Biomedical subjects

H Aramaki

Publications and source records attributed to H Aramaki.

At least 37 records · Page 2Linked to original sources

PubMed1995

Purification and characterization of rat adrenodoxin.

Adrenodoxin was purified from the rat adrenal gland. The A414/A280 value of the purified rat adrenodoxin was 0.90 and the oxidized spectrum showed absorption maxima at 320, 414 and 455 nm, similar to those of bovine adrenodoxin. On SDS-PAGE, the rat adrenodoxin showed a single band with a molecular mass of 11.2 kDa, while the apparent molecular mass by gel filtration through Sephadex G-75 equilibrated with 10 mM K-phosphate (pH 7.5) was 27 kDa. In the reconstituted system, Vmax of NADPH-cytochrome c reduction activity and the Km for the rat adrenodoxin were much the same as those for recombinant bovine adrenodoxin. In the case of cholesterol side-chain cleavage activity, however, these values of the rat adrenodoxin were about half of those of the bovine adrenodoxin. The CD spectrum of the rat adrenodoxin was similar to that of the bovine adrenodoxin but showed a significantly lower ellipticity value in the 195-205 nm region than that of the bovine adrenodoxin. The structural differences may possibly explain differences in the enzymic properties between rat and bovine adrenodoxins.

Adrenal Glands↗

PubMed1995

Residues important for the function of a multihelical DNA binding domain in the new transcription factor family of Cam and Tet repressors.

We report that some prokaryotic repressors including CamR and TetR belong to the same family. CamR and TetR bind to DNA using a multihelical DNA binding domain (DBD) at the N-termini of the proteins, while the C-termini are important for regulating the DNA binding in a manner dependent on their co-factors (camphor for CamR, tetracycline for TetR). In all, 11 important amino acid positions have been identified in the CamR DBD by the systematic substitution of residues by Ala. Of the 11 positions, 10 are either buried in the core, and thus important for creating the hydrophobic environment, or exposed on the surface, and thus important for binding to DNA. The eleventh residue, Gly, seems to be important for a loop structure. The DNA binding mode of this type of DBD and a general mechanism of regulating their DNA binding are discussed in reference to the crystal structure of TetR [Hinrichs et al., (1994) Science, 264, 418-420].

Amino Acid Sequence↗

PubMed1995

Purification and characterization of a cam repressor (CamR) for the cytochrome P-450cam hydroxylase operon on the Pseudomonas putida CAM plasmid.

The cytochrome P-450cam hydroxylase operon of Pseudomonas putida PpG1 (ATCC 17543) encodes proteins responsible for early steps of the degradation of D-camphor. Transcription of this operon is negatively controlled by the cam repressor (CamR), and the expression of camR is autoregulated. CamR was purified from Escherichia coli harboring an overproducing plasmid. The repressor forms a homodimer with a molecular mass of 40 kDa, as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis and gel filtration. CamR protected a specific DNA region from attack by DNase I. This region contains a palindromic operator of the cytochrome P-450cam hydroxylase operon and of the camR gene. Protection was inhibited by the addition of 60 microM D-camphor and also by certain camphor analogs and degradation products, including D-3-bromocamphor, adamantane, 2-adamantanone, 5-exo-hydroxycamphor, and 2,5-diketocamphane. These analogs and degradation products induced cytochrome P-450cam hydroxylase operon expression in vivo.

Base Sequence↗

PubMed1994

Heterologous expression of the cytochrome P450cam hydroxylase operon and the repressor gene of Pseudomonas putida in Escherichia coli.

The cytochrome P450cam hydroxylase operon (camDCAB) of Pseudomonas putida is negatively regulated by a repressor, CamR, which also represses its own gene. The expression in P. putida of both camR and camDCAB is derepressed in the presence of D-camphor. We examined the expression in Escherichia coli of camR and camDCAB by monitoring the enzyme activity of the camD gene product. In the presence or absence of D-camphor in the cell culture, the expression in E. coli of camD was significant and constitutive, suggesting no expression of camR. This lack of expression was confirmed by monitoring the beta-galactosidase activity of camR-lacZ translational fusions. However, S1 nuclease mapping revealed that synthesis of camR mRNA in E. coli was significant and constitutive, as observed in the case of camDCAB mRNA. Thus, it is likely that the expression of camR in E. coli is limited at the translational level.

Camphor↗

PubMed1994

Nucleotide sequence of the gene encoding a repressor for the cytochrome P-450cam hydroxylase operon on the Pseudomonas putida CAM plasmid.

The camR gene of Pseudomonas putida encodes a repressor which regulates expression of the cytochrome P-450cam hydroxylase operon (camDCAB). We determined the nucleotide sequence of 1134 continuous base pairs, including the camR gene. When comparing the amino acid sequence deduced from the open reading frame of the gene sequence with that of amino-terminal five residues of the cam repressor, purified from Pseudomonas putida, we found that the camR gene encodes a protein of 186 amino acids, with a molecular mass of 20.4 kDa. The start codon for the cam repressor is the rare initiation codon GTG. The cam repressor predicted from the camR sequence contained a region similar to that seen in other DNA-binding proteins.

Amino Acid Sequence↗

PubMed1993

Complete nucleotide sequence of the 5-exo-hydroxycamphor dehydrogenase gene on the CAM plasmid of Pseudomonas putida (ATCC 17453).

We determined the complete nucleotide sequence of the first gene of Pseudomonas putida cytochrome P-450cam hydroxylase operon, camD, which encodes 5-exo-hydroxycamphor dehydrogenase. This dehydrogenase apparently consists of 361 amino acids and has a molecular mass of 38.4 kDa. Structural relationships to other zinc-containing alcohol dehydrogenases also became evident.

Alcohol Dehydrogenase↗

PubMed1993

Transcription of the cam operon and camR genes in Pseudomonas putida PpG1.

In Pseudomonas putida carrying the CAM plasmid, the operon (camDCAB) encoding enzymes involved in the degradation pathway of D-camphor is negatively regulated by the CamR protein, and camR is autorepressed. S1 nuclease mapping revealed that camDCAB and camR were divergently transcribed from overlapping promoters, the transcription start sites were separated by 11 bp, and transcriptions of the cam operon (camDCAB) and camR increased about 10- and 4-fold, respectively, immediately after addition of camphor. The transcriptions of camDCAB and camR were negatively regulated through the interaction of the CamR protein with the one operator located in the overlapping promoter region. In vitro transcription experiments were performed to characterize the regulation of cam genes. The camR promoter was initiated by P. putida RNA polymerase containing sigma 70, but transcription from the camDCAB promoter by sigma 70 holoenzyme was not observed. The purified CamR protein repressed in vitro transcription from the camR promoter. This repression was suppressed by camphor. The RNA polymerase binding region of the camR promoter was identified by using DNase I footprinting. In addition, footprinting studies revealed that the CamR protein and RNA polymerase coexisted on the promoter region in a joint nonproductive complex.

Bacterial Proteins↗

PubMed1993

Evidence for autoregulation of camR, which encodes a repressor for the cytochrome P-450cam hydroxylase operon on the Pseudomonas putida CAM plasmid.

The regulatory gene camR on the CAM plasmid of Pseudomonas putida (ATCC 17453) negatively controls expression of the cytochrome P-450cam hydroxylase operon (camDCAB) for the camphor degradation pathway and is oriented in a direction opposite to that of the camDCAB operon. In this study, we examined expression of the camR gene by monitoring the beta-galactosidase activity of camR-lacZ translational fusions in P. putida camR and camR+ strains. We found that the camR gene was autogenously regulated by its own product, CamR. To search for an operator site of the camR gene, a cam repressor (CamR)-overproducing plasmid, pHAOV1, was constructed by placing the camR gene under the control of a pL promoter. The translational initiation codon of CamR was changed by site-directed mutagenesis from GTG to ATG to improve translation efficiency. Judging from sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, the CamR protein was expressed up to about 10% of the soluble protein of CamR-overproducing Escherichia coli JM83/pHAOV1 cells. Results of DNase I footprinting assays using the cell lysate indicated that the CamR repressor covered a single region between the camR gene and the camDCAB operon. Our findings also suggest that the camR gene autogenously regulates its own expression by binding of the gene product, CamR, to the operator, which also serves as an operator of the camDCAB operon.

Base Sequence↗

PubMed1989

A comparative study of the JJC, AJC and UICC T classifications of squamous cell carcinoma of the maxillary sinus.

Seventy-six cases with squamous cell carcinoma of the maxillary sinus were treated with radiotherapy and surgery with or without intra arterial infusion between 1969 to 1985 at the Tokyo Women's Medical College. T classification was made of all cases using the Japan Joint Committee (JJC, 1967), American Joint Committee (AJC, 1977) and Union Internationale Contre le Cancer (UICC, 1987) classifications. 1) By the JJC classification, there were 20 cases in T2, 44 in T3 and 12 in T4. By the AJC classification, there were 9 cases in T2, 58 in T3 and 9 in T4. The classification of 14 cases were changed from JJC T2 to AJC T3, because of extension to the medial nasal meatus. By the UICC classification, there were 27 T2 cases, 32 T3 and 17 T4. 2) Cervical lymph node metastasis was detected in 14 cases (18%) at initial diagnosis and most were found in T3 and T4 cases. 3) The cumulative 5-year survival rates were 67% for T2, 32% for T3 and 25% for T4 by the JJC classification and statistical differences (p less than 0.05) were seen in T2-T3 and T2-T4. In the AJC classification, these were 71% for T2, 37% for T3 and 22% for T4 and no statistical significance was found among them. By the UICC classification, the 5-year survival was 58% for T2, 26% for T3 and 38% for T4 and marked statistical significance (p less than 0.01) was seen in T2-T3 and T2-T4. 4) The cumulative 5-year survival rate in 14 patients with tumor extension to the medial nasal meatus was 55% and it is appropriate to define this stage as T2 in the new UICC classification. These data suggested that UICC classification (1987) is useful and correlated with prognostic analysis.

Adult↗

PubMed1989

Cloning and nucleotide sequences of NADH-putidaredoxin reductase gene (camA) and putidaredoxin gene (camB) involved in cytochrome P-450cam hydroxylase of Pseudomonas putida.

Pseudomonas putida PpGl, which carries the CAM plasmid encoding enzymes involved in the degradation pathway of D-camphor, can utilize D-camphor as a sole carbon source. Cytochrome P-450cam and related enzymes participate in the early oxidation steps of D-camphor degradation metabolism. We cloned from a HindIII DNA library of PpGl a 2.9 kbp CAM segment which carries the major part of camA gene encoding NADH-putidaredoxin reductase and the entire camB gene encoding putidaredoxin. The 2.9 kbp CAM segment was adjacent to the 4.27 kbp HindIII CAM segment which has been previously cloned (Koga et al. (1986) J. Bacteriol. 166, 1089-1095). Thus, the total 7.17 kbp HindIII CAM directed all the genes responsible for early steps of D-camphor degradation, i.e. 5-exo-hydroxycamphor dehydrogenase (camD gene), cytochrome P-450cam (camC), NADH-putidaredoxin reductase (camA), and putidaredoxin (camB). These cam genes form an operon, camDCAB, and are under negative control by the gene camR located immediately upstream from the camD gene. The total number of amino acids deduced from the nucleotide sequence is 422 for putidaredoxin reductase, and 106 for putidaredoxin.

Amino Acid Sequence↗

PubMed1989

[Case report of catecholamine-secreting carotid body tumor].

We encountered a case of catecholamine-secreting carotid body tumor which has never been reported in Japan. The patient was a 20-year old man, who complained of a mass located at the right neck and hypertensive symptoms such as loss of consciousness at extension of the neck, fatigability, etc. Carotid angiography and computed tomography (CT scan) revealed a carotid body tumor. Before operation, a mild hypertension was noted, serum and urinary levels of norepinephrine showed abnormal increase, and phentolamine test was positive. After the tumor was dissected by a surgical procedure associated with carotid reconstruction, blood pressure and norepinephrine levels clearly trended to decrease and phentolamine test returned to negative. Histopathologically, the tumor was a paraganglioma with so-called "organoid" pattern. The tumor was not chromaffin, but the presence of neurosecretory granules was confirmed under electron microscope. In one-year follow up after the operation, no sign suggestive of "latent" or newly arising paraganglioma was noted in assay of catecholamines in serum and urine, nor on adrenal CT scan.

Adult↗

PubMed1986

Preparation and characterization of two major isotypes of parvalbumins from skeletal muscle of bullfrog (Rana catesbeiana).

Two major isotypes of parvalbumins (PA1 and PA2) have been isolated from the skeletal muscle of bullfrog, Rana catesbeiana. The Mr values were estimated to be 10,100 (PA1) and 11,800 (PA2) by SDS/polyacrylamide gel electrophoresis, and the isoelectric points were determined to be 4.78 (PA1) and 4.97 (PA2) by polyacrylamide gel isoelectric focusing. The amino acid compositions and isoelectric points indicate that PA1 corresponds to Rana esculenta pI 4.50 and Rana temporaria pI 4.75 parvalbumins and PA2 to Rana esculenta pI 4.88 and Rana temporaria pI 4.97 parvalbumins, showing that PA1 is genetically a beta-parvalbumin and PA2 an alpha-parvalbumin. However, in terms of the amino acid compositions, PA1 and PA2 are distinctly different from the corresponding parvalbumins of Rana esculenta or Rana temporaria. The ultraviolet spectra of PA1 and PA2 are consistent with their amino acid compositions. An ultraviolet difference spectrum of the Ca2+-loaded form vs. metal-free form indicates that a Tyr and some Phe residues in PA1 are affected by a conformational change associated with the binding of Ca2+. On electrophoresis in polyacrylamide gel in 14 mM Tris and 90 mM glycine, the Ca2+-loaded form of PA1 migrated twice as fast as the Mg2+-loaded form. Both PA1 and PA2 show increased mobility in the Ca2+-loaded forms, like troponin C but different from calmodulin.

Amino Acids↗

PubMed1986

camR, a negative regulator locus of the cytochrome P-450cam hydroxylase operon.

A 4.27-kilobase insert from a HindIII DNA library of Pseudomonas putida carrying the CAM plasmid allowed coordinate expression of genes camD and camC under control of camR, an upstream regulator. The camC gene specifies cytochrome P-450cam, and camD specifies the 5-exo-alcohol dehydrogenase. A 1.38-kilobase deletion from the insert results in the constitutive expression of genes camC and camD; transformation in trans restores the substrate control, indicating that camR is a negative regulator.

Alcohol Dehydrogenase↗

PubMed1986

[Effects of iodine-enriched egg (IE-egg) on nasal allergy: basic and clinical investigations].

The effect of iodine-enriched egg (IE-egg) on nasal allergy was investigated using an experimental allergic model. In addition, the effect of IE-egg was investigated using patients with yearly nasal allergy. IE-egg could suppress the leakage of pontamine sky blue dye in the experimental allergic model. beta-Glucuronidase activity in the perfusate was suppressed with the ingestion of IE-egg. The symptoms of the patients with yearly nasal allergy were mitigated by the ingestion of IE-egg. beta-Glucuronidase activity in the pituita of nasal allergic patients tended to be decreased.

Alkaloids↗

PubMed1971