Cloning and sequence analysis of the StsI restriction-modification gene: presence of homology to FokI restriction-modification enzymes.

StsI endonuclease (R.StsI), a type IIs restriction endonuclease found in Streptococcus sanguis 54, recognizes the same sequence as FokI but cleaves at different positions. A DNA fragment that carried the genes for R.StsI and StsI methylase (M.StsI) was cloned from the chromosomal DNA of S.sanguis 54, and its nucleotide sequence was analyzed. The endonuclease gene was 1,806 bp long, corresponding to a protein of 602 amino acid residues (M(r) = 68,388), and the methylase gene was 1,959 bp long, corresponding to a protein of 653 amino acid residues (M(r) = 76,064). The assignment of the endonuclease gene was confirmed by analysis of the N-terminal amino acid sequence. Genes for the two proteins were in a tail-to-tail orientation, separated by a 131-nucleotide intercistronic region. The predicted amino acid sequences between the StsI system and the FokI system showed a 49% identity between the methylases and a 30% identity between the endonucleases. The sequence comparison of M.StsI with various methylases showed that the N-terminal half of M.StsI matches M.NIaIII, and the C-terminal half matches adenine methylases that recognize GATC and GATATC.     

A case of glucocorticoid-suppressible hyperaldosteronism with aldosterone producing adenoma

A 34-yr-old woman with hypertension (142/102 mmHg), hypokalemia, high plasma and urinary aldosterone and low plasma renin activity was studied. A left adrenal tumor and enlarged right adrenal gland were demonstrated by adrenal venography. During administration of dexamethasone (2 mg daily, for 3 weeks), urinary aldosterone excretion decreased abruptly from 22.5 to 9-11 micrograms/day, serum potassium increased and blood pressure fell to 120-130/80-90 mmHg. After left adrenalectomy, all manifestations improved with no medication. The resected adrenal gland revealed clear cell adenoma and micronodular adrenocortical hyperplasia. The patient was considered to be a rare case of glucocorticoid-suppressible hyperaldosteronism with an aldosterone-producing adenoma.     

Deletion of a phenylalanine in the N-terminal region of human cytochrome P-450(17 alpha) results in partial combined 17 alpha-hydroxylase/17,20-lyase deficiency

Steroid 17 alpha-hydroxylase and 17,20-lyase activities reside within the same polypeptide chain (cytochrome P-450(17 alpha)), and consequently human 17 alpha-hydroxylase deficiencies are characterized by defects in either or both of these activities. Human mutants having these deficiencies represent an excellent source of material for investigation of P-450(17 alpha) structure-function relationships. The CYP17 gene from an individual having partial combined 17 alpha-hydroxylase/17,20-lyase deficiency has been characterized structurally and the homozygous mutation found to be the deletion of the phenylalanine codon (TTC) at either amino acid position 53 or 54 in exon 1. Reconstruction of this mutation into a human P-450(17 alpha) cDNA followed by expression in COS 1 cells led to production of the same amount of immunodetectable P-450(17 alpha) protein as found with expression of the normal human P-450(17 alpha) cDNA. However, 17 alpha-hydroxylase activity of this mutant protein measured in intact cells was less than 37% of that observed upon expression of the wild-type enzyme, whereas 17,20-lyase activity of the mutant was less than 8% of that observed with the normal enzyme. When estimated in intact cells, the Km for 17 alpha-hydroxylation of progesterone was increased by a factor of 2 in the mutant enzyme, whereas the Vmax was reduced by a factor of 3. In order to estimate the kinetic parameters for the 17,20-lyase reaction, microsomes were isolated from transfected COS 1 cells to enrich for this activity. Surprisingly, the specific activity of the mutant 17 alpha-hydroxylase in microsomes was 3-fold less than that observed in intact cells, indicating that the structure of mutant P-450(17 alpha) was dramatically altered upon disruption of COS 1 cells. Apparently the deletion of a single phenylalanine in the N-terminal region of P-450(17 alpha) alters its folding in such a way that both enzymatic activities are dramatically decreased, leading to the partial combined deficiency observed in this individual.     

Molecular cloning, sequence analysis and expression of a cDNA encoding human type-1 angiotensin II receptor.

We isolated a cDNA encoding type-1 angiotensin II receptor from a human liver cDNA library. The cDNA had an open reading frame encoding a protein of 359 amino acid residues with a relative Mr of 41,060. The deduced amino acid sequence of the human angiotensin II (Ang II) receptor was 95.3% and 94.2% identical to those of bovine and rat type-1 Ang II receptors, respectively, and had a significant similarity with the G protein-coupled receptor. The rank order of the binding to the receptor expressed in COS-7 cells was Ang II greater than Ang III greater than Ang I. The expression of the Ang II receptor mRNA was detected in human liver, lung, adrenal and adrenocortical adenomas but not in adrenomedullary tumor, pheochromocytoma, by Northern blot analysis.     

Enzymatic preparation of [1, 3-13C]dihydroxyacetone phosphate from [13C]methanol and hydroxypyruvate using the methanol-assimilating system of methylotrophic yeasts

Dihydroxyacetoone synthase (EC 2.2.1.3), which is a key enzyme of the C₁-compound-assimilating pathway in yeasts, catalyzes transketolation between formaldehyde and hydroxypyruvate, leading to the formation of dihydroxyacetone and CO₂. When [¹³C]formaldehyde was used as a substrate with dihydroxyacytone synthase from Candida boidinii 2201, ¹³C was confirmed to be incorporated to the C-1 and C-3 positions of dihydroxyacetone, and the ¹³C content of each carbon (atoms/100 atoms) was estimated to be 50%. [¹³C]Methanol was also useful for the enrichment of dihydroxyacetone with ¹³C, when alcohol oxidase from a methylotrophic yeast was added for the conversion of methanol to formaldehyde. A fed-batch reaction with periodic addition of the substrates was required for the accumalation of ¹³C-labelled dihydroxyacetone at a higher concentration, because the enzyme system was relatively susceptible to the C donor, formaldehyde or methanol. The optimum conditions for the production gave 160mM (14.4 mg/ml) dihydroxyacetone for 180 min; the molar yield relative to methanol added was 80%. Diyhdroxyacetone kinase (EC 2.7.1.29) from methanol-grown Hansenula polymorpha CBS 4732 was a suitable enzyme for the phosphorylation of dihydroxyacytone. The phosphorylation system, comprising of dihydroxyacetone kinase, adenylate kinase, and ATP, could be coupled with the system for dihydroxyacetone production. A fed-batch reaction afforded 185 mM [1, 3-¹³C]dihydroxyacetone phosphate from [¹³C]methanol; the molar yield of the ester relative to methanol added was 92.5%     

Properties of poly(3-hydroxybutyrate) depolymerase from a marine bacterium, Alcaligenes faecalis AE122.

Alcaligenes faecalis AE122 that used poly(3-hydroxybutyrate) (PHB) as a sole source of carbon was newly isolated from a coastal seawater sample. The strain required seawater for growth on PHB as well as in a nutrient broth, in which seawater could be replaced by an appropriate concentration of NaCl. PHB depolymerase was purified to homogeneity from the culture supernatant of A. faecalis AE122 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme consisted of a monomer subunit with a molecular mass of 95.5 kDa. The N-terminal amino acid sequence was GAWQNNLAGGFNKV. The dimeric and trimeric esters of 3-hydroxybutyrate were the main hydrolysis products of the purified enzyme. The enzyme was most active at pH 9.0 and 55 degrees C and was inhibited by phenylmethylsulfonyl fluoride. Several cations in seawater greatly enhanced the enzyme activity.     

A possible involvement of adrenaline in the facilitation of lordosis behavior in the ovariectomized rat

In order to examine a possible role of adrenaline (AD) or noradrenaline (NA) in the control of lordosis behavior, lordosis quotient (LQ) was observed daily for 8 consecutive days in the ovariectomized rat given daily 1 or 2 microgram/0.1 ml oil of estradiol benzoate (EB) alone or together with 100 microgram/0.1 ml saline of AD or NA. AD but not NA treated together with EB caused a greater change in the daily LQ than the same dose of EB alone and the change in the daily LQ by daily treatment with both 1 microgram EB and 100 microgram AD was equivalent to that by daily treatment with 2 microgram EB alone. A half mg progesterone (P) could induce the lordosis behavior in the ovariectomized rat treated 48 hr prior with both 1 microgram EB and 50 or 100 microgram AD, but not in the one treated with 1 microgram EB alone. While 50, 100 or 200 microgram NA or 10 microgram AD had no effect, 50 or 100 microgram AD pretreated together with 2 microgram EB produced a markedly higher LQ after P than 2 microgram EB alone in the ovariectomized rat. This effect of AD on the induction of lordosis behavior was produced only when AD was pretreated simultaneously with EB and AD priming 24 or 43 hr after EB failed to elicit the effect. Therefore, it is suggested that a change of the brain target site in the estrogen sensitivity produced by AD plays a part in the control of lordosis behavior.     

Neutrophil attractant/activation protein-1 and monocyte chemoattractant protein-1 in rabbit. cDNA cloning and their expression in spleen cells

Rabbit neutrophil attractant/activation protein-1 (NAP-1) and monocyte chemoattractant protein-1 (MCP-1) were investigated. Rabbit spleen cells stimulated with 5 micrograms/ml of Con A produced both neutrophil and monocyte chemotactic activity. Physicochemical characteristics of those activities obtained by HPLC gel filtration and HPLC chromatofocusing were very similar to those of human NAP-1 and MCP-1, suggesting that rabbit spleen cells produce NAP-1 and MCP-1 after Con A stimulation. A cDNA library was constructed from mRNA purified from Con A-stimulated rabbit spleen cells and screened with oligonucleotide probes. By two rounds of screening, NAP-1 and MCP-1 cDNA were cloned. NAP-1 cDNA comprises 1500 bp with an open reading frame that encodes for a 101-amino acid protein highly similar to human NAP-1. MCP-1 cDNA comprises 607 bp with an open reading frame that encodes for a 124-amino acid protein highly similar to human MCP-1. Expression of NAP-1 and MCP-1 mRNA by rabbit spleen cells was studied. Both Con A- and LPS-stimulated spleen cells expressed NAP-1 and MCP-1 mRNA, but the kinetics of expression were different. Con A rapidly induced high NAP-1 and MCP-1 mRNA expression. LPS also rapidly induced NAP-1 mRNA expression, but high MCP-1 mRNA expression was not observed until 15 h after stimulation. Immunoprecipitation of metabolically labeled NAP-1 and MCP-1 with anti-human NAP-1 or MCP-1 polyclonal antibodies was attempted. Immunoprecipitated rabbit NAP-1 with a molecular mass of about 7 kDa was detected by SDS-PAGE and radioautography, but MCP-1 was not. Cloned rabbit NAP-1 and MCP-1 will give us opportunities to study the role of NAP-1 and MCP-1 in vivo.