Evaluation of a simple loop-mediated isothermal amplification test kit for the diagnosis of tuberculosis.

OBJECTIVE: A new loop-mediated isothermal amplification (LAMP) test kit, including a simple DNA extraction device for the detection of Mycobacterium tuberculosis complex, was developed for commercial use and evaluated for its usefulness in diagnosing tuberculosis (TB). DESIGN: The LAMP test was performed using untreated and N-acetyl-L-cysteine (NALC) NaOH-treated sputum specimen. The efficiency of the kit was compared with other conventional laboratory examinations, including other nucleic acid amplification (NAA) tests. RESULTS: The sensitivity of LAMP using raw sputum (direct LAMP) in smear- and culture-positive specimens was 98.2% (95%CI 94.9-99.4), while the sensitivity in smear-negative, culture-positive specimens was 55.6% (95%CI 43.4-68.0). The diagnostic sensitivity of direct LAMP for the diagnosis of individuals with TB was 88.2% (95%CI 81.4-92.7). The sensitivity values of direct LAMP were slightly, but not statistically significantly lower than those of Cobas Amplicor MTB and TRC Rapid MTB, while the sensitivity of the LAMP test using NALC-NaOH treated sputum was significantly lower than other NAA tests (P < 0.05) for smear-negative, culture-positive specimens. The new commercial version of the LAMP kit was easy to handle and yielded results within 1 h of receiving sputum specimens. CONCLUSIONS: This test is considered a promising diagnostic tool for TB, even for peripheral laboratories with limited equipment, such as those in developing countries.

Application of several types of substrates to ficin-catalyzed peptide synthesis.

The capability of ficin, a cystine protease, to form peptide bonds was investigated using several types of N-Boc-amino acid phenyl and naphthyl esters as acyl donor components. Enzyme-catalyzed peptide synthesis was carried out under optimized reaction conditions of pH, acyl acceptor concentration and selection of the best yield organic solvent. It used a condensation of N-Boc-Ala-OpGu and Ala-pNA as a model reaction. The products were obtained in 72-96% yield using 10 different substrates, within a few minutes of reaction time.

Pulmonary Mycobacterium avium complex infection associated with the IVS8-T5 allele of the CFTR gene.

BACKGROUND: The T5 allele in intron 8 (IVS8) on specific haplotype backgrounds (e.g., long TG repeats) causes abnormal splicing in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, and is also known to be associated with chronic airway diseases. OBJECTIVE: To investigate the role of CFTR variations for susceptibility to pulmonary Mycobacterium avium complex (MAC) infection. PARTICIPANTS: Three hundred patients with pulmonary MAC infection (72 males, 228 females; mean age at onset 61.6 + or - 12.4 years) took part in this study. Diagnosis of MAC infection was based on American Thoracic Society criteria. Clinical profiles were collected and blood samples were genotyped for TG repeats, poly-T and M470V polymorphisms. RESULTS: We found significantly higher T5 frequency in MAC patients than in healthy controls from our own study (0.035 and 0.005, respectively, P = 0.023) and other reports. Homozygote for the T5 allele was found in two MAC patients. All T5 alleles were associated with longer TG repeats, the TG12 or TG13 allele. Seventeen of the 21 T5 alleles appeared to be associated with the V470 allele. Other polymorphisms did not show any significant differences in frequency. CONCLUSIONS: These findings suggest that the IVS8 5T allele might be involved in susceptibility to pulmonary MAC infection.

Pulmonary Mycobacterium avium complex infection: association with NRAMP1 polymorphisms.

The present study aimed to elucidate risk factors for nonimmunocompromised pulmonary Mycobacterium avium complex (MAC) infection. Epidemiological data and variations of candidate genes for mycobacterial diseases were analysed in 111 patients with pulmonary MAC infection. Four polymorphisms of the human natural resistance-associated macrophage protein (NRAMP)1 gene, the 5'(GT)n, 469+14 G/C, D543N and the 3'untranslated region (3'TGTG) insertion/deletion, were genotyped using PCR-based methods. Fok I and Taq I polymorphisms of the vitamin D receptor gene and -221 X/Y and codon 54 A/B polymorphisms of the mannose binding lectin gene were also evaluated. Females were more susceptible to MAC infection mainly affecting the right middle lobe or lingular segment of the lung. Patients' residence at the onset of the disease was distributed evenly irrespective of a waterfront or city water supply system. As compared with homozygotes for major alleles of the D543N and TGTG insertion/deletion polymorphism of the NRAMP1 gene, heterozygotes containing minor alleles were less often observed in MAC cases than in controls. This genetic effect was more significant in patients without comorbidity but not in patients with comorbidity. Other polymorphisms did not show any association with the MAC infection. The human natural resistance-associated macrophage protein 1 gene might be involved in susceptibility to pulmonary Mycobacterium avium complex infection.

Regulation of the coronary vasomotor tone: What we know and where we need to go.

The control of coronary blood flow has been studied for decades, but despite our extensive efforts, the critical regulators of flow are largely unknown. One purpose of this review is to summarize some recent concepts about the control of coronary flow and also point out areas where additional knowledge must be acquired. A second purpose of this review is to highlight the need for additional noninvasive measurements of flow that undoubtedly will require further evolution of contemporary technologies, and also application of specific methods toward noninvasive measurements of coronary blood flow. Only after the development of such measurements will the scientific community begin to understand the intricacies of the regulation of coronary flow in human beings.

Chum salmon trypsin-catalyzed preferential formation of peptides containing D-amino acid.

Chum salmon trypsin-catalyzed peptide synthesis has been studied by using nine series of "inverse substrates," i.e., p-amidinophenyl, p- and m-guanidinophenyl, p- and m-(guanidinomethyl)phenyl, and four position isomers of guanidinonaphthyl esters derived from Nalpha-(tertbutyloxycarbonyl)amino acid as acyl donor components. They were found to couple with an acyl acceptor such as L-alanine p-nitroanilide to produce dipeptide in the presence of trypsin. All substrates tested in this study undergo less enantioselective coupling reaction, and the coupling product was the favorably obtained D-series rather than L-series (in the present case; Nalpha-Boc-D-Ala and Nalpha-Boc-L-Ala). The optimum condition for the coupling reaction was studied by changing the organic solvent, buffer solution, pH, and acyl acceptor concentration. It was found that the enzymatic hydrolysis of the resulting product was negligible.

Stenosis differentially affects subendocardial and subepicardial arterioles in vivo.

The presence of a coronary stenosis results primarily in subendocardial ischemia. Apart from the decrease in coronary perfusion pressure, a stenosis also decreases coronary flow pulsations. Applying a coronary perfusion system, we compared the autoregulatory response of subendocardial (n = 10) and subepicardial (n = 12) arterioles (<120 microm) after stepwise decreases in coronary arterial pressure from 100 to 70, 50, and 30 mmHg in vivo in dogs (n = 9). Pressure steps were performed with and without stenosis on the perfusion line. Maximal arteriolar diameter during the cardiac cycle was determined and normalized to its value at 100 mmHg. The initial decrease in diameter during reductions in pressure was significantly larger at the subendocardium. Diameters of subendocardial and subepicardial arterioles were similar 10--15 s after the decrease in pressure without stenosis. However, stenosis decreased the dilatory response of the subendocardial arterioles significantly. This decreased dilatory response was also evidenced by a lower coronary inflow at similar average pressure in the presence of a stenosis. Inhibition of nitric oxide production with N(G)-monomethyl-L-arginine abrogated the effect of the stenosis on flow. We conclude that the decrease in pressure caused by a stenosis in vivo results in a larger decrease in diameter of the subendocardial arterioles than in the subepicardial arterioles, and furthermore stenosis selectively decreases the dilatory response of subendocardial arterioles. These two findings expand our understanding of subendocardial vulnerability to ischemia.

X-ray crystallographic analyses of complexes between bovine beta-trypsin and Schiff base copper(II) or iron(III) chelates.

To establish the structural basis underlying the activity of a novel series of metal-chelate trypsin inhibitors, the structures of p-amidinosalicylidene-l-alaninato(aqua)copper(II) (1a), m-amidinosalicylidene-l-alaninato(aqua)copper(II) (1b), bis(p-amidinosalicylidene-l-alaninato)iron(III) (2a), and bis(m-amidinosalicylidene-l-alaninato)iron(III) (2b) bound to bovine beta-trypsin were studied by X-ray crystallography. The amidinium group of the inhibitor donates hydrogen bonds to Asp189, Gly219 and Ser190, as seen before in trypsin-benzamidine complexes. The copper(II) ion of 1a is situated away from trypsin's catalytic triad residues, and is octahedrally coordinated by a Schiff base and three water molecules. In contrast, the copper(II) ion of 1b is situated close to the catalytic triad and adopts a square pyramidal coordination geometry. The iron(III) ion of 2a is octahedrally coordinated by two Schiff base ligands and, like the copper(II) ion of 1a, is situated away from the catalytic triad. The p-amidinophenyl ring of a second Schiff base ligand of 2a is directed toward a hydrophobic groove formed by Trp215 and Leu99. Finally, the iron(III) ion of 2b appears to be replaced by magnesium(II), which is octahedrally coordinated by a Schiff base, Gln192 and two water molecules. One of the Schiff base ligands seen in the trypsin-2a complex or in the unbound form of 2b is replaced by water molecules and Gln192. His57 and Ser195 form water-mediated interactions with the magnesium(II) ion of 2b, and Ser195 also forms a hydrogen bond with the phenolic oxygen atom of the Schiff base ligand. These structures reveal a novel mode of interaction between metal-chelate inhibitors and serine proteases, thus providing a structural basis for the development of more potent inhibitors against a variety of trypsin-like enzymes.

[DOTS in Japan--Tokyo area].

The resurgence of tuberculosis has required a successful strategy to control TB in Japan. The World Health Organization has recommended the so-called DOTS (Directly Observed Therapy, Short-Course) strategy since 1995 and DOTS has been used with great success not only in many developing countries, but also in many developed countries. In Tokyo, especially in urban areas with a high prevalence of TB, DOT has been started. Using DOT, Sanya in Taito-ku (ward) and Arakawa-ku (ward), have shown high treatment completion--more than 90% since 1997. In 2000, other health care offices in Shinjuku-ku (ward), Yokohama city, Kawasaki city and Nagoya city started DOT for groups of homeless with TB. Presently, DOT is applied only to homeless people. However, the number of people who need DOT is much higher, due to poor adherence. It is also important to reconsider cost-efficiency for TB control in Japan.

Anionic trypsin from chum salmon: activity with p-amidinophenyl ester and comparison with bovine and Streptomyces griseus trypsins.

An anionic trypsin from pyloric caeca of chum salmon (Oncorhynchus keta) was purified by ammonium sulfate and acetone fractionation followed by affinity chromatography, gel-filtration, and DEAE-anion exchange chromatography. The apparent molecular mass was about 24 kDa as determined by SDS-PAGE. The anionic chum salmon trypsin was moderately active toward esterase substrates such as tosyl-L-arginine methyl ester and tosyl-L-lysine methyl ester. Its amidase activity for benzoyl-L-arginine p-nitroanilide was comparative to those of bovine and Streptomyces griseus trypsins. Kinetic characteristics of anionic chum salmon, bovine, and Streptomyces griseus trypsins toward inverse substrate (p-amidinophenyl ester) were compared. Inverse substrate behaved as a specific substrate for anionic chum salmon trypsin with specific binding, efficient acylation, and relatively slow deacylation.

[A clinical study on pulmonary tuberculosis in younger age groups].

In 1997, the number of newly registered patients with pulmonary tuberculosis increased, compared with that in 1996, in Japan. The majority of the increase were occupied by elder patients 70 years of age or higher. But in younger group less than 30 years old, a reduction in the incidence of tuberculosis had been slowed down, until 1996. The purpose of this report is to elucidate the characteristics of these younger patients. 139 cases younger than 30 years of age, who were hospitalized in the tuberculous ward of IMCJ from April 1995 to March 1998, were investigated, and were compared with the control group (557 cases), 30-79 years old who were hospitalized during the same period. In the younger group, the proportion of women cases, discovered by health examination, foreigners, and contact with TB patients in the past was significantly higher than in the control group. But there were no difference between the both groups, concerning the proportion of those spending irregular life or living alone. The proportion of sputa smear negative cases was significantly higher in the younger group than in the control. For early diagnosis of TB among younger group, the application of bronchofiberscopy and nucleic acid diagnostic method, are encouraged.

Effect of dietary control on plasma nitrate level and estimation of basal systemic nitric oxide production rate in humans.

It is of great interest and value to evaluate the systemic nitric oxide (NO) production rate in humans under various conditions. However, the currently available estimation methods are troublesome and time-consuming. We thus aimed at developing a simple method to estimate the basal systemic NO production rate in humans based on a steady-state analysis, i.e., a balance between the systemic NO production rate and the total nitrate elimination rate. Plasma nitrate concentrations of young healthy volunteers (n = 7 in group 1: n = 9 in group 2) were measured for 2 days. In group 1, all subjects had the same meals for 7 days prior to the plasma nitrate measurement. In group 2, all subjects were allowed free diets. The plasma nitrate concentrations were highly influenced by dietary nitrite/nitrate intake in both groups and reached the steady-state levels after 14-h fasting. Accordingly, the basal systemic NO production rates were estimated from the plasma nitrate concentrations after 14-h fasting (group 1, 630 +/- 37 nmol min(-1) = 0.78 +/- 0.03 micromol kg(-1) h(-1); group 2, 597 +/- 45 nmol min(-1) = 0.66 +/- 0.05 micromol kg(-1) h(-1), P = not significant vs group 1). These estimated values were comparable to the values obtained by other methods. In conclusion, the present estimation method with 14-h fasting using a single-compartment analysis was found to be a simple approach to quantitative evaluation and intra- and interindividual comparisons of the basal systemic NO production rates in humans.

Trypsin-catalyzed peptide synthesis with m-guanidinophenyl and m-(guanidinomethyl)phenyl esters as acyl donor component.

Two series of inverse substrates, m-guanidinophenyl and m-(guanidinomethyl)phenyl esters derived from N-(tert-butyloxycarbonyl)-amino acid, were prepared as an acyl donor component for trypsin-catalyzed peptide synthesis. The kinetic behavior of these esters toward tryptic hydrolysis was analyzed. They were found to couple with an acyl acceptor such as L-alanine p-nitroanilide to produce dipeptide in the presence of trypsin. Streptomyces griseus trypsin was a more efficient catalyst than the bovine trypsin. Within the enzymatic peptide coupling methods, this approach was shown to be advantageous, since the resulting peptides are resistant to the enzymatic hydrolysis.

Role of NO and K(+)(ATP) channels in adenosine-induced vasodilation on in vivo canine subendocardial arterioles.

Adenosine (Ado) plays an important role in regulation of coronary vascular tone with nitric oxide (NO) and ATP-sensitive K(+) (K(+)(ATP)) channels. In vitro, it was reported that subendocardial (Endo) arterioles are more sensitive to Ado than subepicardial (Epi) arterioles. The purpose of this study was to observe enhanced vasodilation of Endo arterioles directly and to evaluate possible roles of K(+)(ATP) channels and NO in the different responses of Endo and Epi arterioles to Ado-induced vasodilation. We evaluated dilation of Endo and Epi arterioles (<120 micrometer) of beating canine hearts (n = 19) by Ado (20 and 50 microgram. kg(-1). min(-1) ic) before and after K(+)(ATP) channel blockade (glibenclamide; 200 microgram/kg ic), inhibition of NO synthase [N(G)-nitro-L-arginine methyl ester (L-NAME); 30 microgram. kg(-1). min(-1), 20 min ic], or glibenclamide + L-NAME using a novel needle-probe CCD intravital microscope. Ado induced dose-dependent vasodilation in both Epi and Endo arterioles, but vasodilation was greater in Endo arterioles, i.e., increase at 120 s (maximum dilation) after Ado (50 microgram. kg(-1). min(-1)) was 17% in Endo and 13% in Epi arterioles (P < 0.01). Endo arteriole dilation was attenuated by blockade of K(+)(ATP) channels from 18% (Ado) to 9% (Ado+glibenclamide) increase (P < 0.001) and by inhibition of NO synthase from 17% (Ado) to 9% (Ado+L-NAME) (P < 0.005). Epi arteriole vasodilation was attenuated by blockade of K(+)(ATP) channels from 15 to 9% (P < 0.005) and inhibition of NO from 16 to 10% (P < 0.005). Suppression of vascular response was additive (Endo, 14 to -1%; Epi, 12 to 3%) with glibenclamide + L-NAME. We conclude that 1) the degree of Ado-induced vasodilation was greater in Endo than in Epi arterioles, with higher sensitivity of smaller arterioles in both layers and 2) transmural difference of arteriolar sensitivity to adenosine was abolished or reversed by K(+)(ATP) channel blockade and/or by NO synthase inhibition, indicating crucial involvement of K(+)(ATP) and NO in transmural sensitivity difference.

Hypodontia in primary dentition: a case report.

The prevalence, possible etiological factors and management of hypodontia in primary dentition are reviewed. A case of hypodontia involving the primary lower incisor is reported.

[Clinical investigation on endobronchial tuberculosis].

In order to assess the clinical features and clinical courses of endobronchial tuberculosis, which included trachea to segmental bronchus, we studied 34 cases of patients who were admitted to TB ward of International Medical Center of Japan from 1994 to 1997. We noticed a higher incidence in females and in the main bronchus. Cough was the most common complaint seen in 97% of cases. The duration of symptoms before the initiation of antituberculous chemotherapy was long (on the average 6 months), and they were often treated as bronchial asthma or bronchitis. Bronchoscopic examination is necessary for diagnosis. The scars sometimes gave rise to severe stenosis, especially when the lesion developed to an advanced stage or circumscribed the lumen before treatment. We tried INH inhalation with systemic chemotherapy. Although rapid improvement was suggested by this method, yet no significant difference was seen in the results for the efficacious prevention of stenosis. Five cases required surgical intervension (bronchoplasty and lobectomy) in order to avoid atelectasis or secondary infection. Early diagnosis and appropriate treatment are most important, and bronchoscopic examination is essential in early diagnosis.

Endothelium-derived nitric oxide enhances the effect of intraaortic balloon pumping on diastolic coronary flow.

BACKGROUND: High shear rate with pulsation is one of the major stimuli for the release of endothelium-derived nitric oxide leading to coronary arteriolar dilation. Intraaortic balloon pumping mechanically enhances shear rate and diastolic-to-systolic flow oscillation. We aimed to evaluate whether or not coronary blood flow augmentation during intraaortic balloon pumping is mediated by coronary arteriolar dilation through endothelium-derived nitric oxide release. METHODS: Using a charge-coupled device intravital videomicroscope, we observed epicardial coronary arterioles (40 to 220 microm in diameter) in anesthetized open-chest dogs (n = 10) during 2:1 mode of intraaortic balloon pumping. Endothelium-derived nitric oxide-mediated vasodilatory effects of intraaortic balloon pumping were evaluated by comparing end-diastolic arteriolar diameters between the coupled beats of on and off intraaortic balloon pumping before and after intracoronary endothelium-derived nitric oxide synthesis inhibition with Nomega-nitro-L-arginine (L-NNA, 2 micromol/min) administration. RESULTS: Intraaortic balloon pumping increased coronary arteriolar diameters and coronary blood flow by 11.4%+/-1.8% (p < 0.0001) and 33.4%+/-4.1% (p < 0.001), respectively. Vasodilation was greater in small arterioles (<110 microm; 15.4%+/-2.2%) than in large arterioles (> or =110 microm; 4.2%+/-1.2%, p < 0.0001). L-NNA attenuated the intraaortic balloon pumping-induced vasodilation and augmentation of coronary blood flow to 4.6%+/-1.0% (p < 0.001) and to 20.8%+/-2.1%, (p < 0.05), respectively. Attenuation of vasodilatory effect by L-NNA was observed mainly in small arterioles (from 15.4%+/-2.2% to 5.9%+/-1.2%). CONCLUSIONS: Intraaortic balloon pumping augmented coronary blood flow by dilating coronary arterioles in diastole, more significantly in small arterioles than in large arterioles. Endothelium-derived nitric oxide inhibition markedly attenuated these effects. We conclude that, in a canine model, endothelium-derived nitric oxide contributes to mechanical enhancement of the coronary blood flow with diastolic arteriolar vasodilation during intraaortic balloon pumping.

Application of Schiff base copper(II) and iron(III) chelates to site-specific cleavage of a trypsin.

Amidine-containing Schiff base iron(III) and copper(II) chelates were prepared from alpha-amino acid, metal ion, and salicylaldehyde. These chelates behaved as specific inhibitors of trypsin, with Ki values in the range 10(-5)-10(-6) M. Selective cleavage of the trypsin backbone resulting from specific binding of the chelate to the trypsin active site was investigated. Cleavage was observed when trypsin was incubated with amidine-containing copper(II) or iron(III) chelate, H2O2, and ascorbate. Examination of the three-dimensional structure of trypsin suggests that cleavage occurred at a peptide bond within the Gly195-Ala204 sequence.

Association of chronic infection of Chlamydia pneumoniae and coronary heart disease in the Japanese.

The association of Chlamydia pneumoniae with atherosclerosis of coronary and carotid arteries and the aorta has been demonstrated by seroepidemiology and by detection of the organism in atheromata. We investigated the frequency of C. pneumoniae seropositivity in patients with acute myocardial infarction (AMI). C. pneumoniae-specific antibodies were measured by the microimmunofluorescence test in 160 AMI patients and 160 control subjects matched for age and gender. The odds ratios (ORs) were 2.2 (95% confidence interval (CI), 1.2 to 3.9) for immunoglobulin (Ig)G and 2.7 (95% CI, 1.7 to 4.3) for IgA. After adjustment for other cardiovascular risk factors of age, gender, hypertension, diabetes, cigarette smoking and serum cholesterol, the ORs were essentially unchanged. This study confirmed that the observations of an association between antibody against C. pneumoniae and coronary heart disease in Western nations is also present in Japan. Our results are comparable to the previous seroepidemiological studies reporting ORs of 2.0 or greater.