Deletion analysis of the Taka-amylase A gene promoter using a homologous transformation system in Aspergillus oryzae.

The Taka-amylase A gene (amyB) of Aspergillus oryzae is induced by starch or maltose. The molecular mechanism of the induction was investigated using a fusion of the amyB promoter and the Escherichia coli uidA gene encoding beta-glucuronidase (GUS). To identify the region responsible for high-level expression and regulation within the amyB promoter, a series of deletion promoters was constructed and introduced into the A. oryzae met locus by homologous recombination. Deletion of the region between -377 to -290 (the number indicates the distance in base pairs from the translation initiation point (+1) to the deletion end point) significantly reduced of the GUS activity, but slight reduction of the GUS activity was observed in deletions up to -377. Northern blot analysis showed that reduction of the GUS activity depended upon the expression level of the GUS gene. The region between -377 to -290 is suggested to include the sequence required directly for high-level expression and regulation of the amyB gene.     

Construction of a fusion gene comprising the Taka-amylase A promoter and the Escherichia coli β-glucuronidase gene and analysis of its expression in Aspergillus oryzae

Summary Northern blot analysis of glucose-grown and starch-grown mycelia of Aspergillus oryzae R11340 was conducted using the cloned Taka-amylase A (TAA) gene as a probe. The amount of mRNA homologous to the TAA gene was increased when this fungus was grown with starch as a sole carbon source. In order to analyze the induction mechanism, we inserted the Escherichia coli uidA gene encoding -glucuronidase (GUS) downstream of the TAA promoter and introduced the resultant fusion gene into the A. oryzae genome. Production of a functional GUS protein was induced by starch, but not by glucose. When the effects of various sugars on expression of the fusion gene were examined, the results suggested that the expression of the fusion gene was under control of the TAA gene promoter.     

Gamma-MSH and its related peptides do not augment ACTH-induced steroidogenesis in isolated rat adrenal cells

In a sensitive ACTH bioassay system using isolated rat adrenal cells, we tested the effect of gamma-MSH related peptides on ACTH-induced steroidogenesis. Peptides, including synthetic gamma1-, gamma2-, gamma3- and Lys-gamma3-MSH, exerted no effect in augmenting ACTH-induced steroidogenesis. None of the 16 kilodalton fragment of ACTH/beta-lipotropin precursor and its cleaved fragment had such an activity. The results are in contrast with previous reports concerning ACTH-potentiating activity of gamma-MSH related peptides and, therefore, indicate the necessity of further investigation of the principle involved in this unique biological activity.     

The last three consecutive epidermal growth factor-like structures of human thrombomodulin comprise the minimum functional domain for protein C-activating cofactor activity and anticoagulant activity

We have identified a minimum functional domain of human thrombomodulin for anticoagulant activity using deletion analysis. Four mutants were constructed by site-directed deletion mutagenesis to delete one or more epidermal growth factor (EGF)-like structures from the domain of human thrombomodulin containing six repeated EGF-like structures. These deletion mutants were expressed transiently in COS-1 cells, and their protein C-activating cofactor activities in the culture medium were examined. One mutant protein, E456, which contains the fourth, fifth, and sixth EGF-like structures expresses apparent cofactor activity. However, neither E456-N24 (24 NH2-terminal-residue deletion) nor E456-C16 (16 COOH-terminal-residue deletion) have cofactor activity. E456 was partially purified and its anticoagulant effects on plasma clotting time and platelet aggregation examined. E456 expressed almost the same anticoagulant activities as D123 which contains six consecutive EGF-like structures of thrombomodulin. It was concluded that E456 is the minimum functional domain for both protein C-activating cofactor activity and anticoagulant activity.     

Presence of adrenocorticotropin-potentiating factors in porcine thyroid glands

An extract of porcine thyroid gland in 0.1 N acetic acid exerted dose-dependent potentiation of ACTH-induced corticosterone production in isolated rat adrenal cells. The extract by itself manifested no steroidogenic activity. Upon gel-filtration of the extract, potentiating activities were demonstrated in three main peaks with molecular weights of about 10,000, 5,000 and 2,000. These findings indicate the presence of heterogeneous forms of ACTH-potentiating factors in the thyroid. Significant enhancement of ACTH-induced steroidogenesis was readily apparent with three gel-filtration fractions at a lower concentration of ACTH (4.75 pM). At this concentration, dose-dependent potentiation was observed with these three fractions. Enhanced corticosterone production responses by cells preincubated with the thyroid extract were observed and the results indicated the existence of potentiating mechanisms other than inhibition of ACTH proteolysis. The lack of T4, T3 and thyroglobulin in this activity suggests that the activity resides in other constituents of the thyroid.     

Genetic engineering of a sake yeast producing no urea by successive disruption of arginase gene.

Urea is reported to be a main precursor of ethyl carbamate (ECA), which is suspected to be a carcinogen, in wine and sake. In order to minimize production of urea, arginase-deficient mutants (delta car1/delta car1) were constructed from a diploid sake yeast, Kyokai no. 9, by successive disruption of the two copies of the CAR1 gene. First, the yeast strain was transformed with plasmid pCAT2 (delta car1 SMR1), and strains heterozygous for CAR1 gene were isolated on sulfometuron methyl plates. Successively, the other CAR1 gene was disrupted by transformation with plasmid pCAT1 (delta car1 G418r) and the resulting car1 mutants were isolated on a G418 plate. Arginase assay of the total cell lysate of the mutants showed that 70% of transformants isolated on G418 plates had no detectable enzyme activity, possibly as a result of the disruption of the two copies of the CAR1 gene. Further genomic Southern analysis confirmed this result. We could brew sake containing no urea with the delta car1/delta car1 homozygous mutant. It is of additional interest that no ECA was detected in the resulting sake, even after storage for 5 months at 30 degrees C. This molecular biological study suggests that ECA in sake originates mainly from urea that is produced by the arginase.     

Purification and properties of cytochrome P-450(11beta) from adrenocortical mitochondria.

A cytochrome P-450, which is functional in the steroid methylene 11β-hydroxylation (P-450_11β), has been purified to a protein weight of 85 kg per heme from bovine adrenocortical mitochondria. The purification is accomplished in the presence of deoxycorticosterone as a substrate stabilizer. The procedure involved solubilization of sonicated mitochondrial pellets, ammonium sulfate fractionation, alumina Cγ gel treatment and aniline-substituted Sepharose 4B chromatography.The purified preparation when freed from deoxycorticosterone, has a low spin type absorption spectrum which can rapidly be converted into a typical high spin substrate-bound form by the addition of an 11β-hydroxylatable steroid, either deoxycorticosterone or testosterone. The preparation exhibits high 11β-hydroxylase activity and is free from the cholesterol side-chain cleavage cytochrome P-450 (P-450_scc).The purified P-450_11β, when submitted to SDS-polyacrylamide gel electrophoresis, exhibits a single protein band (molecular weight of 46 kilodaltons) which is clearly distinguished from P-450_scc. As determined by the sedimentation equilibrium method, the molecular weight of the guanidine-treated P-450_11β is estimated to be 43 kilodaltons.     

Cutting edge: Gln22 of mouse MD-2 is essential for species-specific lipopolysaccharide mimetic action of taxol

MD-2 associates with the extracellular domain of Toll-like receptor 4 (TLR4) and greatly enhances LPS signaling via TLR4. Taxol, which mimics the action of LPS on murine macrophages, induces signals via mouse TLR4-MD-2, but not via human TLR4-MD-2. Here we investigated the molecular basis for this species-specific action of Taxol. Expression of mouse MD-2 conferred both LPS and Taxol responsiveness on human embryonic kidney 293 cells expressing mouse TLR4, whereas expression of human MD-2 conferred LPS responsiveness alone, suggesting that MD-2 is responsible for the species-specificity as to Taxol responsiveness. Furthermore, mouse MD-2 mutants, in which Gln(22) was changed to other amino acids, showed dramatically reduced ability to confer Taxol responsiveness, although their ability to confer LPS responsiveness was not affected. These results indicated that Gln(22) of mouse MD-2 is essential for Taxol signaling but not for LPS signaling.     

Mechanism-based inhibition of rat liver microsomal diazepam C3-hydroxylase by mifepristone associated with loss of spectrally detectable cytochrome P450

Since initial studies with the steroids norethindrone and ethynylestradiol, reported by White and Muller-Eberhard in 1977 (Biochem. J. 166, 57-64), there has been continuing interest in xenobiotics that bear terminal or sub-terminal acetylenic groups which can cause catalysis-dependent inhibition of CYP monooxygenases associated either with loss of prosthetic group heme or protein adduct formation. Mifepristone is a synthetic steroid bearing a propyne substitution on carbon 17 and this suggested to us that it may act as a mechanism-based inhibitor of the CYP isoforms responsible for its metabolism. In human and rat liver, CYP3A isoforms have been implicated in mifepristone clearance and mifepristone administration to rats has also been shown to induce CYP3A enzymes and the associated diazepam C3-hydroxylase activity (Cheesman, Mason and Reilly, J. Steroid Biochem. Mol. Biol., 58, 1996, 447-454). With microsomes prepared from the livers of untreated female rats and others in which diazepam C3-hydroxylase has been induced, we show here that mifepristone can cause catalysis-dependent inhibition of this monooxygenase. In addition, incubation of microsomes with mifepristone in the presence, but not in the absence, of NADPH caused loss of spectrally detectable cytochrome P450. These results suggest that heme adduct formation may result from mifepristone metabolism by CYP3A monooxygenases which undergo self-catalysed irreversible inactivation with this drug as substrate. Since mifepristone administration in vivo is able also to cause induction of the synthesis of hepatic CYP3A apoprotein, mifepristone may have the potential in human medicine for complex interactions with other co-administered drugs which are also substrates for CYP3A monooxygenases.