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Biomedical subjects

S M Li

Publications and source records attributed to S M Li.

At least 37 records · Page 2Linked to original sources
PubMed2000

Active expression of the ubiA gene from E. coli in tobacco: influence of plant ER-specific signal peptides on the expression of a membrane-bound prenyltransferase in plant cells.

The ubiA gene from E. coli codes for 4-hydroxybenzoate: polyprenyldiphosphate 3-polyprenyltransferase, an integral membrane protein involved in ubiquinone biosynthesis. This prokaryotic membrane protein was stably expressed in tobacco using Agrobacterium tumefaciens-mediated transformation. Transgenic lines containing a direct fusion of the ubiA structural gene to a 35S-derived promoter gave very low enzyme activity levels (average 0.16 pkat/mg). Inclusion of an N-terminal ER-specific signal peptide from a lectin gene from Phaseolus vulgaris resulted in an average activity of 1.08 pkat/mg in the transgenic tobacco lines. The additional inclusion of a C-terminal HDEL tetrapeptide, responsible for the retention of proteins in the endoplasmic reticulum of eukaryotic cells, increased the activity to 18.6 pkat/mg. When the promotor of this construct was changed from the 35S derivative to the recently described very strong plant promoter (ocs)3mas, the activity increased further to 128.6 pkat/mg. The most active tobacco line showed activities of the introduced enzyme which exceeded those of wild-type E. coli (the source of ubiA) by a factor of 1100. These results demonstrate the efficacy of plant ER-specific signal peptides for the active expression of a prokaryotic membrane protein in plants.

Alkyl and Aryl Transferases↗
PubMed2000

Genetic engineering on shikonin biosynthesis: expression of the bacterial ubiA gene in Lithospermum erythrorhizon.

The naphthoquinone pigment shikonin from Lithospermum erythrorhizon Sieb. et Zucc. (Boraginaceae) was the first plant secondary metabolite produced in industrial scale from plant cell cultures. We have now manipulated the biosynthetic pathway leading to shikonin in L. erythrorhizon by introduction of the bacterial gene ubiA. This gene of Escherichia coli encodes 4-hydroxybenzoate-3-polyprenyltransferase, a membrane-bound enzyme that catalyzes a key step in ubiquinone biosynthesis. Using geranyl diphosphate (GPP) as substrate, it is able to catalyze the formation of 3-geranyl-4-hydroxybenzoate (GBA), a principal step of shikonin biosynthesis. The prokaryotic ubiA gene was fused to two signal sequences for targeting of the resulting peptide to the endoplasmic reticulum (ER). Constructs with different constitutive promoters were introduced into L. erythrorhizon using Agrobacterium rhizogenes-mediated transformation. In the resulting hairy root lines, high UbiA enzyme activities could be observed, reaching 133 pkat mg(-1). Expression of ubiA resulted in an accumulation of GBA in an amount exceeding that of the control culture by a factor of 50. However, the ubiA-transformed lines showed only a marginal (average 22%) increase of shikonin production in comparison to the control lines, and there was no significant correlation of UbiA enzyme activity and shikonin accumulation. This suggests that overexpression of ubiA alone is not sufficient to increase shikonin formation, and that further enzymes are involved in the regulation of this pathway.

Alkyl and Aryl Transferases↗
PubMed2000

Identification of the coumermycin A(1) biosynthetic gene cluster of Streptomyces rishiriensis DSM 40489.

The biosynthetic gene cluster of the aminocoumarin antibiotic coumermycin A(1) was cloned by screening of a cosmid library of Streptomyces rishiriensis DSM 40489 with heterologous probes from a dTDP-glucose 4,6-dehydratase gene, involved in deoxysugar biosynthesis, and from the aminocoumarin resistance gyrase gene gyrB(r). Sequence analysis of a 30.8-kb region upstream of gyrB(r) revealed the presence of 28 complete open reading frames (ORFs). Fifteen of the identified ORFs showed, on average, 84% identity to corresponding ORFs in the biosynthetic gene cluster of novobiocin, another aminocoumarin antibiotic. Possible functions of 17 ORFs in the biosynthesis of coumermycin A(1) could be assigned by comparison with sequences in GenBank. Experimental proof for the function of the identified gene cluster was provided by an insertional gene inactivation experiment, which resulted in an abolishment of coumermycin A(1) production.

Aminocoumarins↗
PubMed2000

Identification of the novobiocin biosynthetic gene cluster of Streptomyces spheroides NCIB 11891.

The novobiocin biosynthetic gene cluster from Streptomyces spheroides NCIB 11891 was cloned by using homologous deoxynucleoside diphosphate (dNDP)-glucose 4,6-dehydratase gene fragments as probes. Double-stranded sequencing of 25.6 kb revealed the presence of 23 putative open reading frames (ORFs), including the gene for novobiocin resistance, gyrB(r), and at least 11 further ORFs to which a possible role in novobiocin biosynthesis could be assigned. An insertional inactivation experiment with a dNDP-glucose 4, 6-dehydratase fragment resulted in abolishment of novobiocin production, since biosynthesis of the deoxysugar moiety of novobiocin was blocked. Heterologous expression of a key enzyme of novobiocin biosynthesis, i.e., novobiocic acid synthetase, in Streptomyces lividans TK24 further confirmed the involvement of the analyzed genes in the biosynthesis of the antibiotic.

Amide Synthases↗
PubMed1999

Detection of circulating uroplakin-positive cells in patients with transitional cell carcinoma of the bladder.

PURPOSE: Although transitional cell carcinoma of the bladder (TCC) metastasizes frequently with devastating consequences, no marker has been available to monitor this process. Uroplakins are a group of specific markers for normal urothelium and are continuously expressed by the majority of TCCs. Detection of uroplakin-positive cells in the circulation would be a strong indication of hematogenous dissemination of tumor cells in patients with TCC. MATERIALS AND METHODS: Total RNAs were extracted from peripheral blood of 60 patients with TCC (50 non-metastatic and 10 metastatic) and 10 healthy controls, reverse-transcribed and subjected to polymerase chain reaction amplification (RT-PCR) using oligonucleotide primers of human uroplakin II gene. A uroplakin-expressing human bladder cancer cell line (RT4) was used as a positive control to establish the sensitivity of the RT-PCR assay. RESULTS: We showed that the PCR-amplification of the mRNA encoding uroplakin II (UPII), a 15-kDa urothelium-specific marker, constitutes a highly sensitive and specific assay for detecting 100% of transitional cell carcinoma tissue, and that this assay can detect a single bladder cancer cell in a 5-ml. blood sample. UPII mRNA was detected in the blood samples of 2 patients with metastatic bladder cancer without chemotherapy and 1 out of 8 such patients with chemotherapy, but not in those of 50 non-metastatic patients or normal controls. CONCLUSIONS: Uroplakin II is a highly specific marker for human TCC and the detection of uroplakin II in the peripheral blood is associated with metastatic spread of bladder cancer cells. The specific and sensitive detection of uroplakin II provides a useful adjunct for detecting bladder cancer metastasis, staging, and monitoring chemotherapeutic response.

Biomarkers, Tumor↗
PubMed1999

[Review on transformed cell and tumorigenicity].

OBJECTIVE: To explore the relationship between characteristics of transformed cell and tumorigenicity. METHODS: Documents about transformed cell and tumorigenicity were reviewed in detail. RESULTS: Normal biological characteristics and cell function could be maintained in non-tumorigenic transformed cell, but it was changed markedly in malignant transformed cell. CONCLUSION: Non-tumorigenic transformed cell can be served as a standard cell line to study the function and growth characteristics of normal cell.

Animals↗
PubMed1998

4-Hydroxybenzoate 3-geranyltransferase from Lithospermum erythrorhizon: purification of a plant membrane-bound prenyltransferase.

Geranyldiphosphate:4-hydroxybenzoate 3-geranyltransferase is a regulatory enzyme in the biosynthesis of shikonin, a phytoalexin and pharmaceutical produced by cell cultures of Lithospermum erythrorhizon Sieb. et Zucc. In Linsmaier-Skoog medium, the activity of this enzyme could be enhanced more than 200-fold by addition of methyl jasmonate, and this culture material was used for the solubilization and purification of the enzyme. Of various detergents examined, digitonin was the most suitable for the solubilization of the enzyme. The solubilized enzyme was purified 800-fold by chromatography over diethylaminoethyl (DEAE)-Sephacel, Heparin-Sepharose, Reactive Green 19-Agarose, and Cholic Acid-Agarose. The purified enzyme required magnesium ions as cofactor and was highly specific for geranyldiphosphate (GPP) and 4-hydroxybenzoate (4HB) as substrates. The K(m) values for 4HB and GPP were calculated by the method of Lineweaver and Burk as 18.4 microM and 13.8 microM, respectively.

Acetates↗
PubMed1998

Effects of metallothionein on action potentials of anoxic and reoxygenated papillary muscles of guinea pigs.

AIM: To study anti-arrhythmic effects of metallothionein (MT). METHODS: Standard microelectrode technique was used to study the effects of MT on action potentials (AP) in anoxic and reoxygenated papillary muscles of guinea pigs. RESULTS: MT (0.02 mmol.L-1) had no effects on AP of the normal papillary muscles; when the muscles were exposed to ischemic solution without MT, there was a marked shortening of action potential duration (APD) at 20%, 50%, and 90% of repolarization (APD20, APD50, APD90) from 82 +/- 7 to 37 +/- 7, 131 +/- 35 to 63 +/- 11, and 167 +/- 12 to 100 +/- 19 ms, respectively, (P < 0.01); and an obvious reduction of resting potential (RP), action potential amplitude (APA), and the maximal upstroke velocity of phase 0 (Vmax) from -92 +/- 9 to -63 +/- 12 mV, 135 +/- 13 to 80 +/- 8 mV, and 286 +/- 55 to 164 +/- 42 V.s-1, respectively (P < 0.01). However, in the presence of MT, the AP parameters (RP, APA, and Vmax) changed from -63 +/- 2 to -82 +/- 1 mV, 80 +/- 8 to 104 +/- 25 mV, and 164 +/- 42 to 237 +/- 43 V.s-1, respectively, (P < 0.01), except that APD20, APD50, and APD90 shortened further from 37 +/- 7 to 12 +/- 3, 63 +/- 11 to 28 +/- 7, and 100 +/- 19 to 82 +/- 11 mV, respectively (P < 0.01). MT decreased the incidence of automaticity during reoxygenation from 91% to 33%. CONCLUSION: MT possesses a calcium regulatory property.

Action Potentials↗
PubMed1997

4-Coumaroyl coenzyme A 3-hydroxylase activity from cell cultures of Lithospermum erythrorhizon and its relationship to polyphenol oxidase.

A 4-coumaroyl-CoA 3-hydroxylase activity was purified 4600-fold from cell cultures of Lithospermum erythrorhizon. The enzyme showed a molecular mass of 42,400 +/- 1700 Da in gel chromatography and required ascorbate, NADH, or NADPH as cofactors. 4-Coumaroyl-CoA, 4-coumarate, p-cresol, and several other phenolic substances, but not tyrosine, were accepted as substrates for the hydroxylation. Besides hydroxylase activity, the enzyme showed diphenol oxidase activity. Both activities were inhibited by diethyldithiocarbamate or beta-mercaptoethanol, although at different concentrations. The enzyme showed striking similarity to a 4-coumaroyl-glucose 3-hydroxylase from sweet potato (Ipomoe batatas) roots, which has reportedly been purified to homogeneity and identified as a specific enzyme of chlorogenic acid biosynthesis. Close examination and comparison to a commercially available polyphenol oxidase, however, suggest that the enzyme activities purified from both Lithospermum and sweet potato are polyphenol oxidases rather than specific enzymes of secondary metabolism.

Adenine Nucleotides↗
PubMed1997

Major origin of mutagenicity of chlorinated drinking water in China: humic acid or pollutants.

Since Loper and Glatz (J. Toxicol. Environ. Health, 4:919-938;1978) discovered the presence of the mutagenicity in drinking water after chlorination in 1978, humic acid (HA) has been considered as an important source. But our research results show that only 1/8 of observed direct frameshift mutagenicity in tap water originated from chlorination of HA isolated from raw water. Contamination from industrial waste and human settlement (night soil) are important potential sources of mutagenicity in chlorinated drinking water. The results show that mutagenicity from night soil after chlorination depended upon pH of sample. Production of mutagenicity at pH 6 is ten times of that at pH 8, and decomposition process is necessary condition for mutagenicity production. Season variation of mutagenicity of tap is also presented in the paper.

China↗
PubMed1997

[Effect of qiangji jianli capsule on metabolism of acetylcholine receptor].

OBJECTIVE: To explore the action mechanism of Qiangji Jianli Capsule (QJJLC) treating myasthenia gravis. METHODS: Internal receptor permeation and degradation test were performed. RESULTS: Volume of receptor permeation in the group with QJJLC at 96 hours was 902.50 +/- 37.48 cpm/mg, while that of control group 738.45 +/- 35.41 cpm/mg. Half-life of receptor degradation in treatment group was 52 hours and that of control 38 hours. Their difference was very significant (P < 0.001). CONCLUSION: QJJLC could accelerate permeation of N-aectylcholine receptor to surface of diaphragm cell membrane and decelerate receptor degradation. It could also increase accumulative amounts of functional receptor in unit time and keep dynamic equilibrium of receptor metabolism in pathologic condition.

Animals↗
PubMed1997

[Effect of puerarin on plasma endothelin, renin activity and angiotensin II in patients with acute myocardial infarction].

OBJECTIVE: To study the changes of endothelin (ET), renin activity (RA) and angiotensin II (AT-II) before and after puerarin treatment in patients with acute myocardial infarction (AMI). METHODS: Forty-three patients with AMI were divided into two groups, and were given puerarin and glucose-insulin-kalium (GIK) treatment respectively. Plasma ET, RA and AT-II were measured by radioimmunoassay (RIA) before and after treatment in different phases. RESULTS: It showed that plasma ET and RA, AT-II levels in AMI were higher than those in control group (P < 0.01). ET level was conversely correlated with RA and AT-II (P < 0.01). After treatment with puerarin, plasma levels of ET, RA and AT-II were recovered to normal in 3 days, but these data recovered to nearly normal until 7-14 days in group with GIK treatment. CONCLUSION: Puerarin might play an important role in regulating the imbalance of ET, RA and AT-II of patients with AMI.

Aged↗
PubMed1996

Effects of long-term administration of octreotide in portal vein-stenosed rats.

The hemodynamic effects of long-term administration of octreotide in portal hypertension has not been established. In addition, whether long-term octreotide treatment prevents the development of portosystemic shunts has not yet been evaluated. Hence, the current study was undertaken to evaluate the effects of long-term administration of octreotide in rats with portal vein stenosis. Immediately after portal vein stenosis or sham operation, rats were given either a long-term octreotide administration of 100 micrograms/kg or a placebo every 12 hours by subcutaneous injection for 14 consecutive days. Systemic hemodynamics and regional blood flows, degree of mesenteric-systemic shunts, and plasma glucagon concentrations were measured after the final dose of octreotide or placebo. A fifth group of portal vein-stenosed rats received hemodynamic and plasma glucagon measurements after 1-day octreotide treatment given at 14 days after surgery. Long-term octreotide treatment modified the hyperdynamic circulation without affecting the degree of mesenteric-systemic shunts, and 1-day octreotide treatment decreased portal tributary blood flow without affecting the portal pressure, systemic hemodynamics, and degree of mesenteric-systemic shunts. Plasma glucagon levels were decreased in portal vein-stenosed rats receiving either long-term or 1-day octreodtide compared with rats receiving placebo. In contrast, chronic octreotide treatment did not affect any of the hemodynamic values or plasma glucagon levels in sham-operated rats. In conclusion, long-term administration of octreotide modified in part the development of portal hypertension and hyperdynamic circulation in portal vein-stenosed rats without affecting the degree of mesenteric-systemic shunts.

Analysis of Variance↗
PubMed1996

Genetic engineering of plant secondary metabolism. Accumulation of 4-hydroxybenzoate glucosides as a result of the expression of the bacterial ubiC gene in tobacco.

The ubiC gene of Escherichia coli encodes chorismate pyruvatelyase, an enzyme that converts chorismate into 4-hydroxybenzoate (4HB) and is not normally present in plants. The ubiC gene was expressed in Nicotiana tabacum L. plants under control of a constitutive plant promoter. The gene product was targeted into the plastid by fusing it to the sequence for the chloroplast transit peptide of the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase. Transgenic plants showed high chorismate pyruvate-lyase activity and accumulated 4HB as beta-glucosides, with the glucose attached to either the hydroxy or the carboxyl function of 4HB. The total content of 4HB glucosides was approximately 0.52% of dry weight, which exceeded the content of untransformed plants by at least a factor of 1000. Feeding experiments with [1,7-13C2]shikimic acid unequivocally proved that the 4HB that was formed in the transgenic plants was not derived from the conventional phenylpropanoid pathway but from the newly introduced chorismate pyruvate-lyase reaction.

Amino Acid Sequence↗
PubMed1996

Hyperglucagonaemia in cirrhotic patients and its relationship to the severity of cirrhosis and haemodynamic values.

Plasma glucagon concentrations were measured in 160 cirrhotic patients (Pugh's grade A in 52 patients, Pugh's grade B in 64 patients and Pugh's grade C in 44 patients). These values were compared with plasma glucagon concentrations in 57 age and sex-matched healthy subjects. Systemic and portal haemodynamic measurements, effective renal plasma flow and creatinine clearance were recorded for each patient. Plasma glucagon levels were significantly increased in cirrhotic patients compared with healthy subjects. In addition, plasma glucagon levels were higher in cirrhotic patients with ascites than in those without ascites and were increased in relation to the severity of cirrhosis as assessed by Pugh's score. Multiple linear regression found that only Child-Pugh's score was estimated to be an independent predictor of hyperglucagonaemia in cirrhotic patients. However, in patients with different degrees of oesophageal varices and in patients without oesophageal varices, plasma glucagon concentrations were no different among the different groups of patients, but were still higher than plasma glucagon concentrations in healthy subjects. In contrast, plasma glucagon levels were negatively correlated with mean arterial pressure and systemic vascular resistance. The results of the present study suggest that impairment of liver function plays, in part, a role in increased plasma glucagon levels observed in patients with cirrhosis. In addition, these data support the hypothesis that hyperglucagonaemia may contribute, at least in part, to the pathogenesis of peripheral arterial vasodilatation in cirrhosis with portal hypertension.

Adult↗
PubMed1996

Screening of microorganisms for biodegradation of poly(lactic-acid) and lactic acid-containing polymers.

The ability of some microorganisms to use lactic acid stereocopolymers and copolymers with glycolic acid as sole carbon and energy sources was studied under controlled or natural conditions. First, 14 filamentous fungal strains were tested in liquid cultures, adopting total lactic acid consumption, nitrogen source exhaustion, and maximal biomass production as selection criteria. Two strains of Fusarium moniliforme and one strain of Penicillium roqueforti were able to totally assimilate DL-lactic acid, partially soluble racemic oligomers (MW = 1,000), and the nitrogen source. Only one strain of F. moniliforme was able to grow on a poly(lactic acid)-glycolic acid copolymer (MW = 150,000) after 2 months of incubation at 28 degrees C on synthetic agar medium. Mycelium development was examined by scanning electron microscopy. F. moniliforme filaments were observed to grow not only at the copolymer surface but also through the bulk of the copolymer. In a second approach, plates made of a racemic poly(lactic acid) were buried in the soil before being incubated in petri dishes containing mineral agar medium under controlled conditions. Five strains of different filamentous fungi were isolated, and their ability to assimilate racemic poly(lactic acid) oligomers was tested in liquid cultures.

Biodegradation, Environmental↗
PubMed1996

Structural characterization and hydrolytic degradation of a Zn metal initiated copolymer of L-lactide and epsilon-caprolactone.

A bioresorbable aliphatic polyester was synthesized by bulk copolymerization of a 1/1 M/M L,L-lactide/epsilon-caprolactone mixture using zinc metal as initiator. The actual composition of the copolymer was found to be 1.5/1 as deduced from 1H NMR spectra obtained in DMSO-d6 solutions where higher resolution was obtained as compared with chlorinated solvents. Resonances due to L-lactyl units (L) exhibited triads stereosensitivity, epsilon-oxycaproyl units (C) being sensitive to dyads. Average lengths of both poly(lactic acid) and polycaprolactone sequences were evaluated and showed the presence of rather long PLA blocks. Furthermore, no CLC triad signal was found, suggesting the absence of transesterification rearrangements. 10 x 10 x 2 mm specimens made of the copolymer were allowed to age in isoosmolar pH = 7.4 phosphate buffer at 37 degrees C. Degradation was monitored by various analytical techniques such as SEC, X-ray diffractometry, DSC, and 1H NMR. Data were compared with the behaviour of PCL and PLA homopolymers allowed to age under similar conditions. Crystallinity and composition changes are discussed in terms of preferential degradation in L- and C-containing amorphous domains, crystallized long PLA blocks being much more resistant.

Absorption↗