Directed mutagenesis in an asporogenous methylotrophic yeast: cloning, sequencing, and one-step gene disruption of the 3-isopropylmalate dehydrogenase gene (LEU2) of Candida boidinii to derive doubly auxotrophic marker strains.

A model system for one-step gene disruption for an asporogenous methylotrophic yeast, Candida boidinii, is described. In this system, the 3-isopropylmalate dehydrogenase gene (C. boidinii LEU2) was selected as the target gene for disruption to derive new host strains for transformation. First, the C. boidinii LEU2 gene was cloned, and its complete nucleotide sequence was determined. Next, the LEU2 disruption vectors, which had the C. boidinii URA3 gene as the selectable marker, were constructed. Of the Ura+ transformants obtained with these plasmids, more than half showed a Leu- phenotype. Finally, the double-marker strains of C. boidinii were derived. When vectors with repeated flanking sequences of the C. boidinii URA3 gene were used for gene disruption, Leu- Ura+ transformants changed spontaneously to a Leu- Ura- phenotype ca. 100 times more frequently than they did when plasmids without the repeated sequences were used. Southern analysis showed that these events included a one-step gene disruption and a subsequent popping out of the C. boidinii URA3 sequence from the transformant chromosome.     

Alpha-elastin coacervate as a protein liquid membrane: effect of pH on transmembrane potential responses.

A protein liquid membrane composed of coacervated alpha-elastin, a chemical fragmentation product of the biological elastic fiber protein, functioned as an amphoteric liquid ion-exchange membrane. Ionic permselectivities of the alpha-elastin coacervate membrane to a series of metal chlorides were investigated for the concentration-cell systems by the ordinary electrochemical measurements. Effects of pH on the transmembrane potential responses for NaCl, CaCl2, and MgCl2 systems were examined. Only in the Ca(2+)-containing system did potential responses stay at constant levels against the pH changes, whereas in the other systems, increasing pH caused potential changes, indicating an improvement of cationic permselectivity across the alpha-elastin coacervate membrane. It was suggested that the characteristic Ca2+ transport mechanisms across the alpha-elastin coacervate membrane are related in some way to the polypeptide backbone interactions specific and selective to Ca2+ ions.     

Are gap junctions necessary for cell-to-cell coupling of smooth muscle?: An update.

Earlier, it was questioned whether gap junctions (GJs) were necessary for cell-cell communication in smooth muscle, and GJs were not seen in some smooth muscles. We reexamined this question in the myometrium and in intestinal smooth muscle, in light of current knowledge of the presence and function of GJs. In the uterus, numerous studies show that an increase in GJ number is associated with the onset of delivery and is required for effective parturition. In all cases, this increase in GJ number and the changes in uterine contractility were correlated with increased electrical and metabolic coupling. Evidence for the much smaller, but detectable, degree of electrical coupling in the preterm uterus is explained by the small (but again detectable) number of GJs present. In the intestine, GJs are readily detected in the circular muscle layer but have not been described in the adjacent longitudinal layer. While our immunohistochemical studies failed to detect GJs in the longitudinal layer, this may not be adequate to prove their absence. Therefore, current knowledge of GJ number and function is adequate to explain cell-cell coupling in the uterus. Although it remains uncertain whether GJs are absent from the longitudinal muscle of the intestine, there is no definitive evidence that cell-cell coupling can occur by means other than GJs.     

Possible involvement of protein kinase C and calcium in GSH efflux from Hep G2 cells.

We investigated the effects of protein kinase C modulations and calcium mobilization on GSH efflux in Hep G2 cells. GSH efflux from Hep G2 cells was increased by a phorbol ester. Staurosporine, an inhibitor of protein kinase C, diminished phorbol ester-stimulated GSH efflux from the cells. GSH efflux was negatively correlated with extracellular calcium concentrations. Verapamil enhanced GSH efflux, whereas ATP decreased GSH efflux. The latter effect was diminished in the absence of extracellular calcium. Protein kinase C and calcium mobilization may be crucial factors in GSH efflux from human hepatocytes.     

Effects of hirsutine, an antihypertensive indole alkaloid from Uncaria rhynchophylla, on intracellular calcium in rat thoracic aorta.

The effects of hirsutine, an indole alkaloid from Uncaria rhynchophylla (MIQ.) Jackson, on cytosolic Ca2+ level ([Ca2+]cyt) were studied by using fura-2-Ca2+ fluorescence in smooth muscle of the isolated rat aorta. Noradrenaline and high K+ solution produced a sustained increase in [Ca2+]cyt. Application of hirsutine after the increases in [Ca2+]cyt induced by noradrenaline and high K+ notably decreased [Ca2+]cyt, suggesting that hirsutine inhibits Ca2+ influx mainly through a voltage-dependent Ca2+ channel. Furthermore, the effect of hirsutine on intracellular Ca2+ store was studied by using contractile responses to caffeine under the Ca(2+)-free nutrient condition in the rat aorta. When hirsutine was added at 30 microM before caffeine treatment, the agent slightly but significantly reduced the caffeine-induced contraction. When added during Ca2+ loading, hirsutine definitely augmented the contractile response to caffeine. These results suggest that hirsutine inhibits Ca2+ release from the Ca2+ store and increases Ca2+ uptake into the Ca2+ store, leading to a reduction of intracellular Ca2+ level. It is concluded that hirsutine reduces intracellular Ca2+ level through its effect on the Ca2+ store as well as through its effect on the voltage-dependent Ca2+ channel.     

The micronucleus test with mouse peripheral blood on N-methyl-N'-nitro-N-nitrosoguanidine and mitomycin C.

The micronucleus test using mouse peripheral blood was conducted with N-methyl-N'-nitro-N-nitro-soguanidine (MNNG) and mitomycin C (MMC) as part of the 5th collaborative study supported by the Environmental Mutagen Society of Japan (CSGMT/MMS.JEMS). Male CD-1 mice were intraperitoneally injected once with 12.5-100 mg/kg of MMC. Peripheral blood was drawn at different intervals after treatment, placed on slides previously coated with acridine orange and the numbers of reticulocytes with micronuclei (MNRETs) were scored. The experiments indicated that the maximum effect of both MNNG and MMC was found about 48 h after treatment, and that the micronucleus test using peripheral blood is useful for the screening of chemicals throughout the experimental period in a single animal.     

Biotransformation of tabersonine in cell suspension cultures of Catharanthus roseus.

To investigate the reactions involved in the biosynthesis of vindoline from tabersonine, the bioconversion products formed when the latter compound was fed to cell suspension cultures of Catharanthus roseus were isolated and characterized. Two biotransformation products of tabersonine were isolated and shown to be lochnericine, which is formed by epoxidation of tabersonine at positions 14, 15, and lochnerinine, the 11-methoxylation product of lochnericine. The bioconversion ratio of the main biotransformation product, lochnericine, reached a value of 80.6% within three days.     

Alteration of mitochondrial genomes containing atpA genes in the sexual progeny of cybrids between Raphanus sativus cms line and Brassica napus cv. Westar

Summary We have investigated the fate of the mitochondrial genomes of cybrids derived from donor-recipient protoplast fusion between X-irradiated Raphanus sativus (cms line) and iodoacetamide-treated Brassica napus cv. Westar. Two out of ten fusion products were male-sterile with the diploid chromosome number of B. napus. The mitochondrial (mt) genomes of the cybrids and their progeny were further analyzed by DNA-DNA hybridizaion using the pea mitochondrial ATPase subunit gene (atpA) as a probe. One cybrid, 18-3, had a 3.0 kb fragment characteristic of B. napus and a 2.0 kb non-parental fragment when the BamHI-digested DNA was hybridized with the probe. In the first-backcrossed progeny of this cybrid, the hybridization pattern was not stably inherited. A 4.0 kb radish fragment, not detectable in the cybrid, appeared in one of the BC₁ generation siblings, and the 2.0 kb non-parental fragment was lost in another. The hybridization patterns in BC₁ progeny siblings of cybrid 12-9 were also varied. The alteration of mtDNA in the cybrid progeny continued to the BC₂ generation. There was no clear evidence of a heteroplasmic state or of sub-stoichiometric molecules in the mt genome of cybrid 18-3. A possible cause of the observed alteration in the mt genome is discussed.     

Ultrastructure of the endolymphatic sac in the mouse.

The ultrastructure of the endolymphatic sac (ES) in the mouse was examined by light and electron microscopy. This organ was divided into three parts: proximal, intermediate and distal. In the proximal portion of the ES, the epithelium consisted of thin squamous cells. The epithelial cells had acquired basolateral processes, numerous small vesicles and well-developed Golgi apparatus. In the intermediate portion, the epithelium consisted of columnar or cuboidal cells. The epithelial cells could be classified into two types: type I and type II. The type I cells had abundant microvilli, pinocytotic vesicles, vacuoles, multivesicular bodies, lysosomes and mitochondria. The type II cells had fewer numbers of these organelles. A few free-floating cells could be observed in the lumen of this intermediate portion, most of which were macrophages. In the distal portion, the epithelium consisted of squamous or cuboidal cells. The epithelial cells had a few cytoplasmic organelles. In the ultrastructural study, each portion of the mouse ES was found to have a very distinct morphological feature. It was suggested that each of these three portions has a different function.     

Production and characterization of N-acyl-D-glutamate amidohydrolase from Pseudomonas sp. strain 5f-1.

N-Acyl-D-glutamate amidohydrolase from Pseudomonas sp. strain 5f-1 was inducibly produced by D isomers of N-acetylglutamate, glutamate, aspartate, and asparagine. The enzyme has been purified to homogeneity by DEAE-cellulose, (NH4)2SO4 fractionation, and chromatofocusing followed by gel filtration on a Sephadex G-100 column. The enzyme was a monomer with molecular weight of 55,000. The enzyme activity was optimal at pH 6.5 to 7.5 and 45 degrees C. The isoelectric point and the pH stability were 8.8 and 9.0, respectively. N-Formyl, N-acetyl, N-butyryl, N-propionyl, N-chloroacetyl derivatives of D-glutamate and glycyl-D-glutamate were substrates for the enzyme. At pH 6.5 in 100 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) buffer at 30 degrees C, a Km of 6.67 mM and a Vmax of 662 mumol/min/mg of protein for N-acetyl-D-glutamate were obtained. None of the metal ions stimulated the enzyme activity. Na+, K+, Mg2+, and Ba2+ acted as stabilizers. Hg2+, Cu2+, Zn2+, Fe3+, and EDTA were strongly inhibitory.