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Expressed sequence tag analysis of Sarcoptes scabiei.

Sarcoptes scabiei is an important parasitic mite in both man and animals. Little is known about the molecular interactions between this pathogen and its host. This is in part explained by the paucity of mite-derived material, including antigens. To extend the knowledge of the molecular repertoire in S. scabiei, we have performed a gene survey by an expressed sequence tag (EST) analysis. A total of 1020 ESTs were generated from an S. scabiei cDNA library. The average sequence read was 510 bp after editing and the overall sequencing success was 89%. Clustering of the sequences resulted in 76 clusters, comprising 36% of the ESTs. Sequence similarity searches showed that almost half of the S. scabiei ESTs could be assigned a putative identity. Many of these transcripts shared similarity with genes involved in basic metabolism and cellular organization. In the data set we also identified several proteases and other types of potential allergens implicated in various disease mechanisms. A relatively large fraction of the ESTs coded for different proteins carrying protease inhibitor-like domains. The clones with no similarity to previously identified genes constituted 11% of our transcripts. The EST data generated in this study will be a valuable resource in further studies of the biology of S. scabiei and in the identification of genes that can serve as potential targets in the control of the parasite.

Animals↗

Differential receptors create patterns diagnostic for ATP and GTP.

Herein we report the combination of a library of resin-bound sensors along with a multicomponent sensor array. This novel combinatorial array sensor system shows selectivity for nucleotide phosphates in solution. The design of the anchored receptor includes a 1,3,5-trisubstituted-2,4,6-triethylbenzene scaffold coupled with peptide libraries. Each chemosensor is placed into a micromachined cavity within a silicon wafer, and the optical changes observed by a charged-coupled device result in near-real-time digital analysis of solutions. A colorimetric displacement assay was performed, and time-dependent imaging studies of the selected sensing ensembles result in a differential responses upon addition of adenosine 5'-triphosphate (ATP), adenosine 5'-monophosphate (AMP), or guanosine 5'-triphosphate (GTP). An advantage to this approach is that it creates an array of sensors that gives a fingerprint response for each analyte. Principal component analysis indicates that the library of chemosensors can differentiate between ATP, GTP, and AMP. On the basis of factor loading values, individual sensors from the library were sequenced to elucidate their chemical composition.

Adenosine Triphosphate↗

An integrated map of human chromosome 6p23.

The human chromosomal band 6p23 is a Giemsa-negative (light) band that may be expected to be relatively gene rich. The genes for spinocerebellar ataxia type 1 (SCA1), guanosine monophosphate reductase (GMPR), DEK involved in a subtype of acute myeloid leukemia (AML), and the folate-sensitive fragile site FRA6A, have already been mapped to 6p23. Recent linkage data have suggested evidence for a susceptibility locus for schizophrenia in the region. We have constructed a single YAC contig of approximately 100 clones spanning the entire 6p23 band from 6p22.3 to 6p24.1 and covering 7.5-8.5 Mb of DNA. The YAC contig contains 55 markers including genetically mapped STSs, physically mapped STSs, anonymous STSs, anonymous ESTs, and ESTs from the genes mapped to the region. The order of the genetically mapped STSs is consistent with their order in the contig and some of the markers not resolved on the genetic map have been resolved by the YACs. Four of the YACs from 6p23 and covering approximately 3 Mb of DNA have been used to isolate approximately 300 cosmids from a flow-sorted human chromosome 6 cosmid library, which have been organized into pockets. The proposed susceptibility locus for schizophrenia is most closely linked to D6S260, which is located within the YAC contig along with genetic markers < or = 5 cM on either side. Therefore, the presented materials are valuable reagents for characterization of the genomic region implicated in schizophrenia.

Base Sequence↗

Preparation of heterodifunctional polyethyleneglycols: network formation, characterization, and cell culture analysis.

Polyethyleneglycols (PEG) are employed extensively in the development of biomaterials; however, the hydroxyl groups in PEG-diols have very limited chemical activity. We developed a synthesis scheme for a library of heterodifunctional PEG (hPEG) with two distinct terminal moieties to improve the reactivity and physicochemical properties of PEG. hPEG were employed in the formulation of polymer networks with various surface physicochemical properties and utilized to study cell-material interaction. Extensive NMR and HPLC analyses confirmed the chemical structure of hPEG. The hydrophilicity of the polymer network was predominantly dependent on the hPEG concentration with the molecular weight and terminal functional group playing lesser roles. Adherent human fibroblast density on the network remained constant with increasing hPEG concentration in the network formulation but decreased rapidly on networks containing 0.8-1.25 g ml(-1) of hPEG. This trend was independent of the hPEG terminal moiety and molecular weight. No adherent cell was observed on all films containing 2.5 g ml(-1) or more of hPEG.

Biocompatible Materials↗

DNA display I. Sequence-encoded routing of DNA populations.

Recently reported technologies for DNA-directed organic synthesis and for DNA computing rely on routing DNA populations through complex networks. The reduction of these ideas to practice has been limited by a lack of practical experimental tools. Here we describe a modular design for DNA routing genes, and routing machinery made from oligonucleotides and commercially available chromatography resins. The routing machinery partitions nanomole quantities of DNA into physically distinct subpools based on sequence. Partitioning steps can be iterated indefinitely, with worst-case yields of 85% per step. These techniques facilitate DNA-programmed chemical synthesis, and thus enable a materials biology that could revolutionize drug discovery.

Anticodon↗

Editorial quality, diffusion and bibliometric indicators of the Revista Espaãnola de Enfermedades Digestivas.

AIM: To evaluate the editorial quality, diffusion, relevance of the scientific content, and the publication practices of the specialised journal Revista Española de Enfermedades Digestivas. MATERIALS AND METHODS: We checked 136 parameters based on ISO standards, the recommendations of scientific and editorial organisations, and studies of scientific editing and international publishing practices for biomedical journals. Diffusion was calculated using national and international databases, specialised libraries in Spain, and Internet sources. The analysis of the scientific content and publication practices was based on bibliometric indicators for the journal, authorship, and contributions. The sample for this study comprised six alternate issues of volume number 88 (1996), the last issue of this volume, and the first issue of volume 89 (1997). The samples used for the bibliometric analysis varied depending on the characteristics of specific indicators and the availability of information. RESULTS: The overall mean value for compliance with standards was 46.1%, while the real mean was calculated at 72.21%. The editorial procedures at the journal are similar to those of analogous international journals. The Revista Española de Enfermedades Digestivas is included in international databases of biomedical journals, and in the interdisciplinary international database SCI. It was found to be present in 70% of the medical libraries of Spanish universities, and in 73% of the hospital libraries studied. Bibliometric indicators showed co-authorship to be 5.5%; the origin of the authors grouped by province and by type of institutional affiliations showed 27.8% of all authors to be from Madrid, and that more were affiliated with general hospitals than with university hospitals. The mean delay between initial receipt of a manuscript and its publication was 300 days. Cocitation analysis gave the journal a central position amongst the 38 Spanish biomedical journals considered representative of the field. The journal's impact factor for 1996 was 0.260. CONCLUSIONS: The Revista Española de Enfermedades Digestivas is a high-quality vehicle of research results, and has acceptable internal editorial procedures. The journal is widely distributed, though its visibility on the Internet should be improved. Co-authorship is similar to that seen in other medical journals. Steps should be taken to make this journal better known within Spain, and to reduce the delay between the initial receipt and the final publication of manuscripts. Its impact factor is increasing steadily.

Authorship↗

The increasing globalization of health librarianship: a brief survey of international trends and activities.

BACKGROUND AND PURPOSE: Throughout his career, Leslie Morton was interested in international developments in health librarianship. In memory of the work he did in this field, the authors examine current developments in international health librarianship and describe some current themes. PROCEDURES: The authors draw from their combined experience in international activities and the published information available from selected library associations and related organizations. FINDINGS: Although many of the major health library associations around the world are tackling agendas specific to their own country, issues of international concern are emerging in common. These are grouped around globalization, partnerships and co-operation, electronic access, especially open access, and working with the developing world in a number of different ways. Of course, the basis of all of these initiatives is to improve the health of the population by providing the best possible access to materials. CONCLUSIONS: Professional associations can provide a useful institutional infrastructure for addressing issues of international interest. Librarians should encourage their associations to develop these international initiatives and to seek out new and innovative ways to work together across international boundaries.

Australia↗

Osler's changing influence. Based on the Osler oration given at the Royal College of Physicians in 1990.

Over 20 years in North America William Osler transformed the pattern of medical education for both undergraduates and specialists. He wrote a textbook that not only was a stimulus to medical students and young doctors, but also inspired the railway baron J D Rockefeller to devote his wealth to the advancement of medical research and education. He encouraged the creation of medical associations and societies because he believed, rightly, that these enhanced not only the quality of medicine but also the relationship of the members of the profession, one with another. He provided a great library of medical history and medical biography which has been and will be an inspiration for years to come. In the 15 years Osler spent in England he created a school of medical science which has continued to burgeon and has formed a foundation for a great clinical and research medical school. He inspired the creation of the post-graduate centres associated with the general hospitals in every town throughout the country; he provided material for a biography which has inspired medical students and practitioners, who now realise that medicine is primarily about sick people, not disease; and he directed another great benefactor, Lord Nuffield, to devote his riches to many aspects of medicine.

Education, Medical↗

High-throughput screening and quantitative structure-efficacy relationship models of potential displacer molecules for ion-exchange systems.

A technique is presented for the high-throughput screening of ion-exchange displacers. Potential displacers were employed to displace proteins in parallel batch ion-exchange experiments. The percentage of protein displaced from a particular stationary phase was then used as a parameter to rank the displacers. By employing this technique, a large number of molecules possessing a range of affinities and properties could be rapidly evaluated. This data was then used together with traditional and electron density-based transferable atom equivalent (TAE) molecular descriptors computed for the displacer molecules to produce quantitative structure-efficacy relationship (QSER) models using a genetic algorithm/partial least squares (GA/PLS) regression approach. The QSER models were generated using a portion of the protein-displacement data, with the remainder serving as a test set. Descriptor selection and model building was accomplished using a genetic algorithm/partial least squares approach. The resulting models were found to have high-correlation coefficients and could be used to accurately predict the behavior of molecules not included in the training set. In addition, the models were employed to examine a virtual library of displacers based on modifications of neomycin to provide further insight into displacer design. The results presented here indicate that it may be possible to design displacers that can dramatically improve the effective selectivity of ion-exchange chromatographic materials.

Algorithms↗

More arrows in the quiver: new pathways and old problems with heavy alkaline earth metal diphenylmethanides.

Progress in the field of sigma-bonded alkaline earth organometallics has been handicapped by numerous complications, such as high reactivity, low solubility, and the limited availability of suitable starting materials. Here we present two synthetic methods, hydrocarbon elimination and desilylation, as alternative routes into this chemistry. A novel barium diphenylmethanide was prepared using these routes delineating that both methods provide a powerful, versatile synthetic access route to an extended library of organometallic alkaline earth derivatives.

Journal Article↗

A constitutive nucleolar protein identified as a member of the nucleoplasmin family.

Using monoclonal antibodies we have localized a polypeptide, appearing on gel electrophoresis with a Mr of approximately 38,000 and a pI of approximately 5.6, to the granular component of the nucleoli of Xenopus laevis oocytes and a broad range of cells from various species. The protein (NO38) also occurs in certain distinct nucleoplasmic particles but is not detected in ribosomes and other cytoplasmic components. During mitosis NO38-containing material dissociates from the nucleolar organizer region and distributes over the chromosomal surfaces and the perichromosomal cytoplasm; in telophase it re-populates the forming nucleoli. With these antibodies we have isolated from a X. laevis ovary lambda gt11 expression library a cDNA clone encoding a polypeptide which, on one- and two-dimensional gel electrophoresis, co-migrates with authentic NO38. The amino acid sequence deduced from this clone defines a polypeptide of 299 amino acids of mol. wt 33,531 which is characterized by the presence of two domains exceptionally rich in aspartic and glutamic acid, one of them flanked by two putative karyophilic signal heptapeptides. Comparison with other protein sequences shows that NO38 is closely related to the histone-binding, karyophilic protein nucleoplasmin: the first 124 amino acids have 58 amino acid positions in common. Protein NO38 also shows striking homologies to the phosphopeptide region of rat nucleolar protein B23 and the carboxyterminal region of human B23. We propose that protein NO38, which forms distinct homo-oligomers of approximately 7S and Mr of approximately 230,000, is a member of a family of karyophilic proteins, the 'nucleoplasmin family'. It is characterized by its specific association with the nucleolus and might be involved in nuclear accumulation, nucleolar storage and pre-rRNA assembly of ribosomal proteins in a manner similar to that discussed for the role of nucleoplasmin in histone storage and chromatin assembly.

Amino Acid Sequence↗

Cloning and expression in Escherichia coli of a dog thyroid cDNA encoding a novel inositol 1,4,5-trisphosphate 5-phosphatase.

In brain and many other tissues, type I inositol 1,4,5-trisphosphate (InsP3) 5-phosphatase is the major isoenzyme hydrolysing the calcium-mobilizing second messenger InsP3. This protein has been purified to apparent homogeneity from a crude soluble fraction of bovine brain, yielding a single major protein band with a molecular mass of 43 kDa after SDS/PAGE. This material was used to determine internal microsequences. A partial DNA sequence has been amplified by PCR by using degenerate primers deduced from two protein sequences (FKAKKYKKV and DENYKSQE). A cDNA clone (BVCT) was isolated by screening a dog thyroid cDNA library. The encoded protein of 412 amino acids has a calculated molecular mass of 47,681 Da. Peptide sequences generated from the bovine brain enzyme were found to be 96% conserved compared with the dog thyroid protein. When clone BVCT was expressed in Escherichia coli, the recombinant protein was shown to hydrolyse both InsP3 and inositol 1,3,4,5-tetrakisphosphate, with apparent Km values of 28 and 3 microM respectively. Enzyme activity was inhibited by EDTA and 2,3-bisphosphoglycerate, both inhibitors of native InsP3 5-phosphatase, but not by EGTA and LiCl, as previously shown for the bovine brain enzyme. Our data show the cloning of type I InsP3 5-phosphatase which, interestingly, does not share any significant sequence identity with the previously cloned type III isoenzyme.

Amino Acid Sequence↗

Genes expressed in the latex of Hevea brasiliensis.

Rubber (cis-1,4-polyisoprene), an important raw material for many industrial uses, is synthesized in the latex of Hevea brasiliensis (Willd. ex A. Juss.) Müll. Arg. We postulated that the genes uniquely or preferentially expressed in the latex may be important for rubber biosynthesis. We constructed cDNA libraries from the latex of H. brasiliensis to investigate the genes expressed in the latex by single-run partial sequencing of the cDNA clones. Sequence analyses identified 245 expressed sequence tags (ESTs), of which 57% showed homology to previously described sequences in public databases. About 16% of the database-matched ESTs encode rubber biosynthesis-related proteins such as rubber elongation factor (REF) and small rubber particle protein (SRPP). The second most frequent transcripts next to rubber biosynthesis-related genes were defense genes and protein metabolism-related genes (12.6% each). About 27% of the database-matched ESTs had sequence homology with genes of unknown function. Among the redundantly expressed genes, REF was the most frequently expressed (6.1%), followed by SRPP (3.7%) and HbLAR (2.9%). Northern blot analyses showed that ten (71%) of the 14 ESTs studied were expressed at a higher level in latex than in leaves.

Journal Article↗

Expressed sequence tags from a root-hair-enriched medicago truncatula cDNA library

The root hair is a specialized cell type involved in water and nutrient uptake in plants. In legumes the root hair is also the primary site of recognition and infection by symbiotic nitrogen-fixing Rhizobium bacteria. We have studied the root hairs of Medicago truncatula, which is emerging as an increasingly important model legume for studies of symbiotic nodulation. However, only 27 genes from M. truncatula were represented in GenBank/EMBL as of October, 1997. We report here the construction of a root-hair-enriched cDNA library and single-pass sequencing of randomly selected clones. Expressed sequence tags (899 total, 603 of which have homology to known genes) were generated and made available on the Internet. We believe that the database and the associated DNA materials will provide a useful resource to the community of scientists studying the biology of roots, root tips, root hairs, and nodulation.

Journal Article↗

Axial dynamics, stability, and interspecies similarity of bacterial community structure in the highly compartmentalized gut of soil-feeding termites (Cubitermes spp.).

The highly compartmentalized gut of soil-feeding termites is characterized by pronounced axial dynamics in physicochemical conditions and microbial processes. In a companion paper (D. Schmitt-Wagner, M. W. Friedrich, B. Wagner, and A. Brune, Appl. Environ. Microbiol. 69:6007-6017, 2003), we demonstrated that the variety of physicochemical conditions in the different gut compartments of Cubitermes spp. is reflected in the diversity of the respective intestinal microbial communities. Here, we used molecular fingerprints of 16S rRNA genes of the bacterial community, obtained by terminal restriction fragment length polymorphism (T-RFLP) analysis, to describe the axial dynamics of the bacterial community structure in the different gut sections. Comparison of the T-RFLP profiles with the predicted terminal restriction fragments of the clones in clone libraries of the gut segments in Cubitermes orthognathus confirmed that all hindgut sections harbored distinct bacterial communities. Morisita indices of community similarity, calculated by comparing the different patterns, revealed large differences between the bacterial communities of soil, gut, and nest material and also among the individual gut sections. By contrast, comparison of the homologous gut segments of different Cubitermes species indicated that the three termite species investigated possessed a similar, gut-specific microbiota that remained comparatively stable even during several months of maintenance in the laboratory.

Animals↗

Immobilization of difunctional building blocks on hydroxysuccinimide activated silica: versatile in situ preparation of chiral stationary phases.

Several brush-type chiral stationary phases (CSPs) based on undecanoyl- or butanoyl-bound (R,R)-1,2-diphenylethane-1,2-diamine (DPEDA) as chiral selector were prepared by an innovative, fast, and less expensive kind of preparation. The key to this method is the immobilization of the enantiomeric pure diamine with only one amino function in a simple substitution reaction on hydroxysuccinimide ester-activated silica. No excess chiral material is lost. Loading can be easily monitored analyzing the filtrate. The free second amino function can subsequently be acylated with different acyl halogenides. Examples with benzoyl- and 3,5-dinitrobenzoyl (DNB) amides show that, based on our new approach, a library of differently acylated Pirkle-type CSPs can easily be obtained. A benzoylated analog of the commercially available ULMO CSP is shown to be very effective in separating enantiomers of N-acyl amino acids.

Journal Article↗

Use of circular permutation and end modification to position photoaffinity probes for analysis of RNA structure.

Photocrosslinking allows first-order structural analysis with relatively small amounts of biological material and can be applied in complex in vitro systems. In this article we describe methods for positioning both arylazide and thionucleotide photoagents within an RNA of interest by end modification of circularly permuted RNAs. Application of this technique provided a library of constraints that, together with biochemical and phylogenetic comparative data, were used to develop a structure model of the bacterial ribonuclease P ribozyme-substrate complex. Circularly permuted genes for in vitro transcription are generated by PCR from tandem genes. Circularly permuted RNA transcripts can be modified with high efficiency at both the 5' and 3' termini with arylazide crosslinking reagents, or transcription can be primed with photoactive nucleotide analog monophosphates such as 6-thioguanosine. These crosslinking agents can be used over a wide range of experimental conditions but remain inert until they are activated by UV light. Crosslinked sites are subsequently mapped by reverse transcriptase primer extension of gel-purified crosslinked species. In addition to providing basic protocols for these methods, we discuss approaches for establishing the relevance of crosslinking data to native RNA structure.

Aminophylline↗