P61 Protein from a Male Sterile Mutant of Rice is an Isoform of the Chloroplast ATPase β Subunit

P61 was a protein identified from chloroplasts of Nongken 58S, a male sterile mutant of rice (Oryza sativa L. ssp. japonica). Microsequence analysis has revealed that its N-terminal sequence was identical to N-termini of ATPase β subunits of chloroplasts from rice and barley. The antiserum produced using ATPase β subunit from maize specifically recognized P61. P61 had the same molecular weight as the chloroplast ATPase β subunit of wild-type rice “Nongken 58”, but had different isoelectric point (pI) from this β　subunit. P61 was more basic than this β subunit. Thus, P61 would be identified as an isoform of the chloroplast ATPase β subunit of rice, named β1. Genetic analysis with a F2 population of Nongken 58SדNongken 58” showed that a single recessive genic gene regulated the formation of β1.     

Crystallization of immunoglobulin Fab fragments specific for DNA

The purification and crystallization of Fab fragments of two mouse monoclonal immunoglobulins specific for different DNA structures are described. In each case, papain digestion of the immunoglobulins produced a mixture of Fab species differing in their isoelectric points. Purification of one of these species was required to obtain suitable crystals. One of these antibodies, Jel 72, is specific for right-handed duplex poly(dG).poly(dC). An Fab fragment of Jel 72 with a pI of 8.8 was purified by anion-exchange chromatography and used to obtain crystals from 56% saturated ammonium sulfate and 50 mM sodium acetate, pH 4.2, that diffract to 2.6-A resolution. They belong to the orthorhombic space group P2(1)2(1)2(1), with cell dimensions of a = 94.6, b = 102.6, c = 92.4 A. The other antibody, Jel 318, binds triple-stranded DNA poly[d(Tm5C)].poly[d(GA)].poly[d(m5C + T)]. Jel 318 Fab fragments with isoelectric points of 7.6 and 7.8 were also purified by anion-exchange chromatography, and crystals were obtained from 12% polyethylene glycol 8000, 50 mM NaCl, and 10 mM Tris.HCl, pH 7.8. These crystals diffract to about 2.4-A resolution and also belong to the orthorhombic space group P2(1)2(1)2(1), with cell dimensions of a = 82.4, b = 139.5, and c = 42.0 A. For both Fab fragments, crystal size and quality improved dramatically upon purification of an individual isoelectric species.     

On Basic Subunits Dissociated from C (11S) Component of Soybean Proteins with Urea

Galactolipase (galactolipid acyl hydrolase, EC 3.1.1.26) was purified 147-fold in good yield (91 %) from rice bran by affinity chromatography, in which the enzyme was adsorbed on a palmitoylated gauze column at pH 5.5 and then was eluted with a buffer solution containing a detergent such as sodium deoxycholate or Triton X–100 at pH 8.0. The preparation obtained was further purified by gel filtration on a Sephadex G–100 column and isoelectric focusing. After electrophoresis, the enzyme separated into four components with different isoelectric points. It seems that galactolipase in rice bran exists in multiple forms. The major component (G–2) with isoelectric point of 7.3, one of them, was purified 268-fold and electrophoretically homogeneous. The enzyme (G–2) hydrolyzed rapidly galactolipid and also slowly phospholipid, but hardly triglyceride.     

Purification and Some Properties of Debranching Enzyme of Germinating Rice Endosperm

A debranching enzyme was extracted from the endosperm of germinating rice seeds and purified through three steps, namely cyclohexaamylose-coupled Sepharose 6B, Ultrogel AcA-44 and Bio-Gel P-150 column chromatography. This disc-electrophoretically homogeneous enzyme showed a specific activity of 43 units/mg of protein (30°C) with a pH optimum of 5.5. The isoelectric point was 4.9, unlike that (pI 3.5) of debranching enzyme of ungerminated rice seeds. Our enzyme hydrolyzed pullulan rapidly, and glutinous rice starch and waxy corn starch moderately. The enzyme was also able to act on phytoglycogen and glycogen unlike debranching enzymes originating in some plants.     

黄淮平原鼬、鼠关系及鼠害防治对策

1984-1986年对黄淮平原农田鼠害的防治进行研究 先后在4省18县放置61822鼠铗日;在7660平方米农田用捕尽佳调查鼠密度,在8县43个样地的75.12平方公里面积河杳黄鼬密度,结果如下： 1．分析八十年代初鼠害大发生的原因。2． 黑线姬鼠胎平均为5．48只(n=343),在淮北平原和豫东南的繁殖指数为14．84和15．52;大仓鼠在上述地区的繁殖指数分别为15.98和32.03;黑线仓鼠在鲁西南的胎平均为6．26只(n=365),繁殖指数仅为8.89 。3．每平方公里害鼠密度高达7963只。4．黄鼬(害鼠天敌)的密度,夏季产仔期每平方公里为0．944±0．65头,初冬为2．68±0．93头。 在鼠类非繁殖胡,对降低鼠密度有一定作甩,应重视害鼠天敌(包括鸮, 家猫、豹猫、蛇类等)的综合作用 5．在黄淮平原,应研究在8周末至9月初进行灭鼠的措施和方法。     

Molecular cloning and characterization of an arginine decarboxylase gene up-regulated by chilling stress in rice seedlings

We cloned a rice cDNA encoding a putative arginine decarboxylase (ADC) protein, a key enzyme involved with putrescine (Put) biosynthesis in plants. The isolated full-length cDNA (OsADC1) contains an insert consisting of 2451 bp. The longest open reading frame within encodes a putative protein of 702 amino acids, with a calculated molecular mass of 74 kDa and an isoelectric point of 4.9. ClustalW alignment revealed that the deduced OsADC1 protein sequence shares overall 60% and 61% identity at the amino acid level with the Pisum sativum and Glycine max ADC proteins, respectively. Additionally, several OsADC1 regions exhibited striking similarity with these two other plant ADC protein sequences, including motifs characteristic of ADC proteins. Further, RNA gel blot analysis revealed markedly increased OsADC1 mRNA levels in rice seedling leaves subjected to chilling stress. Interestingly, this treatment induced a concomitant increase in free Put levels in these samples, coincident with the observed elevated OsADC1 mRNA levels. To our knowledge, this represents the first direct evidence supporting essentially chilling-specific regulation of a rice ADC gene that also potentially influences Put accumulation, a phenomenon previously noted in cold-stressed rice seedlings.     

樟脊网蝽生物学特性观察

樟脊网蝽是香樟的重要害虫,在上海1年发生4代。4月下旬越冬卵始孵,9月下旬开始出现越冬卵。成虫末见期在11月中旬。经室内饲养,樟脊网蝽第三代各龄若虫历期为：一龄61．45±7．8h,二龄48．00±7．6h,三龄43．43±7．6h,四龄51．00±8．0h,五龄70．50±7．5h,整个若虫期历期为281．92±14．8h。第三代野外雌雄比为1：4．07。第三代每雌产2～96粒。日最高产卵量为34粒／头,孤雌可产卵。10％灭百可2000倍稀释液和50％杀螟松2000倍稀释液喷雾防治效果可达93％以上。     

秦岭地区大林姬鼠的酯酶和苹果酸脱氢酶同工酶的研究

用聚丙烯酰胺凝胶等电聚焦电泳分析了大林姬鼠(Apodemus peninsulae)心、肝、脾、肾和腿肌的 α-酯酶,β-酯酶和苹果酸脱氢酶同工酶。结果表明3种同工酶的活性在5种器官组织中均有明显差异,其中以肝组织的酯酶活性最高,不同器官组织的酶谱也有明显差别,如脾的β一酯酶仅有B区带,同一器官组织通常以α-酯酶活性高于β-酯酶。苹果酸脱氢酶在碱性溶液中染色,肝组织有明显的AB医。心肌与腿肌的苹果酸脱氢酶活性略高于其他组织。     

Crystallization and preliminary x-ray diffraction studies of the Fab fragment of a neutralizing monoclonal antibody directed against human rhinovirus serotype 2

We report on the preparation, crystallization, and preliminary x-ray diffraction analysis of the Fab fragment of the monoclonal antibody 8F5 that neutralizes infectivity of human rhinovirus serotype 2 (HRV2). Fab fragments prepared from this antibody by papain digestion were purified to isoelectric homogeneity by ion exchange chromatography and chromatofocusing. Crystals were obtained by the hanging drop vapor diffusion method using ammonium sulfate as precipitant. The crystals belong to the orthorhombic space group P2(1)2(1)2(1) with unit cell dimensions a = 59.9 A, b = 86.3 A, c = 128.2 A and diffract to at least 2.8-A resolution. The cell volume suggests the presence of one molecule per asymmetric unit, and the solvent content is estimated to be 61%.     

Calmodulin isoform-specific activation of a rice calmodulin-binding kinase conferred by only three amino-acids of OsCaM61

The kinase activity of a Ca(2+)/calmodulin (CaM)-binding serine/threonine protein kinase from rice (Oryza sativa) (OsCBK) has been reported to be unaffected by OsCaM1 binding. In this study, we examined whether other rice CaMs can stimulate OsCBK. It was observed that OsCaM61 stimulated OsCBK in a Ca(2+)-dependent manner. In addition, Ala(111), Gly(123) and Ser(127) were identified as critical residues for OsCBK activation. Mutational study and fluorescent spectroscopy analysis indicated that CaM-binding affinity does not correlate with the kinase activity and that these key amino-acids in OsCaM61 play a vital role in suitable changes of OsCBK conformation for kinase activation.     

江豚脊髓的研究

本文研究了江豚脊髓的形态和内部构造,首次报道鲸类脊髓灰质分层和神经核的对应关系,并发现在胸段8—13节 、腰尾段1一6节等白质的侧索中有特殊细胞群,以多极或小三角形细胞为多,也有少数棱形细胞,呈串珠状排列或散在分布,分别与背角I- Ⅴ层相联系,它们显示出与感觉传导系有关,作者认为应分别称之为胸外侧核和腰尾外倒核。     

Rates of carbamylation of specific lysyl residues in bovine alpha-crystallins.

Previous investigations indicate that some forms of cataract may be due to the reactions of isocyanate with lens proteins. The present investigation was directed toward identifying the products of these reactions and determining rate constants for their formation. Bovine alpha-crystallins were incubated with isocyanate and separated into alpha A- and alpha B-crystallins by reversed-phase HPLC (high-performance liquid chromatography). Products of the reaction of isocyanate with alpha-crystallins were analyzed by mass spectrometry and isoelectric focusing. Proteolytic digests of carbamylated alpha A were analyzed by HPLC and fast atom bombardment mass spectrometry to determine the extent of reaction of each of the 7 lysyl residues present in alpha A. These results demonstrate that incubation of alpha-crystallins in 0.1 M KNCO leads to partial carbamylation of all 7 lysines of alpha A-crystallin. The extent of modification after 24 h of incubation varied from 7% at Lys 88 to 61% at Lys 11. Rate constants for the reaction of specific lysyl residues with isocyanate ranged from 5 to 54 x 10(-2) M-1 h-1. The distribution of reaction products, as determined by isoelectric focusing, indicates that the physiologically relevant initial stages of carbamylation of the 7 lysyl residues of alpha A proceed in a noncooperative manner.     

Towards an analysis of the rice mitochondrial proteome

Purified rice (Oryza sativa) mitochondrial proteins have been arrayed by isoelectric focusing/polyacrylamide gel electrophoresis (PAGE), by blue-native (BN) PAGE, and by reverse-phase high-performance liquid chromatography (LC) separation (LC-mass spectrometry [MS]). From these protein arrays, we have identified a range of rice mitochondrial proteins, including hydrophilic/hydrophobic proteins (grand average of hydropathicity = -1.27 to +0.84), highly basic and acid proteins (isoelectric point = 4.0-12.5), and proteins over a large molecular mass range (6.7-252 kD), using proteomic approaches. BN PAGE provided a detailed picture of electron transport chain protein complexes. A total of 232 protein spots from isoelectric focusing/PAGE and BN PAGE separations were excised, trypsin digested, and analyzed by tandem MS (MS/MS). Using this dataset, 149 of the protein spots (the products of 91 nonredundant genes) were identified by searching translated rice open reading frames from genomic sequence and six-frame translated rice expressed sequence tags. Sequence comparison allowed us to assign functions to a subset of 85 proteins, including many of the major function categories expected for this organelle. A further six spots were matched to rice sequences for which no specific function has yet been determined. Complete digestion of mitochondrial proteins with trypsin yielded a peptide mixture that was analyzed directly by reverse-phase LC via organic solvent elution from a C-18 column (LC-MS). These data yielded 170 MS/MS spectra that matched 72 sequence entries from open reading frame and expressed sequence tag databases. Forty-five of these were obtained using LC-MS alone, whereas 28 proteins were identified by both LC-MS and gel-based separations. In total, 136 nonredundant rice proteins were identified, including a new set of 23 proteins of unknown function located in plant mitochondria. We also report the first direct identification, to our knowledge, of PPR (pentatricopeptide repeat) proteins in the plant mitochondrial proteome. This dataset provides the first extensive picture, to our knowledge, of mitochondrial functions in a model monocot plant.     

A rice brassinosteroid-deficient mutant, ebisu dwarf (d2), is caused by a loss of function of a new member of cytochrome P450

We characterized a rice dwarf mutant, ebisu dwarf (d2). It showed the pleiotropic abnormal phenotype similar to that of the rice brassinosteroid (BR)-insensitive mutant, d61. The dwarf phenotype of d2 was rescued by exogenous brassinolide treatment. The accumulation profile of BR intermediates in the d2 mutants confirmed that these plants are deficient in late BR biosynthesis. We cloned the D2 gene by map-based cloning. The D2 gene encoded a novel cytochrome P450 classified in CYP90D that is highly similar to the reported BR synthesis enzymes. Introduction of the wild D2 gene into d2-1 rescued the abnormal phenotype of the mutants. In feeding experiments, 3-dehydro-6-deoxoteasterone, 3-dehydroteasterone, and brassinolide effectively caused the lamina joints of the d2 plants to bend, whereas more upstream compounds did not cause bending. Based on these results, we conclude that D2/CYP90D2 catalyzes the steps from 6-deoxoteasterone to 3-dehydro-6-deoxoteasterone and from teasterone to 3-dehydroteasterone in the late BR biosynthesis pathway.     

Comparison of the multiple molecular forms of bovine adrenocortical P450scc with those of corpus luteum P450scc.

1. Bovine adrenocortical P450scc was resolved into several fractions by chromatography on AH-Sepharose 4B followed by gel filtration on Toyopearl HW55S. All fractions contained P450scc of the same molecular size and the P450scc could be resolved into 3-4 major and more than 10 minor isoelectric point forms by isoelectric focusing on polyacrylamide gel in the presence of Emulgen 913. 2. Both the AH-Sepharose chromatography profile and the isoelectric focusing pattern of the adrenocortical P450scc were more complex than those of the corpus luteum P450scc. The corpus luteum P450scc was practically devoid of the neutral to acidic isoelectric point forms. 3. Three to four P450scc subfractions with different isoelectric focusing pattern were obtained from a purified preparation of adrenocortical P450scc by ion-exchange chromatography on DEAE-Toyopearl 650S or DEAE-Sephadex A25. These P450scc subfractions showed essentially the same spectral properties, catalytic activity, molecular weight and N-terminal amino acid sequence. 4. The most acidic (the latest eluting) subfraction was composed mostly of the neutral to acidic isoelectric point forms. The sedimentation characteristics of this subfraction was also studied. 5. The structural basis of the multiple molecular forms was discussed.     

重组人磷脂酶D2在体内能明显抑制慢性哮喘豚鼠血清中GPI-PLD的表达

 通过建立慢性豚鼠哮喘模型,研究重组人磷脂酶D2(rhPLD2)干预慢性哮喘豚鼠血清中糖基化磷脂酰肌醇特异性磷脂酶D(GPI-PLD)含量的变化,探寻可能的机制. 以rhPLD2干预慢性哮喘豚鼠,用TX-114分相法检测豚鼠血清中GPI-PLD酶活性.与正常状态豚鼠相比较,慢性哮喘状态豚鼠其血清中GPI-PLD酶活性显著升高.但以rhPLD2及地塞米松干预慢性哮喘豚鼠后,该指标显著下降. rhPLD2可通过抑制其血清中GPI-PLD酶活性表达,来抑制哮喘发生过程中的炎症反应.     

Morphological alteration caused by brassinosteroid insensitivity increases the biomass and grain production of rice

The rice (Oryza sativa) dwarf mutant d61 phenotype is caused by loss of function of a rice BRASSINOSTEROID INSENSITIVE1 ortholog, OsBRI1. We have identified nine d61 alleles, the weakest of which, d61-7, confers agronomically important traits such as semidwarf stature and erect leaves. Because erect-leaf habit is considered to increase light capture for photosynthesis, we compared the biomass and grain production of wild-type and d61-7 rice. The biomass of wild type was 38% higher than that of d61-7 at harvest under conventional planting density because of the dwarfism of d61-7. However, the biomass of d61-7 was 35% higher than that of wild type at high planting density. The grain yield of wild type reached a maximum at middensity, but the yield of d61-7 continued to increase with planting density. These results indicate that d61-7 produces biomass more effectively than wild type, and consequently more effectively assimilates the biomass in reproductive organ development at high planting density. However, the small grain size of d61-7 counters any increase in grain yield, leading to the same grain yield as that of wild type even at high density. We therefore produced transgenic rice with partial suppression of endogenous OsBRI1 expression to obtain the erect-leaf phenotype without grain changes. The estimated grain yield of these transformants was about 30% higher than that of wild type at high density. These results demonstrate the feasibility of generating erect-leaf plants by modifying the expression of the brassinosteroid receptor gene in transgenic rice plants.