Successful treatment of atypical mycobacterial meningitis by fluoroquinolone.

We report a case of restricted meningeal infection by atypical mycobacteria, identified by the polymerase chain reaction, in a non-immunocompromised adult successfully treated by multiple antibiotics including fluoroquinolone. New quinolones should be considered as a therapeutic option for such mycobacterial meningitis.

[Comparison of amplified Mycobacterium Tuberculosis Direct Test (MTD), Amplicor Mycobacteria kit (Amplicor) and PCR method for detection Mycobacterium tuberculosis in clinical specimens].

Accuracy amplified Mycobacterium Tuberculosis Direct Test (MTD), Amplicor Mycobacteria kit (Amplicor) and PCR method routinely used in the University of Tokyo Hospital (J. Clin. Microbiol. 31: 446-450 1991) were evaluated and compared with the same samples. The detection limits of MTD, Amplicor and PCR method (University of Tokyo) were 0.01-0.1 CFU/tube, 0.625 CFU/tube and 0.2 CFU/tube respectively, which were almost the same. These were shown to be at least as sensitive as the conventional culture techniques. The Tokyo Univ. method using radioisotope, takes up to 3 days, on the other hand 2 kits take 4-5 hours. These kits become useful tools for the early and rapid detection of M. tuberculosis in uncultured clinical specimens. But the risk of laboratory contamination and false-positive results remain. These must be further improved.

Salt-sensitive hypertension in transgenic mice overexpressing Na(+)-proton exchanger.

Essential hypertension is one of the most common diseases that exacerbate the risk of cardiovascular or cerebrovascular attacks. Although the etiology of essential hypertension remains unclear, recent investigations have revealed that an enhancement of Na(+)-proton (Na(+)-H+) exchange activity is a frequently observed ion transport abnormality in hypertensive patients and animal models. To test the hypothesis that increased Na(+)-H+ exchange causes hypertension, we produced transgenic mice overexpressing Na(+)-H+ exchanger and analyzed their Na+ metabolism and blood pressure. Urinary excretion of water and Na+ was significantly decreased in transgenic mice, and systolic blood pressure was elevated after salt loading. The impaired urinary excretion of Na+ suggested that the Na(+)-H+ exchanger overexpressed in the renal tubules increased reabsorption of Na+, which caused a blood pressure elevation by Na+ retention after excessive salt intake. Our results demonstrate that overexpression of Na(+)-H+ exchanger can be a genetic factor that interacts with excessive salt intake and causes salt-sensitive blood pressure elevation.

Activation of Na(+)-H+ antiporter (NHE-1) gene expression during growth, hypertrophy and proliferation of the rabbit cardiovascular system.

The Na(+)-H+ antiporter is a unique transmembrane protein with multiple roles in cellular functions through intracellular alkalization. It participates in the regulation of intracellular pH, cell volume and intracellular signalling in response to various mitogenic stimuli. To clarify its role as a subcellular signal in cardiovascular remodeling like vascular hyperplasia or cardiac hypertrophy, we determined mRNA levels of the Na(+)-H+ antiporter isoform, NHE-1, in vascular smooth muscles and pressure-overloaded hearts in rabbits. The NHE-1 mRNA levels in rabbit aortas and hearts were developmentally regulated with high levels at embryonic and neonatal stages than in adults. In primary-cultured smooth muscle cells (SMC), the mRNA levels were increased during exponential growth, but decreased to initial levels at confluency. Growth of a mutant SMC line, C5, which is deficient in Na(+)-H+ antiporter activity, was markedly reduced in bicarbonate-free medium. However, when the activity was restored by transfecting cells with a full-length NHE-1 cDNA in an expression vector, the growth rate of C5 was accelerated again. After balloon injury to the vascular wall, the NHE-1 mRNA levels of the injured arteries were also increased, suggesting that Na(+)-H+ antiporter contributes to the network of the growth promoting systems in smooth muscle cells in vivo. Pressure-overload on the ventricle increased the NHE-1 mRNA levels in hearts approximately two-fold of sham-operated rabbits after 3 days and remained for at least two weeks (P < 0.05). We further demonstrated that 3-methylsulfonyl-4-piperidino-benzoyl guanidine mesylate (Hoe 694), a potent antagonist of Na(+)-H+ antiporter, partially inhibited stretch-induced activation of mitogen-activated kinase (MAP kinase) in the cultured cardiomyocytes. From these results, we conclude that activation of the Na(+)-H+ antiporter and its gene expression is involved in molecular mechanisms of both cardiac hypertrophy and vascular smooth muscle cell proliferation, indicating a potential target in developing new therapeutics for cardiovascular diseases.

Nucleotide sequence comparison of the mycobacterial dnaJ gene and PCR-restriction fragment length polymorphism analysis for identification of mycobacterial species.

We recently reported a genus-specific PCR for the mycobacterial dnaJ gene. In the present study, we have determined the nucleotide sequences of the dnaJ gene from 19 mycobacterial species (Mycobacterium tuberculosis, M. bovis, M. bovis BCG, M. africanum, M. microti, M. marinum, M. kansasii, M. gastri, M. simiae, M. scrofulaceum, M. szulgai, M. gordonae, M. avium, M. intracellulare, M. xenopi, M. fortuitum, M. chelonae, M. hemophilum, and M. paratuberculosis). On the basis of the amplified dnaJ gene nucleotide sequences, we constructed a phylogenetic tree of the mycobacterial species by using the neighbor-joining method and unweighted pairwise grouping method of arithmetic average. We found that the phylogenetic relationship inferred within the slowly growing species was in good agreement with the traditional classification, with three major branches corresponding to Runyon's groups I, II, and III. An exception was M. simiae, which was phylogenetically closer to the cluster including members of Runyon's group III than to that of Runyon's group I. On the other hand, the rapid growers, such as M. fortuitum and M. chelonae, did not form a coherent line corresponding to Runyon's group IV, indicating that our phylogenetic analysis based on the dnaJ gene reflects the phenotypic characteristics such as pigmentation but not the growth rate. Finally, we revealed the species-specific restriction sites within the amplified dnaJ gene to differentiate most of the mycobacterial DNA by a combination of PCR with restriction fragment length polymorphism analysis.

Human smooth muscle myosin heavy chain isoforms as molecular markers for vascular development and atherosclerosis.

Smooth muscle myosin heavy chains (MHCs) exist in multiple isoforms. Rabbit smooth muscles contain at least three types of MHC isoforms: SM1 (204 kD), SM2 (200 kD), and SMemb (200 kD). SM1 and SM2 are specific to smooth muscles, but SMemb is a nonmuscle-type MHC abundantly expressed in the embryonic aorta. We recently reported that these three MHC isoforms are differentially expressed in rabbit during normal vascular development and in experimental arteriosclerosis and atherosclerosis. The purpose of this study was to clarify whether expression of human smooth muscle MHC isoforms is regulated in developing arteries and in atherosclerotic lesions. To accomplish this, we have isolated and characterized three cDNA clones from human smooth muscle: SMHC94 (SM1), SMHC93 (SM2), and HSME6 (SMemb). The expression of SM2 mRNA in the fetal aorta was significantly lower as compared with SM1 mRNA, but the ratio of SM2 to SM1 mRNA was increased after birth. SMemb mRNA in the aorta was decreased after birth but appeared to be increased in the aged. To further examine the MHC expression at the histological level, we have developed three antibodies against human SM1, SM2, and SMemb using the isoform-specific sequences of the carboxyl terminal end. Immunohistologically, SM1 was constitutively positive from the fetal stage to adulthood in the apparently normal media of the aorta and coronary arteries, whereas SM2 was negative in fetal arteries of the early gestational stage. In human, unlike rabbit, aorta or coronary arteries, SMemb was detected even in the adult. However, smaller-sized arteries, like the vasa vasorum of the aorta or intramyocardial coronary arterioles, were negative for SMemb. Diffuse intimal thickening in the major coronary arteries was found to be composed of smooth muscles, reacting equally to three antibodies for MHC isoforms, but reactivities with anti-SM2 antibody were reduced with aging. With progression of atherosclerosis, intimal smooth muscles diminished the expression of not only SM2 but also SM1, whereas alpha-smooth muscle actin was well preserved. We conclude from these results that smooth muscle MHC isoforms are important molecular markers for studying human vascular smooth muscle cell differentiation as well as the cellular mechanisms of atherosclerosis.

[Activation of Na(+)-H+ antiporter gene expression during vascular smooth muscle proliferation and cardiac hypertrophy].

To clarify the role of Na(+)-H+ antiporter as a subcellular signal in cardiovascular system, we determined mRNA levels of the Na(+)-H+ antiporter in the vascular smooth muscles and pressure-overloaded hearts in rabbits. The expression of the Na(+)-H+ antiporter gene in rabbit aortas and hearts was much more prominent at embryonic and neonatal stages than in adults. The growth of C5, which is deficient in the Na(+)-H+ antiporter, was accelerated again when the activity was restored by transfecting cells with full-length cDNA for Na(+)-H+ antiporter in an expression vector. In pressure-overloaded hearts, the Na(+)-H+ antiporter mRNA was increased 2.5 fold of sham-operated rabbits at 3 days after aortic constriction, lasting for more than 2 weeks. We conclude from these results that activation of the Na(+)-H+ antiporter gene plays a key role in both cardiac hypertrophy and vascular smooth muscle cell proliferation.

Genus-specific polymerase chain reaction for the mycobacterial dnaJ gene and species-specific oligonucleotide probes.

Identification of tuberculous and nontuberculous mycobacteria by biochemical methods is a long-term process that takes up to 8 weeks for completion and requires expertise to interpret the results. In order to detect and differentiate the major pathogenic mycobacterial species, we developed genus-specific primers that amplify the dnaJ gene from the broad spectrum of mycobacterial species and determined the nucleotide sequences within the dnaJ genes from 19 mycobacterial species (Mycobacterium tuberculosis, M. bovis, M. bovis BCG, M. africanum, M. microti, M. kansasii, M. marinum, M. gastri, M. simiae, M. scrofulaceum, M. szulgai, M. gordonae, M. avium, M. intracellulare, M. xenopi, M. fortuitum, M. chelonei, M. haemophilum, and M. paratuberculosis). On the basis of the dnaJ gene sequences, we developed dot blot hybridization analysis with species-specific oligonucleotide probes for the M. tuberculosis complex. M. avium, M. intracellulare, and M. kansaii, allowing a rapid identification of these species following polymerase chain reaction for the dnaJ gene. We conclude that polymerase chain reaction with the genus-specific primer that amplifies the dnaJ genes and subsequent dot blot analysis with species-specific oligonucleotide probes are most useful for differential diagnosis of tuberculosis and nontuberculous mycobacterial infections.

Identification of three types of PDGF-A chain gene transcripts in rabbit vascular smooth muscle and their regulated expression during development and by angiotensin II.

PDGF-like peptides secreted from smooth muscles have been suggested to be responsible for the smooth muscle growth. In order to elucidate the nature of PDGF-like molecules expressed in vascular smooth muscles, we have isolated and characterized cDNA clones for PDGF-A chain from a rabbit embryonic aorta cDNA library. One of the cDNA clones was found to encode a novel PDGF-A chain, named PDGF-A3 in this report. PDGF-A3 arises from a single PDGF-A chain gene by alternative RNA splicing and differs from the sequences of previously reported endothelial- or the glioma-type transcripts by a 110 bp insertion. Expression of PDGF-A3 mRNA was selectively induced by Angiotensin II in the smooth muscle cell in vitro. Total PDGF-A mRNA is most enriched in embryonic aortas, but its expression is down-regulated with vascular development. PDGF-A mRNA is markedly increased in primary-cultured smooth muscle cells during the log-phase growth. Our results suggest that autocrine production of PDGF-A chains from the smooth muscle cell may play a role in early vascular development and in Angiotensin II-induced smooth muscle cell proliferation.

[Rapid detection and identification of mycobacterial DNA by PCR].

We have developed a highly sensitive and rapid PCR assay for detection and identification of mycobacterial species. Two sets of PCR primers were prepared; one was for the 19 kDa antigen gene and the other for the dna J gene. The PCR for 19 kDa antigen gene was found to be specific to the M. tuberculosis complex; M. tuberculosis, M. bovis and M. africanum. On the other hand, the PCR for the dna J gene was able to detect a broad spectrum of mycobacterial species including M. tuberculosis, M. avium, M. intracellulare and M. kansasii. The sensitivity of PCR was similar to that of the culture method for precultured M. tuberculosis whereas PCR was much more sensitive than the culture for clinical samples. The procedure of decontamination by sodium hydroxide for clinical samples could reduce the viability of M. tuberculosis while it did not affect mycobacterial DNA. The nucleotide sequence homologies among major mycobacteria were also determined following PCR for the dna J gene. This allowed us to make restriction maps for each mycobacterium. We have demonstrated that the PCR-RFLP for the dna J gene will be a useful laboratory test for rapid identification of tuberculous and nontuberculous mycobacterial species.

Allosteric properties, substrate specificity, and subsite affinities of honeybee alpha-glucosidase I.

The substrate specificity of honeybee alpha-glucosidase I, a monomeric enzyme was kinetically investigated. Unusual kinetic features were observed in the cleavage reactions of sucrose, maltose, p-nitrophenyl alpha-glucoside, phenyl alpha-glucoside, turanose, and maltodextrin (DP = 13). At relatively high substrate concentrations, the velocities of liberation of fructose from sucrose, glucose from maltose, p-nitrophenol from p-nitrophenyl alpha-glucoside, and phenol from phenyl alpha-glucoside were accelerated, and so the Lineweaver-Burk plots were convex, indicating negative kinetic cooperativity: the Hill coefficients were calculated to be 0.50, 0.64, 0.50, and 0.67 for sucrose, maltose, p-nitrophenyl alpha-glucoside, and phenyl alpha-glucoside, respectively. For the degradation of turanose and maltodextrin, the enzyme showed a sigmoidal curve in v versus s plots and thus catalyzed the reaction with positive kinetic cooperativity. The Lineweaver-Burk plots were concave and the Hill coefficients were 1.2 and 1.5 for turanose and maltodextrin, respectively. These unique properties cannot be interpreted by the reaction mechanism that Huber and Thompson proposed: (1973) Biochemistry 12, 4011-4020. The rate parameters for the hydrolysis of sucrose, maltose, p-nitrophenyl alpha-glucoside and phenyl alpha-glucoside were estimated by extrapolating the linear part of the Lineweaver-Burk plots at low substrate concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)

Use of L-leucyl-3-carboxy-4-hydroxyanilide as substrate for determining the activity of microsomal aminopeptidase in serum.

We describe a colorimetric method for assay of microsomal aminopeptidase (EC 3.4.11.2) activity in serum. We use a new substrate, L-leucyl-3-carboxy-4-hydroxyanilide, p-xylenol as coupler, and sodium metaperiodate as oxidizing reagent. The colored substance formed by the oxidative condensation between p-xylenol and 5-aminosalicylic acid absorbs maximally at 635 nm, and can be directly measured in serum. In a previous method for this enzyme, L-leucyl-beta-naphthylamide was used as substrate and beta-naphthylamine, a carcinogenic reagent used as a standard in making the assay, was unsuitable for routine use. We found a close correlation between results obtained with the new, safer method and with the previous method.