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Biomedical subjects

Tomoko Yamamoto

Publications and source records attributed to Tomoko Yamamoto.

52 records · Page 3Linked to original sources
PubMed2003

Turnover of FlhD and FlhC, master regulator proteins for Salmonella flagellum biogenesis, by the ATP-dependent ClpXP protease.

In enterobacteria such as Salmonella, flagellar biogenesis is dependent upon the master operon flhDC at the apex of the flagellar gene hierarchy, which is divided into three classes 1, 2 and 3. Previously we reported that depletion of the ClpXP ATP-dependent protease results in dramatic enhancement of class 2 and class 3 gene transcription, whereas the transcription level of the flhDC operon remains normal in Salmonella enterica serovar Typhimurium. This suggests that the ClpXP protease may be responsible for the turnover of the FlhD and FlhC master regulators (Tomoyasu, T., Ohkishi, T., Ukyo, Y., Tokumitsu, A., Takaya, A., Suzuki, M. et al., 2002, J Bacteriol 184:645-653). In this study, to establish the role of the ClpXP protease in the turnover of FlhD and FlhC proteins, we analysed levels of the FlhD and FlhC proteins in wild-type and ClpXP mutant cells using anti-FlhD and anti-FlhC antibodies. The results show that both FlhD and FlhC proteins are markedly accumulated in ClpXP mutant cells and the half-life of FlhC is approximately fivefold longer in the ClpXP mutant, suggesting that the ClpXP protease is responsible for the degradation of FlhD and FlhC. The results also show that the ClpXP protease degrades both proteins in FlhD2FlhC2 complex but does not seem to recognize the respective subunits synthesized individually. Taken together, it is suggested that the cellular concentration of the FlhD2FlhC2 master regulator is tightly controlled at the post-translational level by the ClpXP protease. We also examined the role of other members of the ATP-dependent protease family in the regulation of flagellar biogenesis and concluded that only ClpXP in this family functions as a negative regulator for flagellar biogenesis in Salmonella.

Adenosine Triphosphatases↗
PubMed2003

Aberrant neuronal migration in the brainstem of fukuyama-type congenital muscular dystrophy.

We examined the brainstem of 10 patients with Fukuyama-type congenital muscular dystrophy (FCMD). In the midbrain we noted leptomeningeal glioneuronal heterotopia (LGH) (n = 9) and intramural "micropolygyria" (n = 1) in the tectum, as well as tyrosine hydroxylase-positive ectopic neurons/fibers ventral to the cerebral peduncle (n = 3). In the pontomedullary region, glial fibrillary acidic protein-positive subpial tissue intermingled with neurons and myelinated fibers was present in the ventrolateral pontine surface in all cases and extended over the lateral surface of the upper medulla oblongata. This subpial gliotic band was often contiguous with the extra-pial LGH tissues. The gliotic band protruded from the ventrolateral pontine surface in 3 cases and appeared to include ectopic neurons of the pontine nucleus. Disarrangement of the arcuate nuclei (n = 3) was also noted in the medulla oblongata. We hypothesize that both the radial and tangential neuronal migration systems are disrupted in the FCMD brainstem in addition to altered neuronal migration in the cerebral and cerebellar cortex. Fukutin protein may play a part in the morphogenesis of certain neuronal structures in the brainstem and the dysplastic structure termed "aberrant pyramidal tract" in previous reports may essentially result from an ectopic migration of pontine nucleus neurons.

Adolescent↗
PubMed2003

Oral immunization with ATP-dependent protease-deficient mutants protects mice against subsequent oral challenge with virulent Salmonella enterica serovar typhimurium.

We evaluated the efficacy of mutants with a deletion of the stress response protease gene as candidates for live oral vaccine strains against Salmonella infection through infection studies with mice by using a Salmonella enterica serovar Typhimurium mutant with a disruption of the ClpXP or Lon protease. In vitro, the ClpXP protease regulates flagellum synthesis and the ClpXP-deficient mutant strain exhibits hyperflagellated bacterial cells (T. Tomoyasu et al., J. Bacteriol. 184:645-653, 2002). On the other hand, the Lon protease negatively regulates the efficacy of invading epithelial cells and the expression of invasion genes (A. Takaya et al., J. Bacteriol. 184:224-232, 2002). When 5-week-old BALB/c mice were orally administered 5 x 10(8) CFU of the ClpXP- or Lon-deficient strain, bacteria were detected with 10(3) to 10(4) CFU in the spleen, mesenteric lymph nodes, Peyer's patches, and cecum 1 week after inoculation and the bacteria then decreased gradually in each tissue. Significant increases of lipopolysaccharide-specific immunoglobulin G (IgG) and secretory IgA were detected at week 4 and maintained until at least week 12 after inoculation in serum and bile, respectively. Immunization with the ClpXP- or Lon-deficient strain protected mice against oral challenge with the serovar Typhimurium virulent strain. Both the challenged virulent and immunized avirulent salmonellae were completely cleared from the spleen, mesenteric lymph nodes, Peyer's patches, and even cecum 5 days after the challenge. These data indicate that Salmonella with a disruption of the ATP-dependent protease ClpXP or Lon can be useful in developing a live vaccine strain.

ATP-Dependent Proteases↗
PubMed2003

Lon, a stress-induced ATP-dependent protease, is critically important for systemic Salmonella enterica serovar typhimurium infection of mice.

Studies on the pathogenesis of Salmonella enterica serovar Typhimurium infections in mice have revealed the presence of two prominent virulence characteristics-the invasion of the nonphagocytic cells to penetrate the intestinal epithelium and the proliferation within host phagocytic cells to cause a systemic spread and the colonization of host organs. We have recently demonstrated that the ATP-dependent Lon protease of S. enterica serovar Typhimurium negatively regulates the efficiency of invasion of epithelial cells and the expression of invasion genes (A. Takaya et al., J. Bacteriol. 184:224-232, 2002). This study was performed to reveal the contribution of the Lon protease to the virulence of S. enterica serovar Typhimurium in mice. Determination of 50% lethal doses for the lon disruption mutant and wild-type strain revealed that the mutant was highly attenuated when administered either orally or intraperitoneally to BALB/c mice. The mutant was also found to be able to reach extraintestinal sites but unable to proliferate efficiently within the spleen and cause lethal systemic disease of mice. Macrophage survival assays revealed that the lon disruption mutant could not survive or proliferate within murine macrophages. In addition, the mutant showed extremely increased susceptibility to hydrogen peroxide, which contributes to the bactericidal capacity of phagocytes. The mutant also showed increased sensitivity to acidic conditions. Taken together, the impaired ability of the lon disruption mutant to survive and grow in macrophages could be due to the enhanced susceptibility to the oxygen-dependent killing mechanism associated with respiratory burst and the low phagosomal pH. These results suggest that the Lon protease is essentially involved in the systemic infection of mice with S. enterica serovar Typhimurium, which can be fatal. Of further interest is the finding that the lon disruption mutant persists in the BALB/c mice for long periods without causing an overwhelming systemic infection.

ATP-Dependent Proteases↗
PubMed2003

A new heat shock gene, AgsA, which encodes a small chaperone involved in suppressing protein aggregation in Salmonella enterica serovar typhimurium.

We discovered a novel small heat shock protein (sHsp) named AgsA (aggregation-suppressing protein) in the thermally aggregated fraction from a Salmonella enterica serovar Typhimurium dnaK-null strain. The -10 and -35 regions upstream of the transcriptional start site of the agsA gene are characteristic of sigma(32)- and sigma(72)-dependent promoters. AgsA was strongly induced by high temperatures. The similarity between AgsA and the other two sHsps of Salmonella serovar Typhimurium, IbpA and IbpB, is rather low (around 30% amino acid sequence identity). Phylogenetic analysis suggested that AgsA arose from an ancient gene duplication or amplification at an early evolutionary stage of gram-negative bacteria. Here we show that overproduction of AgsA partially complements the DeltadnaK52 thermosensitive phenotype and reduces the amount of heat-aggregated proteins in both DeltadnaK52 and DeltarpoH mutants of Escherichia coli. These data suggest that AgsA is an effective chaperone capable of preventing aggregation of nonnative proteins and maintaining them in a state competent for refolding in Salmonella serovar Typhimurium at high temperatures.

Amino Acid Sequence↗
PubMed2002

Participation of DnaK in expression of genes involved in virulence of Listeria monocytogenes.

DnaK is required for adaptation to environmental stress and is also involved in bacterial growth under normal conditions. To examine whether DnaK plays a role in the expression of genes related to pathogenicity of Listeria monocytogenes, the transcription of flaA, iap and lmaA in a dnaK mutant was analyzed. Northern blot analysis showed that expression of flaA and lmaA mRNAs was reduced in the dnaK mutant, THY-LK1. A reporter assay revealed that transcription of lmaB in the dnaK mutant, LKS01, constructed in this study was lower than that in the parental strain. In contrast, the dnaK mutation had no effect on the transcription of iap. These results suggest that DnaK is involved in the transcription of flaA and lmaB.

Antigens, Bacterial↗
PubMed2002

Fukutin expression in glial cells and neurons: implication in the brain lesions of Fukuyama congenital muscular dystrophy.

Expression and localization of fukutin, a gene responsible for Fukuyama congenital muscular dystrophy (FCMD), was studied in the central nervous system by in situ hybridization and immunohistochemistry. In control cases, glial cells expressed fukutin and the expression continued from fetuses to adults. Double immunostaining revealed that some of these cells were astrocytes. The glia limitans was stained by immunohistochemistry. In contrast, neuronal expression was decreased with neuronal maturation. The glia limitans formed by endfeet of astrocytes is abnormal in the brain of fetal to adult FCMD cases. These findings suggest an important role of astrocytes for the genesis of FCMD brain, although immature neurons expressed fukutin. In FCMD cases, expression of fukutin looked decreased. In the brain of fetal FCMD cases, decreased expression of fukutin is considered to provoke the disruption of glia limitans. In post-natal FCMD cases, prominent superficial gliosis is observed in the cerebral surface, where fukutin was weakly positive. Reactive increase of astrocytes may be required to maintain the glia limitans for compensating the decrease of fukutin expression in individual astrocytes. In the cerebellum, Bergmann glia, which did not express fukutin in control cases, elongated their cytoplasmic processes to the surface to form glia limitans even in the polymicrogyric area.

Adolescent↗
PubMed2002

Selective formation of certain advanced glycation end products in spinal cord astrocytes of humans and mice with superoxide dismutase-1 mutation.

Recent studies have documented carbonyl stress involvement in the pathogenesis of sporadic amyotrophic lateral sclerosis (ALS). The aim of the present study was to assess a role for carbonyl stress in motor neuron degeneration associated with superoxide dismutase-1 (SOD1) mutant familial ALS and its transgenic mouse model, using an immunohistochemical investigation of advanced glycation end products (AGEs) and advanced lipoxidation end products (ALEs). In the spinal cords from six familial ALS patients with SOD1 A4V mutation and six transgenic mice expressing G93A mutant human SOD1, immunoreactivities for N(epsilon)-(carboxyethyl)lysine, argpyrimidine, pyrraline and N(epsilon)-(carboxymethyl)lysine as AGEs were distinct in almost all of the reactive astrocytes and obscure in the residual neurons, whereas no immunoreactivity for pentosidine as an AGE, or 4-hydroxy-2-nonenal-histidine, malondialdehyde-lysine or acrolein-lysine as ALEs was detectable. Spinal cords from age-matched control humans and mice exhibited no significant immunoreactivities for the examined products. Our results indicate that protein glycation, but not lipid peroxidation, is enhanced in ALS patients with an SOD1 mutation and mutant SOD1 transgenic mice, in which certain AGEs are selectively formed in the spinal cord astrocytes.

Adult↗
PubMed2002

Papillary fibroelastoma of the aortic valve: report of a case.

This report describes a case of aortic papillary fibroelastoma causing transient ischemic attack (TIA), and is followed by a review of the relevant literature. A tumor measuring 1.5 x 1.5 cm was located in the right coronary cusp of the aortic valve, which was found to have a "sea anemone-like" configuration due to collagenous or elastic papillary projections. Aortic fibroelastoma can cause cerebrovascular symptoms, and this patient presented with TIA. Moreover, aortic fibroelastoma usually causes chest symptoms and occasionally results in death despite its small size of generally less than 1 cm. According to the literature, all of the patients who died of fibroelastoma had an aortic valve tumor in the right or left coronary cusp, which indicates that the location of the tumor rather than its size may be an important factor contributing to the case of death. Surgical treatment is usually indicated especially for aortic fibroelastoma because of the high associated risk or cerebrovascular and chest diseases. The recent evolution of echocardiography will promote the chance of establishing a preoperative diagnosis of this lesion. However, because it is not possible to differentiate fibroelastoma from other lesions, including malignancies, by echocardiography alone, this may be another reason for performing surgical removal.

Aortic Valve↗
PubMed2002

Oxidative stress in the human fetal brain: an immunohistochemical study.

Because accumulation of oxidative modification products seems to relate to aging and has not been fully studied in fetal brains, an immunohistochemical examination was performed on nine brains ranging from 22-40 weeks of gestation. These brains did not demonstrate lesions except hypoxic-ischemic changes. Advanced glycation end products and 4-hydroxynonenal are generally reported to be negative in neurons of normal young brains, but, in the present study, distinct positive immunoreaction was observed in neurons of fetal brains. Positive immunoreaction appeared earlier in the medulla oblongata than in the cerebrum, and 4-hydroxynonenal began to accumulate earlier than advanced glycation end products. As for glial cells, advanced glycation end products and 4-hydroxynonenal were positive in reactive astrocytes in mid- to late gestation. Because hypoxic-ischemic changes were observed in most of the patients, it is possible that oxidative stress caused by hypoxic-ischemic may be involved in the accumulation of these products in the fetal brain. 8-Hydroxy-2'-deoxyguanosine was negative even in patients demonstrating positive reaction for advanced glycation end products and 4-hydroxynonenal. In the fetal brain, DNA might be strongly protected from oxidative damage. 4-Hydroxynonenal is generally positive in the cytoplasm but was positive in the nucleus of immature neurons and glial cells in the present study, suggesting a unique metabolism of the fetal brain.

8-Hydroxy-2'-Deoxyguanosine↗
PubMed2002

The ATP-dependent lon protease of Salmonella enterica serovar Typhimurium regulates invasion and expression of genes carried on Salmonella pathogenicity island 1.

An early step in the pathogenesis of Salmonella enterica serovar Typhimurium infection is bacterial penetration of the intestinal epithelium. Penetration requires the expression of invasion genes found in Salmonella pathogenicity island 1 (SPI1). These genes are controlled in a complex manner by regulators in SPI1, including HilA and InvF, and those outside SPI1, such as two-component regulatory systems and small DNA-binding proteins. We report here that the expression of invasion genes and the invasive phenotype of S. enterica serovar Typhimurium are negatively regulated by the ATP-dependent Lon protease, which is known to be a major contributor to proteolysis in Escherichia coli. A disrupted mutant of lon was able to efficiently invade cultured epithelial cells and showed increased production and secretion of three identified SPI1 proteins, SipA, SipC, and SipD. The lon mutant also showed a dramatic enhancement in transcription of the SPI1 genes hilA, invF, sipA, and sipC. The increases ranged from 10-fold to almost 40-fold. It is well known that the expression of SPI1 genes is also regulated in response to several environmental conditions. We found that the disruption of lon does not abolish the repression of hilA and sipC expression by high-oxygen or low-osmolarity conditions, suggesting that Lon represses SPI1 gene expression by a regulatory pathway independent of these environmental signals. Since HilA is thought to function as a central regulator of SPI1 gene expression, it is speculated that Lon may regulate SPI1 gene expression by proteolysis of putative factors required for activation of hilA expression.

ATP-Dependent Proteases↗
PubMed2002

The ClpXP ATP-dependent protease regulates flagellum synthesis in Salmonella enterica serovar typhimurium.

The ClpXP protease is a member of the ATP-dependent protease family and plays a dynamic role in the control of availability of regulatory proteins and the breakdown of abnormal and misfolded proteins. The proteolytic activity is rendered by the ClpP component, while the substrate specificity is determined by the ClpX component that has ATPase activity. We describe here a new role of the ClpXP protease in Salmonella enterica serovar Typhimurium in which ClpXP is involved in the regulation of flagellum synthesis. Cells deleted for ClpXP show "hyperflagellate phenotype," exhibit overproduction of the flagellar protein, and show a fourfold increase in the rate of transcription of the fliC encoding flagellar filament. The assay for promoter activity of the genes responsible for expression of the fliC showed that the depletion of ClpXP results in dramatic enhancement of the expression of the fliA encoding sigma factor final sigma(28), leaving the expression level of the flhD master operon lying at the top of the transcription hierarchy of flagellar regulon almost normal. These results suggest that the ClpXP may be responsible for repressing the expression of flagellar regulon through the control of the FlhD/FlhC master regulators at the posttranscriptional and/or posttranslational levels. Proteome analysis of proteins secreted from the mutant cells deficient for flhDC and clpXP genes demonstrated that the DeltaflhD mutation abolished the enhanced effect by DeltaclpXP mutation on the production of flagellar proteins, suggesting that the ClpXP possibly defines a regulatory pathway affecting the expression of flagellar regulon that is dependent on FlhD/FlhC master regulators.

Adenosine Triphosphatases↗
PubMed2002

Oxidative stress in the brain of Fukuyama type congenital muscular dystrophy: immunohistochemical study on astrocytes.

Astrocytes in the cerebrum and medulla oblongata of cases of Fukuyama type congenital muscular dystrophy were examined by immunohistochemistry of oxidative modification products and free-radical scavenging enzymes because abnormal glia limitans formed by astrocytic end feet is considered to be involved in the genesis of brain lesions of Fukuywama type congenital muscular dystrophy. The study was performed on two fetal cases of Fukuyama type congenital muscular dystrophy of 18 and 20 weeks' gestation and seven patients with Fukuyama type congenital muscular dystrophy ranging in age from 2 to 27 years. Eight age-matched control cases were used. Polymerase chain reaction (PCR) was performed to ascertain the gene phenotype of two child cases, in which prenatal gene analysis was not performed. Astrocytes, especially layer I astrocytes, of postnatal cases of Fukuyama type congenital muscular dystrophy were weakly positivefor Nepsilon-(carboxymethyl)lysine and argpyrimidine, suggesting that they were sensitive to oxidative stress, and the accumulation may be related to the abnormal glia limitans. Secondary increase of manganese (Mn) superoxide dismutase against the increase of free radicals was considered in patients with Fukuyama type congenital muscular dystrophy more than 14 years old considered to be homozygous for founder haplotype: homozygosity was suggested by PCR in two cases. In contrast, expression of Mn superoxide dismutase was decreased in 2- and 6-year-old children with Fukuyama type congenital muscular dystrophy that were heterozygous. Moreover, accumulation of argpyrimidine was exclusively found in astrocytes of the 2-year-old child that exhibited severe brain lesions. Function of astrocytes might be impaired or immature in severe or heterozygous cases. These results may confirm that astrocytes play an important role in the etiology of the brain lesion.

Adolescent↗
PubMed2002

Evaluation of a commercially available serologic assay for antibodies against tuberculosis-associated glycolipid antigen.

A commercially available enzyme immunoassay developed to detect antibodies to a tuberculosis-associated glycolipid antigen was evaluated for serologic diagnosis of tuberculosis. This was a multicenter study comparing the assay with other methods in 78 patients with active pulmonary tuberculosis and in 54 controls with non-tuberculous lung diseases. Sensitivities were highest for sputum culture (91.0%), followed by immunoassay (79.5%), nucleic acid amplification (77.3%), and finally acid-fast staining of sputum smear (60.3%). Immunoassay and amplification, both rapid methods, had similarly high sensitivity in smear-positive subjects (89.4 and 88.9%, respectively); in smear-negative subjects these two techniques showed low sensitivity (64.5 and 60.0%, respectively). Concordance between the two methods was relatively low (72.0%). With regard to specificity, seven out of ten patients with old tuberculosis had positive result by immunoassay (30% specificity). In the control group, 10 out of 54 patients had positive immunoassay result (72.2% specificity), with notably limited specificity in the elderly. The tuberculous glycolipid assay is a rapid method sufficiently sensitive for detection of tuberculosis infection, even in smear-negative patients.

Adolescent↗