Application of a ribosomal DNA integration vector in the construction of a brewer's yeast having alpha-acetolactate decarboxylase activity.

An integration plasmid, pIARL28, containing the ribosomal DNA gene as a homologous recombination sequence was constructed for introduction of the alpha-acetolactate decarboxylase gene into brewer's yeast. The transformation efficiency of pIARL28 was 20- to 50-fold higher than those of the other YIp vectors, as yeast cells had approximately 140 copies of the ribosomal DNA gene. All transformants showed very high alpha-acetolactate decarboxylase activity due to the multiple integrated copies of the plasmid. The transformants were grown in nonselective conditions, and segregants which had maintained the alpha-acetolactate decarboxylase expression cassette but no other vector sequences were isolated. Southern analysis showed that these marker-excised segregants contained more than 20 copies of the alpha-acetolactate decarboxylase gene and were stably maintained under nonselective conditions. Fermentation tests confirmed that the diacetyl concentration was considerably reduced in wort fermented by these marker-excised segregants. The degree of reduction was related to the copy number of the alpha-acetolactate decarboxylase gene.     

Efficient and Reproducible Myogenic Differentiation from Human iPS Cells: Prospects for Modeling Miyoshi Myopathy In Vitro

The establishment of human induced pluripotent stem cells (hiPSCs) has enabled the production of in vitro, patient-specific cell models of human disease. In vitro recreation of disease pathology from patient-derived hiPSCs depends on efficient differentiation protocols producing relevant adult cell types. However, myogenic differentiation of hiPSCs has faced obstacles, namely, low efficiency and/or poor reproducibility. Here, we report the rapid, efficient, and reproducible differentiation of hiPSCs into mature myocytes. We demonstrated that inducible expression of myogenic differentiation1 (MYOD1) in immature hiPSCs for at least 5 days drives cells along the myogenic lineage, with efficiencies reaching 70–90%. Myogenic differentiation driven by MYOD1 occurred even in immature, almost completely undifferentiated hiPSCs, without mesodermal transition. Myocytes induced in this manner reach maturity within 2 weeks of differentiation as assessed by marker gene expression and functional properties, including in vitro and in vivo cell fusion and twitching in response to electrical stimulation. Miyoshi Myopathy (MM) is a congenital distal myopathy caused by defective muscle membrane repair due to mutations in DYSFERLIN. Using our induced differentiation technique, we successfully recreated the pathological condition of MM in vitro, demonstrating defective membrane repair in hiPSC-derived myotubes from an MM patient and phenotypic rescue by expression of full-length DYSFERLIN (DYSF). These findings not only facilitate the pathological investigation of MM, but could potentially be applied in modeling of other human muscular diseases by using patient-derived hiPSCs.     

Increased Renal Iron Accumulation in Hypertensive Nephropathy of Salt-Loaded Hypertensive Rats

Although iron is reported to be associated with the pathogenesis of chronic kidney disease, it is unknown whether iron participates in the pathophysiology of nephrosclerosis. Here, we investigate whether iron is involved in the development of hypertensive nephropathy and the effects of iron restriction on nephrosclerosis in salt- loaded stroke-prone spontaneously hypertensive rats (SHRSP). SHRSP were given either a normal or high-salt diet for 8 weeks. Another subset of SHRSP were fed a high-salt with iron-restricted diet. SHRSP given a high-salt diet developed severe hypertension and nephrosclerosis. As a result, survival rate was decreased after 8 weeks diet. Importantly, massive iron accumulation and increased iron content were observed in the kidneys of salt-loaded SHRSP, along with increased superoxide production, urinary 8-Hydroxy-2′-deoxyguanosine excretion, and urinary iron excretion; however, these changes were markedly attenuated by iron restriction. Of interest, expression of cellular iron transport proteins, transferrin receptor 1 and divalent metal transporter 1, was increased in the tubules of salt-loaded SHRSP. Notably, iron restriction attenuated the development of severe hypertension and nephrosclerosis, thereby improving survival rate in salt-loaded SHRSP. Taken together, these results suggest a novel mechanism by which iron plays a role in the development of hypertensive nephropathy and establish the effects of iron restriction on salt-induced nephrosclerosis.     

O(2)- and H(2)O(2)-dependent verdoheme degradation by heme oxygenase: reaction mechanisms and potential physiological roles of the dual pathway degradation

Heme oxygenase (HO) catalyzes the catabolism of heme to biliverdin, CO, and a free iron through three successive oxygenation steps. The third oxygenation, oxidative degradation of verdoheme to biliverdin, has been the least understood step despite its importance in regulating HO activity. We have examined in detail the degradation of a synthetic verdoheme IXalpha complexed with rat HO-1. Our findings include: 1) HO degrades verdoheme through a dual pathway using either O(2) or H(2)O(2); 2) the verdoheme reactivity with O(2) is the lowest among the three O(2) reactions in the HO catalysis, and the newly found H(2)O(2) pathway is approximately 40-fold faster than the O(2)-dependent verdoheme degradation; 3) both reactions are initiated by the binding of O(2) or H(2)O(2) to allow the first direct observation of degradation intermediates of verdoheme; and 4) Asp(140) in HO-1 is critical for the verdoheme degradation regardless of the oxygen source. On the basis of these findings, we propose that the HO enzyme activates O(2) and H(2)O(2) on the verdoheme iron with the aid of a nearby water molecule linked with Asp(140). These mechanisms are similar to the well established mechanism of the first oxygenation, meso-hydroxylation of heme, and thus, HO can utilize a common architecture to promote the first and third oxygenation steps of the heme catabolism. In addition, our results infer the possible involvement of the H(2)O(2)-dependent verdoheme degradation in vivo, and potential roles of the dual pathway reaction of HO against oxidative stress are proposed.     

Aspergillus oryzae type III polyketide synthase CsyA is involved in the biosynthesis of 3,5-dihydroxybenzoic acid

As a novel superfamily of type III polyketide synthases in microbes, four genes csyA, csyB, csyC, and csyD, were found in the genome of Aspergillus oryzae, an industrially important filamentous fungus. In order to analyze their functions, we carried out the overexpression of csyA under the control of α-amylase promoter in A. oryzae and identified 3,5-dihydroxybenzoic acid (DHBA) as the major product. Feeding experiments using ¹³C-labeled acetates confirmed that the acetate labeling pattern of DHBA coincided with that of orcinol derived from orsellinic acid, a polyketide formed by the condensation and cyclization of four acetate units. Further oxidation of methyl group of orcinol by the host fungus could lead to the production of DHBA. Comparative molecular modeling of CsyA with the crystal structure of Neurospora crassa 2′-oxoalkylresorcylic acid synthase indicated that CsyA cavity size can only accept short-chain acyl starter and tetraketide formation. Thus, CsyA is considered to be a tetraketide alkyl-resorcinol/resorcylic acid synthase.     

A possible role of hydroxyproline-protein in oxygen-sensitive growth

Exposure of Avena coleoptile sections to 8% O₂ brought aboutrespiration decrease, resulting in a decrease of ATP production.The pH at the cell wall surface slightly rose in sections exposedto 8% O₂, while their growth was greatly accelerated. Moreover,this growth acceleration was observed even in sections treatedwith CCCP known to make membranes permeable for protons. Weconcluded that the growth acceleration with reduction of O₂concentration is probably not the result of secretion of H₊ions into cell wall compartments. Results of this study provided evidence to support the hypothesisthat there is an inverse relationship between hydroxyproline-proteinlevel and the ability of a cell to undergo rapid cell elongation.Total labeling of the cell wall fraction with ¹⁴C-proline wasunaffected by 8% O₂ treatment, although the radioactivitiesof hydroxyproline incorporated into this fraction during thetreatments fell to about 45% of the control. Moreover, the radioactivitiesof hydroxyproline incorporated into the SLS-insoluble cell wallfraction of sections exposed to 8% O₂ decreased to about 30%of the control. This decrease of hydroxyproline was also observedin sections treated with cycloheximide, which inhibits the secretionof H₊ ions into the cell wall compartment. Reduction of O₂ concentrationin the surrounding atmosphere affects not only the hydroxylationof peptidyl proline, but also the binding of hydroxyproline-protein(s)to cell wall polysaccharides, and the resulting decrease ofthe protein rigidly bound to them may induce cell elongation. (Received December 5, 1975; )     

Characterization of the carotenoid-binding protein of the Y-gene dominant mutants of Bombyx mori

Carotenoids play important and diverse roles in insects. Recently, we purified and partially characterized a carotenoid-binding protein (CBP) from the wild type of Bombyx mori. In this report, we utilized immunoblotting, ELISA and immunocytochemistry to further characterize and localize the expression of CBP in the larval midgut and silk gland obtained from the wild type and four naturally occurring mutants linked to carotenoids transport. CBP was expressed throughout the 5th stadium, with highest expressions on days 4-5 in the silk gland and days 3-5 in the midgut. Immunoblotting analyses demonstrated the presence of CBP along the middle part of the midgut. Microscopic immunocytochemistry demonstrated that the 33 kDa CBP was uniformly expressed along the brush border of columnar cells in the epithelium of the midgut typifying its function in aiding absorption of dietary carotenoids. Similarly, CBP was highly expressed along the distal membrane of the middle part of the silk gland demonstrating its function in uptake of carotenoids from lipophorin. When the middle silk glands and midguts of the four mutants were incubated with rabbit anti-CBP antibody, only proteins of the Y-gene dominant mutants cross reacted with the antibody further accentuating the hypothesis that the CBP is a Y-gene dependent protein.     

Distribution of growth regulators in relation to the light-induced geotropic responsiveness in Zea roots

Growth regulators were measured in extracts from the upper and lower halves of 7-mm apical segments of horizontally oriented, red-light-irradiated and non-irradiated roots of Zea mays L. cv. Golden Cross Bantam 70 which exhibit a georesponse only after an exposure to light. Abscisic acid (ABA) was measured by gas-liquid chromatography, auxin (indole-3-acetic acid, IAA) by the Avena straight-growth assay, and an unidentified growth inhibitor by a Zea root-growth assay. The ratio of ABA in the upper and lower halves was 1.6 in the irradiated roots and 1.0 in the non-irradiated ones. The total amount of ABA after irradiation was increased by a factor of ca. 1.8. The ratio of IAA in the upper and lower halves of irradiated and non-irradiated roots was 1:3.4 and 1:2.9, respectively. The content (or activity) of an unidentified growth inhibitor was highest in the lower halves of horizontally oriented roots which had been irradiated with red light. The unidentified growth inhibitor, rather than IAA or ABA, may be the major factor in the light-induced geotropic responsiveness in Zea roots.     

Induction of angiogenic response by chemically stable prostacyclin analogs

Angiogenic activities of several chemically stable prostacyclin analogs (isocarbacyclins and 7-fluoro prostacyclin) were evaluated by the chick embryo chorioallantoic membrane assay. These compounds showed potent angiogenic activity at very low concentration (0.1 micrograms/egg 1.0 micrograms/egg), whereas naturally occurring prostaglandins such as prostacyclin and PGE1 were almost ineffective up to 1 microgram/egg. Pretreatment of chorioallantoic membranes with dexamethasone or indomethacin inhibited the angiogenic response induced by these chemically stable prostacyclin analogs. These results indicate that these prostacyclin analogs induce the angiogenic response of chick chorioallantoic membranes via a mechanism involving activation of inflammatory cells, as well as through their direct angiogenic activity.