Production of D-phenylglycine-related amino acids by immobilized microbial cells

Bacterial dihydropyrimidinase was shown to catalyze the hydrolytic cleavage of various 5-substituted hydantoins to the corresponding N-carbamyl-D-amino acids under alkaline conditions. Therefore, an enzymatic method for preparing the D-forms of phenylglycine-related amino acids was developed using immobilized bacterial cells with high enzyme activity. Alkalophilic bacteria were a good enzyme source for this process. The process is simple and economical for use in the production of various amino acids with the D-configuration.     

Glucose uptake in mouse brain regions under hypoxic hypoxia

Glucose uptake of individual structures within the brain was studied by dry-autoradiography with 2-deoxy-D-[¹⁴C(U)]glucose under mild hypoxic hypoxia (12% O₂88% N₂ or 9% O₂91% N₂ for 1 hr). Glucose consumption in the whole brain was estimated by combined gas chromatography-mass spectrometry (GC-MS). Mild hypoxia increased the optical density of the autoradiograph in all regions. The deuterated G-6-P (Glucose-6-phosphate) synthesized from deuterated glucose decreased significantly with 9% O₂ hypoxia (P<0.05). The ratio of the deuterated G-6-P to deuterated glucose, a more appropriate indicator of glucose utilization than the concentration of deuterated G-6-P, decreased significantly with 12% O₂ hypoxia (P<0.01). The hippocampus, white matter, colliculus superior, and corpus geniculatum laterale appeared to be particulary sensitive to hypoxia.     

A case of primary hyperparathyroidism due to an oxyphil cell adenoma.

A 59-year-old woman with primary hyperparathyroidism was found to have a parathyroid adenoma behind the left clavicle. Preoperatively, it appeared as a hypoechoic mass on ultrasonography, as a hot nodule on thallium scintigraphy, and as a high signal on T2-weighted magnetic resonance imaging. Histological, immunohistochemical and ultrastructural studies of the surgically resected tumor revealed a parathyroid adenoma composed mainly of oxyphil cells with production of a parathyroid hormone. Moreover, a multilocular lesion of lymphangiectasia was contained. Hypercalcemia was alleviated postoperatively. These observations corroborated a functioning parathyroid oxyphil cell adenoma. This is the first case report of functioning oxyphil cell adenoma of the parathyroid gland with lymphangiectasia in Japan.     

A sensitive nonisotopic method for the determination of intracellular azidothymidine 5'-mono-, 5'-di-, and 5'-triphosphate.

In order to analyze the efficacy of azidothymidine (AZT), it is important to know intracellular concentrations of AZT metabolites. However, it has been impossible to measure intracellular AZT 5'-monophosphate (AZT-MP), AZT 5'-diphosphate (AZT-DP), and AZT 5'-triphosphate (AZT-TP) without using isotopes. In the present study, we developed a new method to measure intracellular AZT metabolites without radiolabeled compounds. The method employed was a high-performance liquid chromatography (HPLC) system programmed for column switching technique, in which two columns were used: column 1 (TSK-G2000-SW, 300 x 7.5 mm) to preseparate AZT metabolites from major cell components, and column 2 (YMC-A-312-ODS, 150 x 6 mm) to determine the metabolites. The limit of detectability of this system was 3.3 pmol/injection. When MT-4 cells were incubated with various concentrations of AZT, intracellular concentrations of AZT-MP increased in parallel with extracellular AZT. Those of AZT-DP and AZT-TP, however, reached plateaus at 5 and 2 microM of AZT, respectively. In MT-4 and Molt-4 cells incubated with 5 microM AZT, concentrations of AZT-MP increased time dependently, while the AZT-DP/AZT-MP ratios decreased with time. These data suggest that high dose of AZT may not necessarily increase intracellular concentration of AZT-TP. The concentrations of AZT metabolites in peripheral blood mononuclear cells in a patient with AIDS and an asymptomatic carrier were measured; the concentrations were comparable to those in cultured cells. Quantitative analysis of intracellular AZT metabolites without the use of isotopes will increase safety and convenience of measurement, and take an effective step in studying pharmacokinetics of AZT in clinical materials.     

Beta-angonists modulate ACh-inhibition of a K current in intestinal smooth muscle cells.

ACh causes a long-lasting inhibition of STOCs via G proteins in intestinal smooth muscle cells. We examined the effects of isoproterenol (Iso) on the ACh-induced inhibition of STOCs in isolated ileal smooth muscle cells using the G omega-seal whole cell clamp technique. In control, ACh (1 microM) completely suppressed STOCs, which did not desensitize over a period lasting 20 minutes. When Iso (10 microM) was added to the bath in the presence of ACh, the ACh-induced inhibition of STOCs was gradually removed. This effect of Iso was prevented by propranolol (10 microM). Application of Db-cAMP (500 microM) mimicked the Iso effects. Intracellulary applied GTP-gamma S (100 microM) gradually suppressed STOCs in the absence of ACh, which could not be removed by either Iso or Db-cAMP. These results suggest that beta-adrenergic stimulation causes a removal of the muscarinic inhibition of STOCs via a cAMP-dependent process.     

The effect of apo E secretion on lipoprotein uptake in transfected cells.

To investigate the role of apolipoprotein E (apo E) secreted by peripheral tissues in local lipoprotein metabolism, we developed a cell strain that constitutively produced and secreted apo E. A fusion plasmid containing rat apo E genomic DNA under control of mouse metallothionein promotor was constructed and transfected into Chinese hamster ovary cells. A stable transformant designated CHO-MAEII constitutively secreted rat apo E mainly in the form of sialylated free protein. The secretion was further enhanced by metal induction up to 1 micrograms apo E/ml per 12 h. When incubated with 125I-labeled very low density lipoprotein (125I-VLDL) at 37 degrees C, CHO-MAEII took up and degraded 125I-VLDL with higher affinity than control cells. Furthermore, considerable amount of methylated 125I-VLDL was degraded by CHO-MAEII, while no methylated 125I-VLDL was degraded by control cells. No significant differences were found in the uptake of 125I-LDL. The data indicated that apo E molecules secreted by CHO-MAEII were transferred to 125-VLDL particles, which caused a higher affinity of these particles for LDL receptors on the cells. It is suggested that apo E secreted from peripheral tissues enhances the uptake of lipoproteins by themselves or by surrounding cells in the local environment which demand cholesterol and express LDL receptors. CHO-MAEII was a good model for these 'auto- or paracrine-like functions' of apo E.     

Nucleotide sequence of the gene coding for four subunits of cytochrome c oxidase from the thermophilic bacterium PS3

The gene coding for four subunits of cytochrome aa3-type oxidase was isolated from a genomic DNA library of the thermophilic bacterium PS3 and sequenced. The N-terminus of each subunit was also sequenced to verify the initiation site of the reading frame. The deduced amino acid sequences contained 615 amino acid residues for subunit I (CO1/caaB product), 333 residues for subunit II (CO2/caaA product), 207 residues for subunit III (CO3/caaC product), and 109 residues for subunit IV (CO4/caaD product) after processing. Re-examination of the sequencing of caa revealed a longer open reading frame for CO1, which contains 14 transmembrane segments instead of 12 [Sone et al. (1988) J. Biochem. 103, 606-610], although the main portions of the sequences constituting cytochrome a (FeA), cytochrome a3 (FeB), and CuB are correct. PS3 CO2 has an additional sequence for cytochrome c after the CuA binding protein portion with 2 transmembrane segments, which is homologous to the mitochondrial counterpart. PS3 CO3 has DCCD-binding glutamyl residues but contains only 5 transmembrane segments, unlike the mitochondrial counterpart, which has 7 segments. The subunits of PS3 cytochrome oxidase (aa3-type) show clear similarity in amino acid sequences with those of cytochrome bo-type oxidase from Escherichia coli as well, in spite of the difference of hemes. PS3 CO3 and CO4 are much more similar to E. coli CO3 and CO4 than to mitochondrial CO3 and CO4, respectively.     

Evidence for complex formation between GTP binding protein(Gs) and membrane-associated nucleoside diphosphate kinase

When the Gs in rat liver membranes was prelabeled with [32P]NAD and cholera toxin, solubilized with octylglucoside, and then analyzed by sucrose density gradient centrifugation, it was fractionated into two peaks with approximate molecular sizes of 12-13S and 3-4S. Pretreatment without or with GDP beta S of the labeled membranes resulted in a larger peak in the high molecular weight region, whereas pretreatment with glucagon plus GTP gamma S caused almost equal peaks in both regions. The affinity-purified anti-nucleoside diphosphate (NDP) kinase antibodies only precipitated the Gs in high molecular weight region. Under the same condition, small but significant NDP kinase activity was associated with the high molecular weight Gs region although a large portion of the enzyme activity was recovered in fractions where it alone should appear (6.2S). Both Lubrol-PX and digitonin solubilized the Gs in forms insensitive to immunoprecipitation by anti-NDP kinase antibodies although the latter detergent was able to solubilize the Gs in a high molecular weight form, that is, a ternary glucagon-receptor-G protein complex. These results demonstrate that Gs and membrane-associated NDP kinase may exist in part in a complexed form in membranes. Physiological relevance of the complex formation in membrane signal transduction is discussed.     

Catalytic activities of human liver cytochrome P-450 IIIA4 expressed in Saccharomyces cerevisiae

A human liver cytochrome P-450 (P-450) IIIA4 cDNA clone was inserted behind an alcohol dehydrogenase promoter in the plasmid vector pAAH5 and expressed in Saccharomyces cerevisiae (D12 and AH22 strains). A cytochrome P-450 with typical spectral properties was expressed at a level of approximately 8 x 10(5) molecules/cell in either strain of yeast. The expressed P-450 IIIA4 had the same apparent monomeric Mr as the corresponding protein in human liver microsomes (P-450NF) and could be isolated from yeast microsomes. Catalytic activity of the yeast microsomes toward putative P-450 IIIA4 substrates was seen in the reactions supported by cumene hydroperoxide but was often lower and variable when supported by the physiological donor NADPH. The catalytic activity of purified P-450 IIIA4 was also poor in some systems reconstituted with rabbit liver NADPH-P-450 reductase and best when both the detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate and a lipid extract (from liver or yeast microsomes) or L-alpha-1,2-dilauroyl-sn-glycero-3-phosphocholine were present. Under these conditions the expressed P-450 IIIA4 was an efficient catalyst for nifedipine oxidation, 6 beta-hydroxylation of testosterone and cortisol, 2-hydroxylation of 17 beta-estradiol and 17 alpha-ethynylestradiol, N-oxygenation and 3-hydroxylation of quinidine, 16 alpha-hydroxylation of dehydroepiandrosterone 3-sulfate, erythromycin N-demethylation, the 10-hydroxylation of (R)-warfarin, the formation of 9,10-dehydrowarfarin from (S)-warfarin, and the activation of aflatoxins B1 and G1, sterigmatocystin, 7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene (both + and - diastereomers), 3,4-dihydroxy-3,4-dihydrobenz[a]anthracene, 3,4-dihydroxy-3,4-dihydro-7, 12-dimethylbenz[a]anthracene, 9,10-dihydroxy-9,10-dihydrobenzo[b]fluoranthene, 6-aminochrysene, and tris(2,3-dibromopropyl) phosphate to products genotoxic in a Salmonella typhimurium TA1535/pSK1002 system where a chimeric umuC' 'lacZ plasmid is responsive to DNA alkylation. Reaction rates were stimulated by 7,8-benzoflavone and inhibited by rabbit anti-P-450 IIIA (anti-P-450NF), troleandomycin, gestodene, and cimetidine. Evidence was obtained that rates of reduction of ferric P-450 IIIA4 in yeast microsomes and the reconstituted systems are slow and at least partially responsible for the lower rates of catalysis seen in these systems (relative to liver microsomes). The results of these studies with a defined protein clearly demonstrate the ability of P-450 IIIA4 to catalyze regio- and stereoselective oxidations with a diverse group of substrates, and this enzyme appears to be one of the most versatile catalysts in the P-450 family.