Identification of an actin-like protein and of its messenger ribonucleic acid in Saccharomyces cerevisiae.

We have identified a yeast protein that resembles actins from other eucaryotes in its tight binding to pancreatic deoxyribonuclease I, its copolymerizaton with purified muscle actin, its one-dimensional peptide map, and its apparent polymerization into 7-nm filaments. The yeast actin-like protein yielded a single spot on two-dimensional polyacrylamide gel electrophoresis, suggesting that a single protein species was present. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the actin-like protein had an apparent molecular weight of 45,000 compared with 42,000 for muscle actin. In an attempt to identify the messenger ribonucleic acid coding for the actin-like protein, yeast polyadenylic acid-rich ribonucleic acid was translated in wheat germ and reticulocyte cell-free protein-synthesizing systems. The actin-like protein was identified among the translation products of the reticulocyte system by its tight binding to deoxyribonuclease I, its comigration with the in vivo-synthesized actin-like protein during sodium dodecyl sulfate-polyacrylamide gel electrophoresis, an the similarity of its peptide map to that of the in vivo-synthesized protein. A yeast protein synthesized in the wheat-germ system was also found to bind to deoxyribonuclease I and to copolymerize with muscle actin. However, its apparent molecular weight was about 35,000, suggesting that it was a product either of incomplete translation or of proteolytic cleavage of the actin-like protein.     

Stress-free state of the red blood cell membrane and the deformation of its skeleton

The response of a red blood cell (RBC) to deformation depends on its membrane, a composite of a lipid bilayer and a skeleton, which is a closed, twodimensional network of spectrin tetramers as its bonds. The deformation of the skeleton and its lateral redistribution are studied in terms of the RBC resting state for a fixed geometry of the RBC, partially aspirated into a micropipette. The geometry of the RBC skeleton in its initial state is taken to be either two concentric circles, a references biconcave shape or a sphere. It is assumed that in its initial state the skeleton is distributed laterally in a homogeneous manner with its bonds either unstressed, presenting its stress-free state, or prestressed. The lateral distribution was calculated using a variational calculation. It was assumed that the spectrin tetramer bonds exhibit a linear elasticity. The results showed a significant effect of the initial skeleton geometry on its lateral distribution in the deformed state. The proposed model is used to analyze the measurements of skeleton extension ratios by the method of applying two modes of RBC micropipette aspiration.     

One-Step Detection of Aflatoxin-B<Subscript>1</Subscript> Using scFv-Alkaline Phosphatase-Fusion Selected from Human Phage Display Antibody Library

A unique human phage display library was used to successfully generate a scFv to the highly carcinogenic toxin aflatoxin B1. Such an antibody has major potential applications in therapy and diagnostics. To further exploit its analytical capacity, the scFv was genetically fused to alkaline phosphatase, thereby generating a novel and highly sensitive self-indicating reagent. The performance of this reagent was further characterized, demonstrating its efficacy. The sensitivity of scFv-AP fusion was three-fold better than that of the scFv form. The ability of this human library to generate antibodies to a small hapten was clearly demonstrated and this is linked to its intrinsic diversity, which exceeds many existing conventional human libraries. Our results indicate that demography may influence the diversity of the repertoire of the library in terms of its capacity to generate antibodies to specific targets. Equally, the approach demonstrated should also be applicable for other haptens and larger antigens.     

Cloning and expression in Escherichia coli of an extremely thermostable oligo-1,6-glucosidase gene from Bacillus thermoglucosidasius.

The gene for an extremely thermostable oligo-1,6-glucosidase (dextrin-6-alpha-D-glucanohydrolase; EC 3.2.1.10) of obligately thermophilic Bacillus thermoglucosidasius KP1006 was cloned within a 4.2-kilobase HindIII-PvuII fragment of DNA by using the plasmid pUC19 as a vector and Escherichia coli C600 as a host. The gene was transcribed, presumably from its own promoter, in E. coli. E. coli with the hybrid plasmid accumulated oligo-1,6-glucosidase mainly in the cytoplasm. The level of enzyme production was comparable to that observed for B. thermoglucosidasius. The enzyme coincided absolutely with the B. thermoglucosidasius enzyme in its molecular weight (60,000), in its electrophoretic behavior on denaturing and nondenaturing polyacrylamide gels, in the temperature dependency of its stability and activity, and in its antigenic determinants.     

Isolation, purification and cell-free synthesis of calmodulin from the pig anterior pituitary gland.

Calmodulin was extracted and purified from pig anterior pituitary gland. The protein was characterized by its migration on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis in the presence of Ca2+ or EGTA, its U.V. spectrum between 240 and 290 nm and the activation of calmodulin-deficient cyclic AMP phosphodiesterase. The yield was 370 mg/kg wet wt. mRNA was also extracted from the same tissue and translated in a wheat-germ cell-free translation system. Translated calmodulin was identified by its heat-stability, its co-migration with authentic anterior-pituitary calmodulin on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, its acidic isoelectric point (4.15) on flat-bed isoelectric focusing, its Ca2+-dependent binding to fluphenazine-Sepharose 6B, and its co-elution from this gel with authentic unlabelled calmodulin with EGTA buffer. Calmodulin was not translated as a precursor form. In this tissue it was calculated that calmodulin accounted for 0.5-1% of the total translated protein.     

中国林蛙的分类及分布

目的中国林蛙(Rana chensinensis David,1875)是我国林蛙属分布最为广泛的特有种,在上一世纪对其分类问题多有争议,曾被认为广布于中国、蒙古、俄罗斯、日本和朝鲜半岛等。随着对其分类地位的确定,其分布范围也发生了很大变化。为了保护和可持续利用这一重要资源,本文对其分类研究历史和分布进行了总结。方法通过收集、整理和分析研究文献,结合本实验室多年来掌握的野外资料,进行系统的总结。结果中国林蛙自1875年由戴维(中文名为谭卫道)命名以来,曾被划归欧洲林蛙的亚种,并将多种其他林蛙归并入其下。1981年,经过细胞分类学研究,正式恢复为有效种,随后进一步划分出5个亚种。直至本世纪初,经过进一步的分类学研究,厘清了其与东北林蛙和西北林蛙的关系,从而确定其为仅分布于中国的特有种。在此基础上,对其分布也进行了重新确立。结论中国林蛙隶属于蛙科林蛙属中国林蛙种组,目前没有种下分化和亚种形成,分布于我国华北、华中及其周边地区,分布海拔不高于2500m。     

成年大鼠纹状体、边缘区和苍白球的计算机三维结构重建

应用计算机图形技术在大鼠脑的连续冠状切片Nissl染色的基础上通过Onyx2超级图形工作站对大鼠脑的纹状体进行了三维重建。结果提示：大鼠纹状体由尾壳核、苍白球和边缘区三部分组成,其中边缘区位于尾壳核和苍白球之间,被二完全包绕;尾壳核呈近似的内凹半球形,嘴尾径最大的为6.2mm;背腹径最大为4.9mm;宽度（冠状平面上的内外径)为3.5mm。从嘴侧到尾侧随着脑平面的增宽,尾壳核逐渐向外侧（即靠近外轮廓的方向）移位。苍白球呈块形,嘴尾径最大为4.4mm,背腹径最大为2.6mm,宽度（冠状平面上的内外径）最大为1.5mm。位于尾壳核的内侧,除内侧外基它三个方向均被尾壳核包绕。边缘区呈现一个片状扇形结构,嘴侧背腹径大,最大为2.2mm,宽约0.17mm;尾侧背腹径小,为0.8mm,宽约0.13mm。同属壳核和苍白球一样,从嘴侧到尾侧随着脑平面的增宽边缘区亦逐渐向外侧（即靠近外轮廓的方向）移位,其移位的幅度亦明显大于脑平面增宽的幅度;整个边缘区从嘴侧到尾侧呈均匀变化,其片状逐渐变宽,长度（背腹径）逐渐变小,从而形成一个盘状结构。     

New insights into an old protein: the functional diversity of mammalian glyceraldehyde-3-phosphate dehydrogenase.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was considered a classical glycolytic protein examined for its pivotal role in energy production. It was also used as a model protein for analysis of protein structure and enzyme mechanisms. The GAPDH gene was utilized as a prototype for studies of genetic organization, expression and regulation. However, recent evidence demonstrates that mammalian GAPDH displays a number of diverse activities unrelated to its glycolytic function. These include its role in membrane fusion, microtubule bundling, phosphotransferase activity, nuclear RNA export, DNA replication and DNA repair. These new activities may be related to the subcellular localization and oligomeric structure of GAPDH in vivo. Furthermore, other investigations suggest that GAPDH is involved in apoptosis, age-related neurodegenerative disease, prostate cancer and viral pathogenesis. Intriguingly, GAPDH is also a unique target of nitric oxide. This review discusses the functional diversity of GAPDH in relation to its protein structure. The mechanisms through which mammalian cells may utilize GAPDH amino acid sequences to provide these new functions and to determine its intracellular localization are considered. The interrelationship between new GAPDH activities and its role in cell pathologies is addressed.     

Low-cost noninvasive optical CO2 sensing system for fermentation and cell culture

High-throughput bioprocessing is a very promising technique for bioprocess development and optimization because of its high efficiency. The key to its development has been the availability of simple and inexpensive sensors to monitor the bioprocesses conducted in its small-scale bioreactors. Here we report on a low-cost noninvasive CO2 sensing system suitable for any transparent vessel. The system was composed of a CO2 sensing patch, a coaster, an interface, and a computer. The sensing film was prepared using the ion-pair technique. The coaster was a small self-made device with necessary optics and electronics for ratiometric measurements with a component cost of less than dollar 100. Results show that the system was stable and reliable despite its simplicity and low cost. The sensitivity of the CO2 sensing system was not affected by pH, media type, or temperature. It was shown to be stable for at least 10 days, long enough for most bioprocesses.     

Expression, purification, and characterization of a novel methyl parathion hydrolase

The mpd gene coding for a novel methyl parathion hydrolase (MPH) was previously reported and its putative open reading frame was also identified. To further confirm its coding region, the intact region encoding MPH was obtained by PCR and expressed in Escherichia coli as a hexa-His C-terminal fusion protein. The fusion protein was purified to homogeneity by metal-affinity chromatography. The enzyme activity and zymogram assay showed that the fusion protein was functional in degrading methyl parathion. The amino terminal sequencing of the purified recombinant MPH indicated that a signal peptide of the first 35 amino acids was cleaved from its precursor to form active MPH. A rat polyclonal antiserum was raised against the purified mature fusion protein. The results of Western blot and zymogram demonstrated that mature MPH in native Plesiomonas sp. strain M6 was also processed from its precursor by cleavage of a putative signal peptide at the amino terminus. The production of active MPH in E. coli was greatly improved after the coding region for the signal peptide was deleted. HPLC gel filtration of the purified mature recombinant MPH revealed that the MPH was a monomer.     

Occurrence and phenology of parasitic Chalcidoidea on the horse chestnut leafminer,Cameraria ohridella Deschka & Dimic (Lep., Gracillariidae)

Abstract: The parasitoid spectrum of the horse chestnut leafminer, Cameraria ohridella, was examined for its adaptation to a newly introduced host. A total of 15 parasitic species belonging to the suprafamilies Ichneumonoidea and Chalcidoidea was reared, all of them are polyphagous and common on other leafminers in Europe. The abundance of the moth and its natural enemies were studied during one entire vegetation period. The phenology of some of its major parasitic species was investigated and compared with the occurrence of the leafminer with special regard to its possible use in biological pest control.     

Eremomycin--a new antibiotic of the polycyclic glycopeptide group

Eremomycin is a novel antibacterial antibiotic. It was isolated at the Institute of New Antibiotics, the USSR Academy of Medical Sciences from the culture fluid of actinomycete INA-238. By its physico-chemical and biological properties the antibiotic was classified as belonging to the group of polycyclic glycopeptides. Chemical structure of eremomycin was asserted and it was shown to be a new representative of the group close by its structure to vancomycin and differing from it by the carbohydrate composition and structure of tri-phenoxytriaminotricarboxylic acid. By its anti-bacterial spectrum eremomycin was found to be close to ristomycin and vancomycin. Still, its activity was 2-10 times higher. The antibiotic was several times less toxic than vancomycin. Unlike vancomycin and ristomycin, the novel antibiotic induced no tissue necrosis after its intramuscular administration. The chemotherapeutic indices of eremomycin in treatment of staphylococcal and streptococcal sepsis in albino mice exceeded 10 times those of vancomycin. At present eremomycin is under clinical trials.     

Subcloning, nucleotide sequence, and expression of trkG, a gene that encodes an integral membrane protein involved in potassium uptake via the Trk system of Escherichia coli.

The trkG gene encodes a component of the K+ uptake system Trk and is located at 30.5 min inside the lambdoid prophage region rac of the Escherichia coli chromosome. trkG was subcloned, its nucleotide sequence was determined, and its product was identified in a minicell system. The open reading frame of 1,455 bp encodes a hydrophobic membrane protein with a calculated molecular weight of 53,493 that is predicted to contain up to 12 transmembrane helices. The trkG gene product behaved as a hydrophobic membrane protein; it was found exclusively in the membrane fraction of the minicells and its migration in sodium dodecyl sulfate-polyacrylamide gel electrophoresis was anomalous, indicating an apparent molecular weight of 35,000. The trkG gene contains an exceptionally high proportion of infrequently used codons, raising the question of the origin of this gene. trkG does not appear to be a prophage gene since no similarity was observed between the nucleotide sequence of trkG or the amino acid sequence of its product and the sequences of genes or proteins from bacteriophage lambda.     

Longitudinal distribution and ecophysiological characteristics of Hydropsyche exocellata (Trichoptera: Hydropsychidae) in large rivers

The position as a potamic or rhithronic species of Hydropsyche exocellata (Trichoptera Hydropsychmae) was analysed with regard to 1) its metabolism temperature curve and the total amplitude of its respiratory metabolism between 5° and 25°C, n) its distribution in some reaches of the Rhône River basin (France), and Hydropsychmae coexisting species in those reaches to assess its place in the stream continuum Metabolism data indicated clearly that H exocellata was a species adapted to the potamon, but distribution data did not confirm metabolism results The absence of H exocellata, for instance, from the lower and potamic reaches from the Rhône River itself, as well as its frequent coexistence with H pellucidula, a well-established near-rhithronic species regarding metabolism, suggests a more complex definition of its position as a potamic species     

Cyclic diguanylic acid and cellulose synthesis in Agrobacterium tumefaciens.

The occurrence of the novel regulatory nucleotide bis(3',5')-cyclic diguanylic acid (c-di-GMP) and its relation to cellulose biogenesis in the plant pathogen Agrobacterium tumefaciens was studied. c-di-GMP was detected in acid extracts of 32P-labeled cells grown in various media, and an enzyme responsible for its formation from GTP was found to be present in cell-free preparations. Cellulose synthesis in vivo was quantitatively assessed with [14C]glucose as a tracer. The organism produced cellulose during growth in the absence of plant cells, and this capacity was retained in resting cells. Synthesis of a cellulosic product from UDP-glucose in vitro with membrane preparations was markedly stimulated by c-di-GMP and its precursor GTP and was further enhanced by Ca2+. The calcium effect was attributed to inhibition of a c-di-GMP-degrading enzyme shown to be present in the cellulose synthase-containing membranes.     

Relation between interferon-inducing activity of natural interferon inducers and their chemical structure]

The interferon-inducing activity of new derivatives of gossypol, a low molecular weight natural polyphenol, and its model compounds was studied in regard to their chemical structure. The active groups required for development of optimal interferon inductors on the basis of the natural polyphenols were defined. As a result of the studies the new interferon inductor AC-1 which is a condensation product of gossypol, a natural polyphenol, and a cellulose derivative was isolated. The interferon-inducing activity of AC-1 was investigated with respect to its dose and the administration route. High interferon-inducing activity of the interferon inductor after its administration by any route in doses of 10 to 100 mg/kg was demonstrated. The serum interferon titers reached 2560 units/ml.     

外来植物西亚大蒜芥在云南出现并定居

报道了云南省一新记录的外来植物——西亚大蒜芥(Sisymbrium orientaleL.)。该物种在澳大利亚入侵农田、草场,在我国虽已有分布记录,但未见到相关预警性研究报道,目前为止尚未引起重视。西亚大蒜芥种子细小,易随风传播,具有形成大面积扩散的潜力。本次调查发现该物种在云南昆明有扩大分布的趋势。建议对该物种在我国的分布进行全面调查,查清其分布状况和入侵途径,并进行种群动态监测,适时清除,避免不必要的入侵发生。     

Characterization of a solvent resistant and thermostable aminopeptidase from the hyperthermophillic bacterium, Aquifex aeolicus

A leucine aminopeptidase gene of Aquifex aeolicus, a hyperthermophilic bacterium, was cloned and expressed in Escherichia coli, and its expression product was purified and characterized. The expressed protein was purified to homogeneity by using heat to denature contaminating proteins followed by ion-exchange chromatography to purify the heat-stable product. The purified enzyme gave a single band on SDS-PAGE with a molecular weight of 54 kDa. Kinetic studies on the purified enzyme confirmed that it was a leucine aminopeptidase. The optimum temperature for its activity was around 80 degrees C and the optimum pH was in the range from 8.0 to 8.5. It was stable at high temperatures and 27% of its activity was retained after heating at 115 degrees C for 30 min. The purified enzyme had a pH stability range between 4.0 and 11.0. This aminopeptidase was highly resistant to organic solvents such as methanol, ethanol, tetrahydrofuran, dimethyl sulfoxide, acetone, acetonitrile, dimethyl formamide, 1-propanol, 2-propanol, and dioxane.     

一株携带质粒的人两歧双歧杆菌的分离与鉴定

目的:分离携带天然质粒的人双歧杆菌.方法:用自制的改良型Blb双歧杆菌选择培养基,从人新鲜粪便分离双歧杆菌,对初步质粒检测阳性的单菌落通过糖发酵试验、(G C)mol%测定和16S rDNA序列分析,进行菌株鉴定.结果:筛选到一株携带天然质粒的人双歧杆菌,编号B200304,在1.0%琼脂糖凝胶上,测得质粒的相对分子质量约为22 kb.通过对该菌株的形态学观察和糖发酵试验等生理生化特征研究,证明该菌株为两歧双歧杆菌(Bifidobacterium bifidum);HPLC法测得其(G C)mol%为55.6,16SrDNA序列分析进一步证实该菌株为两歧双歧杆菌.结论:分离得到一株携带天然质粒的人两歧双歧杆菌新菌株.     

Structural properties and lipid binding of human apolipoprotein A-IV

The in vivo affinity of human apolipoprotein A-IV (apo-A-IV) for plasma lipoproteins is considerably less than that of other apolipoproteins. We have therefore studied its spectroscopic properties and its association with model chylomicrons to investigate its structural characteristics and to define their influence upon its affinity for lipids. Fluorescence emission spectra of apo-A-IV in dilute aqueous solution revealed that its single tryptophan residue resides in a pH-sensitive hydrophobic domain, which is maximally protected from iodide quenching at pH 7.5. Denaturation of apo-A-IV by guanidine hydrochloride caused a multiphasic fluorescence emission red shift, with an unusual enhancement of quantum yield. Circular dichroism spectroscopy of apo-A-IV demonstrated negative ellipticity maxima at 210 and 222 nm, consistent with 54% alpha-helical structure. The alpha-helicity of apo-A-IV as measured by [theta]222 was also pH-sensitive and displayed a distinctive decrease between pH 7.0 and 8.0. Apo-A-IV was exquisitely sensitive to denaturation by guanidine hydrochloride, and its estimated free energy of stabilization in aqueous solution was near zero. Apo-A-IV bound to the surface of Sf greater than 400 particles of a phospholipid-triglyceride emulsion in a noncooperative, concentration-dependent manner. The affinity of apo-A-IV for these model chylomicrons was influenced by changes in pH or addition of guanidine hydrochloride in a manner which correlated well with the structural changes observed under similar conditions. We conclude that human apolipoprotein A-IV possesses several biophysical properties characteristic of the better studied plasma apolipoproteins, yet, apo-A-IV appears to be marginally stable in aqueous solution and its structural characteristics and lipid binding properties are particularly sensitive to environment.