Intergeneric hybridization betweenMonascus anka andAspergillus oryzae by protoplast fusion

Summary To breed industrially useful strains of a slow-growing, red-pigment-producing strain ofMonascus anka, protoplasts ofM. anka MAK1 (arg) andAspergillus oryzae AOK1 (met, thr) were fused. A mixture of protoplasts prepared from mycelia ofM. anka MAK1 treated with 2% Usukizyme and ofA. oryzae AOK1 treated with 2% Usukizyme and 0.2% NovoZym 234 was incubated with 30% (w/v) polyethylene glycol no. 6000. Heterokaryon fusants complementing the auxotrophies of both mutants were isolated on minimal medium, but segregated into red (MAK1) and white (AOK1) sectors after being cultured on a complete medium. After irradiation with UV light, the fusants gave stable heterozygous diploids that formed long white hyphae. These diploids, which had twice as much DNA in the nucleus as their parents, grew more rapidly than the parent strain YZT1, and produced ethanol earlier than the parents. Production of amylase, protease, and kojic acid by the fusants was intermediate in amount between that of the two parents.     

Genetic Analysis of the Saccharomyces Cerevisiae Rho3 Gene, Encoding a Rho-Type Small Gtpase, Provides Evidence for a Role in Bud Formation

RHO3 encodes a Rho-type small GTPase of the yeast Saccharomyces cerevisiae. We isolated temperature-sensitive alleles and a dominant active allele of RHO3. Ts(-) rho3 cells lost cell polarity during bud formation and grew more isotropically than wild-type cells at nonpermissive temperatures. In contrast, cells carrying a dominant active mutant RHO3 displayed cold sensitivity, and the cells became elongated and bent, often at the position where actin patches were concentrated. These phenotypes of the rho3 mutants strongly suggest that RHO3 is involved in directing the growing points during bud formation. In addition, we found that SRO6, previously isolated as a multicopy suppressor of rho3, is the same as SEC4. The sec4-2 mutation was synthetic lethal with temperature-sensitive rho3 mutations and suppressed the cold sensitivity caused by a dominant active mutant RHO3. The genetic interactions between RHO3 and SEC4, taken together with the fact that the Rab-type GTPase Sec4p is required to fuse secretory vesicles together with plasma membrane for exocytosis, support a model in which the Rho3p pathway modulates morphogenesis during bud growth via directing organization of the actin cytoskeleton and the position of the secretory machinery for exocytosis.     

Effect of shock-loading of heavy metals on total organic carbon and phosphate removal in an anaerobic-aerobic activated sludge process

The effect of shock-loading of zinc, copper and cadmium ions on the removal of total organic carbon (TOC) and phosphate in an anaerobic-aerobic activated sludge process was investigated. TOC removal was not sensitive to shock-loading of Zn²⁺ and Cd²⁺ ions, and complete removal was achieved even at 20 mg Zn²⁺/l and 20 mg Cd²⁺/l. However, with over 1 mg Cu²⁺/1 TOC removal efficiency decreased. PO _inf4 ^sup3- removal, in contrast, was extremely sensitive to these metal ions, with the threshold being 1 mg Zn²⁺/l and 1 mg Cd²⁺/l. Higher concentrations adversely affected PO _inf4 ^sup3- removal. Copper again proved detrimental; no PO _inf4 ^sup3- removal was achieved even at 1 mg Cu/l. These results highlight the sensitivity of the removal efficiencies of TOC and PO _inf4 ^sup3- to shock loadings of these heavy metals.Y.P. Ting is with the Department of Chemical Engineering, National University of Singapore, Kent Ridge, 0511, Singapore; H. Imai and S. Kinoshita are with the Department of Chemical Process Engineering, Hokkaido University, Sapporo 060, Japan.     

Effects of body and head positions on bilateral difference in tympanic temperatures

We have examined the nonparallel changes in tampanic membrane temperatures (T _ty) from the two ears in response to various changes in body and head positions. Upon assuming a lateral recumbent position, the T _ty on the lower side increased while that on the upper side decreased. Pressure application over a wide area of the lateral chest only caused inconsistent and obscure asymmetric changes in T _ty. A lateral flexion of the head with the subject sitting upright and a rotation of the head to the side in a supine position induced an increase in the T _ty on the lower side compared to that on the upper side. The temperature and blood flow of the forehead often decreased on the lower side and increased on the upper side, although such responses were not always concomitant with the asymmetric changes in T _ty. A dorsal flexion of the head with the subject in a reclining position caused a slight increase in the T _ty, whereas raising the head upright induced a slight decrease in them. Two additional experiments were carried out with single photon emission computed tomography using ^99mTc-hexamethylpropyleneamine oxime as tracer, and a slight, relative decrease in counts was noted in the right hemisphere during rotation of the head to the right. These results would strongly suggest that unilateral increases and decreases in T _ty could have been caused by one-sided decreases and increases, respectively, in blood flow to the brain and/or the tympanic membrane, induced by a vasomotor reflex involving vestibular stimulation.     

Expression of an ABA-induced gene of tomato in transgenic tobacco during periods of water deficit

The gene le25 is an abscisic acid (ABA)-induced gene of tomatowhich is expressed both in wilted vegetative organs and developingseeds. Spatial and temporal expression was analysed in tobaccoplants transformed with a chimeric gene in which 5'-upstreamDNA sequences of le25 were fused to the E. coli uidA gene, whichencodes ß-glucuronidase (GUS). Histochemical stainingrevealed that GUS was expressed in all tissues of vegetativeorgans in response to water deficit. Exogenous ABA induced expressionto a lesser extent, even though ABA content was the same asdroughtstressed leaves, indicating a difference in responseto endogenous ABA compared to exogenous ABA. Water-deficit-inducedGUS expression in floral tissues was examined in pre-anthesisfloral buds from four different stages (I–IV; 11, 16,33, 49 mm bud length, respectively). While non-stressed floralorgans showed no GUS activity except in pollen at stages IIIand IV, GUS activity was water-deficit-induced in sepals ofall stages, petals of stage II, and stigmas of stage II andIII. In seeds, GUS activity was detected in both the embryoand endosperm at 15 d post-anthesis, which coincided with alarge increase in the concentration of ABA in the seed. In transgenicplants, the le25 5'-flanking DNA drove expression of GUS duringwater deficit in two modes: non-tissue-specific expression invegetative organs, and tissue-specific expression in reproductiveorgans. The location of GUS activity indicated that ABA concentrationis elevated throughout the tissues of the leaf during periodsof water deficit. Key words: Tomato, ABA, drought stress, lea gene, water deficit     

Unique tissue distribution of a mouse macrophage C-type lectin

We examined mouse tissue for the expression of macrophage galactose/N-acetylgalactosamine-specificC-type lectin using a rat monoclonal antibody (mAb) specificfor this lectin (mAb LOM-14). The binding of mAb LOM-14 wasdetected in detergent extracts from tissue by means of immunoblottinganalysis. It was shown that this mAb did not cross-react withmouse hepatic lectins, a structural homologue. The macrophagelectin was widely distributed among various mouse tissues asjudged by the affinity isolation followed by the immunochemicaldetection. The exceptions were brain, liver, kidney, small intestine,and peripheral blood. Extracts from these organs exhibited,at best, very weak signals upon mAb LOM-14 binding, despitethe presence of cells expressing macrophage markers. The mostintense signal was observed in the extract from skin, suggestingthat cells expressing this lectin are abundant in skin. Thetissues shown to contain this lectin were further investigatedby immunohistochemical staining of the sections. Cells weredistributed in the connective tissue and in the interstice,particularly the dermis and subcutaneous layer of skin. Cellslocalized in the epithelium of skin (epidermis) or other epitheliathat we examined were not stained. Perivascular localizationof cells stained with mAb LOM-14 was also demonstrated in cardiacand skeletal muscle tissues. Immunoelectron microscopy revealedthe presence of this lectin along the rough endoplasmic reticulum.In conclusion, the distribution of C-type lectin specific forgalactose/N-acetylgalactosamine in mice was unique. The connectivetissue-specific distribution should provide important informationon the biological role of this lectin. lectin macrophage calcium-type lectin connective tissue     

Further cholinergic aspects of carotid body chemotransduction of hypoxia in cats

Fitzgerald, Robert S., Machiko Shirahata, and Tohru Ide.Further cholinergic aspects of carotid body chemotransduction ofhypoxia in cats. J. Appl. Physiol.82(3): 819-827, 1997.From the 1930s into the 1970s, the role ofacetylcholine (ACh) in the carotid body's chemotransduction of hypoxiawas debated. Since the late 1970s, the issue has been pursued onlyintermittently or not at all. The purpose of this study was to testagain with a new preparation the hypothesis that ACh is an excitatoryneurotransmitter in the cat carotid body's chemotransduction ofhypoxia. We tested the effect of the specific nicotinic blockermecamylamine and the muscarinic blocker of all five muscarinicreceptors, atropine. We further tested the effects ofM₁ andM₂ muscarinic-receptor blockers.The carotid body region was selectively perfused with hypoxicKrebs-Ringer bicarbonate (KRB) solutions that were blocker free orcontained varying doses of the blockers. Both mecamylamine and atropinereduced the response to hypoxic KRB in a dose-related manner. TheM₂ muscarinic-receptor blockersgallamine and AFDX 116 increased the response to hypoxic KRB, whereasthe M₁ muscarinic-receptor blockerpirenzepine reduced the response to hypoxic KRB. These data areconsistent with an excitatory role for ACh in the carotid bodychemotransduction of hypoxia in the cat.

     

The release of monovalent counterions by addition of divalent countemons to aqueous solutions of maleic acid copolymer

Divalent cation binding and the release of monovalent cations accompanying the cation binding were experimentally studied by ion-selective electrode methods in aqueous solutions of copolymer of maleic acid and ethyl vinyl ether. It was found that in the process of Ca2+ addition, all the Ca2+ added was bound to polyions and the initially condensed Na+ was released in proportion to the concentration of the added Ca2+ up to the critical concentration of added Ca2+ at which the condensation of Ca2+ ceases. Values of the structural charge density parameter xi(s), were determined from the end-points of condensation of Ca2+. The process of Na+ release by adding Ca2+ was analyzed on the basis of the counterion condensation theory by using these xi(s) values. In addition, the relationship between the activity coefficient gamma-- of Ca2+ and degree of neutralization alpha in salt-free solutions was obtained from the Manning theory. Agreement between the calculated and experimental values was excellent in both cases.     

Porcine 80-kDa diacylglycerol kinase is a calcium-binding and calcium/phospholipid-dependent enzyme and undergoes calcium-dependent translocation.

We attempted to assess the regulatory role of EF-hand motifs recently detected in the primary structure of porcine 80-kDa diacylglycerol kinase (DGK) (Sakane, F., Yamada, K., Kanoh, H., Yokoyama, C., and Tanabe, T. (1990) Nature 344, 345-348). By using 80-kDa DGK purified from porcine thymus cytosol, we found that this isozyme indeed bound 2 mol Ca2+ per mol enzyme with high affinity (apparent dissociation constant, kd = 0.3 microM). The Ca2+ binding was cooperative with a Hill coefficient of 1.4. We next studied the effect of 1 x 10(-5) M Ca2+ on the kinetic properties of DGK employing a beta-octyl glucoside mixed micellar assay system. In the absence of Ca2+, phosphatidylserine, so far used as an enzyme activator in various assay systems, was rather inhibitory, and Ca2+ alone activated enzyme to a limited extent. However, phosphatidylserine plus Ca2+ markedly activated the enzyme, giving approximately 4-fold higher Vmax and 10-fold less Km values for ATP. In contrast, the apparent Km values for diacylglycerol were not significantly affected (approximately 3 mol %). Furthermore, by immunoblotting using anti-80 kDa DGK antibodies we found that the soluble DGK in the homogenate of porcine thymocytes was translocated to membranes in a Ca2(+)-dependent manner. Indeed we noted the presence of a 33-residue amphipathic alpha-helix in the DGK sequence, which may account for the protein-lipid interaction. The results demonstrate that Ca2+ plays a key role in the regulation of DGK action by controlling enzyme interaction with membrane phospholipids.     

An increased level of sperm abnormalities in mice with a partial deletion of the Y chromosome

Two congenic lines of mice, one with a partial deletion of the Y chromosome, differ in the percentage of spermatozoa with abnormal heads: B10.BR/SgSn males give 22.6% and B10.BR-Ydel/Ms males give 64.2% abnormal sperm. The F1s resulting from crosses of B10.BR/SgSn males with females of five common inbred strains exhibited significantly lower levels of abnormal sperm than the parental strains, as opposed to F1 hybrids sired by B10.BR-Ydel/Ms mutant males, where very high levels of abnormal spermatozoa were found. About 30% of abnormal spermatozoa, produced by males with deletion on the Y chromosome, were characterized by a flat acrosomal cap. This class of abnormality was never observed in non-mutant males, suggesting a mutant-specific defect. These results demonstrate the important role of the Y chromosome in spermatogenesis.