Biomedical subjects

W Shi

Publications and source records attributed to W Shi.

At least 19 recordsLinked to original sources

PubMed1999

Expression of bvgAS of Bordetella pertussis represses flagellar biosynthesis of Escherichia coli.

BvgAS is a two-component system of Bordetella pertussis involved in the reciprocal regulation of the virulence genes and the flagellar biosynthesis. In this study, we found that expression of bvgAS in Escherichia coli also results in reduced motility. The repression was relieved by the addition of known chemical modulators of BvgAS such as MgSO4 and nicotinic acid, indicating that functional BvgAS proteins are required for the negative control of E. coli motility. In addition, BvgAS repressed the transcription of the flhDC master operon of E. coli, which consequently caused non-flagellation on the cell surface. However, expression of BvgAS had no effect on stress-resistant motile mutants of E. coli. These data suggest that E. coli may have BvgA-like protein(s) involved in the regulatory interactions between the stress response and the flagellar biosynthesis.

Bacterial Proteins↗

PubMed1999

Outcome for cancer patients requiring mechanical ventilation.

PURPOSE: To describe hospital survival for cancer patients who require mechanical ventilation. MATERIALS AND METHODS: A prospective, multicenter observational study was performed at five academic tertiary care hospitals. Demographic and clinical variables were obtained on consecutive cancer patients at initiation of mechanical ventilation, and information on vital status at hospital discharge was acquired. RESULTS: Our analysis was based on 782 adult cancer patients who met predetermined inclusion criteria. The overall observed hospital mortality was 76%, with no statistically significant differences among the five study centers. Seven variables (intubation after 24 hours, leukemia, progression or recurrence of cancer, allogeneic bone marrow transplantation, cardiac arrhythmias, presence of disseminated intravascular coagulation, and need for vasopressor therapy) were associated with an increased risk of death, whereas prior surgery with curative intent was protective. The predictive model based on these variables had an area under the receiver operating characteristic curve of 0.736, with Hosmer-Lemeshow goodness-of-fit statistics of 7.19; P = .52. CONCLUSION: This model can be used to estimate the probability of hospital survival for classes of adult cancer patients who require mechanical ventilation and can help to guide physicians, patients, and families in deciding goals and direction of treatment. Prospective independent validation in different medical settings is warranted.

Critical Care↗

PubMed1998

KCNQ2 and KCNQ3 potassium channel subunits: molecular correlates of the M-channel.

The M-current regulates the subthreshold electrical excitability of many neurons, determining their firing properties and responsiveness to synaptic input. To date, however, the genes that encode subunits of this important channel have not been identified. The biophysical properties, sensitivity to pharmacological blockade, and expression pattern of the KCNQ2 and KCNQ3 potassium channels were determined. It is concluded that both these subunits contribute to the native M-current.

Adult↗

PubMed1998

Cloning of a mammalian elk potassium channel gene and EAG mRNA distribution in rat sympathetic ganglia.

1. Three new members of the EAG potassium channel gene family were identified in rat and the complete coding sequence of one of these genes (elk1) was determined by cDNA cloning. 2. The elk1 gene, when expressed in Xenopus oocytes, encodes a slowly activating and slowly deactivating potassium channel. 3. The elk1 gene is expressed in sympathetic ganglia and is also expressed in sciatic nerve. 4. Six of the seven known EAG genes were found to be expressed in rat sympathetic ganglia, suggesting an important functional role for these channels in the sympathetic nervous system.

Animals↗

PubMed1998

OCI-5/GPC3, a glypican encoded by a gene that is mutated in the Simpson-Golabi-Behmel overgrowth syndrome, induces apoptosis in a cell line-specific manner.

OCI-5/GPC3 is a member of the glypican family. Glypicans are heparan sulfate proteoglycans that are bound to the cell surface through a glycosyl-phosphatidylinositol anchor. It has recently been shown that the OCI-5/GPC3 gene is mutated in patients with the Simpson-Golabi-Behmel Syndrome (SGBS), an X-linked disorder characterized by pre- and postnatal overgrowth and various visceral and skeletal dysmorphisms. Some of these dysmorphisms could be the result of deficient growth inhibition or apoptosis in certain cell types during development. Here we present evidence indicating that OCI-5/GPC3 induces apoptosis in cell lines derived from mesothelioma (II14) and breast cancer (MCF-7). This induction, however, is cell line specific since it is not observed in NIH 3T3 fibroblasts or HT-29 colorectal tumor cells. We also show that the apoptosis-inducing activity in II14 and MCF-7 cells requires the anchoring of OCI-5/GPC3 to the cell membrane. The glycosaminoglycan chains, on the other hand, are not required. MCF-7 cells can be rescued from OCI-5/GPC3-induced cell death by insulin-like growth factor 2. This factor has been implicated in Beckwith-Wiedemann, an overgrowth syndrome that has many similarities with SGBS. The discovery that OCI-5/GPC3 is able to induce apoptosis in a cell line- specific manner provides an insight into the mechanism that, at least in part, is responsible for the phenotype of SGBS patients.

3T3 Cells↗

PubMed1998

Rhodopsin arginine-135 mutants are phosphorylated by rhodopsin kinase and bind arrestin in the absence of 11-cis-retinal.

Arginine-135, located at the border between the third transmembrane domain and the second cytoplasmic loop of rhodopsin, is one of the most highly conserved amino acids in the family of G protein-coupled receptors. The effect of mutation at Arg-135 on the ability of rhodopsin to undergo desensitization was investigated. Four mutants, R135K, R135Q, R135A, and R135L, were examined for their ability to be phosphorylated by rhodopsin kinase, to bind arrestin, and to activate the rod cell G protein, transducin (Gt). All of the mutants were phosphorylated, bound arrestin, and were able to activate Gt when reconstituted with 11-cis-retinal. Surprisingly, several of the mutants could be phosphorylated by rhodopsin kinase and could bind arrestin in the absence of 11-cis-retinal but were not able to activate Gt. These observations represent the first demonstration of a mutant G protein-coupled receptor that assumes a conformation able to interact with its G protein-coupled receptor kinase and arrestin, but not with its G protein, in the absence of ligand.

Animals↗

PubMed1998

The allosteric regulation of pyruvate kinase by fructose-1,6-bisphosphate.

BACKGROUND: Yeast pyruvate kinase (PK) catalyzes the final step in glycolysis. The enzyme therefore represents an important control point and is allosterically activated by fructose-1,6-bisphosphate (FBP). In mammals the enzyme is found as four different isozymes with different regulatory properties: two of these isozymes are produced by alternate splicing. The allosteric regulation of PK is directly related to proliferation of certain cell types, as demonstrated by the expression of an allosterically regulated isozyme in tumor cells. A model for the allosteric transition from the inactive (T) state to the active (R) state has been proposed previously, but until now the FBP-binding site had not been identified. RESULTS: We report here the structures of PK from yeast complexed with a substrate analog and catalytic metal ions in the presence and absence of bound FBP. The allosteric site is located 40 A from the active site and is entirely located in the enzyme regulatory (C) domain. A phosphate-binding site for the allosteric activator is created by residues encoded by a region of the gene corresponding to the alternately spliced exon of mammalian isozymes. FBP activation appears to induce several conformational changes among active-site sidechains through a mechanism that is most likely to involve significant domain motions, as previously hypothesized. CONCLUSIONS: The structure and location of the allosteric activator site agrees with the pattern of alternate genetic splicing of the PK gene in multicellular eukaryotes that distinguishes between a non-regulated isozyme and the regulated fetal isozymes. The conformational differences observed between the active sites of inactive and fully active PK enzymes is in agreement with the recently determined thermodynamic mechanism of allosteric activation through a 'metal relay' that increases the affinity of the enzyme for its natural phosphoenolpyruvate substrate.

Allosteric Regulation↗

PubMed1998

Polyreactive antigen-binding B cells are the predominant cell type in the newborn B cell repertoire.

Polyreactive antibodies bind to a variety of different self and non-self antigens. The B cells that make these antibodies express the polyreactive lg receptor on their surface. To determine the frequency of polyreactive antigen-binding B cells in peripheral blood, we incubated two different antigens, one (insulin) labeled with fluorescein isothiocyanate and the other (beta-galactosidase) with phycoerythrin, with peripheral B cells. The percentage of cells that bound these antigens was determined with the fluorescence-activated cells sorter. Approximately 21% of adult B cells bound insulin, 28% bound beta-galactosidase, and 11% bound both antigens. In contrast to B cells in the adult repertoire, 49% of B cells in cord blood bound insulin, 54% bound beta-galactosidase, and 33% bound both antigens. The properties of polyreactive antigen-binding B cells in adult and cord blood were similar, except for the fact that almost all the polyreactive antigen-binding B cells in cord blood were CD5 positive (93%), whereas only 40% of the polyreactive antigen-binding B cells in adult peripheral blood were CD5 positive, indicating that the CD5 marker is not directly linked to polyreactivity. The percentage of polyreactive antigen-binding B cells in patients with Sjögren's syndrome, systemic lupus erythematosus and rheumatoid arthritis was equal to or slightly below that found in the normal adult B cell repertoire. It is concluded that polyreactive antigen-binding B cells are a major constituent of the normal adult B cell repertoire and are the predominant cell type in the newborn B cell repertoire.

Adolescent↗

PubMed1998

Differential responses of pulmonary arteries and veins to histamine and 5-HT in lung explants of guinea-pigs.

1. The mechanisms by which histamine and 5-HT differentially contract pulmonary arteries and veins are unclear. In lung explants from 26 guinea-pigs, we compared responses of pulmonary arteries and vein to histamine, 5-HT and KCI, and examined potential determinants for the differential responses. Lungs were filled with agarose, sectioned into approximately 1 mm thick slices, and vascular luminal areas measured by image analysis. 2. Histamine and 5-HT produced a concentration-dependent constriction in arteries and veins, greater in the latter. KCl constricted arteries and veins equally. 3. The histamine H1 antagonist chlorpheniramine (10(-4) M) abolished contractions to histamine; the H2 antagonist cimetidine enhanced maximal responses and sensitivity of arteries and veins to histamine, and diminished the differences between their maximal responses; the NO synthase inhibitor Nomega-nitro-L-arginine (L-NOARG) increased the maximal responses of arteries and veins, and the differences between their responses; indomethacin had no effect. 4. Contractions to 5-HT were abolished in arteries and markedly reduced in veins by the 5-HT2 antagonist ketanserin (10(-4) M); L-NOARG potentiated the maximal responses of arteries but not of veins; indomethacin increased the maximal responses of arteries but reduced them in veins. 5. By morphometry, arteries had a greater medial thickness and luminal diameter than veins. 6. The data suggest that in guinea-pigs, H2 receptors are responsible for the differential contractile responses of pulmonary arteries and veins to histamine, whereas endothelium-derived vasoactive substances are responsible for their differential contractile responses to 5-HT.

Animals↗

PubMed1998

The octreotide suppression test and [111In-DTPA-D-Phe1]-octreotide scintigraphy in neuroendocrine tumours correlate with responsiveness to somatostatin analogue treatment.

OBJECTIVE: The somatostatin analogue octreotide (Sandostatin, Novartis, Basie) significantly improves the syndromes suffered by most patients with neuroendocrine tumours (NETs). The use of [111In-DTPA-D-Phe1]-octreotide scintigraphy ([111In]-pentetreotide) to predict the response to octreotide treatment has been described. Short-term hormone inhibition by a single injection of octreotide has also been reported. This study aimed to compare the effects of the suppression test with the response to long-term somatostatin analogue treatment, and to seek a correlation between the short-term suppression test, [11In]-pentetreotide observations and long-term somatostatin analogue treatment. DESIGN AND MEASUREMENTS: Short octreotide suppression test and octreotide scintigraphy. Blood samples were collected before (0900, 0930 h), at (1000 h), and after (1030, 1100, 1200, 1300 h) the injection of 50 micrograms octreotide subcutaneously. Plasma hormones relevant to the syndrome were analysed by radioimmunoassay. The short suppression effects, the [111In]-pentetreotide observations and the response to long-term treatment with somatostatin analogue were evaluated and compared. PATIENTS: Twenty-six patients with metastatic NETs were evaluated, including 14 carcinoid tumours, 10 pancreatic endocrine tumours and 2 medullary carcinomas of thyroid (MCTs). Twelve patients had received octreotide treatment before the study, another 4 patients were treated subsequently with somatostatin analogue. RESULTS: During the short suppression test, hormones relevant to the syndromes were suppressed in 24 patients (those with carcinoids and pancreatic endocrine tumours). There was no suppression in the 2 patients with MCT. [111In]-pentetreotide observations closely correlated with the short suppression response to octreotide. Fourteen patients were treated with somatostatin analogue, and responded clinically; they had a positive short inhibition test and positive tumour uptake. Two patients with MCT did not respond to the treatment and had a negative suppression test and a negative [111In]-pentetreotide. CONCLUSION: Our results suggest that a consistent relationship exists between the short suppression test and the response to somatostatin analogue treatment in the majority of the patients with neuroendocrine tumours. The octreotide suppression test and octreotide scintigraphy together will be helpful in selecting appropriate patients for clinical treatment with somatostatin analogues.

Adult↗

PubMed1998

A new set of chemotaxis homologues is essential for Myxococcus xanthus social motility.

Myxococcus xanthus cells aggregate and develop into multicellular fruiting bodies in response to starvation. A new M. xanthus locus, designated diffor defective in fruiting, was identified by the characterization of a mutant defective in fruiting body formation. Molecular cloning, DNA sequencing and sequence analysis indicate that the dif locus encodes a new set of chemotaxis homologues of the bacterial chemotaxis proteins MCPs (methyl-accepting chemotaxis proteins), CheW, CheY and CheA. The dif genes are distinct genetically and functionally from the previously identified M. xanthus frz chemotaxis genes, suggesting that multiple chemotaxis-like systems are required for the developmental process of M. xanthus fruiting body formation. Genetic analysis and phenotypical characterization indicate that the M. xanthus dif locus is required for social (S) motility. This is the first report of a M. xanthus chemotaxis-like signal transduction pathway that could regulate or co-ordinate the movement of M. xanthus cells to bring about S motility.

Amino Acid Sequence↗

PubMed1998

Rapid and quantitative detection of Streptococcus mutans with species-specific monoclonal antibodies.

Streptococcus mutans is known to be a prime etiologic agent for the initiation and progression of human dental caries. Rapid, accurate, and quantitative detection of S. mutans will help us better understand the pathogenic mechanisms of dental caries and will help to develop methods for caries diagnosis and risk assessment. This study describes the development of three highly species-specific monoclonal IgG antibodies against S. mutans. The antibodies were used to develop a number of methods that quantitatively detect S. mutans in less <1 min and are sensitive enough to detect a single bacterial cell. These methods could be widely used in basic and clinical studies related to S. mutans and in the clinical diagnosis and treatment of caries in humans.

Antibodies, Bacterial↗

PubMed1998

Localization of neuroendocrine tumours with [111In] DTPA-octreotide scintigraphy (Octreoscan): a comparative study with CT and MR imaging.

A wide variety of neuroendocrine tumours express somatostatin receptors, and can be visualized by radiolabelled somatostatin analogue scintigraphy. To investigate the value of [111In]-octreotide scintigraphy (Octreoscan), 48 patients (37 with proven carcinoid, pancreatic endocrine and medullary carcinoma of thyroid tumours, 11 with neuroendocrine syndromes multiple endocrine neoplasia (MEN-I) and Zollinger-Ellison syndrome (ZES) were examined with 111In-DTPA-D-Phe1-octreotide. Scintigrams were obtained at 24 and 48 h, and the results were compared with CT and magnetic resonance imaging (MRI). Thirty-five of 48 patients had positive [111In]-octreotide scintigraphy (23/25 (92%) carcinoids, 8/9 (89%) PETs, 4/11 (36%) MEN-I & ZES). Of the 42 lesions located by conventional imaging techniques, 37 (88%) were also identified by Octreoscan. Unexpected lesions (40 sites), not detected by CT or MR imaging were found in 24/48 (50%) patients. [111In]-octreotide scintigraphy has a higher sensitivity for tumour detection, and is superior to MR imaging and CT scanning in the identification of previously unsuspected extraliver and lymph node metastases. It may also be helpful for the localization of clinically suspected tumours in patients with MEN-I and ZES.

Adolescent↗

PubMed1998

A DnaK homolog in Myxococcus xanthus is involved in social motility and fruiting body formation.

Myxococcus xanthus is a gram-negative soil bacterium which exhibits a complex life cycle and social behavior. In this study, two developmental mutants of M. xanthus were isolated through Tn5 transposon mutagenesis. The mutants were found to be defective in cellular aggregation as well as in sporulation. Further phenotypic characterization indicated that the mutants were defective in social motility but normal in directed cell movements. Both mutations were cloned by a transposon-tagging method. Sequence analysis indicated that both insertions occurred in the same gene, which encodes a homolog of DnaK. Unlike the dnaK genes in other bacteria, this M. xanthus homolog appears not to be regulated by temperature or heat shock and is constitutively expressed during vegetative growth and under starvation. The defects of the mutants indicate that this DnaK homolog is important for the social motility and development of M. xanthus.

Bacterial Proteins↗

PubMed1998

Chemotaxis in Borrelia burgdorferi.

Borrelia burgdorferi is a motile spirochete which has been identified as the causative microorganism in Lyme disease. The physiological functions which govern the motility of this organism have not been elucidated. In this study, we found that motility of B. burgdorferi required an environment similar to interstitial fluid (e.g., pH 7.6 and 0.15 M NaCl). Several methods were used to detect and measure chemotaxis of B. burgdorferi. A number of chemical compounds and mixtures were surveyed for the ability to induce positive and negative chemotaxis of B. burgdorferi. Rabbit serum was found to be an attractant for B. burgdorferi, while ethanol and butanol were found to be repellents. Unlike some free-living spirochetes (e.g., Spirochaeta aurantia), B. burgdorferi did not exhibit any observable chemotaxis to common sugars or amino acids. A method was developed to produce spirochete cells with a self-entangled end. These cells enabled us to study the rotation of a single flagellar bundle in response to chemoattractants or repellents. The study shows that the frequency and duration for pausing of flagella are important for chemotaxis of B. burgdorferi.

Adaptation, Physiological↗

PubMed1998

Methylation of FrzCD defines a discrete step in the developmental program of Myxococcus xanthus.

Myxococcus xanthus is a gram-negative soil bacterium which undergoes fruiting body formation during starvation. The frz signal transduction system has been found to play an important role in this process. FrzCD, a methyl-accepting taxis protein homologue, shows modulated methylation during cellular aggregation, which is thought to be part of an adaptation response to an aggregation signal. In this study, we assayed FrzCD methylation in many known and newly isolated mutants defective in fruiting body formation to determine a possible relationship between the methylation response and fruiting morphology. The results of our analysis indicated that the developmental mutants could be divided into two groups based on their ability to show normal FrzCD methylation during development. Many mutants blocked early in development, i.e., nonaggregating or abnormally aggregating mutants, showed poor FrzCD methylation. The well-characterized asg, bsg, csg, and esg mutants were found to be of this type. The defects in FrzCD methylation of these signaling mutants could be partially rescued by extracellular complementation with wild-type cells or addition of chemicals which restore their fruiting body formation. Mutants blocked in late development, i.e., translucent mounds, showed normal FrzCD methylation. Surprisingly, some mutants blocked in early development also exhibited a normal level of FrzCD methylation. The characterized mutants in this group were found to be defective in social motility. This indicates that FrzCD methylation defines a discrete step in the development of M. xanthus and that social motility mutants are not blocked in these early developmental steps.

Bacterial Proteins↗

PubMed1997

Identification of two nervous system-specific members of the erg potassium channel gene family.

Two new potassium channel genes, erg2 and erg3, that are expressed in the nervous system of the rat were identified. These two genes form a small gene family with the previously described erg1 (HERG) gene. The erg2 and erg3 genes are expressed exclusively in the nervous system, in marked contrast to erg1, which is expressed in both neural and non-neural tissues. All three genes are expressed in peripheral sympathetic ganglia. The erg3 channel produces a current that has a large transient component at positive potentials, whereas the other two channels are slowly activating delayed rectifiers. Expression of the erg1 gene in the sympathetic nervous system has potential implications for the etiology of the LQT2 form of the human genetic disease long QT syndrome.

Animals↗

PubMed1997

Sensing antimonite and arsenite at the subattomole level with genetically engineered bioluminescent bacteria.

A highly sensitive and selective optical sensing system for antimonite has been developed using genetically engineered bacteria. The basis of this system is the ability of certain bacteria to survive in environments that are contaminated with antimonite, arsenite, and arsenate. The survival is conferred to the bacteria by the ars operon, which consists of five genes that code for three structural proteins, ArsA, ArsB, and ArsC, and two regulatory proteins, ArsD and ArsR. ArsA, ArsB, and ArsC form a protein pump system that extrudes antimonite, arsenite, and arsenate once these anions reach the cytoplasm of the bacterium. A method was developed for monitoring antimonite and arsenite by using a single plasmid that incorporates the regulatory gene of the extrusion system, arsR, and the genes of bacterial luciferase, luxA and luxB. In the designed plasmid, ArsR regulates the expression of bacterial luciferase in a manner that is dependent on the concentration of antimonite and arsenite in the sample. Thus, the bioluminescence emitted by luciferase can be related to the concentration of antimonite and arsenite in the sample. Concentrations for antimonite and arsenite in the order of 10(-5) M, which corresponds to subattomole levels, can be detected. This bacterial-based sensing system is highly selective for antimonite and arsenite.

Adenosine Triphosphatases↗