Processing of multi-dimensional NMR data with the new software PROSA

Summary The new program PROSA is an efficient implementation of the common data-processing steps for multi-dimensional NMR spectra. PROSA performs linear prediction, digital filtering, Fourier transformation, automatic phase correction, and baseline correction. High efficiency is achieved by avoiding disk storage of intermediate data and by the absence of any graphics display, which enables calculation in the batch mode and facilitates porting PROSA on a variety of different computer systems; including supercomputers. Furthermore, all time-consuming routines are completely vectorized. The elimination of a graphics display was made possible by the use of a new, reliable automatic phase-correction routine. CPU times for complete processing of a typical heteronuclear three-dimensional NMR data set of a protein vary between less than 1 min on a NEC SX3 supercomputer and 40 min on a Sun-4 computer system.     

A DNA barcode library for Germany′s mayflies,stoneflies and caddisflies (Ephemeroptera,Plecoptera and Trichoptera)

Mayflies, stoneflies and caddisflies (Ephemeroptera, Plecoptera and Trichoptera) are prominent representatives of aquatic macroinvertebrates, commonly used as indicator organisms for water quality and ecosystem assessments. However, unambiguous morphological identification of EPT species, especially their immature life stages, is a challenging, yet fundamental task. A comprehensive DNA barcode library based upon taxonomically well‐curated specimens is needed to overcome the problematic identification. Once available, this library will support the implementation of fast, cost‐efficient and reliable DNA‐based identifications and assessments of ecological status. This study represents a major step towards a DNA barcode reference library as it covers for two‐thirds of Germany's EPT species including 2,613 individuals belonging to 363 identified species. As such, it provides coverage for 38 of 44 families (86%) and practically all major bioindicator species. DNA barcode compliant sequences (≥500 bp) were recovered from 98.74% of the analysed specimens. Whereas most species (325, i.e., 89.53%) were unambiguously assigned to a single Barcode Index Number (BIN) by its COI sequence, 38 species (18 Ephemeroptera, nine Plecoptera and 11 Trichoptera) were assigned to a total of 89 BINs. Most of these additional BINs formed nearest neighbour clusters, reflecting the discrimination of geographical subclades of a currently recognized species. BIN sharing was uncommon, involving only two species pairs of Ephemeroptera. Interestingly, both maximum pairwise and nearest neighbour distances were substantially higher for Ephemeroptera compared to Plecoptera and Trichoptera, possibly indicating older speciation events, stronger positive selection or faster rate of molecular evolution.     

Insights in metabolism and toxin production from the complete genome sequence of Clostridium tetani

The decryption of prokaryotic genome sequences progresses rapidly and provides the scientific community with an enormous amount of information. Clostridial genome sequencing projects have been finished only recently, starting with the genome of the solvent-producing Clostridium acetobutylicum in 2001. A lot of attention has been devoted to the genomes of pathogenic clostridia. In 2002, the genome sequence of C. perfringens, the causative agent of gas gangrene, has been released. Currently in the finishing stage and prior to publication are the genomes of the foodborne botulism-causing C. botulinum and of C. difficile, the causative agent of a wide spectrum of clinical manifestations such as antibiotic-associated diarrhea. Our team sequenced the genome of neuropathogenic C. tetani, a Gram-positive spore-forming bacterium predominantly found in the soil. In deep wound infections it occasionally causes spastic paralysis in humans and vertebrate animals, known as tetanus disease, by the secretion of potent neurotoxin, designated tetanus toxin. The toxin blocks the release of neurotransmitters from presynaptic membranes of interneurons of the spinal cord and the brainstem, thus preventing muscle relaxation. Fortunately, this disease is successfully controlled through immunization with tetanus toxoid, a formaldehyde-treated tetanus toxin, but nevertheless, an estimated 400,000 cases still occur each year, mainly of neonatal tetanus. The World Health Organization has stated that neonatal tetanus is the second leading cause of death from vaccine preventable diseases among children worldwide. This minireview focuses on an analysis of the genome sequence of C. tetani E88, a vaccine production strain, which is a toxigenic non-sporulating variant of strain Massachusetts. The genome consists of a 2,799,250 bp chromosome encoding 2618 open reading frames. The tetanus toxin is encoded on a 74,082 kb plasmid, containing 61 genes. Additional virulence-related factors as well as an insight into the metabolic strategy of C. tetani with regard to its pathogenic phenotype will be presented. The information from other clostridial genomes by means of comparative analysis will also be explored.     

Aggregate Clearance of α-Synuclein in Saccharomyces cerevisiae Depends More on Autophagosome and Vacuole Function Than on the Proteasome

Parkinson disease is the second most common neurodegenerative disease. The molecular hallmark is the accumulation of proteinaceous inclusions termed Lewy bodies containing misfolded and aggregated α-synuclein. The molecular mechanism of clearance of α-synuclein aggregates was addressed using the bakers' yeast Saccharomyces cerevisiae as the model. Overexpression of wild type α-synuclein or the genetic variant A53T integrated into one genomic locus resulted in a gene copy-dependent manner in cytoplasmic proteinaceous inclusions reminiscent of the pathogenesis of the disease. In contrast, overexpression of the genetic variant A30P resulted only in transient aggregation, whereas the designer mutant A30P/A36P/A76P neither caused aggregation nor impaired yeast growth. The α-synuclein accumulation can be cleared after promoter shut-off by a combination of autophagy and vacuolar protein degradation. Whereas the proteasomal inhibitor MG-132 did not significantly inhibit aggregate clearance, treatment with phenylmethylsulfonyl fluoride, an inhibitor of vacuolar proteases, resulted in significant reduction in clearance. Consistently, a cim3-1 yeast mutant restricted in the 19 S proteasome regulatory subunit was unaffected in clearance, whereas an Δatg1 yeast mutant deficient in autophagy showed a delayed aggregate clearance response. A cim3-1Δatg1 double mutant was still able to clear aggregates, suggesting additional cellular mechanisms for α-synuclein clearance. Our data provide insight into the mechanisms yeast cells use for clearing different species of α-synuclein and demonstrate a higher contribution of the autophagy/vacuole than the proteasome system. This contributes to the understanding of how cells can cope with toxic and/or aggregated proteins and may ultimately enable the development of novel strategies for therapeutic intervention.     

A novel type of replicative enzyme harbouring ATPase,primase and DNA polymerase activity

Although DNA replication is a process common in all domains of life, primase and replicative DNA polymerase appear to have evolved independently in the bacterial domain versus the archaeal/eukaryal branch of life. Here, we report on a new type of replication protein that constitutes the first member of the DNA polymerase family E. The protein ORF904, encoded by the plasmid pRN1 from the thermoacidophile archaeon Sulfolobus islandicus, is a highly compact multifunctional enzyme with ATPase, primase and DNA polymerase activity. Recombinant purified ORF904 hydrolyses ATP in a DNA-dependent manner. Deoxynucleotides are preferentially used for the synthesis of primers approximately 8 nucleotides long. The DNA polymerase activity of ORF904 synthesizes replication products of up to several thousand nucleotides in length. The primase and DNA polymerase activity are located in the N-terminal half of the protein, which does not show homology to any known DNA polymerase or primase. ORF904 constitutes a new type of replication enzyme, which could have evolved independently from the eubacterial and archaeal/eukaryal proteins of DNA replication.     

狭义小奥德蘑属（膨瑚菌科,蘑菇目）的一个新系统

本文对狭义小奥德蘑属Oudemansiella s. str.的概念做了修订,在修订后的属中,狭义干蘑属Xerula s. str.的物种不纳入其中。在狭义小奥德蘑属下,提出了一个包含4个组O. sect. Oudemansiella、Mucidula、Dactylosporina 和 Radicatae的新系统。小奥德蘑组sect. Oudemansiella包括热带至南温带的一些物种,如新热带小奥德蘑O. platensis、澳洲小奥德蘑O. australis、旧热带小奥德蘑O. canarii和宽褶小奥德蘑O. crassifolia,这些物种的菌盖表皮为粘栅栏型,由菌丝组成,但其中常夹杂有链状排列的膨大细胞。粘蘑组sect. Mucidula包含北半球温带至亚热带的一些物种,如粘小奥德蘑O. mucida、网褶小奥德蘑O. venosolamellata和近粘小奥德蘑O. submucida,其菌盖表皮为粘子实层-栅栏型,由近棒状的顶端膨大细胞组成。小奥德蘑组和粘蘑组的物种,在外形和小生境上有相似之处,其担子果皆生于地表外的腐木上,菌柄上有或无菌环。刺孢组sect. Dactylosporina包含中南美洲那些孢子表面有指状凸起的物种。长根组sect. Radicatae由长根小奥德蘑O. radicata及其近缘种为代表,是该属中最大的组,包括该属其他三组之外的所有种。北美的O. americana、欧洲的O. caussei 和东亚的O. hongoi曾被置于小奥德蘑属中的白毛组O. sect. Albotomentosae或干蘑属的亮毛组X. sect. Hyalosetae,在本系统中它们没有纳入小奥德蘑属,因为它们可能代表一个单独的属。本文还提出了1新等级、32个新组合和1个新名称。     

人脊髓创伤后神经元病变的神经丝免疫组织化学研究

本文用神经丝（ＮＦ）免疫组织化学方法在１５例人体尸检材料中研究了脊髓创伤后生存２ｈ～９Ｗ的脊髓神经元胞体和轴突的病理学改变。结果表明：脊髓创伤后２ｈ,神经丝免疫组织化学反应即可显示ＮＦ阳性反应产物在轴突内聚集。创伤后第４天,病变的前角运动神经元胞体内神经丝反应异常地增强。以上结果表明：神经丝免疫组织化学方法比常规显示轴突的染色方法能更早更清晰地显示脊髓内轴突的病变,并进一步证实了创伤后细胞骨架紊乱在神经元的病理发病机理中起重要作用。     

An extraction method for nematodes in decomposition studies using the minicontainer-method

The minicontainer-method is a new method developed to study biological processes related to soil litter decomposition. An adaptation of the classical Baermann-funnel technique is described which can be used, in association with the minicontainer method, to investigate the role of Nematoda in litter decomposition. The use of the extraction method is illustrated in a study of the effects of different tillage systems on the decomposition of rye straw and on the nematode density in minicontainers with different mesh sizes of 20 µm, 500 µm and 2 mm. Three tilled plots (conventional deep plough, cultivator and two-layer plough) and an untilled control were compared after periods of 4 weeks and 38 weeks. On both sample dates there were significant main effects of treatment and mesh size on the nematode density, and additionally, after 38 weeks significant treatment x soil depth interactions. After 4 weeks, there were significant main effects of treatment and soil depth on the decomposition, but no mesh size effects, whereas after 38 weeks, all experimental factors had a significant effect on the decomposition of the straw. Due to the small volume of litter substrate used in the minicontainer method, the efficiency of nematode extraction is high and the lack of oxygen in the minicontainers presents no serious problem during the extraction process. The method also allows the simultaneous extraction of a large number of samples within a short period of time. Our results indicate that the method is suitable to study the microdistribution of nematode activity within the soil profile and improves the possible applications of the minicontainer-method.     

Ein bisher unbekanntes Neurohämalorgan von Craspedosoma rawlinsii leach (Diplopoda,Nematophora)

Zusammenfassung Das Neurohämalorgan von Craspedosoma rawlinsii ist paarig und von kugeliger Form. Es liegt lateral von der Antenuenbasis der Gena innen an. Ventral sitzt es einer Speicheldrüse auf. Die übrige Oberfläche grenzt an einen Hämolymphsinus. Es wird von zwei Nerven gebildet, deren Aufzweigungen bier blind endigen. Es ist rings von Perineurium umhüllt dessen Zellen sich häufig überlappen. Der eine Nerv des Neurohälorgans besteht aus Axonen neurosekretorischer Zellen des Protocerebrallobus, der andere aus Axonen neurosekretorischer Zellen des Tritocerebrum. Gliazellen sind nicht vorhanden. Alle Axone enthalten Neurosekret. Es können 4 Typen neurosekretorischer Elementargranula unterschieden werden. Das Neurosekret scheint an Stellen ausgeschüttet zu werden, an denen die Axone vom Hölymphsinus nur durch die Basalmembran des Perineurium getrennt sind. Die Abgabe erfolgt wahrscheinlich durch Exocytose. Synaptoide Vesikel werden nirgends gefunden.

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A new neurohaemal organ in Craspedosoma rawlinsii leach (Diplopoda, Nematophora)

A neurohaemal organ of the head in millipeds is described. Its ultrastructure is compared with those of related organs especially in insects.

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Herrn Prof. Dr. Manfred Gersch zu seinem 62. Geburtstag gewidmet.