Identification ofStreptomyces odor spectrum

The chemical composition of odors produced by nine strains ofStreptomyces was determined. StrainsStreptomyces aureofaciens, S. avermitilis, S. cinamomensis, S. coelicolor, S. griseus, S. lividans, S. rimosus, S. spectabilis, S. virginiae (as representatives of producers of biologically active compounds) were cultivated at the same time statically in dishes and in shaken flasks at similar cultivation conditions. According to the GC-MS analysis of odor compounds, more than twenty noteworthy volatile chemical individuals were identified. As the main component of odor spectrum geosmin and homologues of oxolones (dihydrofuranones) were found; the other compounds (pyrazine derivatives, acetoin and its homologues, aromatic esters, furan derivatives,etc.) were in minority. An erratum to this article is available at .     

Structural modifications and in vitro pharmacological evaluation of 4-pyridyl-piperazine derivatives as an active and selective histamine H3 receptor ligands

A novel series of 4-pyridylpiperazine derivatives with varying alkyl linker length and eastern part substituents proved to be potent histamine H₃ receptor (hH₃R) ligands in the nanomolar concentration range. While paying attention to their alkyl linker length, derivatives with a six methylene linker tend to be more potent than their five methylene homologues. Moreover, in the case of both phenoxyacetyl- and phenoxypropionyl- derivatives, an eight methylene linkers possess lower activity than their seven methylene homologues. However, in global analysis of collected data on the influence of alkyl linker length, a three methylene homologues appeared to be of highest hH₃R affinity among all described 4-pyridylpiperazine derivatives from our group up to date. In the case of biphenyl and benzophenone derivatives, compounds with para- substituted second aromatic ring were of higher affinity than their meta analogues. Interestingly, benzophenone derivative 18 showed the highest affinity among all tested compounds (hH₃R K_i = 3.12 nM). The likely protein-ligand interactions, responsible for their high affinity were demonstrated using molecular modeling techniques. Furthermore, selectivity, intrinsic activity at H₃R, as well as drug-like properties of selected ligands were evaluated using in vitro methods.     

Strepsirhine dichotomy from a human perspective (Primates: Lorisoidea, Lemuroidea)

Using monospecific, polyclonal antisera against 69 human plasma proteins, 128 antigenic determinants from 40 cross-reacting homologues were characterized in representatives of the prosimian genera Lemur, Eulemur, Varecia (Lemuridae) and Otolemur (Galagidae). Seventeen determinants from 16 different proteins were absent in homologues of the gagalo but were shared by lemurs and several platyrrhines, cercopithecids, and hominoids. Smaller locus samples for potto(Perodicticus potto) and slow loris (Nycticebus coucang) confirmed the more distant immunological relationship of lorises than of lemurs to anthropoids. If evolutionary rates are constant and equal in lorisiform and lemuriform prosimians, this patern of character distribution indicates strepsirhine paraphyly, lorises diverging earlier (possibly some 6 times 10⁶ years) than lemurs from anthropoid ancestors. If this is so, lorises rather than true lemurs should be elected to root the polarity of character evolution in Primates. As an alternative, galagine proteins evolve more rapidly than lemurine homologues.     

Analysis of non-peptidic compounds as potential malarial inhibitors against Plasmodial cysteine proteases via integrated virtual screening workflow

Falcipain-2 (FP-2) and falcipain-3 (FP-3), haemoglobin-degrading enzymes in Plasmodium falciparum, are validated drug targets for the development of effective inhibitors against malaria. However, no commercial drug-targeting falcipains has been developed despite their central role in the life cycle of the parasites. In this work, in silico approaches are used to identify key structural elements that control the binding and selectivity of a diverse set of non-peptidic compounds onto FP-2, FP-3 and homologues from other Plasmodium species as well as human cathepsins. Hotspot residues and the underlying non-covalent interactions, important for the binding of ligands, are identified by interaction fingerprint analysis between the proteases and 2-cyanopyridine derivatives (best hits). It is observed that the size and chemical type of substituent groups within 2-cyanopyridine derivatives determine the strength of protein–ligand interactions. This research presents novel results that can further be exploited in the structure-based molecular-guided design of more potent antimalarial drugs.     

Chemical semisynthesis of aprotinin homologues and derivatives mutated inp′ positions

An extended concept for the replacement of amino acids in theP' region of aprotinin by chemical semisynthesis is presented. Either fragment condensation with dipeptides protected as tert-butyl ester or stepwise introduction of two single amino acid-tert-butyl esters into a partially esterified aprotinin derivative (with free Lys¹⁵-carboxyl group) lacking the amino acids Ala¹⁶ and Arg¹⁷ leads to aprotinin homologues and derivatives mutated in theP ₁ andP ₂ position. This method may complement the recently reported enzymatic synthesis by enabling access to aprotinin homologues and derivatives, which cannot be prepared enzymatically. The synthesis of [Ala¹⁷]BPTI and [seco-17/18]BPTI is described in detail.     

冬虫夏草菌菌丝体中的氨基酸及其衍生物的代谢组学分析

为探究冬虫夏草菌（Ophiocordyceps sinensis）菌丝体氨基酸及其衍生物的差异性,采用超高效液相色谱串联质谱（Ultra performance liquid Chromatography, tandem mass spectrometry, UPLC-MS/MS）技术对3株冬虫夏草菌（玉树菌株XSH、达日菌株DR、贵德菌株LJ）菌丝体的氨基酸及其衍生物进行靶向定量检测,利用主成分分析（Principal component analysis, PCA）和正交偏最小二乘判别分析（Orthogonal signal correction and partial least squares-discriminant analysis, OPLS-DA）考察样品分类情况、筛选差异代谢物。结果表明,3株菌株菌丝体中均检测到72种氨基酸及其衍生物,发现除组氨酸外可合成蛋白质的19种氨基酸,菌株XSH的总氨基酸及必需氨基酸绝对含量均高于其他菌株。菌株XSH相比菌株DR和菌株LJ分别含有29种和28种差异氨基酸及其衍生物,菌株DR相比菌株LJ含有10种差异氨基酸及其衍生物,此外,3株菌株菌丝体含有3个共有的差异氨基酸及其衍生物,分别为高精氨酸、N-乙酰-L-谷氨酰胺、肌氨酸。京都基因与基因组百科全书（Kyoto encyclopedia of genes and genomes,KEGG）通路富集分析显示,差异氨基酸及其衍生物在精氨酸和脯氨酸代谢通路中更活跃,不同产区冬虫夏草菌株的氨基酸含量可能受精氨酸和脯氨酸代谢通路的影响。基于研究结果发现不同产区的冬虫夏草菌株的氨基酸及其衍生物存在较大的差异。     

The Resemblance of Bryozoan Gizzard Teeth to “Annelid-like” Setae

Abstract The gizzard teeth of a bryozoan (Bowerbankia imbricata) are found to be the structural homologues of certain epidermal derivatives, popularly known as “annelid-like” setae.     

AtHVA22 gene family in Arabidopsis: phylogenetic relationship,ABA and stress regulation,and tissue-specific expression

HVA22 is an ABA- and stress-inducible gene first isolated from barley (Hordeum vulgare L.). Homologues of HVA22 have been found in plants, animals, fungi and protozoa, but not in prokaryotes, suggesting that HVA22 plays a unique role in eukaryotes. Five HVA22 homologues, designated AtHVA22a, b, c, d and e, have been identified in Arabidopsis. These five AtHVA22 homologues can be separated into two subfamilies, with AtHVA22a, b and c grouped in one subfamily and AtHVA22d and e in the other. Phylogenetic analyses show that AtHVA22d and e are closer to barley HVA22 than to AtHVA22a, bandc, suggesting that the two subfamilies had diverged before the divergence of monocots and dicots. The distribution and size of exons of AtHVA22 homologues and barley HVA22 are similar, suggesting that these genes are descendents of a common ancestor. AtHVA22 homologues are differentially regulated by ABA, cold, dehydration and salt stresses. These four treatments enhance AtHVA22a, d and e expression, but have little or even suppressive effect on AtHVA22c expression. ABA and salt stress induce AtHVA22b expression, but cold stress suppresses ABA induction of this gene. Expression of AtHVA22d is the most tightly regulated by these four treatments among the five homologues. In general, AtHVA22 homologues are expressed at a higher level in flower buds and inflorescence stems than in rosette and cauline leaves. The expression level of these homologues in immature siliques is the lowest among all tissues analyzed. It is suggested that some of these AtHVA22 family members may play a role in stress tolerance, and others are involved in plant reproductive development.     

New hybrids between Agropyron and wheat

Summary Intergeneric hybrids of Triticum aestivum (2n=42,AABBDD) with Agropyron ciliare (2n= 28,SSYY), A. trachycaulum (2n=28,SSHH), A. yezoense (2n=28,SSYY) and A. scirpeum (2n=28) are reported for the first time. F₁ hybrids of T. aestivum were also produced with A. intermedium (2n=42,E₁E₁E₂E₂Z₁Z₁) and A. junceum (2n=14,JuJu). All wheat-Agropyron hybrids were obtained by embryo rescue technique. Cultivars and reciprocal crosses differed for seed set, seed development and F₁ plant production. The F₁ hybrids were sterile. Attempts to obtain amphiploids were unsuccessful. However, backcross derivatives were obtained with wheat as the recurrent parent.The level of chromosome pairing in A. trachycaulum x wheat, A. yezoense x wheat and wheat x A. junceum hybrids provided no evidence of homologous or homoeologous pairing. Mean pairing frequencies in A. ciliare x wheat, wheat x A. scirpeum and wheat x A. intermedium hybrids indicated homoeologous or autosyndetic pairing. Ph gene was more effective in regulating homoeologous pairing in A. yezoense x wheat hybrids than in A. ciliare x wheat hybrid. Chromosome pairing data of BC₁ derivatives indicated that either some of the wheat chromosomes were eliminated or Agropyron chromosomes caused reduced pairing of wheat homologues.Contribution No. 82-653-J, Department of Plant Pathology, Kansas State Agricultural Experiment Station, Manhattan, Kan, USA     

The cytokinin activities of 6-α-alkylbenzyloxypurines

The homologous series of 6-α-n-alkylbenzyloxypurines with alkyl groups (—(CH₂)_nCH₃) from n = 0 to n = 11 have been prepared and examined in three tests for cytokinin activity. The parent benzyloxypurine was also included in the tests. Substituting methyl (n = 0) into the methylene group of 6-benzyloxypurine removed activity almost completely; thereafter, increasing the size of the alkyl group (n = 1–5) gave compounds which were active in all tests, the butyl (n = 3) and pentyl (n = 4) derivatives being more active than 6-benzyloxypurine itself. Activity then fell as the series was ascended; the higher homologues (n = 8–11) being completely inactive. The results are discussed in relation to steric and other considerations.     

Antisense suppression of the putative ribosomal protein S3A gene disrupts ovarian development in Drosophila melanogaster

The Drosophila melanogaster homologue of the Anopheles gambiae C3 cDNA has been isolated and characterized by sequence analysis. The encoded protein was localized by immunochemical and immunocytochemical methods. The Drosophila C3 protein is highly similar to homologues of disputed function, which have previously been identified in fungi, plants and animals. The protein is ubiquitous and localized in the cytoplasm. Cell fractionation followed by detection with a specific antibody preparation shows that the protein is associated with the 40S ribosomal subunit. The C3 gene is located in section 101F of chromosome 4. Antisense transgenic analysis shows that this gene is essential for oogenesis. The most prominent phenotype resulting from antisense depletion of C3 RNA is disappearance of the follicular cells of the ovary (where the concentration of C3 protein is normally high) and abnormalities of the associated germline derivatives, leading to failure of egg production. Received: 13 January 1997 / Accepted: 13 June 1997     

The yeast gene MSH3 defines a new class of eukaryotic MutS homologues

We have identified a gene in Saccharomyces cerevisiae, MSH3, whose predicted protein product shares extensive sequence similarity with bacterial proteins involved in DNA mismatch repair as well as with the predicted protein product of the Rep-3 gene of mouse. MSH3 was obtained by performing a polymerase chain reaction on yeast genomic DNA using degenerate oligonucleotide primers designed to anneal with the most conserved regions of a gene that would be homologous to Rep-3 and Salmonella typhimurium mutS. MSH3 seems to play some role in DNA mismatch repair, inasmuch as its inactivation results in an increase in reversion rates of two different mutations and also causes an increase in postmeiotic segregation. However, the effect of MSH3 disruption on reversion rates and postmeiotic segregation appears to be much less than that of previously characterized yeast DNA mismatch repair genes. Alignment of the MSH3 sequence with all of the known MutS homologues suggests that its primary function may be different from the role of MutS in repair of replication errors. MSH3 appears to be more closely related to the mouse Rep-3 gene and other similar eukaryotic mutS homologues than to the yeast gene MSH2 and other mutS homologues that are involved in replication repair. We suggest that the primary function of MSH3 may be more closely related to one of the other known functions of mutS, such as its role in preventing recombination between non-identical sequences.     

High-frequency S-layer protein variation in Campylobacter fetus revealed by sapA mutagenesis

Campylobacter fetus utilizes paracrystalline surface (S-) layer proteins that confer complement resistance and that undergo antigenic variation to facilitate persistent mucosal colonization in ungulates. C. fetus possesses multiple homologues of sapA, each of which encode full-length S-layer proteins. Disruption of sapA by a gene targeting method (insertion of kanamycin (km) resistance) caused the loss of C. fetus cells bearing full-length S-layer proteins and their replacement by cells bearing a 50 kDa truncated protein that was not exported to the cell surface. After incubation of the mutants with serum, the survival rate was approximately 2 × 10^-2. Immunoblots of survivors showed that phenotypic reversion involving high-level production of full-length (98, 127 or 149 kDa) S-layer proteins had occurred. Revertants were serum resistant but caused approximately 10-fold less bacteraemia in orally challenged mice than did the wild-type strain. Southern hybridizations of the revertants showed rearrangement of sapA homologues and retention of the km marker. These results indicate that there exists high-frequency generation of C. fetus sapA antigenic variants, and that intracellular mechanisms acting at the level of DNA reciprocal recombination play key roles in this phenomenon.     

Expansion of the <Emphasis Type="Italic">Ago</Emphasis> gene family in the teleost clade

AGO proteins are universal effectors of eukaryotic small RNA-directed regulatory pathways. In this study, we used a comparative genomics approach to explore the AGO sub-family in the teleost clade. We identified five Ago homologues in teleost genomes, one more than encoded in other vertebrate clades. The additional teleost homologue was preserved most likely due to the differential retention of regulatory elements following the fish-specific genome duplication event that occurred approximately 350 million years ago. Analysis of all five Ago genomic loci in teleosts revealed that orthologues contain specific, conserved sequence elements in non-coding regions indicating that the teleost Ago paralogues are differentially regulated. This was supported by qRT-PCR analysis that showed differential expression of the zebrafish homologues across development and between adult tissues indicating stage and tissue-specific function of individual AGO proteins. Multiple sequence alignments showed not only that all teleost homologues possess critical residues for AGO function, but also that teleost homologues contain multiple orthologue-specific features, indicative of structural diversification. Notably, these are retained throughout the vertebrate lineage arguing these may be important for orthologue-specific functions.     

Phylogenetic analysis of archaeal PCNA homologues

Proliferating cell nuclear antigen (PCNA) is an essential component of the DNA replication and repair machinery in the domain Eucarya. Eukaryotes and euryarchaeotes, which belong to one subdomain of Archaea, possess a single PCNA homologue, whereas two distinct PCNA homologues have been identified from Sulfolobus solfataricus, which belongs to the other archaeal subdomain, Crenarchaeota. We have cloned and sequenced two genes of PCNA homologues from the thermoacidophilic crenarchaeon Sulfurisphaera ohwakuensis. These genes, referred to as the Soh PCNA A gene and the Soh PCNA B gene, were found to encode 245 amino acids (aa) (27 kDa) and 248 aa (27 kDa), respectively. In deduced amino acid sequences of both PCNA homologues, the motif L/I-A-P-K/R, implicated in binding of PCNA with replication factor C (RFC), was identified. Phylogenetic analysis of all available archaeal PCNA homologues suggests that crenarchaeal homologues are divided into two groups. Group A consists of Soh PCNA A, one of the S. solfataricus PCNA homologues, and one of the Aeropyrum pernix PCNA homologues. The other crenarchaeal homologues form group B. Crenarchaeal PCNA homologues constitute a monophyletic subfamily. These results suggest that the evolution of crenarchaeal PCNA homologues has been characterized by one or two gene duplication events, which are assumed to have occurred after the split of the crenarchaeal and euryarchaeal lineages. Received: July 10, 2000 / Accepted: September 26, 2000     

Microbiological production of tocopherols: current state and prospects

Tocopherols are antioxidants that prevent various diseases caused by oxidative stress. Tocochromanols comprise four isoforms of tocopherols and four isoforms of tocotrienols but α-tocopherol is the most abundant and active isoform in human and animal tissues. Tocopherols are used as dietary supplements for human, as food preservatives, in manufacture of cosmetics, and for fortification of animal feed. Only photosynthetic cells are known to accumulate detectable concentrations of tocopherols. Tocopherols can be extracted and purified or concentrated from vegetable oils and other higher plant materials. However, the concentrations in these higher plant materials are very low and there are high proportions of the less-active homologues of tocopherols. Among the many strains of photosynthetic microorganisms known to accumulate tocopherols, Euglena gracilis is promising for commercial production of α-tocopherol. The growth rate and α-tocopherol contents are relatively high and α-tocopherol comprise more than 97% of all the tocopherols accumulated by Euglena gracilis. Although a lot of work has been done to increase the contents and composition of tocopherols in higher plants through genetic and metabolic engineering, work on genetic modification of microorganisms for increased tocopherol accumulation is scarce. Many cultivation systems have been investigated for efficient production of tocopherol by Euglena gracilis. However, those that involve heterotrophic metabolism are more promising. Bubble columns and flat-plate photobioreactors are more suitable for commercial production of tocopherols, than the tubular, internally illuminated, and open-air photobioreactors.     

Pyocin-mediated antagonistic interactions in Pseudomonas spp. isolated in James Ross Island,Antarctica

Interactions within bacterial communities are frequently mediated by the production of antimicrobial agents. Despite the increasing interest in research of new antimicrobials, studies describing antagonistic interactions among cold-adapted microorganisms are still rare. Our study assessed the antimicrobial interactions of 36 Antarctic Pseudomonas spp. and described the genetic background of these interactions in selected strains. The overall bacteriocinogeny was greater compared to mesophilic Pseudomonas non-aeruginosa species. R-type tailocins were detected on transmission electron micrographs in 16 strains (44.4%); phylogenetic analysis of the corresponding gene clusters revealed that the P. prosekii CCM 8878 tailocin was related to the Rp3 group, whereas the tailocin in Pseudomonas sp. CCM 8880 to the Rp4 group. Soluble antimicrobials were produced by eight strains (22.-2%); gene mining found pyocin L homologues in the genomes of P. prosekii CCM 8881 and CCM 8879 and pyocin S9-like homologues in P. prosekii CCM 8881 and Pseudomonas sp. CCM 8880. Analysis of secretomes confirmed the production of all S- and L-type pyocin genes. Our results suggest that bacteriocin-based inhibition plays an important role in interactions among Antarctic soil bacteria, and these native, cold-adapted microorganisms could be a promising source of new antimicrobials.     

Allele mining in <Emphasis Type="Italic">Solanum</Emphasis>: conserved homologues of <Emphasis Type="Italic">Rpi-blb1</Emphasis> are identified in <Emphasis Type="Italic">Solanum stoloniferum</Emphasis>

Allele mining facilitates the discovery of novel resistance (R) genes that can be used in breeding programs and sheds light on the evolution of R genes. Here we focus on two R genes, Rpi-blb1 and Rpi-blb2, originally derived from Solanum bulbocastanum. The Rpi-blb1 gene is part of a cluster of four paralogues and is flanked by RGA1-blb and RGA3-blb. Highly conserved RGA1-blb homologues were discovered in all the tested tuber-bearing (TB) and non-tuber-bearing (NTB) Solanum species, suggesting RGA1-blb was present before the divergence of TB and NTB Solanum species. The frequency of the RGA3-blb gene was much lower. Interestingly, highly conserved Rpi-blb1 homologues were discovered not only in S. bulbocastanum but also in Solanum stoloniferum that is part of the series Longipedicellata. Resistance assays and genetic analyses in several F1 populations derived from the relevant late blight resistant parental genotypes harbouring the conserved Rpi-blb1 homologues, indicated the presence of four dominant R genes, designated as Rpi-sto1, Rpi-plt1, Rpi-pta1 and Rpi-pta2. Furthermore, Rpi-sto1 and Rpi-plt1 resided at the same position on chromosome VIII as Rpi-blb1 in S. bulbocastanum. Segregation data also indicated that an additional unknown late blight resistance gene was present in three populations. In contrast to Rpi-blb1, no homologues of Rpi-blb2 were detected in any material examined. Hypotheses are proposed to explain the presence of conserved Rpi-blb1 homologues in S. stoloniferum. The discovery of conserved homologues of Rpi-blb1 in EBN 2 tetraploid species offers the possibility to more easily transfer the late blight resistance genes to potato varieties by classical breeding.     

Non-LTR retrotransposons (LINEs) as ubiquitous components of plant genomes

During the course of work aimed at isolating a rice gene from Oryza australiensis by PCR, the oligonucleotide primers used were found to generate a fragment that showed sequence homology to the endonuclease (EN) region of the maize non-LTR retrotransposon (LINE) Cin4. We carried out further PCRs using oligonucleotide primers that hybridized to these sequences, and found that they amplified several fragments, each with homology to the EN regions, from Oryza sativa cv. Nipponbare as well as O. australiensis. We mapped the approximate locations of two rice LINE homologues by screening clones in a YAC library made from a rice (O. sativa) genome, and found that each homologue was present in a low copy number apparently at nonspecific regions on rice chromosomes. We then carried out PCR using degenerate oligonucleotide primers which hybridized to the rice LINE homologues and Cin4 to ascertain whether LINE homologues are present in a variety of members of the plant kingdom, including angiosperms, gymnosperms, bracken, horsetail and liverwort. Cloning and nucleotide sequencing revealed that 53 clones obtained from 27 out of 33 plant species contained LINE homologues. In addition to these homologues, we identified four homologues with EN regions in the Arabidopsis thaliana genome by a computer search of databases. The nucleotide sequences of almost all the LINE homologues were greatly diverged, but the derived amino acid sequences were well conserved, and all contained glutamic acid and tyrosine residues at almost the same relative positions as in the the active site regions of AP (apurinic/apyrimidinic)-endonucleases. The EN regions in the LINE homologues from closely related plant species show a closer phylogenetic relationship, indicating that sequence divergence during vertical transmission has been a major influence upon the evolution of plant LINEs. Received: 13 July 1998 / Accepted: 13 October 1998