Rapid detection of cholera toxin gene of Vibrio cholerae O1 by polymerase chain reaction.
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Cholera Toxin↗
Biomedical subjects
B Yan
Publications and source records attributed to B Yan.
[Prevention and control of tuberculosis and AIDS].
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Acquired Immunodeficiency Syndrome↗
[Detection of rpoB gene mutation in Mycobacterium tuberculosis by PCR "cold" SSCP].
OBJECTIVE: To evaluate the applicability of detection of rpoB gene mutation in M. tuberculosis susceptibility testing. METHODS: 87 M. tuberculosis isolates and 22 sputum specimens from patients with active pulmonary tuberculosis were detected by PCR-SSCP. RESULTS: The sensitivity of PCR for rpoB gene amplification was 100 pg DNA and 5000 organisms. The rpoB gene could be detected in the all isolates tested. In comparison with conventional susceptibility testing methods, the sensitivity and specificity of PCR-"cold" SSCP analysis for detecting rifampin resistance in 87 M. tuberculosis isolates was 89.6% and 100%, respectively. Among 22 smear- and culture-positive sputum specimens, only 1 (4.5%) was positive by PCR, however, 6 (27.3%) of them were positive by nested-PCR. The "cold" SSCP results of these 6 specimens were corresponding to that of the susceptibility testing. CONCLUSIONS: The PCR-"cold" SSCP described here can easily and rapidly detect rifampin resistance of M. tuberculosis. After increasing the primer specificity and amplification sensitivity, the technique might be used for detection of M. tuberculosis rifampin resistance in clinical specimen directly.
Antibiotics, Antitubercular↗
Spectral tuning in bacteriorhodopsin in the absence of counterion and coplanarization effects.
The basis for wavelength regulation in bacteriorhodopsin (BR) and retinylidene proteins in general has been studied for decades but is still only partially understood. Here we report the preparation and spectroscopic characterization of BR analogs aimed at investigating the existence of spectral tuning mechanisms other than the two widely accepted mechanisms, weakened counterion interactions and ring/chain coplanarization. We synthesized two novel retinal analogs containing a saturated 13-14 bond, which interrupts the interaction of the protein counterions with the chromophore conjugation system. Furthermore, one of the analogs has a planar polyene system so that the contribution to the red shift of BR by retinal ring/chain coplanarization is also absent. We incorporated these analogs into bacterioopsin and discovered a sizable amount of red shift, which can be accounted for by interactions between the polar or polarizable groups of the protein and the retinal polyene chain. Our results suggest that the wavelength regulation in BR is achieved by synergistic chromophore/protein interactions including ring/chain coplanarization, excited state stabilization by polar or polarizable protein side chains located along the polyene chain, and weakened counterion interactions near the Schiff base positive charge.
Bacteriorhodopsins↗
Effects of substrate binding and pH on the secondary structure of carnitine acetyltransferase.
Carnitine acetyltransferase (CAT) exists as a monomer in solution as demonstrated by dynamic light scattering measurements. Under these conditions, interactions between CAT and its substrates, L-carnitine and acetyl-CoA, were studied by circular dichroism (CD) and fluorescence spectroscopy over a wide range of substrate concentrations. CD data indicated that the binding of L-carnitine and acetyl-CoA caused changes in the secondary structure of the protein. Quenching of the intrinsic protein fluorescence upon binding of either substrate corroborated these findings. Analysis of the binding data suggests that binding of both substrates to CAT is specific and saturable, and that there is a single binding site (or multiple identical and independent binding sites) on CAT for each substrate. Estimated L-carnitine/CAT dissociation constants were 506 +/- 58 microM and 236 +/- 27 microM in the absence or presence of acetyl-CoA, respectively. The dissociation constant for acetyl-CoA/CAT was estimated at 19 +/- 7 microM. The effect of pH on the secondary structure of the protein was determined in order to investigate the structural cause for the pH-dependent enzymatic activity of CAT. Loss of alpha-helices and a reduction of thermal stability in CAT was detected at both acidic and basic pH. Thus, the reduced catalytic activity of CAT at acidic or basic pH may be due to pH-induced protein unfolding.
Acetyl Coenzyme A↗
Rat serum carboxylesterase. Cloning, expression, regulation, and evidence of secretion from liver.
Multiple forms of carboxylesterase have been identified in rat liver, and five carboxylesterases (designated hydrolases A, B, C, S, and egasyn) have been cloned. Hydrolases A, B, C, and egasyn all have a C-terminal consensus sequence (HXEL) for retaining proteins in the endoplasmic reticulum, and these carboxylesterases are found in rat liver microsomes. In contrast, hydrolase S lacks this C-terminal consensus sequence and is presumed to be secreted. In order to test this hypothesis, a polyclonal antibody was raised against recombinant hydrolase S from cDNA-directed expression in Escherichia coli. In addition to hydrolases A, B, and C (57-59 kDa), this antibody recognized a 67-kDa protein in rat liver microsomes and a 71-kDa protein in rat serum. The 71-kDa protein detected in rat serum was also detected in the extracellular medium from primary cultures of rat hepatocytes. Non-denaturing gel electrophoresis with staining for esterase activity showed that a serum carboxylesterase comigrated with the 71-kDa protein. Immunoprecipitation of the 71-kDa enzyme from rat serum decreased esterase activity toward 1-naphthylacetate and para-nitrophenylacetate. The 71-kDa protein immunoprecipitated from rat serum had an N-terminal amino acid sequence identical to that predicted from the cDNA encoding hydrolase S, providing further evidence that hydrolase S is synthesized in and secreted by the liver. The levels of the 67-kDa protein in rat liver microsomes and the levels of the 71-kDa protein in rat serum were co-regulated. Deglycosylation of microsomes and serum converted the 67- and 71-kDa proteins to a 58-kDa peptide, which matches the molecular mass calculated from the cDNA for hydrolase S. These results suggest that the 67-kDa protein in liver microsomes is a precursor form of hydrolase S that undergoes further glycosylation before being secreted into serum. In rats, liver appears to be the only source of hydrolase S because no mRNA encoding hydrolase S could be detected in several extrahepatic tissues. Serum carboxylesterases have been found to play an important role in lipid metabolism and detoxication of organophosphates, therefore, the secretion of hydrolase S and the modulation of its expression by xenobiotics may have physiological as well as toxicological significance.
Animals↗
Cloning and expression of hydrolase C, a member of the rat carboxylesterase family.
Using polymerase chain reaction (PCR), we have isolated a cDNA that encodes a rat liver carboxylesterase. This novel enzyme, designated hydrolase C, is structurally very similar to hydrolase B, a microsomal carboxylesterase expressed in rat liver and kidney. Hydrolase B and C are 96% identical in nucleotide sequence and 93% identical in deduced amino acid sequence. Both enzymes have an 18-amino-acid signal peptide at the N-terminus. The C-terminus of hydrolase B and C contains an HXEL consensus sequence for retaining proteins in the endoplasmic reticulum. As expected, when the cDNA encoding hydrolase C was expressed in a baculovirus/Sf21 cell system, the recombinant enzyme was localized in the endoplasmic reticulum. Hydrolase B and C both have putative N-linked glycosylation sites at Asn1 and Asn61. The active site of hydrolase B and C appears to be composed of a nucleophile, Ser203, a basic residue, His448, and an acidic residue, either Asp97 or Glu228. Based on cloning experiments, restriction endonuclease mapping and Northern blotting, hydrolase B is expressed in both rat liver and kidney, whereas hydrolase C is expressed predominantly, perhaps exclusively, in liver. When expressed in Escherichia coli, hydrolase C was catalytically inactive and unstable, but when expressed in the baculovirus/Sf21 cell system hydrolase C it was stable and catalytically active toward 1-naphthylacetate and esters of para-nitrophenol. Hydrolase C is the fourth member of the rat carboxylesterase family to be cloned and sequenced. In terms of nucleotide and deduced amino acid sequence, hydrolase C is highly similar to hydrolase B, but differs from hydrolase B in terms of its catalytic activity and tissue distribution. Recombinant hydrolase C has properties similar to those described for esterase RL2, which was purified from rat liver microsomes by Hosokawa et al. (Arch. Biochem. Biophys. 277, 219-227, 1990), although additional studies will be required to establish conclusively the identity of this enzyme. The high degree of sequence identity (96%) between hydrolase B and C, particularly in the 3' untranslated region, suggests that the genes encoding these two carboxylesterases evolved by duplication and divergence of a common ancestral gene.
Amino Acid Sequence↗
Rat testicular carboxylesterase: cloning, cellular localization, and relationship to liver hydrolase A.
We recently purified from rat liver microsomes a carboxylesterase, designated hydrolase A, that catalyzes the hydrolysis of para-nitrophenylacetate with high affinity (Km approximately 25 microM) and is very sensitive to the inhibitory effects of phenylmethylsulfonyl fluoride (PMSF). Based on its catalytic properties, isoelectric point, and N-terminal amino acid sequence, hydrolase A corresponds to the pI 6.1 esterase cloned from a rat liver cDNA library by Robbi et al. (Biochem. J. 269, 451-458, 1990). A PMSF-sensitive esterase with high affinity toward para-nitrophenylacetate is also present in testicular microsomes at levels that slightly exceed those in liver microsomes. Antibody against purified hydrolase A recognizes a 57-kDa protein in both liver and testicular microsomes, suggesting that hydrolase A is expressed to a high degree in both tissues. To determine whether the testicular carboxylesterase is identical to hydrolase A, a rat testicular cDNA library was constructed and screened with antibody against hydrolase A. A 709-bp cDNA was isolated from immunopositive clones. Screening the same cDNA library by polymerase chain reaction (PCR) with one primer based on the sequence of the 709-bp cDNA and one primer based on the sequence of the adjoining lambda gt11 arm yielded a 1.1-kb cDNA that overlapped with the 709 bp-sequence. Together these two cDNA fragments spanned a 1792-bp sequence with an opening reading frame encoding 518 amino acids, which corresponds to approximately 95% of the C-terminal sequence of the liver pI 6.1 esterase (i.e., hydrolase A). Except for four nucleotide differences at positions 479, 855, 1335, and 1350, the sequence of the testicular cDNA was identical to the cDNA sequence of the liver pI 6.1 esterase reported by Robbi et al. None these changes results in an amino acid substitution. However, these four base substitutions were not observed when a cDNA encoding hydrolase A was isolated from a rat liver cDNA library by PCR. These results establish that the same carboxylesterase, namely, hydrolase A, is expressed in rat liver and testis. The levels of mRNA for hydrolase A in various rat tissues was estimated from Northern blots probed with the 709-bp cDNA isolated from the rat testicular cDNA library. A approximately 2-kb mRNA for hydrolase A was detected in liver, testis, lung, and prostate, which confirms the tissue distribution of hydrolase A based on catalytic activity and Western immunoblotting.(ABSTRACT TRUNCATED AT 400 WORDS)
Animals↗
Kinetic, circular dichroism and fluorescence studies on heterologously expressed carnitine palmitoyltransferase II.
Km estimates for carnitine and palmitoyl-CoA of heterologously expressed rat liver carnitine palmitoyl-transferase-II (rCPT-II) were 950 +/- 27 microM and 34 +/- 6 microM, respectively. Vmax for the enzyme was 1.8 mumol/min/mg purified protein. Consistent with an ordered reaction mechanism in which palmitoyl-CoA binds first, SDZ CPI 975, a reversible carnitine palmitoyltransferase inhibitor containing both carnitine and alkyl moieties, inhibited rCPT-II competitively with carnitine and uncompetitively with palmitoyl-CoA. Substrate-enzyme interactions were examined by circular dichroism (CD) and fluorescence. Both carnitine and palmitoyl-CoA alone induced conformational changes in the enzyme; dissociation constant estimates by CD for carnitine and palmitoyl-CoA were 41 +/- 5 microM and 7 +/- 2 microM, respectively.
Animals↗
[Effect of Cladonia alpestris on Trichomonas vaginalis in vitro].
In this paper, an experimental research is reported on the effect of water extract of Cladonia alpestris and S-(-) usnic acid on Trichomonas vaginalis in vitro. The results showed that both the water extract of Cladonia alpestris and S-(-)usnic acid exhibited a strong effect against Trichomonas vaginalis in vitro. As the time of action of the agents was prolonged, the mortality of Trichomonas vaginalis increased. For S-(-) usnic acid, 0.4 mg/ml was the lowest effective concentration against Trichomonas vaginalis in vitro. No remarkable differences were found in the effect of the S-(-)usnic acid and metronidazole at concentrations of 0.4 mg/ml and 0.6 mg/ml.
Animals↗
Advancement of tuberculosis control and research in China.
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China↗
[The detection and significance of HBV-DNA in serum of pulmonary tuberculous patients].
Hepatitis B virus deoxyribonucleic acid (HBV-DNA) in the serum and the liver function from 268 patients with pulmonary tuberculosis were investigated. The patients have been followed up for 3.5 years. In the course of antituberculous chemotherapy, the abnormal liver function rate turned to be 95% in the positive HBV-DNA group but only 10% and 5.1% in the negatives, HBV-DNA group and pulmonary tuberculosis group respectively (P < 0.01, P < 0.01, P > 0.05). The abnormal liver function rate turned to be 49% in the positive HBV-M group but only 5.1% and 10% in the negative control group and pulmonary tuberculosis group (P < 0.01, P < 0.01, P > 0.05) respectively. The presence of HBV-DNA in patients with pulmonary tuberculosis was associated with high mortality rate and the cause of death was related to hepatic failure. The results suggest that to detect serum HBV-DNA is important in pulmonary tuberculous patients. Antiviral therapy for patients with pulmonary tuberculosis who had detectable HBV-DNA may improve prognosis.
Adult↗
Rat kidney carboxylesterase. Cloning, sequencing, cellular localization, and relationship to rat liver hydrolase.
We recently purified from rat liver microsomes a carboxylesterase, designated hydrolase B, that catalyzes the hydrolysis of para-nitrophenylacetate with low affinity (Km approximately 400 microM) and is relatively insensitive to the inhibitory effects of phenylmethylsulfonyl fluoride. A carboxylesterase with identical properties is also present in rat kidney microsomes, at levels comparable to those in liver microsomes. The kidney enzyme is immunochemically indistinguishable from hydrolase B by Western immunoblotting and Ouchterlony double diffusion analysis. This study describes the cloning and sequencing of hydrolase B. A 1809-base pair (bp) cDNA was isolated from a rat kidney cDNA library screened with antibody against hydrolase B. Screening the same cDNA library by two-step polymerase chain reaction with external and internal primers based on the sequence of the 1809-bp cDNA and a primer based on the sequence of the adjoining lambda gt11 arm yielded a 279-bp cDNA that overlapped by 179 bp with the 1809-bp-sequence. Together these two cDNAs spanned a 1909-bp sequence with an opening reading frame encoding 561 amino acids, which includes all 543 amino acid residues in the mature protein plus an 18-amino acid signal peptide at the N terminus. The mature protein encoded by this kidney cDNA matches perfectly the N-terminal amino acid sequence of purified hydrolase B for 30 amino acid residues, as determined by automated Edman degradation. The mature protein contains 5 cysteine residues, two potential N-linked glycosylation sites, and a C-terminal tetrapeptide (His-Asn-Glu-Leu) that matches the HXEL consensus sequence for retaining proteins in the lumen of the endoplasmic reticulum. Based on alignment of conserved amino acid sequences in several mammalian carboxylesterases, and based on the mechanism of catalysis of serine proteases, the catalytic triad in hydrolase B is apparently composed of the nucleophile Ser203, the basic amino acid His448, and the acidic amino acid Asp97 or Glu228. Northern blots probed with the 1809-bp cDNA identified high levels of a approximately 2-kilobase mRNA for hydrolase B in liver and kidney. Little or no mRNA for hydrolase B was detected in testis, lung, prostate, brain, and heart, which confirms the tissue distribution of hydrolase B based on catalytic activity and Western immunoblotting. Immunocytochemical studies established that hydrolase B is localized in the centrilobular region of the liver and in the proximal tubules of the kidney, where it presumably plays a role in the metabolism of xenobiotics and possibly endogenous lipids, although a precise physiological role for hydrolase B remains to be determined.
Amino Acid Sequence↗
Purification and characterization of two rat liver microsomal carboxylesterases (hydrolase A and B).
The enzymatic hydrolysis of para-nitrophenylacetate by rat liver microsomes is predominantly catalyzed by two esterases: one with high affinity (Km approximately 25 microM) and one with low affinity (Km approximately 400 microM) for the substrate. Two kinetically distinct esterases were similarly detected in liver microsomes from mouse, hamster, guinea pig, rabbit, cat, cynomolgus monkey, and human, but only the high-affinity enzyme was detectable in dog liver microsomes. The tissue distribution of these kinetically distinct esterases was examined in rats. High-affinity (Km 20-35 microM esterase activity toward para-nitrophenylacetate was detected in testis, lung, prostate, and pancreas. The activity in testicular microsomes was comparable to that in liver microsomes. Low-affinity (Km 200-700 microM) esterase activity was detected in kidney, small intestine, lung, spleen, heart, and brain. The activity in kidney microsomes was comparable to that in liver microsomes. The high-affinity esterase in testicular and liver microsomes was highly sensitive to the inhibitory effects of phenylmethylsulfonyl fluoride (PMSF), whereas the low-affinity esterase in kidney and liver microsomes was relatively resistant. These results suggested that rat liver microsomes contain two esterases with high activity toward para-nitrophenylacetate, a PMSF-sensitive esterase with high substrate affinity, and a PMSF-insensitive esterase with low substrate affinity. In support of the hypothesis, we have purified and characterized two esterases, designated hydrolases A and B, which appear be the only abundant enzymes in rat liver microsome that rapidly hydrolyze para-nitrophenylacetate. Hydrolase A hydrolyzed para-nitrophenylacetate with high affinity (Km approximately 25 microM), and was inhibited by extremely low concentrations of PMSF (IC50 approximately 100 nM). In contrast, hydrolase B hydrolyzed para-nitrophenylacetate with low affinity (Km approximately 400 microM) and was inhibited only by relatively high concentrations of PMSF (IC50 approximately 100 microM Paraoxon, the active metabolite of parathion, and cresylbenzodioxaphosphorin oxide, the active metabolite tri-ortho-tolylphosphate, completely inhibited the hydrolysis of pra-nitrophenylacetate by rat liver microsomes and by hydrolases A and B, whereas the sulfhydryl agent, para-chloromercurobenzoate, was not inhibition. These results suggest that hydrolases A and B are both serine esterases. The N-terminal amino acid sequence of hydrolases A and B were similar but distinct (23 the first 30 amino acid residues were identical), indicating that these two esterases are isozymes.(ABSTRACT TRUNCATED AT 400 WORDS)
Amidohydrolases↗
Regulation of two rat liver microsomal carboxylesterase isozymes: species differences, tissue distribution, and the effects of age, sex, and xenobiotic treatment of rats.
The preceding paper described the purification of two rat liver microsomal carboxylesterases, designated hydrolases A and B, that have high affinity (Km approximately 25 microM) and low affinity (Km approximately 400 microM) for para-nitrophenylacetate, respectively. The present study describes the preparation and purification of polyclonal antibodies against these purified enzymes. Each antibody was subjected to immunoabsorption chromatography to remove antibodies against epitopes common to both hydrolases A and B. The resulting isozyme-specific antibodies were used to study the regulation of hydrolases A and B by Western immunoblotting and Ouchterlony immunodiffusion. Liver microsomes from mouse, hamster, rabbit, guinea pig, cat, dog, cynomolgus monkey, and humans contained one or more proteins that were immunochemically related and similar in size (M(r) approximately 60 kDa) to hydrolase A and/or hydrolase B. These proteins were preferentially recognized by the antibody against hydrolase A, except for cat liver microsomal esterase, which was preferentially recognized by antibody against hydrolase B. In rats, the levels of hydrolases A and B in liver microsomes were coregulated as a function of age, sex, and xenobiotic treatment of rats. The levels of both enzymes were very low in 1- and 2-week-old rats, but increased abruptly at 3 weeks of age in both male and female rats. Treatment of mature male rats with 11 known microsomal enzyme inducers caused little (< 35%) or no induction of hydrolase A or B, whereas treatment of rats with beta-naphthoflavone, pregnenolone- 16 alpha-carbonitrile or dexamethasone suppressed the levels of both enzymes. The kinetic analysis of para-nitrophenylacetate hydrolysis described in the preceding paper identified a high-affinity esterase (Km 20-35 microM) in rat liver, testis, lung, prostate, and pancreas and identified a low-affinity enzyme (Km 300-800 microM) in liver, kidney, small intestine, lung, brain, spleen, and heart. Immunoblot analysis established that hydrolase A was present in liver, testis, lung, and prostrate at concentrations that accounted for the high-affinity esterase activity in these tissues. Hydrolase A was not detected in the pancreas, even though this tissue contained low levels of a high-affinity esterase. Hydrolase B was detected in liver and kidney at concentrations that accounted for the low-affinity esterase activity in these tissues. Hydrolase B was not detected in the other tissues examined, some of which (e.g., small intestine) contained high levels of a low-affinity esterase. These results indicate that hydrolases A and B are independently expressed in a wide variety of extrahepatic tissues in rats.(ABSTRACT TRUNCATED AT 400 WORDS)
Age Factors↗
Steric constraints in the retinal binding pocket of sensory rhodopsin I.
Steric constraints in the retinal binding pocket of sensory rhodopsin I (SR-I) are analyzed by studying effects of sample temperature and retinal analogs. The flash-induced yield of the earliest detected intermediate S610, which corresponds to the K intermediate in the bacteriorhodopsin (BR) photocycle, decreases below 220 K and reaches zero at 100 K, while K formation is independent of temperature. The reduced S610 formation at low temperatures indicates a more restricted retinal binding pocket in SR-I during primary photochemical events. Introduction of bulky substituents on the retinal polyene chain in four retinal analogs greatly retards or blocks the final step of chromophore binding to the apoprotein of SR-I. Except for the 14-methyl substitution, these modifications exhibit little or no effect on chromophore binding to BR apoprotein. These results corroborate that the retinal polyene chain binding domain in SR-I is more sterically constrained than that of the retinal pocket in BR. Deletion of the beta-ionone ring renders the analog SR-I pigments nonfunctional, as does deletion of the 13-methyl group, but the corresponding BR analogs are both photochemically and physiologically active. In contrast to the corresponding BR analog, photolysis of the analog SR-I reconstituted with 13-desmethylretinal does not produce an S610-like intermediate at room temperature. The above results and the previous findings that protein constraints inhibit the accommodation of a stable 13-cis-retinal configuration in SR-I suggest a model in which the 13-methyl group functions as a fulcrum to permit movement of one or both ends of retinal to overcome an energy barrier against isomerization.
Bacteriorhodopsins↗
A new hypothesis about the relationship between free radical reactions and hemorheological properties in vivo.
This paper is concerned with a hypothesis that disturbance of free radical reactions may lead to abnormality of hemorheological properties in vivo, and so the free radicals generated in vivo may damage certain tissue cells indirectly by reducing the supply of oxygen and nutrients to these cells through slowing the circulation of blood. This hypothesis is based on the following evidence: A. We have found that the whole blood viscosity at low shear rate correlates to the lipid peroxidation in the patients suffering from certain cardio- or cerebrovascular diseases, and in dogs during liver ischemia reperfusion or hemorrhagic pancreatitis. B. Reports have shown that several alterations of hemorheological properties may take place as a result of free radical reactions, such as lipid peroxidation. For instance, lipid peroxidation may lead to decrease of deformability of red cells, increase of aggregation of red cells, formation of liquid thrombin, etc. C. We have demonstrated that some alterations of hemorheological properties involve the role of free radicals in rats suffering from intestinal ischemia/reperfusion. As evidence for this conclusion, superoxide dismutase (SOD) used as a specific scavenger of superoxide anion radical (O2-) can significantly prevent the intestinal ischemia/reperfusion induced changes of lipid peroxidation, red cell aggregation, Cassion's viscosity and whole blood viscosity at low shear rate in rats.
Animals↗
Comment on "Radiative and nonradiative recombination of bound excitons in GaP:N. I. Temperature behavior of zero-phonon line and phonon sidebands of bound excitons" and " Radiative and nonradiative recombination of bound excitons in GaP:N. IV. Formation of phonon sidebands of bound excitons"
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Journal Article↗