基于可见光测定的山羊支原体山羊肺炎亚种抗原定量

山羊支原体山羊肺炎亚种(Mycoplasma capricolum subsp. capripneumoniae, Mccp)是山羊传染性胸膜肺炎(contagious caprine pleuropneumonia, CCPP)的病原,可用灭活疫苗和荚膜多糖(capsular polysaccharide, CPS)间接血凝试剂进行预防和血清学检测,但高昂的培养成本和复杂的抗原定量一直困扰着生产人员。为解决生产实际中出现的这些问题,本研究基于Mccp代谢组学的前期理论基础,通过改变初始pH值的方法,初步筛选出初始pH值为7.8的可以同时提高2种抗原产量的糖发酵培养基。利用紫外可吸收光谱可识别酚红,以及十六烷基三甲基溴化铵(cetyltrimethylammonium bromide, CTAB)可与阴离子荚膜多糖结合的理论依据,建立了利用紫外光谱分析Mccp达到的培养阶段,以及利用CTAB沉淀法相对定量发酵液荚膜多糖抗原产量的方法。通过紫外图谱观察的方法可对应Mccp生长曲线进行指导生产,大大节省传统颜色变化单位(color change unit, CCU)法的监测时间,提高了原肉眼观察方法的精确度。建立的CTAB沉淀法可在5 h内完成对CPS含量的监测,与传统的差值法相比大大缩短了时间,并且其准确度得到苯酚-硫酸法的验证。本研究优化的一种培养基和建立的两种相关性比较方法,可有效降低Mccp生产成本,提高生产效率,这些方法已在本实验室的研究阶段得到应用,为进一步改进CCPP灭活疫苗和荚膜多糖的生产工艺以及快速定量提供了实验数据。     

The protein composition of Mycoplasma capricolum

Summary The whole cell proteins and the ribosomal proteins of Mycoplasma capricolum ATCC 27343 have been analyzed by two-dimensional polyacrylamide gel electrophoresis. The M. capricolum cell is relatively rich in basic proteins. The number of total protein spots detected was approximately 355, which is less than one-third of that of Escherichia coli or Bacillus subtilis. In contrast, the number (30 and 20 protein species have been found to be present in the 50S and 30S ribosomal subunits, respectively) and the size of the ribosomal proteins in the M. capricolum do not seem to be significantly different from those of typical eubacteria.     

Organization of ribosomal RNA genes in Mycoplasma capricolum

Summary DNA segments carrying rRNA genes of Mycoplasma capricolum have been cloned and characterized by restriction endonuclease mapping, DNA-RNA hybridization and nucleotide sequencing. The M. capricolum genome has two sets of rRNA gene clusters, where the arrangement is in the order of (5)16S-23S-5S(3). The spacer region between 16S and 23S rDNA is extremely rich in AT and does not carry any tRNA genes. Present address: Division of Hematology and Immunology of Internal Medicine, Kanazawa Medical University, Uchinada-Cho, Kahoku-Gun Ishikawa Pref. 920-02, Japan     

Evidence of a Restriction/Modification System in Lactobacillus delbrueckii subsp. lactis CNRZ 326

Lactobacillus delbrueckii subsp. lactis (Lb. lactis) CNRZ 326 is widely used in the propagation of Lb. delbrueckii bacteriophages. In this study, evidence is presented that this strain possesses a restriction-modification (R/M) system. The mitomycin C-induced temperate bacteriophage lb539 has a reduced efficiency of plaquing (EOP) on CNRZ 326 cells (EOP = 10⁻³), but after several passages on this strain, or on the indicator strain Lb. lactis LKT, the recovered phages (phages lb539.326 and lb539.LKT) have an EOP equal to 1. Restrictive development on CNRZ 326 was also observed after phage lb539.326 was propagated on the strain Lb. lactis CRL 934. The R/M system was also active against the virulent Lb. delbrueckii phage ll-h. Plasmid DNA was not detected in CNRZ 326, which suggests that the R/M system described is chromosomally encoded. Received: 11 September 1997 / Accepted: 21 October 1997     

Revision of genus Melamphaes (Melamphaidae). II. Multi-Raker species: M. polylepis, M. falsidicus sp. nova, M. pachystomus sp. nova, M. macrocephalus, M. leprus

The second part of the publication is devoted to the Melamphaes species (family Melamphaidae), which are characterized by 20 and more rakers on the first gill arch, by seven soft rays in the ventral fin, by absence of a temporal spine, by 14–15 rays in the pectoral fin, and by 11 abdominal vertebrae. M. polylepis is characterized by circumtropical range (Atlantic Ocean, Indian Ocean, western and central Pacific Ocean). Newly described species M. falsidicus is described from the northern Atlantic Ocean, where it was sampled between 34°N and 58°N. Before, this species was defined as M. microps. Another newly described species, M. pachystomus, is described along the Peruvian Coast. M. macrocephalus is redescribed. This species inhabits the eastern tropical Pacific Ocean (approximately between 30°N and 23°S). One of the studied specimens of M. macrocephalus was characterized by larger body size (SL = 128 mm) than was described before for this species. M. leprus is known currently by single findings from the eastern tropical Atlantic Ocean (between 11°N and 4°S). This species was also found in the samples obtain in the Gulf of Guinea.     

First report on the molecular prevalence of Mycoplasma capricolum subspecies capripneumoniae (Mccp) in goats the cause of contagious caprine pleuropneumonia (CCPP) in Balochistan province of Pakistan

Contagious caprine pleuropneumonia (CCPP) caused by Mycoplasma capricolum subspecies capripneumoniae (Mccp) is a disease of goats which causes high morbidity and mortality and is reported in many countries of the world. There are probably no reports on the molecular prevalence of Mccp, Mycoplasma capricolum subsp. capricolum (Mcc) and Mycoplasma putrefaciens (Mp) in Balochistan and any other part of Pakistan. Thirty goats (n = 30) with marked respiratory symptoms were selected and procured from forty goat flocks in Pishin district of Balochistan in 2008. The genomic deoxyribonucleic acid (DNA) from the lung samples (n = 30) of the slaughtered goats was purified and subjected to polymerase chain reaction (PCR) assays for the presence of Mycoplasma mycoides cluster members and Mp. The PCR-RFLP (restriction fragment length polymorphism) was also used to further confirm the Mccp. Of the thirty lung samples 17 (56.67%) were positive for the molecular prevalence of Mcc, Mccp and Mp. In total the molecular prevalence was observed as 17.65% for Mccp (n = 3), 70.59% for Mcc (n = 12) and 11.76% for Mp (n = 2). The RFLP profile has also validated the PCR results of Mccp by yielding two bands of 190 and 126 bp. The results of PCR-RFLP coupled with the presence of fibrinous pleuropneumonia and pleurisy during postmortem of goats (n = 3) strongly indicated the prevalence of CCPP in this part of world. Moreover the prevalence of Mcc and Mp is also alarming in the study area. We report for the very first time the molecular prevalence of Mcc, Mccp, and Mp in the lung tissues of goats in the Pishin district of Balochistan, Pakistan.     

Restriction enzyme-mediated DNA integration in Coprinus cinereus

Restriction enzyme-mediated DNA integration (REMI) has recently received attention as a new technique for the generation of mutants by transformation in fungi. Here we analyse this method in the basidiomycete Coprinus cinereus using the homologous pab1 gene as a selectable marker and the restriction enzymes BamHI, EcoRI and PstI. Addition of restriction enzymes to transformation mixtures results in an earlier appearance of transformants and influences transformation rates in an enzyme- and concentration-dependent manner. Low concentrations of restriction enzyme result in increased numbers of transformants compared to no addition of enzymes. Transformation rates decrease with higher enzyme concentrations. If protoplasts are made from cells stored in the cold, the transformation rates drop drastically even in the presence of low amounts of enzyme. In several transformants, plasmid integration directly correlated with the action of restriction enzyme at random chromosomal restriction sites. In some cases, restriction enzymes appear to reduce the number of integration events per transformant. Simultaneously, mutation rates can be enhanced due to the presence of restriction enzymes. Although restriction enzymes clearly promote plasmid integration into the host genome they also have cytotoxic and possibly mutagenic effects that result from processes other than plasmid integration. In consequence, for any given enzyme used in REMI mutagenesis, the enzyme concentration that gives the highest number of transformants must be defined experimentally. Such optimal transformation conditions should give the highest probability of obtaining mutations caused by a single restriction enzyme-mediated integration of the selection marker. Received: 2 September 1996 / Accepted: 7 April 1997     

Refined, Circular Restriction Map of the Bacillus thuringiensis subsp. israelensis Plasmid Carrying the Mosquito Larvicidal Genes

All the genetic elements responsible for the mosquito larval toxicity of Bacillus thuringiensis subsp. israelensis are located on one of its largest plasmids, nicknamed pBtoxis. Two linkage groups (with sizes of about 75 and 55 kb) have previously been mapped partially with respect to SacI and BamHI restriction sites (Ben-Dov et al., 1996), but linking them to a single circular plasmid unambiguously was impossible with the available data. To finalize the plasmid map, another rare cutting restriction endonuclease, AlwNI, was used in addition. The two linkage groups and the fragments generated by AlwNI were aligned on the circular plasmid, and known insertion sequences were localized on the refined map. Pulsed-field electrophoresis revealed that the total size of pBtoxis (137 kb) was larger than thought before.     

金钟藤分类考证及补充描述

旋花科植物金钟藤在中国华南地区已蔓延暴发成为"森林杀手",已引起各界的高度关注。该文对金钟藤的系统归隶、基名与异名、模式标本、分类文献,以及相关种进行考证和分析,澄清了一些误解和标本鉴定的错误,论证了金钟藤变种黄毛金钟藤在中国仅产于广西,而云南产的黄毛金钟藤实为铜钟藤,并把Merremia biosiana var.sumatrana van Ooststr.作黄毛金钟藤的新异名处理。     

Restriction and modification in B. subtilis

Summary The content of 5-methylcytosine (5MC) and 6-methyladenine (6MA) in modified and nonmodified DNAs from B. subtilis and B. subtilis phage SPP1 were determined. Nonmodified SPP1 · O DNA contains about 15 5MC residues/molecule. Each modified SPP1 ·R DNA molecule carries 190 modification specific methyl groups. This number is sufficient to account for modification of the 80 restriction sites in SPP1 DNA (Bron and Murray, 1975) against endo R · Bsu R, assuming each modified site contains two 5MC residues. Resistance of SP01 DNA against endo R · Bsu R restriction both in vivo and in vitro is probably not due to methylation of endo R·Bsu R recognition sites.     

Restriction and modification in B. subtilis

Summary The effects of restriction in vivo by competent B. subtilis R cells and in vitro by purified endonuclease BsuR on transformation and transfection with native and denatured DNA were investigated.The results show that transformation by either native, or denatured DNA is not affected by restriction, whereas transfection both with native and denatured SPP1 DNA is severely restricted.In contrast to the results obtained in vivo, the biological activity of native and denatured transforming DNA is destroyed by BsuR in vitro, as is the transfecting activity of native and denatured SPP1 DNA. The sensitivity of denatured DNA, either with mixtures of the complementary strands or with separated single strands¹ alone, is significantly lower than that of native DNA.The results are discussed in the context of possible mechanisms underlying the different responses of transforming and transfecting DNA to in vivo restriction by B. subtilis R cells.Abbreviations EGTA ethyleneglycolbis-(aminoethylether)tetraacetic acid - m⁺ modified - m^- non-modified - moi multiplicity of infection - r⁺ m⁺ restricting and modifying - r^- m^- mon-restricting and non-modifying - SSC 0.15M NaCl+0.015 M trisodium citrate - SDS sodium dodecyl sulphate     

Food plant specialization and feeding efficiency in the tent caterpillars Malacosoma disstria and M. americanum

Larvae of Malacosoma americanum (F.) an oligophagous species that feeds primarily on Prunus and other rosaceous trees, were compared to larvae of the more highly polyphagous congener M. disstria Hb., with respect to the efficiency of utilization of the foliage of a common host plant, Prunus serotina Ehrh. We obtained values similar to those reported for other Lepidoptera for the commonly used measures of the fraction of ingested food that was assimilated, and for the growth in dry weight per unit of food ingested or assimilated. Moreover, the two species did not differ in any measure of efficiency. Our results are compatible with the conclusion that specialized phytophagous insects do not use their host plants with greater physiological efficiency than do generalized insects.

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Zusammenfassung Raupen von Malacosoma americanum, einer oligophagen Art, die sich vor allem auf Prunus und andern baumartigen Rosaceen. entwickelt, wurden mit Raupen der polyphageren Verwandten M. disstria in Hinblick auf die Verwertung der Blätter ihres gemeinsames Wirtes Prunus serotina verglichen. Wirerhielten ähnliche Werte wie sie früher für andere Lepidopteren publiziert worden waren und zwar in Bezug auf die üblichen Messwerte, Anteil verwertete Nahrung, Wachstum in Trockengewicht pro Einheit gefressene oder verwertete Nahrung. Zudem unterschieden sich die beiden Arten in keiner Masszahl für Effizienz. Unsere Resultate sind im Einklang mit der Folgerung, das spezialisierte phytophage Insekten ihre Wirtspflanzen nicht besser ausnützen als as polyphage Arten tun.

     

Cardenolides from Asclepias asperula subsp. Capricornu and A. Viridis

6′-O-(E-4-hydroxycinnamoyl) Desglucouzarin, the first cardenolide containing a cinnamoyl ester moiety, has been isolated from the ethanolic extract of the milkweed, Asclepias asperula. In addition, five known cardenolides were isolated and identified from A. asperula and A. viridis.     

Morphological, chemical and genetic differentiation of two subspecies of Cistus creticus L. (C. creticus subsp. eriocephalus and C. creticus subsp. corsicus)

Cistus creticus L., an aromatic species from the Mediterranean area, contains various diterpenes bearing the labdane skeleton. The production of essential oil from this species has potential economic value, but so far, it has not been optimized. In order to contribute to a better knowledge of this species and to its differentiation, the morphological characters, volatile chemical composition and genetic data of two subspecies (C. creticus subsp. eriocephalus and C. creticus subsp. corsicus) were investigated. The leaf trichomes were studied using scanning electron microscopy. The chemical composition of Corsican essential oil (C. creticus subsp. corsicus) has been reported using GC, GC/MS and ¹³C NMR; the main constituents were oxygenated labdane diterpenes (33.9%) such as 13-epi-manoyl oxide (18.5%). Using plant material (54 samples) collected from 18 geographically distinct areas of the islands of Corsica and Sardinia, the basis of variation in the headspace solid-phase microextraction volatile fraction and an inter-simple sequence repeat genetic analysis were also examined. It was shown that the two subspecies of C. creticus differed in morphology, essential oil production, volatile fraction composition and genetic data.