Application of a microfluidic device for counting of bacteria.

AIMS: To develop a miniaturized analytical system for counting of bacteria. METHODS AND RESULTS: Escherichia coli cells were used throughout the experiments. The system consists of a microfluidic chamber, a fluorescence microscope with a charge-coupled device (CCD) camera and syringe pumps. The chamber was made of a silicone rubber (30 x 30 mm and 4 mm high). The E. coli cells were flowed from a micro-nozzle fabricated in the chamber and detected with the CCD camera. The individual cells were indicated as signal peaks on a computer. The cell counts showed a good correlation compared with that of a conventional plate counting method, and results of the simultaneous detection of live and dead cells were also presented. CONCLUSIONS, SIGNIFICANCE AND IMPACT OF THE STUDY: The system having a small disposable nozzle has the advantages for low cost and safe medical or environmental analysis, when compared with a conventional flow cytometer. This is the first step of the development of a one-chip microbe analyzer.

[A case report of pulmonary Mycobacterium kansasii infection found regular medical examination in our medical college of nursing].

A 20-year-old female was checked by chest X-ray film just before starting practical nurse training in the hospital. She was diagnosed as tuberculosis in the initial phase of treatment. In Japan, the number of newly registered tuberculosis has been increasing since 1997, and the stop-tuberculosis campaign is organized by the Ministry of Health and Welfare. The incidence rate of tuberculosis announced officially by the Ministry of Public Welfare was 33.9 per 100,000 in 1997, while that of nontuberculous mycobacteriosis has been increasing year by year, and it was 2.45 in 1997. The one out of 4 nontuberculous mycobacteriosis is caused by M. kansasii. Six colonies of Mycobacterium kansasii were detected by gastric juice culture from this patient. Untreated strains of M. kansasii are susceptible to rifampicin, isoniazid, ethambutol, ethionamide, streptomycin and cycloserine at concentrations readily available in the serum with usual therapeutic doses. Isolates are usually resistant to available serum level of pyrazinamide. The patient was treated with rifampicin, isoniazid and ethambutol for 6 months. Pyrazinamide was stopped at 1 month and 10 days treatment due to liver dysfunction and resistance to the organism. Pulmonary infiltration with cavity disappeared during follow-up examination. Nowadays we must take into account not only tuberculosis but also primary nontuberculous mycobacteriosis at regular medical check of young female.

Isolation of a chymotrypsinogen B-like enzyme from the archaeon Natronomonas pharaonis and other halobacteria.

A protease of a molecular mass of approximately 30kDa was isolated and purified from the haloalkaliphilic archaeon Natronomonas (formerly Natronobacterium) pharaonis. The enzyme hydrolyzed synthetic peptides, preferentially at the carboxyl terminus of phenylalanine or leucine, as well as large proteins. Hydrolysis occurred over the range of pH from 6 to 12, with an optimum at pH 10. The temperature optimum was 61 degrees C. The enzyme was nearly equally active over the range of salt concentration from 0.5 to 4M (NaCl or KCl). A strong cross-reaction with a polyclonal antiserum against human chymotrypsin was observed. Enzymatic activity was inhibited by typical serine protease inhibitors. There was significant homology between N-terminal and internal sequences from autolytic fragments and the sequence of bovine chymotrypsinogen B; the overall amino acid composition was similar to that of vertebrate chymotrypsinogens. Evidence for a zymogen-like processing of the protease was obtained. Cell extracts from other halobacteria exhibited similar proteolytic activity and immunoreactivity. The data suggested a widespread distribution of a chymotrypsinogen B-like protease among halo- and haloalkaliphilic Archaea.

The subunit structure of nitrite reductase purified from the denitrifier Achromobacter cycloclastes.

The copper-containing nitrite reductase of Achromobacter cycloclastes has been considered to be a homotrimer with three identical subunits both in the crystal and in solution. In this study, however, the enzyme was found to be a heterotrimer consisting of two subunits with molecular masses of 37 kDa and 36.2 kDa, and the 37 kDa subunit was 6 amino acid residues longer than the smaller subunit. Signal-peptide cleavage sites in its N-terminal region are discussed.

Analysis of the nitrous oxide reduction genes, nosZDFYL, of Achromobacter cycloclastes.

The structural gene, nosZ, for the monomeric N2O reductase has been cloned and sequenced from the denitrifying bacterium Achromobacter cycloclastes. The nosZ gene encodes a protein of 642 amino acid residues and the deduced amino acid sequence showed homology to the previously derived sequences for the dimeric N2O reductases. The relevant DNA region of about 3.6 kbp was also sequenced and found to consist of four genes, nosDFYL based on the similarity with the N2O reduction genes of Pseudomonas stutzeri. The gene product of A. cycloclastes nosF (299 amino acid residues) has a consensus ATP-binding sequence, and the nos Y gene encodes a hydrophobic protein (273 residues) with five transmembrane segments, suggesting the similarity with an ATP-binding cassette (ABC) transporter which has two distinct domains of a highly hydrophobic region and ATP-binding sites. The nosL gene encodes a protein of 193 amino acid residues and the derived sequence showed a consensus sequence of lipoprotein modification/processing site. The expression of nosZ gene in Escherichia coli cells and the comparison of the translated sequences of the nosDFYL genes with those of bacterial transport genes for inorganic ions are discussed.

Alpha- and beta-subunits of a V-type membrane ATPase in a hyperthermophilic sulfur-dependent archaeum, Thermococcus sp. KI.

The genes encoding alpha- and beta-subunits of a V-type ATPase in a sulfur-dependent hyperthermophilic archaeum, Thermococcus sp. KI, were cloned and sequenced. The deduced amino acid sequences were approximately 60, 50 and 25% identical to those of other archaeal, eukaryotic V-type and E. coli F-type ATPase, respectively. Phylogenetic analysis revealed that Thermococcus ATPase was closely related to that of Thermus, and those of Methanosarcina and Halobacterium.

Vacuolar-type ATPase in a hyperthermophilic archaeum, Thermococcus sp. KI.

Membrane ATPase was purified from a hyperthermophilic heterotrophic archaeum, Thermococcus sp. KI, which grew anaerobically at 90 degrees C in the presence of sulfur. The purified enzyme had an optimal temperature of 90 degrees C and its molecular mass was estimated to be 600 kDa. It consisted of 4 subunits with molecular masses of 70, 60, 29 and 15 kDa. While the ATPase activity was resistant to most ATPase inhibitors, the activity was reduced by nitrate, an inhibitor of the vacuolar (V)-type ATPase. N-terminal amino acid sequence of the 70 kDa subunit was similar to those of catalytic subunit of V-type ATPases. This indicates that hyperthermophilic heterotrophs have a V-type ATPase in their membranes.

ATP-dependent H+ -pump activity in inverted vesicles of Methanosarcina mazei Gö1 and characterization of membrane ATPase.

ATP-dependent H+ -pump activity was found in inverted vesicles of Methanosarcina mazei Gö1 by using acridine orange as a fluorescent probe. The H+ -pump activity specifically required both Mg and sulfite ions, but azide, an inhibitor of F0F1-ATPase, did not inhibit the activity. The membranes prepared from M. mazei also had an Mg-ATPase activity, and at least the presence of vacuolar-type ATPase was detected.

Membrane ATPase from the aceticlastic methanogen Methanothrix thermophila.

A new isolate of the aceticlastic methanogen Methanothrix thermophila utilizes only acetate as the sole carbon and energy source for methanogenesis (Y. Kamagata and E. Mikami, Int. J. Syst. Bacteriol. 41:191-196, 1991). ATPase activity in its membrane was found, and ATP hydrolysis activity in the pH range of 5.5 to 8.0 in the presence of Mg2+ was observed. It had maximum activity at around 70 degrees C and was specifically stimulated up to sixfold by 50 mM NaHSO3. The proton ATPase inhibitor N,N'-dicyclohexylcarbodiimide inhibited the membrane ATPase activity, but azide, a potent inhibitor of F0F1 ATPase (H(+)-translocating ATPase of oxidative phosphorylation), did not. Since the enzyme was tightly bound to the membranes and could not be solubilized with dilute buffer containing EDTA, the nonionic detergent nonanoyl-N-methylglucamide (0.5%) was used to solubilize it from the membranes. The purified ATPase complex in the presence of the detergent was also sensitive to N,N'-dicyclohexylcarbodiimide, and other properties were almost the same as those in the membrane-associated form. The purified enzyme revealed at least five kinds of subunits on a sodium dodecyl sulfate-polyacrylamide gel, and their molecular masses were estimated to be 67, 52, 37, 28, and 22 kDa, respectively. The N-terminal amino acid sequences of the 67- and 52-kDa subunits had much higher similarity with those of the 64 (alpha)- and 50 (beta)-kDa subunits of the Methanosarcina barkeri ATPase and were also similar to those of the corresponding subunits of other archaeal ATPases. The alpha beta complex of the M. barkeri ATPase has ATP-hydrolyzing activity, suggesting that a catalytic part of the Methanothrix ATPase contains at least the 67- and 52-kDa subunits.

[A case of recurrent pulmonary alveolar proteinosis treated by pulmonary lavage: were the remissions due to the natural course or due to the lavage?].

We report a 39-year-old male with pulmonary alveolar proteinosis. Although no abnormal shadows were seen on regular check-up chest roentgenogram one year previously, diffuse alveolar filling shadows were noted on admission, suggesting fairly acute progression of the disease. He was treated with bronchoalveolar lavage of each segment of both lungs by flexible fiberoptic bronchoscope under local anesthesia. Following this treatment, the shadows on X-ray film and shortness of breath resolved. Oxygen tension of arterial blood, pulmonary function, and serum CEA recovered to almost within normal ranges. The shadows deteriorated twice, but therapeutic lavage performed at the outpatient clinic was effective on each occasion. He thus received 3 series of bronchoalveolar lavage over a period of more than 3 years. We followed the clinical course of this patient for 6 years, and he remained well 3 years after the final treatment.

[A case of diffuse panbronchiolitis, performed an open lung biopsy after improvement with 6 years medication].

A 51-year-old male was hospitalized in June 1983, complaining of productive cough and dyspnea. Diffuse panbronchiolitis (DPB) was diagnosed on the basis of the physical examination, chest roentgenogram, chest CT and transbronchial lung biopsy (TBLB). The patient underwent surgery for chronic sinusitis and deviated nasal septum, and received Pseudomonas aeruginosa vaccine, ampicillin and erythromycin. He revealed a posterior mediastinal tumor in March 1989. The clinical findings of DPB improved but open lung biopsy was performed on the occasion of surgery for the posterior mediastinal tumor. Pathologically, fibrosis and mild infiltration of mononuclear cells localized in the walls of respiratory bronchioli and in surrounding areas was recognized in addition to slight accumulation of foamy macrophages in interstitial spaces. These morphological findings, as well as the clinical findings, might suggest repair of DPB lesions.

Sequential changes of perivascular edema cuffs in models of permeability and hemodynamic pulmonary edema.

The areas of perivascular edema cuffs surrounding pulmonary arteries and veins were sequentially measured as an index of the fluid transport unit in lung interstitium (FTULI) in epinephrine-induced and oleic acid-induced pulmonary edema in rats. The former edema represents a model of hemodynamic edema and the latter, permeability pulmonary edema, respectively. In epinephrine-induced pulmonary edema, both the ratio of edema cuff area to cross-sectional area of the pulmonary artery (Rr) and the ratio of lung weight to body weight (L/B) were increased in parallel, reached maximum levels at 0.5 h after the treatment, and returned to the control levels after 3 h. In oleic acid-induced pulmonary edema, the changes in Rr and L/B were not parallel, and the maximum levels were reached at different times, Rr at 3 h and L/B at 1.5 h. Rr returned close to the control level in 24 h but L/B remained elevated so that rate of recovery was delayed. The cuffs around the veins appeared similar to those around the arteries, but were very slight in both models. The difference in the time course of Rr and L/B in the two models may suggest that the recruitment of FTULI is insufficient in oleic acid-induced pulmonary edema; this limitation seems to be an important factor which makes the permeability edema refractory to treatment, together with the damage to the blood gas barrier.

Dicyclohexylcarbodiimide-binding protein is a subunit of the Methanosarcina barkeri ATPase complex.

Membrane ATPase of Methanosarcina barkeri was inhibited by N, N'-dicyclohexylcarbodiimide (DCCD), whereas the extrinsic alpha beta complex of the same enzyme was not. Consistent with this finding, a 6,000 dalton (6 kDa) membrane protein was preferentially labeled with radioactive DCCD. The DCCD-sensitive ATPase was solubilized from the membranes with octylglucoside and purified in the presence of this detergent. The purified ATPase contained the alpha and beta subunits and also at least four additional proteins (40, 27, 23 and 6 kDa). The 6 kDa protein in the purified enzyme reacted with DCCD, indicating that it is a subunit of an integral part of the M. barkeri ATPase complex.

Amino acid sequence of the alpha and beta subunits of Methanosarcina barkeri ATPase deduced from cloned genes. Similarity to subunits of eukaryotic vacuolar and F0F1-ATPases.

The atpA and atpB genes coding for the alpha and beta subunits, respectively, of membrane ATPase were cloned from a methanogen Methanosarcina barkeri, and the amino acid sequences of the two subunits were deduced from the nucleotide sequences. The methanogenic alpha (578 amino acid residues) and beta (459 amino acid residues) subunits were highly homologous to the large and small subunits, respectively, of vacuolar H+-ATPases; 52% of the residues of the methanogenic alpha subunit were identical with those of the large subunit of vacuolar enzyme of carrot or Neurospora crassa, respectively, and 59, 60, and 59% of the residues of the methanogenic beta subunit were identical with those of the small subunits of N. crassa, Arabidopsis thaliana, and Sacharomyces cerevisiae, respectively. The methanogenic subunits were also highly homologous to the corresponding subunits of Sulfolobus acidocaldarius ATPase. The methanogenic alpha and beta subunits showed 22 and 24% identities with the beta and the alpha subunits of Escherichia coli F1, respectively. Furthermore, important amino acid residues identified genetically in the E. coli enzyme were conserved in the methanogenic enzyme. This sequence conservation suggests that vacuolar, F1, methanogenic, and S. acidocaldarius ATPases were derived from a common ancestral enzyme.

Sarcoma associated with pregnancy.

A case is reported of sarcoma associated with pregnancy in which the patient underwent a cesarean section with myomectomy and was delivered of a living infant. A pathologic examination indicated sarcoma.

[A study of muscle pressure in making functional impression. Horizontal and vertical forces on oral vestibulum of maxilla with full natural dentition].

The purpose of this thesis was to establish objectively a technique for muscle training in functional impression of denture space. Ten normal subjects in their twenties were employed. Small transducers were placed in labial and buccal sides of gingival mucosa of the maxilla. The horizontal and vertical muscular forces of the molar, premolar and anterior regions were recorded simultaneously during rest position, during the application of Myomonitor, during pronunciation and during the fourteen types of functional movements relating to muscle training. The data were statistically analyzed and the following results were obtained. 1. The average muscular force during rest position was minimal in all areas measured and could be used as standard for other functional movements. 2. Each type of functional movement had its distinct muscular force in all areas measured and there were significant differences among the areas of measurement and among the directions of measurement. 3. The functional movements that showed maximum muscular force were, in the anterior region, suction of the index finger for both vertical and horizontal directions, in the premolar region, suction of the index finger for horizontal direction and rotation of the cheek for vertical direction, in the molar region, inward rolling of upper lip for both directions. 4. The number of functional movement which showed significant differences in the muscular forces among directions tend to increase as one move from anterior to premolar and to molar region. In this case the horizontal forces became larger than the vertical forces. Also both the horizontal and vertical forces tend to increase as one move anteriorly. 5. The muscular forces obtained from the rotation movement made by the subject were larger than the forces of the rotation movement made by the clinicians in all areas measured. 6. The average muscular force during the application of Myo-monitor was minimal and similar to the force during rest position. 7. There was a large difference among individuals in the muscular forces during pronunciation, and all were below the average force of the fourteen functional movements. 8. From the above results it is suggested that a technique for muscle training be established objectively for the functional impression of maxillary labial and buccal denture space.

[Regional distribution of perfusion and ventilation in hamartoangiomyomatosis of the lung].

We investigated regional distribution of perfusion and ventilation in three cases of hamartoangiomyomatosis (HAM) by 133Xe gas and 99mTc-MAA. In two cases, 133Xe washout were dominantly delayed in middle lung field and in the third case, it was delayed in upper lung field. This distribution was different from the result in the other chronic obstructive pulmonary disease (COPD). In most cases of COPD, 133Xe washout was prominently delayed in the lower lung field. Distribution of pulmonary perfusion in HAM were consistent with that of ventilation as in COPD.

Isolation of subunits from Methanosarcina barkeri ATPase: nucleotide-binding site in the alpha subunit.

The alpha (62,000-dalton) and beta (49,000-dalton) subunits of Methanosarcina barkeri ATPase were purified to homogeneity. The subunits and ATPase complex were trypsinized in the presence of various nucleotides. ATP and ADP changed the trypsin sensitivity of the alpha subunit in the complex and isolated forms, suggesting the presence of a nucleotide-binding site in the alpha subunit.