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Biomedical subjects

S F Yang

Publications and source records attributed to S F Yang.

At least 37 records · Page 2Linked to original sources

Differential induction of seven 1-aminocyclopropane-1-carboxylate synthase genes by elicitor in suspension cultures of tomato (Lycopersicon esculentum).

The key enzyme of ethylene biosynthesis, ACC synthase, is encoded by a multigene family. We describe three new DNA sequences encoding members of the ACC synthase family of the tomato. One of these sequences encodes a novel ACC synthase, LE-ACS6, which is phylogenetically related to the ACC synthases LE-ACS1A and LE-ACS1B. Gene-specific probes for seven tomato ACC synthase genes were prepared. They were used for RNase protection assays to study the accumulation of ACC synthase transcripts in suspension-cultured tomato cells after the addition of an elicitor. The ACC synthase genes LE-ACS2, LE-ACS5 and LE-ACS6 were strongly induced by the elicitor. In contrast, the genes LE-ACS1B, LE-ACS3 and LE-ACS4 were constitutively expressed and LE-ACS1B was present at all times at a particularly high level. Thus, there are two groups of ACC synthase transcripts expressed in these cells, either elicitor-induced or constitutive. A transcript of LE-ACS1A was not detected. Despite the presence of LE-ACS1B, LE-ACS2, LE-ACS3, LE-ACS4 and LE-ACS5, there was only little ethylene produced in the absence of the elicitor. Increased ethylene production is usually correlated with the accumulation of ACC synthase transcripts, indicating that ethylene production is controlled via the transcriptional activation of ACC synthase genes. However, the abundance of several ACC synthase mRNAs studied was not strictly correlated with the rate of elicitor-induced ethylene production. Our data provide evidence that the activity of these ACC synthases may not solely be controlled by the transcriptional activation of ACC synthase genes.

Amino Acid Sequence↗

A novel bifunctional fusion enzyme catalyzing ethylene synthesis via 1-aminocyclopropane1-carboxylic acid.

A C terminus truncated soybean 1-aminocyclopropane-1-carboxylic acid (ACC) synthase (466 aa) was fused to an N terminus truncated tomato ACC oxidase (312 aa) to create a 778-amino acid fusion polypeptide. This ACC synthase-ACC oxidase fusion enzyme (ACSO) was expressed in a heterologous prokaryotic Escherichia coli system, which is capable of converting endogenous S-adenosyl-L-methionine (AdoMet) to ethylene. The molecular weight of the fusion enzyme, ACSO, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was 90 +/- 3 kDa. Gel filtration analysis indicates that the native ACSO is oligomeric and is capable of converting exogenously supplied AdoMet to ethylene. The ethylene production rate of ACSO fusion enzyme was determined to be 6.0 nmol h-1 mg-1 under our assaying conditions using the partially purified enzyme extract. In the enzyme reaction mixture, an increase in ethylene production catalyzed by the bifunctional ACSO was accompanied by a decrease in ACC accumulation. Similarly, in E. coli cells, the level of ACC, produced as an intermediate during the sequential reactions from AdoMet to ethylene, was also found to arise earlier than that of ethylene. Because ACSO could produce ethylene from the ubiquitous AdoMet in living cell and the method commonly used to measure gaseous ethylene is simple, fast, and sensitive, we anticipate this bifunctional fusion enzyme to be useful as a reporter and for research in molecular biology, developmental biology, fermentation, and genetic engineering.

Amino Acid Oxidoreductases↗

Characterization of the ferrous ion binding sites of apple 1-aminocyclopropane-1-carboxylate oxidase by site-directed mutagenesis.

The putative ferrous ion binding sites (H177, D179, and H234) of apple fruit 1-aminocyclopropane-1-carboxylate oxidase determined by homology comparisons with enzymes which required non-haem Fe2+ for activity were altered by site-directed mutagenesis. The activities of mutants (H177F, D179H, D179A, H234F, and H234D) were completely lost. SDS-PAGE and western immunoanalysis confirmed that loss of enzyme activity in mutants was not due to impaired enzyme expression. These results strongly suggest that H177, D179, and H234 are the Fe2+ binding site.

Amino Acid Oxidoreductases↗

Effects of N-terminal deletions on 1-aminocyclopropane-1-carboxylate synthase activity.

A series of nested N-terminal deletions were made on the full-length (wt) and C-terminal deleted (Cdel) 1-aminocyclopropane-1-carboxylate synthase cDNAs. These wt and mutant ACC synthases were over-expressed in a heterologous E. coli expression system. It was found that removal of an amino acid region (residues 2-12) from the non-conserved N-termini of wt and Cdel ACC synthases led to a slight increase in both in vivo ACC production and in vitro ACC synthase activity. Further deletion of 11 amino acids through Glu-23 from the N-termini of both wt and Cdel ACC synthases resulted in a substantial reduction in both in vivo ACC production and in vitro enzyme activity. Deletion of an amino acid region, residues 3 through 27, from the N-terminus of ACC synthase abolished enzyme activity completely. Kinetic analysis of a highly purified double-deletion mutant (NCdel-1) of ACC synthase demonstrated that the Km of this mutant is 42 microM, which is much smaller than that of the corresponding Cdel (280 microM) and closer to that of wt (22 microM) reported previously, suggesting a clear effect of the non-conserved N-terminal region on its ACC synthase function.

Amino Acid Sequence↗

Canal debridement: effectiveness of sodium hypochlorite and calcium hydroxide as medicaments.

The action of chemicals such as calcium hydroxide (Ca(OH)2) and sodium hypochlorite (NaOCl) that are used as tissue solvents may be enhanced by prolonged contact. The objective of this study was to determine if sealing Ca(OH)2 and NaOCl into the canal space would improve debridement of both the main canal and areas inaccessible to files. Mesial root canals of 75 freshly extracted mandibular molars were step-back hand-instrumented. Another six molars were controls. Either Ca(OH)2, NaOCl, or no medication was sealed in the canals for 1 or 7 days. Canals were finally irrigated with H2O and prepared for histological evaluation. The cleanliness of main canals and inaccessible areas (isthmi and fins) at the apical, middle, and coronal thirds was examined, scored, and compared by nonparametric statistical analysis. Results showed no significant differences among different groups in either the 1-day or 7-day time intervals in either the main canal or inaccessible areas. Instrumentation combined with NaOCl irrigation alone accounted for the removal of tissue in the main canal. In conclusion, in this system, prolonged contact with Ca(OH)2 and NaOCl was similarly ineffective; neither contributed significantly to canal debridement.

Analysis of Variance↗

Increased risk of Helicobacter pylori associated with birth in wartime and post-war Japan.

BACKGROUND: Helicobacter pylori infection is now widely recognized as a cause of stomach cancer. We assessed trends in H. pylori infection in Japan, a population with high rates of gastric malignancy. METHODS: Using an enzyme-linked immunosorbent assay (ELISA), we tested sera collected between 1980 and 1993 from Tokyo University Hospital patients for anti-H. pylori IgG. Patients ranged in age from 0 to 94 years. Helicobacter pylori prevalence was then assessed for age and/or birth cohort effects. RESULTS: Of 1207 sera, 470 (38.9%) were positive for H. pylori IgG. By univariate analysis, both older age and birth in an earlier decade were associated with an increased risk of infection. Age-specific prevalence of H. pylori by birth cohort suggested increases in infection during the decades from 1900 to 1959, and age-specific decreases since 1960. In multivariate analysis, H. pylori infection increased with age and was most prevalent among those born in the 1940s and 1950s. CONCLUSION: Relative to other birth cohorts, people born in the 1940s and 1950s have a higher prevalence of H. pylori. This increased prevalence of infection among those born in wartime Japan likely attests to the impact of compromised living conditions on acquisition of H. pylori, and may portend continued high rates of gastric cancer in forthcoming years.

Adolescent↗

Analysis of LE-ACS3, a 1-aminocyclopropane-1-carboxylic acid synthase gene expressed during flooding in the roots of tomato plants.

The plant hormone ethylene is produced in response to a variety of environmental stresses. Previous work has shown that flooding or anaerobic stress in the roots of tomato plants caused an increase in the production of the ethylene precursor 1-aminocyclopropane-1-carboxylate (ACC) in the roots, due to flooding-induced activity of ACC synthase (EC 4.4.1.14). RNA was extracted from roots and leaves of tomato plants flooded over a period of 48 h. Blot analysis of these RNAs hybridized with probes for four different ACC synthases revealed that the ACC synthase gene LE-ACS3 is rapidly induced in roots. LE-ACS2 is also induced, but at later times. The genomic clone for LE-ACS3 was isolated and sequenced. At all time points, the probe from the LE-ACS3 coding region hybridized to two bands in the RNA blots. Hybridization using the first and third introns of LE-ACS3 separately as probes indicate that flooding may inhibit processing of the LE-ACS3 transcript. Sequence homology analysis identified three putative cis-acting response elements in the promoter region, corresponding to the anaerobic response element from the maize adh1 promoter, the root-specific expression element from the cauliflower mosaic virus 35S promoter and a recognition element for chloroplast DNA binding factor I from the maize chloroplast ATP synthase promoter.

Amino Acid Sequence↗

Vertical root fracture in nonendodontically treated teeth.

Vertical root fractures have been reported to occur primarily in endodontically treated teeth due to condensation forces and/or with post placement. This study describes 11 Chinese patients with 12 molars that developed vertical root fractures without endodontic or post procedures. These showed characteristics of a true vertical root fracture as confirmed after extraction. Fractured teeth showed a consistent pattern. The majority were severely attrited mandibular molars in males. All had clinically intact crowns with no or minimal restorations.

Aged↗

Anaerobic tissue-dissolving abilities of calcium hydroxide and sodium hypochlorite.

Closed root canals likely have an oxygen-free environment; most bacteria in canals are anaerobic. These bacteria and other debris are difficult to remove. Unknown is tissue dissolution with chemicals under these anaerobic conditions. This study evaluated and compared dissolving properties of calcium hydroxide (Ca(OH)2) and sodium hypochlorite (NaOCl) on bovine pulp tissue in aerobic and anaerobic environments. Sixty bovine pulp specimens were dried, then randomly divided into six groups. Groups A and B were immersed in Ca(OH)2 + water solution, whereas group C and D were in 2.5% NaOCl. Groups E and F (controls) specimens were placed in distilled water. Groups A, C, and E were incubated anaerobically, and groups B, D, and F were incubated under regular atmospheric conditions, all for 7 days. Percentages of weight loss were compared between groups. Results showed the following: (a) both chemicals partially dissolved pulp tissue, (b) anaerobic environment did not alter tissue-dissolving properties of Ca(OH)2 or NaOCl, and (c) Ca(OH)2 and NaOCl were equal and more effective than water.

Anaerobiosis↗

Ribotyping of clinical Vibrio vulnificus isolates.

Restriction fragment length polymorphism analysis of rRNA genes (ribotyping) was used to differentiate Vibrio vulnificus isolates. Among the 10 restriction enzymes tested, HindIII was shown to provide the most discriminatory patterns. Stul was used for further analysis of strains that were indistinguishable with HindIII. Thirteen clinical V. vulnificus strains were analyzed for their ribotypes with HindIII, as well as Stul when necessary. Four of the clinical strains were isolated from different samples collected from the same patient, and were shown to have identical ribotypes. All the others gave unique ribotypes, indicating the large genetic divergence in V. vulnificus clinical isolates. The ribotype of V. vulnificus by HindIII remained unchanged after successive in vitro and in vivo passages. HindIII gave rise to five bands which were shown in every V. vulnificus strain but not in other vibrio species tested, suggesting that ribotyping with this restriction enzyme may be useful for confirming the identification of this bacterium.

Bacterial Typing Techniques↗

Expression of apple 1-aminocyclopropane-1-carboxylate synthase in Escherichia coli: kinetic characterization of wild-type and active-site mutant forms.

The pyridoxal phosphate-dependent enzyme 1-aminocyclopropane-1-carboxylate synthase (ACC synthase; S-adenosyl-L-methionine methylthioadenosine-lyase, EC 4.4.1.14) catalyzes the conversion of S-adenosylmethionine (AdoMet) to ACC and 5'-methylthioadenosine, the committed step in ethylene biosynthesis in plants. Apple ACC synthase was overexpressed in Escherichia coli (3 mg/liter) and purified to near homogeneity. A continuous assay was developed by coupling the ACC synthase reaction to the deamination of 5'-methylthioadenosine by adenosine deaminase (adenosine aminohydrolase, EC 3.5.4.4) from Aspergillus oryzae. The enzyme is dimeric, with kcat = 9s-1 per monomer and Km = 12 microM for AdoMet. The pyridoxal phosphate-binding site of ACC synthase appears to be highly homologous to that of aspartate aminotransferase, suggesting similar roles for corresponding residues. Site-directed mutagenesis of Lys-273, Arg-407, and Tyr-233 (corresponding to residues 258, 386, and 225 in aspartate aminotransferase) and kinetic analyses of the mutants confirms their importance in the ACC synthase mechanism. The Lys-273 to Ala mutant has no detectable activity, supporting the identification of this residue as the base catalyzing C alpha proton abstraction. Mutation of Arg-407 to Lys results in a precipitous drop in kcat/Km and an increase in Km for AdoMet of at least 20-fold, in accordance with its proposed role as principal ligand for the substrate alpha-carboxylate group. Replacement of Tyr-233 with Phe causes a 24-fold increase in the Km for AdoMet and no change in kcat, suggesting that this residue plays a role in orienting the pyridoxal phosphate cofactor in the active site.

Amino Acid Sequence↗

Pulmonary inflammatory cell response to sustained endotoxin administration.

We have developed a model of human sepsis in sheep. Twenty-four hours after continuous infusion of Escherichia coli endotoxin (lipopolysaccharide) (10 ng.kg-1.min-1) was begun, pulmonary transvascular fluid flux was almost five times the baseline values, cardiac output was nearly doubled, and mean arterial pressure was reduced by approximately 20 mmHg. At this time, the animals were killed and their lungs were fixed by endotracheal installation of 2.5% glutaraldehyde at 25 cmH2O pressure. Morphometry was performed by point counting, and data were expressed as relative volume density. Pulmonary edema and congestion were observed in sheep receiving lipopolysaccharide, whereas sham controls appeared normal. There was an increase in interstitial volume density. There was a significant increase (P < 0.01) in volume density of the pulmonary intravasculature (180%), interstitial macrophages (270%), and mast cells (240%). The volume densities of intravascular and interstitial polymorphonuclear neutrophils also showed a small insignificant increase.

Animals↗

Efficacy of cefazolin in promoting ovine tracheal epithelial repair.

Inhalation of toxic materials has been shown injurious to the upper airways. Because secondary infection can slow the repair process, we undertook this study to determine the efficacy of antibiotic therapy in promoting the healing process of acute tracheal wounds. In 18 anesthetized sheep, a portion of the cervical trachea was exposed to smoke from smoldering cotton cooled to 38 degrees C. Nine received the broad-spectrum antibiotic, cefazolin, while none received saline placebo. At 13 days after injury, nonciliated and ciliated cell counts were 75% and 33% respectively of noninjured trachea receiving placebo, while in those receiving cefazolin, nonciliated and ciliated cell counts increased to 93% and 114% of paired noninjured trachea, respectively. We suggest that cefazolin therapy allows cell proliferation and differentiation to proceed more rapidly than observed with placebo therapy and thus may be a helpful augmentor to the healing of an acute airway injury.

Animals↗

Structure and expression of cDNAs encoding 1-aminocyclopropane-1-carboxylate oxidase homologs isolated from excised mung bean hypocotyls.

By screening a mung bean (Vigna radiata L.) hypocotyl cDNA library using a combination of apple (pAE12) and tomato (pTOM13) 1-aminocyclopropane 1-carboxylate (ACC)-oxidase cDNAs as probes, putative ACC-oxidase clones were isolated. Based on restriction-enzyme map and DNA-sequencing analyses, they can be divided into two homology classes, represented by pVR-ACO1 and pVR-ACO2. While pVR-ACO1 and pVR-ACO2 exhibit close homology in their coding regions, their 3'-noncoding regions are divergent. pVR-ACO1 is a 1312-bp full-length clone and contains a single open reading frame encoding 317 amino acids (MW = 35.8 kDa), while pVR-ACO2 is 1172 bp long and is a partial cDNA clone encoding 308 amino acids. These two deduced amino-acid sequences share 83% identity, and display considerable sequence conservation (73-86%) to other ACC oxidases from various plant species. Northern blot analyses of RNAs isolated from hypocotyl, leaf, and stem tissues using gene-specific probes indicate that the pVR-ACO1 transcript is present in all parts of the seedling and that the expression in hypocotyls is further increased following excision. The maximum induction of ACC-oxidase transcripts occurred at about 6 h after excision, while the maximum enzyme activity was observed at 24 h. When excised hypocotyls were treated with ethylene a further enhanced level of transcripts was observed. Aminooxyacetic acid, an inhibitor of ACC-synthase activity, and 2,5-norbornadiene, an inhibitor of ethylene action, suppressed the wound-induced accumulation of ACC-oxidase mRNA, while an addition of ethylene in these tissues restored the accumulation of ACC-oxidase mRNA.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Oxidoreductases↗

Activation of 1-aminocyclopropane-1-carboxylate oxidase by carbon dioxide.

1-Aminocyclopropane-1-carboxylate (ACC) oxidase requires CO2/HCO3- as an essential activator for its activity. Taking advantage that the equilibrium concentrations of CO2 and HCO3- vary with pH and that the interconversions of CO2 and HCO3- are slower at low temperature, we identified CO2 rather than HCO3- as the active species involved in the activation process. Preincubation of the enzyme with a saturating concentration of CO2 resulted in increased activation of the enzyme when preincubation pH was raised, indicating that CO2 reacted with an enzyme group having an alkaline pKa. It is suggested that the CO2 activation of ACC oxidase involves the formation of a carbamate. CO2 increases the Vmax of the reaction but decreases the affinity of the enzyme toward its substrate ACC. A plausible reaction scheme accounting for the CO2 activation process is presented.

Amino Acid Oxidoreductases↗

Microleakage of endodontic temporary restorative materials.

This study compared the sealing of Caviton, Cavit, and IRM at two powder to liquid ratios of 6 g/ml and 2 g/ml. Standard endodontic access preparations were made in 140 noncarious, nonrestored crowns of extracted human molars. They were divided into six groups, including positive and negative controls. Microleakage assessment was evaluated by basic fuchsin dye penetration after thermal cycling (5 to 55 degrees C for 100 cycles). The results indicated that Caviton provided the best seal, followed by Cavit. Cavit demonstrated better sealing than IRM at the powder to liquid ratio of 6 g/ml and 2 g/ml. There was a statistically significant difference in leakage between the Cavit group and IRM (powder to liquid = 6 g/ml), between the Cavit group and IRM (powder to liquid = 2 g/ml) group (p < 0.05). However, no statistically significant difference between the two different powder to liquid ratio groups of IRM was disclosed.

Analysis of Variance↗

Influence of detergent aerosol on lung microvascular permeability.

Noncardiogenic edema fluid often contains high levels of plasma proteins, which may inhibit the function of the lung surfactant complex and thus decrease interstitial hydrostatic pressures. We questioned whether, in the awake and standing animal, displacement of the alveolar surface lining would alter the permeability of the thin and sparsely supported pulmonary capillaries. Sheep prepared with lung lymph fistulae were given an aerosolized detergent (Det) to displace the lung surfactant complex. After the sheep were given Det, protein flux was significantly increased (P < 0.05). To validate the suggested permeability increase, pulmonary vein occluders were surgically implanted and experiments repeated with pulmonary arterial pressures elevated 10 mmHg above baseline. After 2 h of elevated pulmonary arterial pressure, lung lymph increased fivefold. At this time, lymph-to-plasma total protein concentration ratios for air and saline-plus-ethanol vehicle were significantly lower (P < 0.01) than baseline ratios (0.26 +/- 0.06 and 0.34 +/- 0.07, respectively). No significant difference could be shown in lymph-to-plasma ratios after the sheep were given Det. We conclude that disruption of the alveolar lining can cause a detectable increase in protein flux due, in part, to an increase in microvascular permeability.

Aerosols↗

[Postoperative hypoglycemia after pheochromocytoma resection].

A case of severe hypoglycemia (30 mg/dL) after resection of unilateral pheochromocytoma is reported. Consciousness regained after 20 gm dextrose water was given intravenously. Rebound insulin storm is highly suspected as the main mechanism for the development of post-operative hypoglycemia. Administration of alpha and beta adrenergic blockers may also contribute to the severity of the hypoglycemia. Closely monitoring blood sugar level during the perioperative period is the only way to prevent the occurrence of such a catastrophe.

Adrenal Gland Neoplasms↗