Systematic sequencing of the Bacillus subtilis genome: progress report of the Japanese group.
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Bacillus subtilis↗
Biomedical subjects
K Yamane
Publications and source records attributed to K Yamane.
Determination of a 21548 bp nucleotide sequence around the 24 degrees region of the Bacillus subtilis chromosome.
A 21548 bp nucleotide sequence around the 24 degrees region of the Bacillus subtilis chromosome, located 306 kb downstream from the zero point of the physical map, was determined. Twenty-one putative ORFs were identified: two ORFs (Orf1 and Orf20) were consistent with the nucleotide and amino acid sequence of B. subtilis OrfJ, whose function is not known, and Pcp, respectively; four were found to display significant similarities to known proteins in data banks, i.e. 5-keto-3-deoxyglucarate dehydratase (Orf2), aldehyde dehydrogenase (Orf3) and two glucarate dehydratases (Orf4 and 5); three had considerable similarity to the sensor-regulator proteins of bacterial two-component signal transduction systems (Orf1, 11 and 12); two had considerably high levels of similarity to parts of known proteins (Orf13 and 19); and five showed low levels of similarity to known proteins in the data banks. The remaining six ORFs showed no similarity to known proteins.
Aldehyde Dehydrogenase↗
A 10 kb nucleotide sequence at the 5' flanking region (32 degrees) of srfAA of the Bacillus subtilis chromosome.
The nucleotide sequence of approximately 10 kb at the 5' flanking region (32 degrees) of srfAA of the Bacillus subtilis chromosome was determined. Eleven putative ORFs were identified. Three of them (orf6, orf7 and orf8) coincided with known B. subtilis genes (comJ, comI and tlpC) encoding a competence-specific protein, a DNA-entry nuclease and a transducer-like protein, respectively. The products of two other ORFs showed similarity to GlnP of Escherichia coli (orf1) and beta-glucosidase A of B. polymyxa (orf5).
Amino Acid Transport Systems, Basic↗
The nasB operon and nasA gene are required for nitrate/nitrite assimilation in Bacillus subtilis.
Bacillus subtilis can use either nitrate or nitrite as a sole source of nitrogen. The isolation of the nasABCDEF genes of B. subtilis, which are required for nitrate/nitrite assimilation, is reported. The probable gene products include subunits of nitrate/nitrite reductases and an enzyme involved in the synthesis of siroheme, a cofactor for nitrite reductase.
Anion Transport Proteins↗
New tumor clips for the removal of large or deep-seated tumors: technical note.
We describe newly developed tumor-holding clips, which have been applied in more than 50 patients with large or deep-seated tumors. We devised various kinds of standard tumor clips by modifying Sugita aneurysm clips. Our new tumor clips permit gentle, steady, and easy retraction of the tumor in any direction without disturbance of the operative field. With the use of these clips, a surgeon can operate with both hands. From our experience of removing more than 50 tumors, we confirm that our tumor clips are useful for the removal of large or deep-seated tumors.
Brain Neoplasms↗
Four aromatic residues in the active center of cyclodextrin glucanotransferase from alkalophilic Bacillus sp. 1011: effects of replacements on substrate binding and cyclization characteristics.
Three-dimensional structures of cyclodextrin glucanotransferases (CGTases) have revealed that four aromatic residues, which are highly conserved among CGTases but not found in alpha-amylases, are located in the active center. To analyze the roles of these aromatic residues, Phe-183, Tyr-195, Phe-259, and Phe-283 of Bacillus sp. 1011 CGTase were replaced by site-directed mutagenesis, and the effects of this procedure were examined. Y195L-CGTase, in which Tyr-195 was replaced by a leucine residue, underwent a drastic change in its cyclization characteristics: it produced considerably more gamma-cyclodextrin than the wild-type enzyme and virtually no alpha-cyclodextrin. Y195L-CGTase had increased Km values for cyclodextrins, whereas the values for a linear maltooligosaccharide donor were insignificantly changed. Taken together with the structural information of CGTase crystals soaked with substrates, we propose that Tyr-195 plays an important role in the spiral binding of substrate. Replacing either Phe-183 or Phe-259 with leucine induced increased Km values for acceptors. Furthermore, the double mutant F183L/F259L-CGTase had considerably decreased cyclization efficiency, but the intermolecular transglycosylation activity remained normal. These results indicated that Phe-183 and Phe-259 are cooperatively involved in acceptor binding, and that they play a critical role in cyclization when the nonreducing end of amylose binds to the active center of CGTase. Replacing Phe-283 with a leucine residue induced a decrease in kcat and in affinity for acarbose, suggesting that Phe-283 is involved in transition-state stabilization.
Acarbose↗
Functional interaction between human histocompatibility leukocyte antigen (HLA) class II and mouse CD4 molecule in antigen recognition by T cells in HLA-DR and DQ transgenic mice.
Studies in vitro have suggested that a species barrier exists in functional interaction between human histocompatibility leukocyte antigen (HLA) class II and mouse CD4 molecules. However, whether mouse CD4+ T cells restricted by HLA class II molecules are generated in HLA class II transgenic mice and respond to peptide antigens across this barrier has remained unclear. In an analysis of T cell responses to synthetic peptides in mice transgenic for HLA-DR51 and -DQ6, we found that DR51 and DQ6 transgenic mice acquired significant T cell response to influenza hemagglutinin-derived peptide 307-319 (HA 307) and Streptococcus pyogenes M12 protein-derived peptide 347-397 (M6C2), respectively. Inhibition studies with several monoclonal antibodies showed that transgenic HLA class II molecules presented these peptides to mouse CD4+ T cells. Furthermore, T cell lines specific for HA 307 or M6C2 obtained from the transgenic mice could respond to the peptide in the context of relevant HLA class II molecules expressed on mouse L cell transfectants that lack the expression of mouse MHC class II. These findings indicate that interaction between HLA class II and mouse CD4 molecules is sufficient for provoking peptide-specific HLA class II-restricted T cell responses in HLA class II transgenic mice.
Amino Acid Sequence↗
The Bacillus subtilis SRP54 homologue, Ffh, has an intrinsic GTPase activity and forms a ribonucleoprotein complex with small cytoplasmic RNA in vivo.
B. subtilis Ffh is a homologue of SRP54, which is one component of the mammalian signal recognition particle. B. subtilis Ffh was expressed in E. coli as a derivative with a hexa histidine tag at the COOH terminus and purified to near homogeneity. The purified Ffh had intrinsic GTPase activity as predicted from its amino acid sequence. Using antiserum against Ffh, we also demonstrated that B. subtilis Ffh forms a complex with scRNA which is a B subtilis homologue of the RNA component of SRP in vivo, and that half of the resulting complex is found in the peripheral fraction of the cytoplasmic membrane where the initiation of protein translocation occurs. These findings provide evidence of a ribonucleoprotein complex in B. subtilis, reminiscent of SRP.
Animals↗
Crystallization and preliminary X-ray studies of cyclodextrin glucanotransferase from alkalophilic Bacillus sp. 1011.
Large crystals of cyclodextrin glucanotransferase (CGTase) from alkalophilic Bacillus sp. 1011, a typical alkalophilic enzyme, have been obtained at room temperature using polyethylene glycol 3000 and 2-propanol as precipitant. They belong to the triclinic space group P1 with the following unit cell constants: a = 64.93 A, b = 74.45 A, c = 79.12 A, alpha = 85.2 degrees, beta = 105.0 degrees and gamma = 101.0 degrees. The crystallographic asymmetric unit seems to contain two molecules of CGTase, with crystal volume per protein mass (Vm) of 2.41 A3/Da and solvent content of 49% by volume. The crystals diffract to at least 2.0 A resolution and they are suitable for X-ray analysis.
Bacillus↗
The transglycosylation reaction of cyclodextrin glucanotransferase is operated by a Ping-Pong mechanism.
A new photometric assay of the disproportionation activity of cyclodextrin glucanotransferase (CGTase) using 3-ketobutylidene-beta-2-chloro-4-nitrophenyl-maltopentaoside as the donor, proved that the transglycosylation reaction of CGTase was operated by a Ping-Pong Bi Bi mechanism. The values of the kcat/Km(acceptor) proved that the same configurations of free hydroxyl groups with those of D-glucopyranose at C2, C3 and C4 positions were required for the acceptors used by CGTase. The structure around C6 on acceptors was not essential for acceptor function, but it was recognized by CGTase, since the values of kcat/Km for D-xylose were smaller than that for D-glucose. The value of kcat/Km for maltose was about 20-times larger than that for D-glucose, indicating that at least two glucopyranosyl rings are recognized by the acceptor binding sites.
Bacillus↗
Diffusion anisotropy of cerebral ischaemia.
Focal cerebral ischemia was produced by occlusion of the middle cerebral artery with a silicone cylinder in Wistar rats. Diffusion-weighted echo-planar images (DW-EPIs) using the motion-probing gradient (MPG) method were acquired at 1-3 hours and 24-48 hours after occlusion. Apparent diffusion coefficients (ADCs) were calculated from these images in ischemic lesions and in normal unoccluded regions. Results were as follows. 1. Ischemic lesions could be detected on the DW-EPIs at 1 hour after occlusion. 2. The ADC of water in the brain tissue was smaller than that of free water as a result of restricted diffusion. 3. Anisotropic diffusion that probably can be attributed to the myelin sheath was observed in the normal deep white matter. 4. In the ischemic lesions, the ADC decreased rapidly within 1-3 hours after occlusion and then slightly further declined after 24-48 hours. In the ischemic deep white matter, diffusion anisotropy disappeared at 24-48 hours after occlusion. Diffusion-weighted imaging may have applications in the examination of pathophysiological mechanisms in cerebral ischemia by means of evaluation of ADC and diffusion anisotropy.
Animals↗
Acute brain swelling in cerebral embolization model of rats. I: Epidural pressure monitoring.
The authors measured epidural pressure of rats using a microballoon to monitor brain swelling after cerebral embolism. Rats with embolization of the major cerebral artery frequently demonstrated acute brain swelling. Within 5 hours of embolization, the increase in epidural pressure was 29.7 +/- 20.3 mm Hg (mean +/- SD), ranging from 3 to 68 mm Hg. The increase in epidural pressure tended to reach a near-maximal value within 30 minutes of embolization and thereafter stayed at that point until the end of the experiment. These results suggest that measuring epidural pressure can quantitatively assess acute brain swelling in rats.
Acute Disease↗
Primary pineal melanoma with long-term survival: case report.
A rare case of primary pineal melanoma is reported. The patient was a 53-year-old woman who complained of a severe headache. Computed tomography and magnetic resonance images revealed obstructive hydrocephalus caused by a mass lesion in the pineal region. A biopsy was performed through an occipital transtentorial approach. A black pigmented solitary tumor was seen without leptomeningeal dissemination. Histologic examination revealed melanoma. Chemotherapy consisting of dacarbazine, ACNU, vincristine, and interferon was used. Follow-up imaging studies showed dramatic reduction of the tumor without recurrence for 4 years. This report demonstrates that a solitary primary intracranial melanoma without leptomeningeal dissemination and with rare mitoses may yield a good result with chemotherapy.
Antineoplastic Combined Chemotherapy Protocols↗
Acute brain swelling in cerebral embolization model of rats. II: Biochemical study.
To investigate the mechanism of acute brain swelling after cerebral embolism in rats, the authors evaluated the regional changes in tissue pH, ATP, blood-brain barrier function, and water content. The deep cerebrum--thalamus, hypothalamus, and hippocampus--had alkaline change in tissue pH accompanied with the disruption of the blood-brain barrier, decrease of ATP, and increase of water content. The cortex and the caudate putamen, on the other hand, did not show edema though acidic change and ATP decrease were seen. These results suggest that an early vasogenic edema in the deep cerebrum is a main factor for evolving acute brain swelling in this embolization model of rats.
Acute Disease↗
Inhibition of 15-hydroxy prostaglandin dehydrogenase activity in rabbit gastric antral mucosa by 13-hydroperoxyoctadecadienoic acid.
The effect of 13-hydroperoxyoctadecadienoic acid (13-HPODE), a hydroperoxy adduct of linoleic acid (LA), on the activities of prostaglandin (PG) synthesizing and catabolizing enzymes in rabbit gastric antral mucosa was examined. 13-HPODE had no effect on the synthesis of PGE2, PGF2 alpha and PGD2 from exogenous arachidonic acid in the microsomal fraction of the gastric mucosa at concentrations ranging from 5-20 microM. On the other hand, at 1-10 microM, it inhibited the activity of 15-hydroxy PG dehydrogenase (PGDH), which catalyzes the initial step of catabolism of PGs, in a dose-dependent manner. The concentration required for 50% inhibition was approximately 1 microM. Experiments utilizing LA, 13-hydroperoxyoctadecadienoic acid and Fe2+ indicated the requirement of the hydroperoxy moiety for the inhibitory effect of 13-HPODE on the PGDH activity. These results suggest that 13-HPODE has the potential to increase the levels of biologically active PGs in gastric mucosa by preventing their inactivation and may have functional effects within the stomach.
Animals↗
A truncated Bacillus subtilis SecA protein consisting of the N-terminal 234 amino acid residues forms a complex with Escherichia coli SecA51(ts) protein and complements the protein translocation defect of the secA51 mutant.
Although wild-type Bacillus subtilis SecA barely complements the growth and protein translocation defect of Escherichia coli secA51(ts) at the non-permissive temperature, an N-terminal peptide of B. subtilis SecA complements the defects. To elucidate the mechanism of this complementation, a series of plasmids encoding truncated SecA proteins was constructed and their products were analyzed in E. coli cells. The truncated B. subtilis SecA protein consisting of the N-terminal 234 amino acid residues (BN234) complemented the growth and protein translocation defects of E. coli secA51 but not those of another secA amber mutant, E. coli secA13(ts). BN234 existed in both a soluble form, possibly as a homodimer, and a higher-molecular-weight complex in E. coli strain MM52 (secA51). The purified complex, consisting of at least BN234, SecA51, and ATP-dependent protease La, was held together by a cross-linking reagent, EDAC. The other truncated proteins consisting of the N-terminal 584 or 396 amino acid residues and the C-terminal 607 residues of B. subtilis SecA did not complement the two E. coli mutants or form a complex with SecA51. These results suggest that BN234 and SecA51 proteins form a functional complex in vivo and complement the defects of E. coli MM52.
Adenosine Triphosphatases↗
Small cytoplasmic RNA of Bacillus brevis: transcriptional and phylogenetic analysis.
Using a DNA fragment of Bacillus subtilis scRNA as a probe, a Bacillus brevis gene encoding the small cytoplasmic RNA was cloned and characterized. B. brevis scRNA consists of 273 nucleotides; the sequence has comparatively low homology (approximately 70%) with other Bacillus sequences. Phylogenetic analysis indicated that B. brevis forms a line of descent distinct from other Bacillus species. However, despite the low overall homology, both functional nucleotide sequence and secondary structural features defined among signal recognition particle (SRP) RNA family members were well conserved.
Bacillus↗
Structural requirements of Bacillus subtilis small cytoplasmic RNA for cell growth, sporulation, and extracellular enzyme production.
Bacillus subtilis small cytoplasmic RNA (scRNA; 271 nucleotides) is a member of the signal recognition particle (SRP) RNA family, which has evolutionarily conserved primary and secondary structures. The scRNA consists of three domains corresponding to domains I, II, and IV of human SRP 7S RNA. To identify the structural determinants required for its function, we constructed mutant scRNAs in which individual domains or conserved nucleotides were deleted, and their importance was assayed in vivo. The results demonstrated that domain IV of scRNA is necessary to maintain cell viability. On the other hand, domains I and II were not essential for vegetative growth but were preferentially required for the RNA to achieve its active structure, and assembled ribonucleoprotein between Ffh and scRNA is required for sporulation to proceed. This view is highly consistent with the fact that the presence of domains I and II is restricted to sporeforming B. subtilis scRNA among eubacterial SRP RNA-like RNAs.
Bacillus subtilis↗