Streptococcus mutans isolated from a 4-year-old girl diagnosed with infective endocarditis.

Objectives: Infective endocarditis (IE) has an extremely high fatality rate. In this study, we isolated a strain of Streptococcus mutans, which we called HM, from the blood drawn from a 4-year-old girl diagnosed with IE. We aimed to fully type the HM strain and investigate its biological properties, including its virulence with respect to IE. Material and methods: A 16S rRNA phylogenetic tree and glucosyltransferase gene sequences were used to type HM. Serotyping was performed using the Ouchterlony method. Morphological observations were made using phase contrast and electron microscopy. Fibrinogen adhesion and biofilm formation were investigated to examine the tissue colonization properties of HM, whereas its bodily origin was determined from its fingerprinting pattern. Results: The isolated strain was S. mutans serotype e. However, its morphology was observed to be short chains, unlike that of the NCTC 10449 reference strain. Fibrinogen adhesion and biofilm formation were more apparent than in NCTC 10449. The fingerprinting pattern showed that HM came from the patient's saliva. Conclusions: HM differs from NCTC 10449 in its higher fibrinogen affinity. HM was also found to be derived from the oral cavity. These results highlight the importance of good oral hygiene for the prevention of IE in children.

Properties of acetylcholine-induced hyperpolarization in smooth muscle cells of the mouse mesenteric artery.

The properties of smooth muscle cell hyperpolarization produced by acetylcholine (ACh) were investigated in mesenteric arteries isolated from mice. The resting membrane potential of the smooth muscle cells was about -60 mV. When ACh (10 microM) was applied for 1 min, the membrane hyperpolarized with a peak amplitude of about 5 mV which was reached in about 1 min, following which the potential slowly reverted to the resting level over about 7 min following withdrawal of ACh from the superfusate (recovery component). Exposure of the artery to 0.5 mM Ba(2+), an inhibitor of inward rectifier K-channels, depolarized the membrane by about 13 mV, increased the amplitude of the ACh-induced hyperpolarization to about 10 mV, and facilitated the visualization of the recovery component. Indomethacine (10 microM), an inhibitor of cyclooxygenase, inhibited the recovery component and as a consequence reduced the duration of the hyperpolarization. The ACh-induced response was not markedly altered by either N(omega)-nitro-L-arginine (10 microM), an inhibitor of nitric oxide (NO) production, or catalase (130 U/ml), a super oxide scavenger. Exogenously applied hydrogen peroxide (H(2)O(2), 300 microM) hyperpolarized the membrane by about 5 mV, which was abolished by catalase. These results suggest that in the mouse mesenteric artery, the ACh-induced hyperpolarization has two components, both an indomethacin-sensitive and an indomethacin-insensitive component. The former component may be produced by prostanoids, while the latter may be produced by factors other than NO or H(2)O(2). The results also suggested that the inward rectifier K-channels may be important for producing the resting membrane potential, but they may not be the main contributor to the ACh-induced hyperpolarization of smooth muscle cell membranes in the mouse mesenteric artery.

Draft Genome Sequence of a Clinical Isolate of Streptococcus mutans Strain HM.

We report the draft genome sequence of Streptococcus mutans strain HM isolated from a 4-year-old girl with infective endocarditis. The genomics information will provide information on the genetic diversity and virulence potential of S. mutans strain HM.

[Cavernous hemangioma growing continuously from the spinal epidural space into the paravertebral muscles: case report].

A 19-year-old male was admitted to our hospital with a 5 year history of right low back pain. MR images showed an epidural mass which located on the posterolateral side at the T12-L1 level and extended through the right intervertebral foramen into the paraspinal muscles. Excision of the intramuscular and epidural lesions was performed and his symptom quickly improved. Histopathological examination of the mass revealed a cavernous hemangioma. It may affect any part of neuraxis, however cavernous hemangiomas located at the spinal epidural space are rare and particularly the lesions which grow continuously into the paravertebral muscles are very rare. Radiological presentation of this case was confusing in the preoperative diagnosis. It is important that we know the clinical, developmental and radiological characteristics of cavernous hemangiomas.

Association between palatal morphogenesis and Pax9 expression pattern in CL/Fr embryos with clefting during palatal development.

The CL/Fr mouse strain develops cleft lip and palate (CLP) spontaneously. In this study, Pax9 mRNA expression was investigated in the palatal shelves during palatal morphogenesis to assess the correlation between secondary palatal morphogenesis and Pax9 expression of CL/Fr embryos with spontaneous cleft lip and palate. The expression of Pax9 mRNA was characterised using whole mount in situ hybridisation with a digoxygenin-labelled probe. In the control strain of C57BL/6 and CL/Fr normal embryos, Pax9 was expressed in the palate, especially along the medial edge (ME), on embryonic day 13.5 (E13.5) and E14.5 when the palatal shelves grew vertically down the side of the tongue and subsequently elevated to a horizontal position, and was down regulated on E15.5 when the palatal shelves met and began fusing. In the cleft embryo, Pax9 was expressed in the ME region but was not down regulated on E15.5. Furthermore, whole mount in situ hybridisation was performed after organ culture, using CL/Fr-N and CL/Fr-BCL palatal shelves dissected and approximated by pairs on E13.5. This showed that Pax9 was still expressed in the ME region in separated palatal shelves of CL/Fr-N and CL/Fr-BCL embryos, while Pax9 expression was down regulated in paired palatal shelves. These expression patterns of Pax9 in normal and cleft embryos during palatal fusion indicate that Pax9 expression is altered in spontaneous cleft lip and palate, and concludes that there is a direct correlation between Pax9 expression and palatal fusion.

Effect of ammonium hexafluorosilicate application for arresting caries treatment on demineralized primary tooth enamel.

Ammonium hexafluorosilicate (AHF) has been applied to arrest caries without discoloration. The purpose of this study was to observe structural and elemental changes of demineralized and AHF applied primary tooth enamel. Enamel from the labial surface of 20 primary canines was divided into an unground side and ground side at the center of the tooth, and demineralized with 35% phosphoric acid for 6 min. The teeth were divided into 4 groups according to a 3-min application of AHF and 1 week of soaking in artificial saliva, as follows: group A (neither AHF nor saliva), group B (only saliva), group C (only AHF), and group D (AHF and saliva), and then subdivided according to whether the enamel was ground or unground. Specimens were analyzed with scanning electron microscopy (SEM) and energy dispersive X-ray spectrometry (EDS). The data were statistically analyzed using ANOVA and Fisher's PLSD test at α = 0.05. In groups A and B, prism structures were seen, however, in groups C and D, enamel surfaces were covered with spherical particles. Ca/P ratio was significantly higher in groups C and D than in groups A and B. There was no significant difference between ground and unground enamel in the content of any element. The values for F, Na, Mg and Si persents and Ca/P ratio were significantly higher for the enamel surface than for points 10-30 µm beneath the surface. Results of this study suggest the possibility that AHF treatment arrests caries, although further study will be required to confirm this result.

Dental manifestations of a pediatric patient with hyperimmunoglobulin e syndrome: a case report.

A 7-year-old girl with hyperimmunoglobulin E syndrome (HIES) presented to our clinic with tooth agenesis in both primary and permanent dentitions. The patient's serum immunoglobulin E level was elevated at 17,091 IU/ml, and her medical history indicated the occurrence of HIES, numerous skin abscesses, and recurrent infection by bacteria and/or fungi such as Candida from birth. She also suffered from heart disease. Dental manifestations included extensive caries, impaired root resorption of primary mandibular central incisors and absence of primary mandibular canines and permanent mandibular lateral incisors. Intraoral phenotypes in HIES patients have already been reported in detail, but no previous report has described abnormalities in the number of primary teeth in HIES patients. The purpose of this report was to describe the dental manifestations in the primary dentition of a hyperimmunoglobulin E syndrome patient and to emphasize the importance of preventive dental management from early childhood in compromised children, such as those with HIES.

Paralogous murine Nudt10 and Nudt11 genes have differential expression patterns but encode identical proteins that are physiologically competent diphosphoinositol polyphosphate phosphohydrolases.

We previously described paralogous human genes [NUDT10 and NUDT11 [where NUDT is (nucleoside diphosphate attached moiety 'X')-type motif, also known as the 'nudix'-type motif]] encoding type 3 diphosphoinositol polyphosphate phosphohydrolases (DIPP3) [Hidaka, Caffrey, Hua, Zhang, Falck, Nickel, Carrel, Barnes and Shears (2002) J. Biol. Chem. 277, 32730-32738]. Normally, gene duplication is redundant, and lacks biological significance. Is this true for the DIPP3 genes? We address this question by characterizing highly-conserved murine Nudt10 and Nudt11 homologues of the human genes. Thus these genes must have been duplicated prior to the divergence of primates and sciurognath rodents, approx. 115 million years ago, greatly exceeding the 4 million year half-life for inactivation of redundant paralogues; our data therefore indicate that the DIPP3 duplication is unusual in being physiologically significant. One possible functional consequence is gene neofunctionalization, but we exclude that, since Nudt10 and Nudt11 encode identical proteins. Another possibility is gene subfunctionalization, which we studied by conducting the first quantitative expression analysis of these genes. We demonstrated high Nudt10 expression in liver, kidney and testis; Nudt11 expression is primarily restricted to the brain. This differential, but complementary, expression pattern indicates that subfunctionalization is the evolutionary consequence of DIPP3 gene duplication. Our kinetic data argue that diphosphoinositol polyphosphates are more physiologically relevant substrates for DIPP3 than are either diadenosine hexaphosphate or 5-phosphoribosyl 1-pyrophosphate. Thus the significance of the Nudt10/Nudt11 duplication is specific hydrolysis of diphosphoinositol polyphosphates in a tissue-dependent manner.

The importance to chondrocyte differentiation of changes in expression of the multiple inositol polyphosphate phosphatase.

It is important to both physiological and pathological osteogenesis to understand the significance of changes in gene expression in growth-plate chondrocytes that transit between the proliferative and hypertrophic states. MINPP is one such gene of interest. The Minpp protein dephosphorylates highly phosphorylated inositol signaling molecules InsP(5) and InsP(6). We show here that the ATDC5 chondrocyte progenitor cell line can recapitulate developmentally specific changes in MINPP expression previously only seen in longitudinal bone growth plates-both an initial 2-3-fold increase and a subsequent decrease back to initial levels during transition to hypertrophy. The increase in MINPP expression was accompanied by a 40% decrease in InsP(6) levels in ATDC5 cells. However, InsP(5) levels were not modified. Furthermore, throughout the hypertrophic phase, during which MINPP expression decreased, there were no alterations in InsP(5) and InsP(6) levels. We also created an ATDC5 line that stably overexpressed Minpp at 2-fold higher levels than in wild-type cells. This had no significant effect upon cellular levels of InsP(5) and InsP(6). Thus, substantial changes in MINPP expression can occur without a net effect upon InsP(5) and InsP(6) turnover in vivo. On the other hand, Minpp-overexpressing cells showed impaired chondrogenesis. We noted that the expression of alkaline phosphatase activity was inversely correlated with the expression of MINPP. The ATDC5 cells that overexpress Minpp failed to show an insulin-dependent increase in alkaline phosphatase levels, which presumably affects phosphate balance [J. Biol. Chem. 276 (2001) 33995], and may be the reason cellular differentiation was impaired. In any case, we conclude that Minpp is important to chondrocyte differentiation, but in a manner that is, surprisingly, independent of inositol polyphosphate turnover.

An adjacent pair of human NUDT genes on chromosome X are preferentially expressed in testis and encode two new isoforms of diphosphoinositol polyphosphate phosphohydrolase.

Combinatorial expression of the various isoforms of diphosphoinositol synthases and phosphohydrolases determines the rates of phosphorylation/dephosphorylation cycles that have been functionally linked to vesicle trafficking, stress responses, DNA repair, and apoptosis. We now describe two new 19-kDa diphosphoinositol polyphosphate phosphohydrolases (DIPPs), named types 3alpha and 3beta, which possess the canonical Nudix-type catalytic motif flanked on either side by short Gly-rich sequences. The two enzymes differ only in that Pro-89 in the alpha form is replaced by Arg-89 in the beta form, making the latter approximately 2-fold more active in vitro. Another Nudix substrate, diadenosine hexaphosphate, was hydrolyzed less efficiently (k(cat)/K(m) = 0.2 x 10(5) m(-1) s(-1)) compared with diphosphoinositol polyphosphates (k(cat)/K(m) = 2-40 x 10(5) m(-1) s(-1)). Catalytic activity in vivo was established by individual overexpression of the human (h) DIPP3 isoforms in HEK293 cells, which reduced cellular levels of diphosphoinositol polyphosphates by 40-50%. The hDIPP3 mRNA is preferentially expressed in testis, accompanied by relatively weak expression in the brain, contrasting with hDIPP1 and hDIPP2 which are widely expressed. The hDIPP3 genes (NUDT10 encodes hDIPP3alpha; NUDT11 encodes hDIPP3beta) are only 152 kbp apart at p11.22 on chromosome X and probably arose by duplication. Transcription of both genes is inactivated on one of the X chromosomes of human females to maintain appropriate gene dosage. The hDIPP3 pair add tissue-specific diversity to the molecular mechanisms regulating diphosphoinositol polyphosphate turnover.

Role of the PLC-related, catalytically inactive protein p130 in GABA(A) receptor function.

The protein p130 was isolated from rat brain as an inositol 1,4,5-trisphosphate-binding protein with a domain organization similar to that of phospholipase C-delta1 but lacking PLC activity. We show that p130 plays an important role in signaling by the type A receptor for gamma-aminobutyric acid (GABA). Yeast twohybrid screening identified GABARAP (GABA(A) receptor-associated protein), which is proposed to contribute to the sorting, targeting or clustering of GABA(A) receptors, as a protein that interacts with p130. Furthermore, p130 competitively inhibited the binding of the gamma2 subunit of the GABA(A) receptor to GABARAP in vitro. Electrophysiological analysis revealed that the modulation of GABA-induced Cl- current by Zn2+ or diazepam, both of which act at GABA(A) receptors containing gamma subunits, is impaired in hippocampal neurons of p130 knockout mice. Moreover, behavioral analysis revealed that motor coordination was impaired and the intraperitoneal injection of diazepam induced markedly reduced sedative and antianxiety effects in the mutant mice. These results indicate that p130 is essential for the function of GABA(A) receptors, especially in response to the agents acting on a gamma2 subunit.