Internal transcribed spacer (ITS)-PCR identification of MRSA.

Polymerase chain reaction (PCR) analysis of the 16S-23S rRNA gene internal transcribed spacer (ITS) followed by microchip gel electrophoresis was useful for identification of staphylococci and for strain delineation of Staphylococcus aureus. In the study presented in this chapter, 74 ITS patterns were demonstrated among 1,188 isolated colonies of S. aureus: 55 patterns for methicillin-susceptible S. aureus (MSSA), 4 patterns for methicillin-resistant S. aureus (MRSA), and 15 patterns for both MSSA and MRSA, highlighting the inability of ITS pattern analysis to differentiate the MSSA and MRSA strains. To overcome this problem, simultaneous PCR amplification of the ITS region and mecA gene was applied to isolated colonies of staphylococcus species and positive-testing blood culture bottles.

Discovery and evaluation of selective N-type calcium channel blockers: 6-unsubstituted-1,4-dihydropyridine-5-carboxylic acid derivatives.

A structure-activity relationship study of 6-unsubstituted-1,4-dihydropyridine and 2,6-unsubstituted-1,4-dihydropyridine derivatives was conducted in an attempt to discover N-type calcium channel blockers that were highly selective over L-type calcium channel blockers. Among the tested compounds, (+)-4-(3,5-dichloro-4-methoxy-phenyl)-1,4-dihydro-pyridine-3,5-dicarboxylic acid 3-cinnamyl ester was found to be an effective and selective N-type calcium channel blocker with oral analgesic potential.

Asymmetric synthesis and biological evaluations of (+)- and (-)-6-dimethoxymethyl-1,4-dihydropyridine-3-carboxylic acid derivatives blocking N-type calcium channels.

An efficient asymmetric synthesis of 1,4-dihydropyridine derivatives is described. The key step is the stereoselective Michael addition using t-butyl ester of L-valine as a chiral auxiliary to achieve good ee (>95% for all the tested experiments) and moderate yield. With this method, (+)-4-(3-chlorophenyl)-6-dimethoxymethyl-2-methyl-1,4-dihydropyridine-3,5-dicarboxylic acid cinnamyl ester was obtained and was characterized as a promising N-type calcium channel blocker with improved selectivity over L-type compared to its (-)- and racemic isomers.

Rapid identification of gram-negative bacteria with and without CTX-M extended-spectrum ß-lactamase from positive blood culture bottles by PCR followed by microchip gel electrophoresis.

We evaluated the usefulness of PCR analysis of the 16S-23S rRNA gene internal transcribed spacer (ITS) and the CTX-M extended-spectrum ß-lactamase (ESBL) followed by microchip gel electrophoresis (MGE) for direct identification and CTX-M detection of Gram-negative bacteria (GNB) from positive blood culture bottles. Of 251 GNB isolated from blood cultures containing a single bacterium, 225 (90%) were correctly identified at the species level directly from positive blood culture bottles by comparing the ITS-PCR patterns of the sample strain with those of the control strains. There were no cases of incorrect identification. Limitations encountered included the inability to detect mixed cultures (four bottles) as well as some species (Enterobacter species and Klebsiella oxytoca) demonstrating identical ITS-PCR patterns. A total of 109 ESBL-producing isolates from various clinical materials obtained between January 2005 and December 2008 were examined for bla(CTX-M), bla(SHV), and bla(TEM) genes by PCR and sequences of PCR products. CTX-M ESBL was detected in 105 isolates, and SHV ESBL was detected in two isolates. The remaining two isolates (K. oxytoca) were shown to harbor bla(OXY.) Twenty (19%) of 104 Escherichia coli isolates from blood cultures were suspected to produce ESBL by the combination disk method, and these isolates were shown to harbor CTX-M ESBL by PCR-MGE. The results were obtained within 1.5 h at a calculated cost of $6.50 per specimen. In conclusion, simultaneous detection of ITS length polymorphisms and bla(CTX)-(M) by single PCR followed by MGE is useful for rapid, cost-effective, and reliable species-level identification of CTX-M ESBL-producing GNB responsible for bloodstream infections.

Restrictive mitral annuloplasty for functional mitral regurgitation: acute hemodynamics and serial echocardiography.

BACKGROUND: Long-term effects of restrictive mitral annuloplasty (RMA), especially on hemodynamics and left ventricular (LV) function in patients with functional mitral regurgitation (MR), have not been fully investigated. METHODS AND RESULTS: From 1999 to 2008, 44 patients with refractory heart failure and functional MR underwent RMA with stringent downsizing of the mitral annulus. Serial echocardiography was performed to evaluate LV function (reverse remodeling), estimated systolic pulmonary artery pressure (PAP) and mitral valve geometry at baseline and at discharge, and annually thereafter. Cardiac catheterization was performed at baseline, and at discharge to evaluate acute hemodynamic change. There were 3 early deaths, and the 5-year survival rate was 78 ± 8%. In 41 survivors the clinical symptoms, stratified according to New York Heart Association class, significantly improved after surgery. Postoperative cardiac catheterization showed significant unloading for left ventricle, as well as improvement in LV systolic function. Serial echocardiography showed that improvements in LV function and systolic PAP were sustained in the majority of patients. Multivariate Cox regression analysis identified preoperative pulmonary hypertension (systolic PAP>60 mm Hg) as the significant predictor for postoperative adverse cardiac events. CONCLUSIONS: RMA for functional MR resulted in sustained improvement of hemodynamics and LV function over time. Additional studies are needed to define the negative impact of preoperative pulmonary hypertension in patients with this condition.

Properties and reactivity of the adenosine radical generated by radiation-induced oxidation in aqueous solution.

The formation of oxidation products of DNA bases induced by hole injection into DNA duplexes is closely related to the characteristics of the radical species generated, especially those from purine bases possessing lower oxidation potentials than pyrimidines. To investigate the reactivities of adenosine base radicals generated from the radical cations of adenosine (Ado), we have conducted extensive pulse radiolysis and steady-state X-radiolysis investigations of Ado under the conditions generating oxidizing radical species such as the sulfate radical anion (SO(4)(-*)). Kinetic analysis of the transient absorption spectra demonstrated that the redox-neutral radical of adenosine [Ado(-H)(*)] decays via a homogeneous bimolecular reaction, recombines with an alkyl alcohol radical, or abstracts hydrogen from the phosphate ion, with rate constants of 2k = 4.2 x 10(8) M(-1) s(-1) and k = 6.4-8.5 x 10(8) and 10(3)-10(4) M(-1) s(-1), respectively. High-dose pulsed electron beam irradiation, which generates high concentrations of Ado(-H)(*) during the radiolysis, yielded 8-oxo-7,8-dihydroadenosine (8-oxo-Ado) with a G value of 0.018 x 10(-7) mol J(-1). It is thus proposed that the disproportionation of Ado(-H)(*) might lead to the formation of 8-oxo-Ado as a minor process, which is in addition to the widely accepted mechanism in which the addition of hydroxyl radical to Ado initiates its formation.

[Serologic diagnosis of fungal infections].

Serologic markers such as fungal antigens and beta-D-glucan are becoming increasingly important for early diagnosis of invasive fungal infections (IFI). Fungal antigens detected with commercially available kits include mannan from Candida spp., galactomannan from Aspergillus spp., and galactoxylomannan from Cryptococcus spp. Measurement of (1,3)-beta-D-glucan in blood may be useful as a preliminary screening tool for IFI, despite the fact that beta-D-glucan can be detected in a number of other fungi. The clinical sensitivity and specificity of serologic assays for diagnosing IFI are somewhat variable. These variations are mainly related to the patient population examined, the test conditions and the frequency of the serologic survey. A large number of cases should be evaluated to clarify the characteristics of the assay kits.

The structure-activity relationship study on 2-, 5-, and 6-position of the water soluble 1,4-dihydropyridine derivatives blocking N-type calcium channels.

In order to find an injectable and selective N-type calcium channel blocker, we have performed the structure-activity relationship (SAR) study on the 2-, 5-, and 6-position of 1,4-dihydropyridine-3-carboxylate derivative APJ2708 (2), which is a derivative of Cilnidipine and has L/N-type calcium channel dual inhibitory activities. As a consequence of the optimization, 6-dimethylacetal derivative 7 was found to have an effective inhibitory activity against N-type calcium channels with more than 170-fold lower activity for L-type channel compared to that of APJ2708.

Internal transcribed spacer (ITS)-PCR identification of MRSA.

Polymerase chain reaction (PCR) analysis of the 16S-23S rRNA gene internal transcribed spacer (ITS) followed by microchip gel electrophoresis was useful for identification of staphylococci and for strain delineation of Staphylococcus aureus. In the study presented in this chapter, 40 ITS patterns were demonstrated among 228 isolated colonies of S. aureus: 26 patterns for methicillin-susceptible S. aureus (MSSA); 11 patterns for methicillin-resistant S. aureus (MRSA); and 3 patterns for both MSSA and MRSA, highlighting the inability of ITS pattern analysis to differentiate the MSSA and MRSA strains. To overcome this problem, simultaneous PCR amplification of the ITS region and the mecA gene was applied to isolated colonies of staphylococcus species and positive-testing blood culture bottles.

Catheter-related fungemia due to fluconazole-resistant Candida nivariensis.

We report on a case of fungemia due to fluconazole-resistant Candida nivariensis (MIC, > or =128 microg/ml). Internal transcribed spacer PCR followed by microchip gel electrophoresis with a blood culture that tested positive revealed a unique pattern different from those of other pathogenic yeasts.

Product and pulse radiolysis studies on radical-ion splitting of N(1)-C(5')-linked dimer hydrates of 5-substituted uracils by one-electron reduction in anoxic aqueous solution.

Steady-state gamma-radiolysis, pulse radiolysis, and cyclic voltammetry have been performed to identify the mechanism by which N(1)-C(5')-linked homodimer hydrates [1-(6'-hydroxy-5',6'-dihydrothymin-5'-yl)thymine (2a) and [1-(5'-fluoro-6'-hydroxy-5',6'-dihydrouracil-5'-yl)-5-fluorouracil (2b)], N(1)-C(6')-linked dimer hydrate [1-(5'-hydroxy-5',6'-dihydrothymin-6'-yl)thymine (3a)], and N(1)-C(5')-linked heterodimer hydrate [1-(6'-hydroxy-5',6'-dihydrothymin-5'-yl)-5-fluorouracil (2ba)] undergo radiolytic reductive splitting to regenerate the parent monomers in anoxic aqueous solution. Radiolytic reductions of the thymine homodimer hydrates 2a and 3a by hydrated electrons (e(aq)-) regenerated the parent thymine (1a) almost quantitatively, while the 5-fluorouracil homodimer hydrates cis-2b and trans-2b afforded 1-(uracil-5'-yl)-5-fluorouracil efficiently along with a small amount of the parent 5-fluorouracil (1b). In contrast to 2b, the heterodimer hydrate analogue 2ba with noneliminating 5'-methyl substituent releases 5-fluorouracil 1b almost quantitatively in the radiolytic reduction. The pulse radiolysis studies suggested that the electron adducts are produced primarily at the thymine and 5-fluorouracil structural unit in the dimer hydrates 2a,b, respectively, in which the resulting dimer hydrate radical anion of 2b (2b*-) was more stable than that of 2a (2a*-). As characterized by pulse radiolysis and cyclic voltammetry, the 5-fluorouracil homodimer hydrate 2b bearing F-substituent at C(5') undergoes one-electron reduction to eliminate exclusively fluoride ion along with the formation of dimer hydrate C(5') radical (2b(-F)*) with oxidizing property. The formation of a possible dimer hydrate radical intermediate 2b(-F)* was also supported by the effect of amines as the reducing additives on the yields of 1b and 4b in the radiolytic reduction of 2b.

Occurrence of the Fimbria Gene hifA in clinical isolates of nonencapsulated Haemophilus influenzae.

The adherence of Haemophilus influenzae to epithelial cells plays a crucial role in infections. However, little is known about the occurrence of fimbriae. In this study, we examined the distribution of the fimbria gene (hifA) by PCR among 167 H. influenzae strains isolated from patients with respiratory infections. Almost all (163; 98%) of the isolates were nonencapsulated strains. The carriage rate of hifA by the nonencapsulated strains was 18.4%. Electron microscopy showed that fimbriae were abundantly present on the cell surface of hifA-positive strains tested. Only four (2.4%) isolates were encapsulated, all of which were type b and did not possess hifA. The present work suggests that fimbriae may play a considerable role as adhesins in nonencapsulated H. influenzae strains.

Discovery, structure-activity relationship study, and oral analgesic efficacy of cyproheptadine derivatives possessing N-type calcium channel inhibitory activity.

Antiallergic drug cyproheptadine (Cyp) is known to have inhibitory activities for L-type calcium channels in addition to histamine and serotonin receptors. Since we found that Cyp had an inhibitory activity against N-type calcium channel, Cyp was optimized to obtain more selective N-type calcium channel blocker with analgesic action. As a consequence of the optimization, we found 13 with potent N-type calcium channel inhibitory activity which had lower inhibitory activities against L-type calcium channel, histamine (H1), and serotonin (5-HT2A) receptors than those of Cyp. 13 showed an oral analgesic activity in rat formalin-induced pain model.

Evaluation of a newly developed down-flow immunoassay for detection of serum mannan antigens in patients with candidaemia.

A down-flow immunoassay has been developed to detect serum mannan antigens, and the test was recently marketed as the Unimedi Candida monotest. Using 251 serum samples from 105 patients with candidaemia, a comparison of the Unimedi Candida monotest with the Cand-Tec latex agglutination test and 2 microplate enzyme immunoassay tests (Platelia Candida Ag test and Unimedi Candida) was conducted. One hundred and seventy-five febrile patients without clinical and microbiological evidence of fungal infections and pneumocytosis were examined as controls. The Cand-Tec test had a sensitivity of 38% and a specificity of 82%. The sensitivity and specificity of the Platelia Candida Ag test, the Unimedi Candida and the Unimedi Candida monotest were 53 and 92%, 69 and 89% and 82 and 96%, respectively. The sensitivity of the Unimedi Candida monotest was significantly (P<0.01) higher than that of the Plateria Candida Ag test for diagnosing candidaemia caused by Candida parapsilosis. The beta-D-glucan assay had a high sensitivity of 95%, with a specificity of 84%. Of 74 patients with candidaemia whose sera were available before or on positive blood culture sampling, 29 (39%), 38 (51%) and 48 (65%) patients had antigenemia detected using the Platelia Candida Ag test, the Unimedi Candida and the Unimedi Candida monotest, respectively. The Unimedi Candida monotest seems to be a promising tool for the early diagnosis of invasive candidiasis, because the test was sensitive, simple, rapid (approx. 1 h) and cost-effective.

Structure-activity relationship study of 1,4-dihydropyridine derivatives blocking N-type calcium channels.

Cilnidipine is a 1,4-dihydropyridine derived L/N-type calcium channel dual blocker possessing neuroprotective and analgesic effects which are related to its N-type calcium channel inhibitory activity. In order to find specific N-type calcium channel blockers with the least effects on cardiovascular system, we performed structure-activity relationship study on APJ2708, which is a derivative of cilnidipine, and found a promising N-type calcium channel blocker 21b possessing analgesic effect in vivo with a 1600-fold lower activity against L-type calcium channels than that of cilnidipine.

Rapid identification of staphylococcal strains from positive-testing blood culture bottles by internal transcribed spacer PCR followed by microchip gel electrophoresis.

PCR analysis of the 16S-23S rRNA gene internal transcribed spacer (ITS) followed by microchip gel electrophoresis (MGE) was evaluated for its usefulness in identification of staphylococci. Forty ITS PCR patterns were demonstrated among 228 isolated colonies of Staphylococcus aureus: 26 patterns for methicillin-susceptible S. aureus (MSSA; 91 strains), 11 patterns for methicillin-resistant S. aureus (MRSA; 99 strains), and 3 patterns for both MSSA and MRSA (38 strains). Thirty-seven control strains of coagulase-negative staphylococci (CNS) representing 16 species showed unique ITS PCR patterns (24 patterns) at the species and subspecies levels: two patterns for S. caprae, S. cohnii, S. haemolyticus, and S. saprophyticus; three patterns for S. lugdunensis; four patterns for S. capitis; and one pattern for each of the other CNS species. The combined PCR-MGE method was prospectively adapted to the positive blood culture bottles, and this method correctly identified MSSA and MRSA in 102 (89%) of 114 blood cultures positive for S. aureus on the basis of the ITS PCR patterns. Eight ITS PCR patterns were demonstrated from 166 blood culture bottles positive for CNS. The most frequent CNS species isolated from blood cultures were S. epidermidis (76%), S. capitis (11%), and S. hominis (8%). Overall, all 280 blood culture bottles shown to contain a single Staphylococcus species by routine phenotypic methods were correctly identified by the PCR-MGE method at the species level, whereas the organism failed to be identified in 8 culture bottles (3%) with mixed flora. The PCR-MGE method is useful not only for rapid identification ( approximately 1.5 h) of staphylococci in positive blood culture bottles, but also for strain delineation of S. aureus.

Neuronal Ca2+ channel blocking action of an antihypertensive drug, cilnidipine, in IMR-32 human neuroblastoma cells.

Although the anti-sympathetic mechanisms of the antihypertensive drug cilnidipine have been analyzed in neuronal cells derived from rodents, there is little information regarding the effects of cilnidipine in human neuronal cells. We investigated the effects of cilnidipine on N-type Ca2+ channels in IMR-32 human neuroblastoma cells using fura-2-based microfluorimetry. The ratio of the intensities of the emitted fluorescence at an excitation wavelength of 340 nm to that at 380 nm was calibrated to estimate the intracellular concentration of Ca2+. Stimulation of IMR-32 cells by 40 mmol/l KCl immediately increased the intensities ratio. In the presence of 10 micromol/l of nifedipine to block L-type Ca2+ channels, omega-conotoxin GVIA, a selective N-type Ca2+ channel blocker, in a concentration of 1 micromol/l suppressed the elevation of the intensities ratio induced by 40 mmol/l KCl. Similarly, cilnidipine in a concentration of 10 micromo/l suppressed the elevation of the ratio induced by 40 mmol/l KCl, and this suppression was effectively inhibited after the treatment with omega-conotoxin GVIA. These results suggest that cilnidipine potentially inhibits N-type Ca2+ channels in human neuronal cells and might be applied as a prospective therapeutic tool to provide neuronal protection as well as its antihypertensive effects and anti-sympathetic actions.