Two-dimensional NMR, circular dichroism, and fluorescence studies of PP-50, a synthetic ATP-binding peptide from the beta-subunit of mitochondrial ATP synthase.

PP-50, a peptide based on residues 141-190 of the beta-subunit of mitochondrial F1-ATPase, contains the GX4GKT consensus region for nucleoside triphosphate binding and has been shown to bind ATP [Garboczi, D.N., Shenbagamurthi, W.K., Hullihen, J., & Pedersen, P.L. (1988) J. Biol. Chem. 263, 812-816]. At pH 4.0, appropriate for NMR studies, PP-50 retains the ability to bind ATP tightly (KD = 17.5 microM) with a 1:1 stoichiometry as shown by titrations measuring the partial quenching of ATP fluorescence by PP-50. CD spectra of PP-50 at pH 4.0 and at low ionic strength show 5.8% helix, 30.2% beta-structure, and 64% coil. ATP binding increases the structure of PP-50, changing the CD to 7.5% helix, 44.5% beta-structure, and 48% coil. Increasing the ionic strength to 50 mM KCl also increases the structure, changing the CD to 7.4% helix, 64.4% beta-structure, and 28.2% coil. The 600-MHz proton NMR spectrum of PP-50, at pH 4.0 and low ionic strength, has been assigned by 2D methods (TOCSY, DQF-COSY, and NOESY with jump-return water suppression). Based on strong d alpha N NOEs, J alpha N values, and NH chemical shifts differing from random coil values, regions of extended structure are detected from residues 1-7 and 43-48. Based on dNN, dNN(i,i+2), and d alpha N(i,i+2) NOEs and 3J alpha N values, possible type I' and type I turns are found from residues 11-14 and 31-34, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evidence that Glyceraldehyde-3-Phosphate Dehydrogenase Is Involved in Age-Induced Apoptosis in Mature Cerebellar Neurons in Culture

Abstract: Under typical culture conditions, cerebellar granule cells die abruptly after 17 days in vitro. This burst of neuronal death involves ultrastructural changes and internucleosomal DNA fragmentations characteristic of apoptosis and is effectively arrested by pretreatment with actinomycin-D and cycloheximide. The level of a 38-kDa protein in the particulate fraction is markedly increased during age-induced cell death and by pretreatment with NMDA, which potentiates this cell death. Conversely, the age-induced increment of the 38-kDa particulate protein is suppressed by actinomycin-D and cycloheximide. N-terminal microsequencing of the 38-kDa protein revealed sequence identity with glyceraldehyde-3-phosphate dehydrogenase (GAPDH). A GAPDH antisense oligodeoxyribonucleotide blocks age-induced expression of the particulate 38-kDa protein and effectively inhibits neuronal apoptosis. In contrast, the corresponding sense oligonucleotide of GAPDH was completely ineffective in preventing the age-induced neuronal death and the 38-kDa protein overexpression. Moreover, the age-induced expression of the 38-kDa protein is preceded by a pronounced increase in the GAPDH mRNA level, which is abolished by actinomycin-D, cycloheximide, or the GAPDH antisense, but not sense, oligonucleotide. Thus, our results suggest that overexpression of GAPDH in the particulate fraction has a direct role in age-induced apoptosis of cerebellar neurons.     

Inhibition of Excitatory Amino Acid-Induced Phosphoinositide Hydrolysis as a Possible Mechanism of Nitroprusside Neurotoxicity

Abstract: Inclusion of sodium nitroprusside {Na₂[Fe²⁺-(CN)₅NO]} into the culture medium is toxic to cultured rat cerebellar granule neurons. A possible underlying mechanism may be the inhibition of phosphoinositide (PI) response to excitatory amino acids (EAAs) because activation of glutamate receptors can be neuroprotective and neurotrophic in differentiating neurons. Sodium nitroprusside selectively inhibited the PI response to EAAs (NMDA > glutamate = quisqualate > kainate) without affecting that to carbachol or KCI. In contrast, S-nitroso-N-acetylpenicillamine (SNAP), another nitric oxide (NO) donor, potentiated NMDA-induced PI hydrolysis. Hemoglobin reversed the effects of nitroprusside and SNAP. However, NO may not be involved because NO solution was without effect and N-acetylpenicillamine, a SNAP analogue that does not contain a NO moiety, also potentiated NMDA-induced PI hydrolysis in a hemoglobin-sensitive manner. Furthermore, the metabolites of NO (nitrate and nitrite), l -arginine, reduced glutathione, 8-bromo-cyclic guanosine 3′:5′-cyclic monophosphate (8-Br-cGMP), and atrial natriuretic peptide, which accelerates the production of cGMP independent of NO, were ineffective as modulators. However, potassium ferrocyanide {K₄[Fe²⁺(CN)₆]}, but not potassium ferricyanide {K₃[Fe³⁺(CN)₆]}, inhibited NMDA-induced PI hydrolysis as effectively as nitroprusside, but this inhibition was not reversed by hemoglobin. Cyanide, a product from the disintegration of nitroprusside, potentiated rather than inhibited NMDA-induced PI hydrolysis. Taken together, these results suggest that the parent molecule itself, nitroprusside, contributes primarily in inhibiting EAA-induced PI hydrolysis. Inhibition of EAA-induced PI hydrolysis may in part mediate the mechanisms of nitroprusside toxicity in primary cultures of differentiating cerebellar granule neurons.     

Two new loci for hybrid sterility in cultivated rice (Oryza sativa L.)

Female gamete abortion in Indica-Japonica crosses of rice was earlier identified to be due to an allelic interaction at the S-5 locus on chromosome 6. Recently, in other crosses of rice, similar allelic interactions were found at loci designated as S-7 and S-8, located on chromosomes 7 and 6 respectively. All of them are independent of each other. At the S-5 locus, Indica and Japonica rice have S-5 ⁱ and S-5 ^j alleles respectively and Javanicas, such as Ketan Nangka, have a neutral allele S-5 ⁿ .The S-5 ⁱ /S-5 ^j genotype is semi-sterile due to partial abortion of female gametes carrying S-5 ^j , but both the S-5 ⁿ /S-5 ⁱ and S-5 ⁿ /S-5 ^j genotypes are fertile. The S-5 ⁿ allele is thus a wide-compatibility gene (WCG), and parents homozygous for this allele are called wide-compatible varieties (WCV). Such parents when crossed with Indica or Japonica varieties do not show F₁ hybrid sterility. Wide-compatible parents have been used to overcome sterility barriers in crosses between Indica and Japonica rice. However, a Javanica variety, Ketan Nangka (WCV), showed typical hybrid sterility when crossed to the Indian varieties N22 and Jaya. Further, Dular, another WCV from India, showed typical hybrid sterility when crossed to an IRRI line, IR2061-628-1-6-4-3(IR2061-628). By genetic analyses using isozyme markers, a new locus causing hybrid sterility in crosses between Ketan Nangka and the Indicas was located near isozyme loci Est-1 and Mal-1 on chromosome 4, and was designated as S-9. Another new locus for hybrid sterility in the crosses between Dular and the IR2061-628 was identified and was found linked to four isozyme loci, Sdh-1, Pox-2, Acp-1 and Acp-2, on chromosome 12. It was designated as S-15. On the basis of allelic interactions causing female-gamete abortion, two alleles were found at S-9, S-9 ^kn in Ketan Nangka and S-9 ⁱ in N22 and Jaya. In the heterozygote, S-9 ^kn /S-9 ⁱ , which was semisterile, female gametes carrying S-9 ^kn were aborted. The hybrid of Dular and IR2061-628, with a genetic constitution of S-15 ^Du /S-15 ⁱ , was semi-sterile and the female gametes carrying S-15 ^Du were aborted. A Japonica tester variety, Akihikari, and an Indica variety, IR36, were found to have neutral alleles, S-9 nand S-15 n, at these loci, in addition to S-7 nand at S-7. The accumulation of three neutral alleles into a breeding line should help solve the hybrid sterility problem in wide crosses of rice.     

棉铃虫致病菌BT—931菌株的应用研究

１９９２－１９９３年,作者从菏泽、聊城棉田采集的自然罹病死亡棉铃虫幼虫体内,分离到２个较高毒效的Ｂｔ菌株,编号为ＢＴ—９３１和ＢＴ—０２１,经室内毒力测定和田间药效试验表明,防效及保蕾效果接近或超过化学农药,菌药混剂３—５天平均防治效果达８７．０－９１．６％,增效作用显著。经１９９４年大田防治示范表明,利用ＢＴ—９３１菌剂。配合化学农药防治棉铃虫,具有成本低、保护天敌、持效期长、增产效益高等优点。本研究对菌剂的田间应用技术进行了试验示范。     

Expression and secretion of human lipocortin-1 by promoter and signal sequence of STA1 from Saccharomyces diastaticus

Summary For the secretion of human lipocortin-1 (LC-1) in yeast, a expression and secretion vector was constructed by using the promoter and signal sequence of glucoamylase gene (STA1) of Saccharomyces diastaticus. After the cDNA of human LC-1 was ligated with the secretion vector, the resulting hybrid plasmid was transformed into S. diastaticus. When the recombinant S. diastaticus was cultivated in YPD medium, LC-1 was expressed and secreted into the extracellular medium, yielding LC-1 protein at a concentration of 2.5 g/mL.     

磁水对蕃茄酯酶同工酶的影响

蕃茄（ＬｙｃｏｐｅｒｓｉｃｏｍｅｓｃｕｌｅｎｔｕｍＭｉｌｌ）在各生长发育期酯酶同工酶谱带无明显变化,但经磁水处理后各生长发育阶段功能叶中酯酶同工酶酶谱及磷素含量与对照组相比存在显著差异,且具有酶谱负极端带数增加,正极端带数减少的趋势。总磷含量测定结果表明处理组均比对照组显著提高。这些变化在磁水处理著前后代中不存在,故认为磁水只是致使非编码顺序局部复制引起ＤＮＡ分子高级结构改变或是通过活化调节基因来调控酯酶同工酶,活化磷代谢,达到促进生长和增产的效果,而并非导致可遗传变异的结果。     

Interpretation of gas residence time distributions in large airlift tower loop reactors

Gas-residence time distribution (RTD) response curves measured in a 23 m high pilot plant airlift tower loop reactor, which consisted of a riser, a special downcomer construction and at the top of the riser a large head. The measurements were evaluated by means of a deterministic dispersion model, which yielded the following particular parameters for the riser, downcomer and the head: Gas-Bo numbers, gas-mean residence times, gas holdups, liquid velocities, gas and liquid circulation times as well as a fraction of the large and small bubbles in a model medium (water) and during cultivation of baker's yeast.List of Symbols A cross section - Bo Bodenstein number - Bo _d (= l _d w _G,d /D _d ) - Bo _h (= l _h w _G,h /D _h ) - Bo _r (= l _r w _G,r /D _r ) - D longitudinal dispersion coefficient - E gas holdup - E(t) RTD-density function - L, l length parameter - q fraction of the gas throughput which is not recirculated (approximately equal to fraction of the large bubbles) - r fraction of the throughput which is recirculated (approximately equal to the fraction of the small bubbles) - t _c circulation time - t _cL liquid circulation time - t _c,L ^* liquid circulation time calculated from the measured w _Ld in the downcomer - V ^h hydrodynamical calculated gas-liquid volume - V _d ^h (=V _d+0.75/2 V _k ) - V _k ^h =(0.25V _k ) - V _r ^h = (V _r+0.75/2 V _k ) - V _L liquid volume - V _G dispersed gas volume - V _G ^* gas throughput at the gas distributor (given in m³/h) under standard conditions, 1 bar and 25°C) - V _G,d ^* gas throughput in downcomer (=V _G ^* ) - V _G,h ^* gas throughput in head (=V _G ^* ) - V _G,r ^* gas throughput in riser (V _G ^* (1+) - w _g gas velocity - w _G,rel relative gas velocity with respect to the liquid velocity w _L - w _G,d gas velocity in the downcomer (=w _G,rel –w _Ld ) - w _G,h gas velocity in the head (=w _G,rel ) (since wLh = o) - w _G,r gas velocity in the riser (=w _G,rel +w _Lr ) - w _L liquid velocity - w _L,d liquid velocity in the downcomer measured with mass flow meter - w _sg ·w _SL superficial gas and liquid velocities - first moment of the response curve - mean residence time Indices d downcomer - G gas phase - h head - L liquid phase - r riser - h hydrodynamic (upper position) Dedicated to the 65th birthday of Proffessor Fritz Wagner.The authors gratefully acknowledge the financial support by the Krupp Industrietechnik, Grevenbroich and the support of Pleser Co, Darmstadt. H. M. Rüffer thanks the Verband der Chemischen Industrie for a Fond der Chemie scholarship, and W. Liwei thanks the government of Lower Saxony for a graduate scholarship.     

Differentiation of immortalized epithelial cells derived from cystic fibrosis airway submucosal glands

Summary Cystic fibrosis (CF) involves abnormalities in mucus production and secretion of the airway. Studies of the regulation of airway mucin production and secretion has been difficult due to the lack of in vitro models of the airway epithelial cells which express functional differentiation. Because the majority of the mucin in the airway is apparently produced by the submucosal glands, we have focused our attention on the development of cell culture models of human airway submucosal glands. This report describes the propagation of CF airway submucosal gland epithelial cells which continue to express mucin production. The CF bronchus was obtained from a 31-yr-old patient who received a double lung transplant. The glands were dissected out and primary cultures prepared by the explant/outgrowth procedure. The cells were immortalized by infection with Adl2-SV40 hybrid virus. The cultures are maintained in serum-free keratinocyte basal medium supplemented with insulin (5μg/ml), hydrocortisone (0.5μg/ml), epidermal growth factor (10 ng/ml), bovine pituitary extract (25μg/ml), and antibiotics. Cultures were passaged using 0.125% trypsin in Ca⁺² and Mg⁺²-free Hanks’, balanced salt solution. Polymerase chain reaction (PCR) analysis demonstrated that the cells were homozygous for the ΔF508 mutation. Morphologic observations showed that the cells were epithelial and were interconnected by sparsely distributed desmosomes. Their cytoplasm contained secretory-type structures including abundant Golgi, rough endoplasmic reticulum, and secretory vesicles. Immunofluorescent studies determined that all cells were positive for cytokeratins, mucin glycoconjugates, and cystic fibrosis transmembrane conductance regulator. The cultures secreted substantial amounts of mucin glycoproteins and expressed the MUC-2 mucin gene. Patch clamp experiments revealed that the cells expressed defective Cl⁻ channels which were not activated by Forskolin.     

Ar^＋,远红外激光,γ射线单一及复合处理水稻的诱变效果

用Ar~+、远红外两种激光和~(60)Co—r射线单一或复合处理两个籼稻品种的干种子,分析和比较了不同处理对水稻的当代生物学效应及处理二代的变异频率。结果表明,两种激光对当代的几个性状均表现为刺激效应,并有减轻,射线辐射损伤的作用,复合处理二代的变异频率和变异类型数明显高于相应的单一处理。说明复合处理是提高激光育种效果的有效方法。