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Biomedical subjects

S Shin

Publications and source records attributed to S Shin.

At least 73 records · Page 4Linked to original sources
PubMed2000

Global regulatory mutations in csrA and rpoS cause severe central carbon stress in Escherichia coli in the presence of acetate.

The csrA gene encodes a small RNA-binding protein, which acts as a global regulator in Escherichia coli and other bacteria (T. Romeo, Mol. Microbiol. 29:1321-1330, 1998). Its key regulatory role in central carbon metabolism, both as an activator of glycolysis and as a potent repressor of glycogen biosynthesis and gluconeogenesis, prompted us to examine the involvement of csrA in acetate metabolism and the tricarboxylic acid (TCA) cycle. We found that growth of csrA rpoS mutant strains was very poor on acetate as a sole carbon source. Surprisingly, growth also was inhibited specifically by the addition of modest amounts of acetate to rich media (e.g., tryptone broth). Cultures grown in the presence of >/=25 mM acetate consisted substantially of glycogen biosynthesis (glg) mutants, which were no longer inhibited by acetate. Several classes of glg mutations were mapped to known and novel loci. Several hypotheses were examined to provide further insight into the effects of acetate on growth and metabolism in these strains. We determined that csrA positively regulates acs (acetyl-coenzyme A synthetase; Acs) expression and isocitrate lyase activity without affecting key TCA cycle enzymes or phosphotransacetylase. TCA cycle intermediates or pyruvate, but not glucose, galactose, or glycerol, restored growth and prevented the glg mutations in the presence of acetate. Furthermore, amino acid uptake was inhibited by acetate specifically in the csrA rpoS strain. We conclude that central carbon flux imbalance, inhibition of amino acid uptake, and a deficiency in acetate metabolism apparently are combined to cause metabolic stress by depleting the TCA cycle.

Acetates↗
PubMed2000

Experimental infection of ponies with Borrelia burgdorferi by exposure to Ixodid ticks.

Seven specific-pathogen-free (SPF) ponies, 1-5 years old, were exposed to Borrelia burgdorferi-infected adult ticks while being treated with dexamethasone over 5 consecutive days. One SPF pony (pony No. 178) was first exposed to laboratory-reared nymphs without B. burgdorferi infection and 3 weeks later was exposed to B. burgdorferi-infected adult ticks with concurrent dexamethasone treatment for 5 consecutive days. Four uninfected ponies treated with dexamethasone, exposed to laboratory-reared ticks without B. burgdorferi infection served as uninfected controls. Clinical signs, bacteriologic culture, polymerase chain reaction (PCR) for bacterial DNA, immunologic responses, and gross lesions and histopathologic changes were investigated during the experiment or at necropsy 9 months after tick exposure. In all of the seven challenged ponies, infection with B. burgdorferi was detected from monthly skin biopsies and various tissues at postmortem examination by culture and by PCR. However, pony No. 178 exposed to laboratory-reared nymphs (without B. burgdorferi infection) and challenged with B. burgdorferi-infected adult ticks 2 months later did not develop a B. burgdorferi infection. All of the infected ponies seroconverted. Control ponies and pony No. 178 were negative by culture, PCR, and serology. Except for skin lesions, we failed to induce any significant histopathologic changes in this study. This is the first report of successful tick-induced experimental infection in ponies by exposure to B. burgdorferi-infected ticks. This Lyme disease model will be very useful to evaluate efficacy of vaccines against the Lyme agent and the effect of antibiotic therapy on horses infected with B. burgdorferi.

Animals↗
PubMed2000

Deletion of SMN and NAIP genes in Korean patients with spinal muscular atrophy.

Childhood-onset proximal spinal muscular atrophies (SMAs) are an autosomal recessive, clinically heterogeneous group of neuronopathies characterized by selective degeneration of anterior horn cells. The causative genes to be reported are survival motor neuron (SMN) and neuronal apoptosis inhibitory protein (NAIP) genes. The deletion of telomeric copy of SMN (SMN(T)) gene was observed in over 95% of SMAs. The deletion rate of NAIP gene is 20-50% according to disease severity. The objective of this article is to genetically characterize the childhood-onset spinal muscular atrophy in Koreans. Five Korean families (14 constituents containing 5 probands) with SMA were included in this study. Polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) were used for the deletion analysis of SMN(T). Multiplex PCR method was used for NAIP analysis. Four probands showed deletion of SMNT gene. Deletion of SMN(C) (centromeric SMN) gene was found in one proband who did not show the deletion of SMN(T) gene and in the father of one proband who showed the deletion of SMN(T) gene. The deletion of NAIP gene was not found among all the studied individuals. The extent of deletion in Koreans was smaller than that in other studied population. PCR-RFLP deletion analysis can be applied to diagnose SMA and make a prenatal diagnosis.

Adolescent↗
PubMed1999

Quorum sensing controls expression of the type III secretion gene transcription and protein secretion in enterohemorrhagic and enteropathogenic Escherichia coli.

Enterohemorrhagic Escherichia coli O157:H7 and enteropathogenic E. coli cause a characteristic histopathology in intestinal cells known as attaching and effacing. The attaching and effacing lesion is encoded by the Locus of Enterocyte Effacement (LEE) pathogenicity island, which encodes a type III secretion system, the intimin intestinal colonization factor, and the translocated intimin receptor protein that is translocated from the bacterium to the host epithelial cells. Using lacZ reporter gene fusions, we show that expression of the LEE operons encoding the type III secretion system, translocated intimin receptor, and intimin is regulated by quorum sensing in both enterohemorrhagic E. coli and enteropathogenic E. coli. The luxS gene recently shown to be responsible for production of autoinducer in the Vibrio harveyi and E. coli quorum-sensing systems is responsible for regulation of the LEE operons, as shown by the mutation and complementation of the luxS gene. Regulation of intestinal colonization factors by quorum sensing could play an important role in the pathogenesis of disease caused by these organisms. These results suggest that intestinal colonization by E. coli O157:H7, which has an unusually low infectious dose, could be induced by quorum sensing of signals produced by nonpathogenic E. coli of the normal intestinal flora.

Adhesins, Bacterial↗
PubMed1999

Vaccination against lyme disease with recombinant Borrelia burgdorferi outer-surface protein A (rOspA) in horses.

Eight 1-year-old ponies were vaccinated with recombinant OspA (ospA gene derived from B. burgdorferi B31) with adjuvant (aluminium hydroxide). Four ponies were used as non-vaccinated controls with adjuvant. One hundred and twelve days after the first vaccination, the vaccinated and non-vaccinated ponies were challenged by exposure to B. burgdorferi-infected adults tick (Ixodes scapularis) collected from Westchester County, New York (tick infection rate >/=60%). Protection from infection was evaluated by culture for B. burgdorferi from three monthly skin biopsies taken near the site of tick bites. B. burgdorferi was not isolated from any of the vaccinated ponies. In contrast, three of four control ponies challenged by tick exposure were skin culture positive. At the time of tick exposure, vaccinated ponies had antibody to B. burgdorferi demonstrable by KELA (kinetic-ELISA), western blot and a serum growth inhibition assay. Antibodies in the challenge control ponies were only detectable by two to three months after tick exposure and remained at intermediate levels until termination of the study. By western blot analysis, antibodies to OspA first appeared in the sera of vaccinated ponies three weeks after the first vaccination. The absence of additional bands, known to develop when the animal is infected, suggests that infection was blocked after tick exposure of vaccinated ponies. Results from this study show that vaccination with recombinant OspA protected ponies against infection after experimental challenge with B. burgdorferi-infected ticks.

Animals↗
PubMed1999

Regions of Byr4, a regulator of septation in fission yeast, that bind Spg1 or Cdc16 and form a two-component GTPase-activating protein with Cdc16.

In the fission yeast Schizosaccharomyces pombe, septation and constriction of the actomyosin ring for cell division are positively regulated by the Spg1 GTPase, a member of the Ras superfamily. Spg1 is negatively regulated by Byr4 and Cdc16, which together form a two-component GTPase-activating protein for the Spg1 GTPase. To better understand how Byr4 regulates septation, Byr4 mutants were tested for in vitro functions. This analysis revealed that Byr4 contained one Cdc16-binding site and four Spg1-binding sites (SBS), designated SBS1-SBS4. Although mutants with a single SBS bound Spg1 and inhibited GTP dissociation, the equilibrium binding affinity of these mutants was 28-280-fold weaker than Byr4. Because some Byr4 mutants with multiple SBSs bound Spg1 tighter than the corresponding mutants with a single SBS, multiple SBSs probably interact to cause the high affinity binding of Byr4 to Spg1. A region of Byr4 that bound Spg1, SBS4, and the region that bound Cdc16, Cdc16-binding site, was necessary and sufficient to form Cdc16-dependent Spg1GAP activity that was similar to that of wild-type Byr4 with Cdc16.

Binding Sites↗
PubMed1999

Genetic analysis of human immunodeficiency virus type 1 strains in Kenya: a comparison using phylogenetic analysis and a combinatorial melting assay.

We surveyed human immunodeficiency virus (HIV) subtype distribution from peripheral blood mononuclear cells (PBMCs) collected in 1995 from 24 HIV-1-infected Kenyan residents (specimens from predominantly male truck drivers and female sex workers near Mombasa and Nairobi). Processed lysates from the PBMC samples were used for env amplification, directly sequenced, and analyzed by phylogenetic analysis. Envelope amplification products were also used for analysis in a polymerase chain reaction (PCR)-based assay, called the combinatorial melting assay (COMA). Results of the two tests were compared for assignment of subtype for this Kenyan cohort. The COMA, a PCR capture technique with colorimetric signal detection, was used with HIV reference subtype strains as well as regional (East Africa) HIV strains for subtype identification. Performance of the COMA was at 100% concordance (24 of 24) as compared with DNA sequencing analysis. Phylogenetic analysis showed 17 isolates to be subtype A, 3 subtype D, and 4 subtype C viruses. This may represent an increase in subtype C presence in Kenya compared with previously documented reports. The COMA can offer advantages for rapid HIV-1 subtype screening of large populations, with the use of previously identified regional strains to enhance the identification of local strains. When more detailed genetic information is desired, DNA sequencing and analysis may be required.

Amino Acid Sequence↗
PubMed1999

Purification and characterization of glutamate dehydrogenase as another isoprotein binding to the membrane of rough endoplasmic reticulum.

Glutamate dehydrogenase (GDH) was purified from rough endoplasmic reticulum (RER) in rat liver using anion-exchange and affinity chromatography. As GDH has been known as an enzyme that exists mainly in the matrix of mitochondria, the properties of purified GDH were compared with those of mitochondrial GDH. The GDH activity in 0. 1% Triton X-100-treated RER subcellular fraction was nearly the same as intact RER, whereas that of the mitochondrial fraction increased by 50% after the detergent treatment. In kinetic values, in addition, mitochondrial GDH had a higher K(m) value for NADP(+) than NAD(+), whereas the K(m) value for NAD(+) was higher than that for NADP(+) in the case of GDH of RER, which showed a difference in specificity to cofactors. Moreover, when two GDH isoproteins were incubated at 42 degrees C or treated with trypsin, GDH from RER was more stable against heat inactivation and less susceptible to proteolysis than mitochondrial GDH in both cases. In addition, GDH of RER had at least five amino acids different from mitochondrial GDH when sequences of N-terminal and several internal peptide fragments were analyzed. These results showed that GDH of RER is another isoprotein of GDH, of whose properties are different from those of mitochondrial GDH.

Amino Acid Sequence↗
PubMed1999

Revascularization and new bone formation in heat-treated bone grafts.

Human imnmunodeficiency virus (HIV) infection is one of the possible serious complications associated with bone allografts. In order to prevent infection, grafted bone is sterilized by various treatments. Heat treatment has attracted attention as a simple and practical method. We carried out a histological study of the influence of heat treatment on autogenic bone grafts. To eliminate the problem of antigenicity of grafted bone, we used autografts, not allografts. Three types of heat-treated autografts were employed: heat-treated at 60 degrees C for 30 min, at 80 degrees C for 10 min, and at 100 degrees C for 5 min; as a control, fresh autografts were replaced in the rabbits' ilium. One, 2, 4 and 8 weeks after grafting, we performed microangiography and prepared two types of samples: transparent and haematoxylin-eosin (H&E) stained. Then, using an image analyzer, we quantitatively measured revascularization and new bone formation in the grafted bone. The grafts heat-treated at 60 degrees C showed early and good revascularization and new bone formation, from 1 to 8 weeks. The grafts heat-treated at 80 degrees C showed relatively good revascularization and new bone formation. However, the grafts heat-treated at 100 degrees C showed unsatisfactory revascularization and bone formation, less than 40% of control 8 weeks after grafting. Therefore, heat treatment at 60-80 degrees C does not seriously affect revascularization and new bone formation.

Animals↗
PubMed1999

Characterization of antimicrobial agents extracted from Asterina pectinifera.

The extracts from the starfish Asterina pectinifera obtained using various organic solvents and distilled water were tested for their antimicrobial activities against fungi and yeasts. The methanol and water extracts were found to be the most active and Aspergillus spp. and Cryptococcus neoformans proved to be sensitive species. The extracts are polar, stable to a wide range of pH and a high temperature, and possess a very distinctive UV spectrum. The colour reactions for functional groups of antimicrobial substances indicate the presence of some distinctive chemical properties common to steroidal oligoglycosides which results suggest that the antimicrobial activity is due to structures common to steroidal oligoglycosides.

Animals↗
PubMed1999

Involvement of phospholipase A2 activation in anthrax lethal toxin-induced cytotoxicity.

The molecular mechanism of cytotoxic effect exerted by the lethal toxin (LeTx) of Bacillus anthracis is not well understood. In the present study, using primary culture of mouse peritoneal macrophages, we have investigated possible cytotoxic mechanisms. LeTx was not found to induce high levels of nitric oxide (NO) production for NO-mediated toxicity. Fragmentation of DNA, a biochemical marker of apoptosis, was not observed in LeTx-treated cells. Pretreatment of cells with antioxidants such as melatonin and dehydroepiandrosterone (DHEA) did not protect the LeTx-induced cytotoxicity. However, addition of phospholipase A2 (PLA2) inhibitors (quinacrine, p-bromophenacyl bromide, manoalide, butacaine) to the culture medium resulted in the inhibition of cytotoxicity of LeTx in a dose-dependent manner. LeTx-induced cytotoxicity was also inhibited by the tyrosine-specific protein kinase inhibitor genistein, but not by the protein kinase C inhibitors staurosporine or H-7. The results of these studies indicate a role for PLA2 and protein kinase in the cytotoxic mechanism of macrophages by anthrax lethal toxin.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1999

Acetate metabolism in a pta mutant of Escherichia coli W3110: importance of maintaining acetyl coenzyme A flux for growth and survival.

In order to study the physiological role of acetate metabolism in Escherichia coli, the growth characteristics of an E. coli W3100 pta mutant defective in phosphotransacetylase, the first enzyme of the acetate pathway, were investigated. The pta mutant grown on glucose minimal medium excreted unusual by-products such as pyruvate, D-lactate, and L-glutamate instead of acetate. In an analysis of the sequential consumption of amino acids by the pta mutant growing in tryptone broth (TB), a brief lag between the consumption of amino acids normally consumed was observed, but no such lag occurred for the wild-type strain. The pta mutant was found to grow slowly on glucose, TB, or pyruvate, but it grew normally on glycerol or succinate. The defective growth and starvation survival of the pta mutant were restored by the introduction of poly-beta-hydroxybutyrate (PHB) synthesis genes (phbCAB) from Alcaligenes eutrophus, indicating that the growth defect of the pta mutant was due to a perturbation of acetyl coenzyme A (CoA) flux. By the stoichiometric analysis of the metabolic fluxes of the central metabolism, it was found that the amount of pyruvate generated from glucose transport by the phosphoenolpyruvate-dependent phosphotransferase system (PTS) exceeded the required amount of precursor metabolites downstream of pyruvate for biomass synthesis. These results suggest that E. coli excretes acetate due to the pyruvate flux from PTS and that any method which alleviates the oversupply of acetyl CoA would restore normal growth to the pta mutant.

Acetates↗
PubMed1999

Early region 1 transforming functions are dispensable for mammary tumorigenesis by human adenovirus type 9.

Some human adenoviruses are tumorigenic in rodents. Subgroup A and B human adenoviruses generally induce sarcomas in both male and female animals, and the gene products encoded within viral early region 1 (E1 region) are both necessary and sufficient for this tumorigenicity. In contrast, subgroup D human adenovirus type 9 (Ad9) induces estrogen-dependent mammary tumors in female rats and requires the E4 region-encoded ORF1 oncoprotein for its tumorigenicity. Considering the established importance of the viral E1 region for tumorigenesis by adenoviruses, we investigated whether this viral transcription unit is also necessary for Ad9 to generate mammary tumors. The nucleotide sequence of the Ad9 E1 region indicated that the gene organization and predicted E1A and E1B polypeptides of Ad9 are closely related to those of other human adenovirus E1 regions. In addition, an Ad9 E1 region plasmid demonstrated focus-forming activity in both low-passage-number and established rat embryo fibroblasts, whereas a large deletion within either the E1A or E1B gene of this plasmid diminished transforming activity. Surprisingly, we found that introducing the same transformation-inactivating E1A and E1B deletions into Ad9 results in mutant viruses that retain the ability to elicit mammary tumors in rats. These results are novel in showing that Ad9 represents a unique oncogenic adenovirus in which the E4 region, rather than the E1 region, encodes the major oncogenic determinant in the rat.

Adenovirus E1 Proteins↗
PubMed1999

Characteristics of monochorionic-diamniotic growth-retarded twins during the third trimester.

The goal of this study was to assess the characteristics of monochorionic-diamniotic (MD) growth-retarded twin infants with twin-twin transfusion syndrome (TTTS) compared with those without TTTS during the third trimester. Retrospective analyses of the growth patterns and amniotic fluid volumes were performed on 5 MD twin pregnancies in which one or both twins showed growth retardation with TTTS, and the results weve compared with those without TTTS. Eighty-three percent of growth-retarded twin infants without TTTS in MD twin gestation showed an asymmetric growth pattern, while all TTTS cases showed a symmetric pattern (p < 0.05). Polyhydramnios of the co-twin was found in 80% of TTTS cases, while no polyhydramnios was found in patients without TTTS (p < 0.05). Assessment of growth patterns and amniotic fluid volume may be useful to exclude the possibility of TTTS in MD growth-retarded twin pregnancies during the third trimester.

Adult↗
PubMed1999

Spinocerebellar ataxia type 2 in seven Korean families: CAG trinucleotide expansion and clinical characteristics.

Studies on spinocerebellar ataxias (SCA) have been hampered by a lack of disease markers. Clinical and pathological heterogeneity also made the classification unreliable. Linkage studies established that there are multiple subtypes of SCA. Five types are found to have unstable CAG expansion; the diagnosis can be established by molecular genetic study. Therefore, we systemically screened degenerative ataxia patients for these five SCA types, and identified eight patients with SCA2 (seven from six families and one sporadic case). This paper presents the clinical information on the seven patients, whose clinical information was available in detail. CAG repeat expansion in the patients ranged from 38 to 47 (normal control, 19 to 27). The onset ages ranged from 16 to 41 with 27.1 years as the mean, which correlated inversely with repeat lengths. All patients presented dysarthria and gait ataxia. Upper limb dysmetria or dysdiadochokinesia appeared later but progressed, causing severe disability. Slow saccade (4 patients in 7) and decreased DTR (4 in 7) were common. MRIs showed severe atrophy of the brainstem and cerebellum in all patients. We conclude that SCA2 is the most frequent type in Korea and carries rather pure cerebellar syndrome, slow saccade, and hyporeflexia.

Adolescent↗
PubMed1998

Experimental infection of the human granulocytic ehrlichiosis agent in horses.

Human blood collected from two patients from Westchester County, New York with human granulocytic ehrlichia (HGE) infection was inoculated into two ponies. Inoculated ponies developed clinical signs similar to a previous report (Madigan et al., 1995). Histopathological changes involved follicular hyperplasia of lymphoid tissues. HGE DNA was detected by PCR in muscle, fascia, peritoneum, and adrenal gland after the ponies produced a high level of antibodies to HGE. We suggest that HGE may reside in poorly vascularized connective tissues, where the antibodies may have some difficulties to penetrate, resulting in persistent infection. Since HGE and E. equi cause very similar diseases in both humans and horses, they may be the same organism with minor genetic differences.

Adrenal Glands↗