry--a comparison with liquid chromatography.

A thin-layer chromatography (TLC) method using UV and fluoresecence densitometry is described for the assay and purity control of minocycline (MC). With a mobile phase dichloromethane-methanol-water (57:35:8, v/v/v) and a silica gel thin-layer, previously sprayed with 10% m/v sodium edetate adjusted to pH 9.0, 4-epiminocycline and 7-didemethylminocycline were well separated from MC and from each other, 7-monodemethylminocycline and 6-deoxy-6-demethyltetracycline (6-DODMTC) were not separated from each other and were only partially separated from minocycline. 6-DODMTC was selectively determined by fluorescence densitometry, while quantification of other impurities and the assay of MC were performed by UV densitometry. Results obtained with qualitative TLC were compared with those obtained by a liquid chromatography (LC) method using a poly(styrene-divinylbenzene) copolymer stationary phase. The correlation coefficient for TLC and LC results was > 0.999. For TLC the relative standard deviation for the assay of MC at 1.25 mg ml-1 was < 3.0% (n = 4), while for LC it was < 1.0% (n = 4).

Anti-Bacterial Agents↗
PubMed1994

Cysteine 254 can cooperate with active site cysteine 247 in reactivation of 5,5'-dithiobis(2-nitrobenzoic acid)-inactivated rhodanese as determined by site-directed mutagenesis.

Sulfhydryl substitution mutants of rhodanese (thiosulfate:cyanide sulfurtransferase; EC 2.8.1.1) were used to determine whether the 4 cysteine residues in the native structure could cooperate in reactions. The sulfhydryl reactivity of persulfide-containing (ES) rhodanese was not significantly changed when cysteine residues at positions 63, 254, and 263 were replaced by serine, either individually or in combination. However, the sulfhydryl reactivity of persulfide-free (E) rhodanese was enhanced when Cys-254 was mutated. One sulfhydryl group, presumably the active site Cys-247, reacted rapidly in the E forms of these proteins with 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) or 4,4'-dipyridyl disulfide (4-PDS). After reaction with DTNB or 4-PDS, proteins with Cys-254 retained > 95% of their original activities as compared with Ser-254-containing proteins, which retained < 6% of their activities. Cyanide treatment could release thionitrobenzoate from rhodanese-thionitrobenzoate complexes with an approximate 1:1 stoichiometry. After this treatment, only the wild-type and C263S enzymes were fully active. Cyanide-treated rhodanese-thionitrobenzoate complexes of the C254S and C254S/C263S mutants could be fully reactivated using an exogenously added thiol, beta-mercaptoethanol. These results are consistent with the formation of a Cys-247-thiocyano derivative that is inactive but capable of being reactivated by intramolecular transfer of cyanide to Cys-254. In the absence of Cys-254, beta-mercaptoethanol can serve as the transferring sulfhydryl group.

Animals↗
PubMed1994

Phosphorylation of aorta caldesmon by endogenous proteolytic fragments of protein kinase C.

Endogenous caldesmon kinase activity in sheep aorta smooth muscle was purified and characterized. The enzyme was identified as a proteolytic fragment of protein kinase C by cross-reactivity with anti-protein kinase C antibodies, autophosphorylation, substrate specificity and the primary structure of the sites of phosphorylation on caldesmon. The enzyme phosphorylated aorta caldesmon both in native thin filaments and in the isolated state. Up to 2.9 mols of phosphate per mol of caldesmon were transferred. Prolonged incubation of caldesmon with the kinase resulted in phosphorylation of Ser-127, Ser-587, Ser-600, Ser-657, Ser-686, and Ser-726 (numbering corresponds to chicken gizzard caldesmon sequence). Ser-600 and Ser-587 were the major sites of phosphorylation containing more than 30% of phosphate transferred. Phosphorylation did not significantly affect the interaction of caldesmon with Ca(2+)-calmodulin. However, phosphorylation of both intact caldesmon and of its C-terminal fragment (658C), containing residues 658-756, significantly decreased their ability to inhibit acto-heavy meromyosin ATPase. This seems to be partially due to a decrease in the binding of caldesmon and 658C to actin-tropomyosin and partly due to an uncoupling of the binding-inhibition relationship.

Actin Cytoskeleton↗
PubMed1994

Capsid assembly and involved function analysis of twelve core protein mutants of duck hepatitis B virus.

The roles of different regions of the duck hepatitis B virus (DHBV) core protein on viral capsid assembly and related functions were examined. Twelve deletion and insertion mutations which covered 80% of the DHBV C open reading frame were constructed and expressed in Escherichia coli. The N-terminal region (amino acids 3 to 66) of DHBV core protein was important for its tertiary structure and function in E. coli. The expressed core mutants without this region apparently inhibited E. coli growth. The results of transmission electron microscopy of E. coli thin sections, capsid agarose gel, and sucrose gradient sedimentation demonstrated that a few DHBV core mutants with insertion in the N terminus and deletion in the C terminus retained the ability to form core-like particles in E. coli. However, other mutations in most of N-terminal and central regions strongly inhibited the self-assembly ability of DHBV core protein in E. coli. In addition, the mutant with a C-terminal region deletion (amino acids 181 to 228) lost most of the nucleic acid-binding activity of the DHBV core protein.

Amino Acid Sequence↗
PubMed1993

Identification of casein kinase II as a major endogeneous caldesmon kinase in sheep aorta smooth muscle.

A caldesmon kinase activity was detected in an ATP extract of the myofibril-like pellet from sheep aorta. The enzyme was purified 745-fold and was identified as casein kinase II on the basis of molecular size, substrate specificity, and high sensitivity to heparin inhibition. Casein kinase II phosphorylated isolated caldesmon and caldesmon incorporated into native thin filaments, and transferred about 1 mol of phosphate per mol of caldesmon-h. Ser-73 was the main site phosphorylated by casein kinase II in chicken gizzard caldesmon. Phosphorylation of caldesmon reduced its affinity for smooth muscle myosin but had no effect upon the ability of caldesmon to inhibit the ATPase activity of actomyosin.

Animals↗
PubMed1992

Cholinergic differentiation in neurogenic basal forebrain cultures.

To study early events in the central nervous system (CNS) cholinergic development, cells from rat basal forebrain tissue were placed in culture at an age when neurogenesis in vivo is still active [embryonic day (E) 15]. The rapid mortality of these cells in defined medium, with 50% mortality after 5-10 h, was blocked completely by soluble proteins from the olfactory bulb (a basal forebrain target), extending earlier observations (Lambert, Megerian, Garden, and Klein, 1988). Treated cultures were capable of incorporating thymidine into DNA, and most cells incorporating 3H-thymidine (greater than 90%) also stained positive for neurofilament, confirming neuronal proliferation in the supplemented cultures. A small percentage of 3H-thymidine labelled cells were glial fibrillary acidic protein (GFAP) positive, but growth factors that support astroglial proliferation [epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), and insulin-like growth factor (IGF-1)] were not sufficient for neuronal support. After 5 culture days with supplemented medium, almost 50% of the cells showed choline acetyltransferase (ChAT) immunofluorescence. The cholinergic neurons typically formed clusters separate from noncholinergic cells. These mature cultures did not develop if young cultures were treated with aphidicolin to block DNA synthesis. The data show that cultures of very young rat basal forebrain cells can be neurogenic, giving rise to abundant cholinergic neurons, and that early cell proliferation is essential for long-term culture survival.

Acetylcholinesterase↗
PubMed1992

Mass balance strategy for protein sequencing. Application to a protein with an extended DNPNNP repeating motif.

The value of the mass balance strategy is illustrated in the sequence determination of S. aureus V8 protease. Capillary electrophoresis, electrospray mass spectrometry, and high performance tandem mass spectrometry are used as well as proteolysis and Edman degradation. The carboxy terminus is found to contain 17 irregularly repeating units of the triptych motifs NNP and DNP, which provide a challenge to any strategy involving mapping, sequencing, and overlapping of hydrolytic peptides.

Amino Acid Sequence↗
PubMed1991

Gas chromatographic determination of aldicarb and its metabolites in urine.

A method is described for the determination of aldicarb and its metabolites (the sulphoxide and sulphone) in urine by gas chromatography with flame photometric detection (GC-FPD). The sample was concentrated with a column containing activated charcoal and Florisil, and then eluted with dichloromethane-acetone (1:1, v/v). The aldicarb and aldicarb sulphoxide in the eluate solution were oxidized to aldicarb sulphone and the total sulphone concentration was determined by GC-FPD after extraction with dichloromethane and clean-up with an activated charcoal column. The detection limit was 0.0024 mg/l. The mean recoveries from spiked urine in the range 0.04-0.12 mg/l were 90.9%, 86.6%, 92.6% for aldicarb, aldicarb sulphoxide and aldicarb sulphone, respectively.

Aldicarb↗
PubMed1991

Assay and purity control of metacycline by thin-layer chromatography combined with UV and fluorescence densitometry--a comparison with liquid chromatography.

A thin-layer chromatographic (TLC) method involving UV and fluorescence densitometry is described for the assay and purity control of metacycline. With a mobile phase dichloromethane-methanol-water (58:35:7, v/v/v) and a silica gel thin-layer, previously sprayed with 10% sodium edetate solution adjusted to pH 9.0, all the potential impurities of metacycline were well separated from the main component and from each other. Results obtained with UV densitometry (TLC-UV) and fluorescence densitometry (TLC-F) were compared with those obtained by a liquid chromatography (LC) method using a poly(styrene-divinylbenzene) stationary phase. The correlation coefficients (r) for TLC-UV and LC or TLC-F and LC were better than 0.9999. For TLC-UV the relative standard deviation (RSD) for the assay of the main component was less than 2%, for TLC-F less than 3.0% and for LC less than 1.0%.

Anti-Bacterial Agents↗
PubMed1967

Protein components of bacteriophages lambda and lambda virulent.

Parallel studies have been made of the protein coats of the temperate bacteriophage lambda and of a deletion mutant, lambda virulent. A new method for preparing ghosts of both phages by the action of Cu(++) is described. Protein ghosts of both phages can be dissolved in citrate at pH values below 3, more rapidly in the presence of 8 m urea. Both phages yielded three apparently identical protein components which can be separated by thin-layer gel filtration and thin-layer gel electrophoresis. The protein of molecular weight 47,000 +/- 1,500 represents about 55% of the protein of the ghosts and is therefore likely to be the subunit of the head. The other proteins of molecular weight 30,000 +/- 1,500 and 16,000 +/- 1,500 represent approximately 25% and 20% of the protein, respectively. Amino acid analyses of the ghosts from the two phages have been carried out and show no significant differences. The buoyant density of phage lambda virulent is 0.016 g/ml less than that of lambda. Since no differences have been found in the protein components of the two phages, this indicates that the virulent mutant contains approximately 16% less deoxyribonucleic acid than the temperate phage.

Amino Acids↗
PubMedDate not supplied

Cloning by functional complementation in Trypanosoma brucei.

A procyclic Trypanosoma brucei double-knockout mutant lacking the ornithine decarboxylase (ODC) gene was transfected with a T. brucei genomic library in the expression vector pTSO-HYG4, which utilizes the PARP promoter and replicates extrachromosomally by virtue of a minicircle origin of replication. Transfectants which grew in the absence of exogenous putrescine, the product of the ODC-catalyzed reaction, were obtained at a frequency of 1.6 x 10(-7) and shown to restore ODC protein synthesis and enzymatic activity. Restriction enzyme patterns and Southern blot analysis of plasmids recovered from these cells and propagated in E. coli showed that the inserts contained a single copy of the T. brucei ODC gene. These results demonstrate for the first time the feasibility of identifying novel T. brucei genes by direct complementation of mutant T. brucei cell lines.

Animals↗