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PubMed1996

Transforming growth factor-beta inhibits interferon-gamma-induced HLA-DR expression by cultured human fibroblasts.

This study shows the induction of HLA-DR (DR) in fibroblasts by IFN-gamma and investigates the molecular mechanisms involved in the further DR down-regulation by TGF-beta 1. Kinetics of DR induction on human dermal fibroblasts by IFN-gamma showed that 1 hr of exposure was required to induce detectable levels of DR, and maximal DR expression was achieved only after 2 days of exposure to IFN-gamma. TGF-beta 1 inhibited DR induction by IFN-gamma, although complete inhibition never could be achieved, even with high concentrations of TGF-beta 1 and low concentrations of IFN-gamma. Inhibition was not accounted for by reduction in cell numbers, as TGF-beta 1 stimulated growth of the fibroblasts. Inhibition of DR induction was seen only if TGF-beta 1 was added during the first 24 hr of IFN-gamma treatment. TGF-beta 1 inhibited equally well if the cells were pretreated for as little as 1 hr and then washed before addition of IFN-gamma. TGF-beta 1 did not cause an overall suppression of protein synthesis. Northern blot analysis revealed that TGF-beta 1 greatly reduced the steady-state level of DR beta mRNA induced by IFN-gamma at 24 hr, and then DRP transcripts became undetectable at later stages. It is concluded that early intracellular signals must build up to stimulate maximum DR synthesis, which, later on, are inactivated or degraded by the action of TGF-beta 1. We suggest that these mechanisms regulating DR gene transcription involve the action of genes coding for specific IFN-gamma-inducible transcriptional factors that are turned on and off in an expeditious manner.

Cells, Cultured↗
PubMed2006

Conserved small non-coding RNAs that belong to the sigmaE regulon: role in down-regulation of outer membrane proteins.

Enteric bacteria respond to misfolded proteins by activating the transcription of "heat shock" genes. These genes are arranged in two major regulons controlled by the alternative sigma factors sigmaH and sigmaE. The two transcription factors coordinate the stress response in different cellular compartments; the sigmaH regulon is induced by stress in the cytoplasm whereas the sigmaE regulon is activated by stress signals in the cell envelope. In Escherichia coli sigmaE plays a central role in maintaining cell envelope integrity both under stress conditions and during normal growth. Previous work established that sigmaE is essential for viability of the bacterium and up-regulates expression of approximately 100 protein-encoding genes that influences nearly every aspect of the cell envelope. Moreover, the expression of several outer membrane proteins is down-regulated upon sigmaE activation. Here, we show that two Hfq-binding small RNAs, MicA and RybB, are under positive control of sigmaE. Transient induction of RybB resulted in decreased levels of the mRNAs encoding OmpC and OmpW. sigmaE -mediated regulation of ompC and ompW expression was abolished in strains lacking RybB or Hfq. Recently MicA was shown to act in destabilizing the ompA transcript when rapidly grown cells entered the stationary phase of growth. Also, the alternative sigma factor down-regulates this message in a small non-coding RNA-dependent fashion. These findings add the sigmaE regulon to the growing list of stress induced regulatory circuits that include small regulatory RNAs and provide insight in a homeostatic loop that prevent a build-up of unassembled outer membrane proteins in the envelope.

Bacterial Outer Membrane Proteins↗
PubMed1997

A cassette for high-level expression in the mouse salivary glands.

Expression in the mouse salivary glands may be used as a model system for studies involving oral cavity delivery of gene products. Previously, sequences from the mouse Psp gene were used to build a minigene construct denoted 'Lama'. This construct was used as a cassette for expression of human factor VIII light chain in mouse saliva. However, whereas the endogenous Psp mRNA is the most abundant protein-coding transcript in the parotid glands, the Lama mRNA was expressed below 1% of the level of Psp mRNA in these glands. Here, we show that a 25-kb cosmid-derived DNA fragment (PspX25) carrying the structural gene and large flanking areas of Psp is expressed in all 14 analysed lines in the parotid glands. The average level of transgene expression was estimated to be 45% of that of the endogenous Psp gene. More importantly, it was possible to transfer PspX25's ability for high-level parotid gland expression to the Lama construct.

Animals↗
PubMed2000

BodyMap: a collection of 3' ESTs for analysis of human gene expression information.

BodyMap is a collection of site-directed 3' expressed sequence tags (ESTs) (gene signatures, GSs) that contains the transcript compositions of various human tissues and was the first systematic effort to acquire gene expression data. For the construction of BodyMap, cDNA libraries were made, preserving abundance information and histologic resolutions of tissue mRNAs. By sequencing 164,000 randomly selected clones, 88,587 GSs that represent chromosomally coded transcripts have been collected from 51 human organs and tissues. They were clustered into 18,722 independent 3' termini from transcripts, and more than 3000 of these were not found among ESTs assembled in UniGene (Build 75). Assessment of the prevalence of polyadenylation signals and comparison with GenBank cDNAs indicated that there was no significant contamination by internally primed cDNAs or genomic fragments but that there was a relatively high incidence (12%) of alternative polyadenylation sites. We evaluated the sensitivity and resolution of expression information in BodyMap by in silico Northern hybridization and selection of tissue-specific gene probes. BodyMap is a unique resource for estimation of the absolute abundance of transcripts and selection of gene probes for efficient hybridization-based gene expression profiling.

3' Untranslated Regions↗
PubMed2004

Genome wide in silico SNP-tumor association analysis.

BACKGROUND: Carcinogenesis occurs, at least in part, due to the accumulation of mutations in critical genes that control the mechanisms of cell proliferation, differentiation and death. Publicly accessible databases contain millions of expressed sequence tag (EST) and single nucleotide polymorphism (SNP) records, which have the potential to assist in the identification of SNPs overrepresented in tumor tissue. METHODS: An in silico SNP-tumor association study was performed utilizing tissue library and SNP information available in NCBI's dbEST (release 092002) and dbSNP (build 106). RESULTS: A total of 4865 SNPs were identified which were present at higher allele frequencies in tumor compared to normal tissues. A subset of 327 (6.7%) SNPs induce amino acid changes to the protein coding sequences. This approach identified several SNPs which have been previously associated with carcinogenesis, as well as a number of SNPs that now warrant further investigation CONCLUSIONS: This novel in silico approach can assist in prioritization of genes and SNPs in the effort to elucidate the genetic mechanisms underlying the development of cancer.

Databases, Genetic↗
PubMed1989

[Good Manufacturing Practice (GMP) in the food industry].

The GMP-Rules, which were formulated by the WHO in 1969, were passed into German national lay by April 1st, 1985 as "Betriebsverordnung für pharmazeutische Unternehmer". The aim of this law is to guarantee constant quality of medical supplies. The food producing industry is facing similar requirements formulated either within the Codex Alimentarius or within the Code of Hygienic Practice of FDA (General Requirements for Food Manufacture). The degree of realization for these concepts of quality assurance however is varying extremely between different areas of food production. The requirements with regard to buildings, production plants, process technology, distribution and personnel hygiene as well as the principles of the HAAC-concept are described using examples from soft cheese, milk powder- and sweets industry.

Food-Processing Industry↗
PubMed1982

Regulation of phosphoenolpyruvate carboxykinase (GTP) synthesis in rat liver cells. Rapid induction of specific mRNA by glucagon or cyclic AMP and permissive effect of dexamethasone.

Isolated rat liver cells maintained in suspension culture for 4 to 5 h synthesize the gluconeogenic cytosolic enzyme phosphoenolpyruvate carboxykinase at a rate approximately 5-fold lower than the in vivo hepatic rate. Glucagon rapidly re-induces phosphoenolpyruvate carboxykinase synthesis in such cells. The rate of enzyme synthesis doubles in 40 min and plateaus at a level 6- to 13-fold higher than in control cells 120 min after glucagon addition at maximal concentration. Consistent with the presumed role of cyclic AMP as a mediator of enzyme induction, the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine, added simultaneously with glucagon, shifts the hormone dose-response curve 2 log units to the left. Moreover, cyclic AMP supplied exogenously to the cells mimics the inductive effect of glucagon. Total cellular RNA isolated from hepatocytes induced by glucagon contains an increased level of mRNA coding for phosphoenolpyruvate carboxykinase, as determined by translational assay. The kinetics and extent of the rise in mRNA level are adequate to explain the stimulation of enzyme synthesis. Although glucagon on its own induces a build-up of phosphoenolpyruvate carboxykinase mRNA and a commensurate stimulation of enzyme synthesis, the glucagon induction is very markedly amplified when the cells are first preincubated with dexamethasone. The glucocorticoid by itself, however, does not have any substantial effect on the level of phosphoenolpyruvate carboxykinase mRNA or on the rate of enzyme synthesis. Its role can therefore be characterized as permissive.

Animals↗
PubMedDate not supplied

When cost-saving strategies are unacceptable.

With health care redesign, nurses in every setting are challenged to provide more cost-effective care. While quality models for health care have been developed, nurses are at times asked to implement unsafe practices in the name of cost reduction or profit building. Both legal and ethical dilemmas arise when an emphasis on economic considerations supersedes patient well-being. Nurses must recognize their primary duty as patient advocates, and work to modify unsafe plans. The ANA's Code for Nurses and Guidelines for Reporting Incompetent, Unethical or Illegal Practices, and the Nursing Practice Act are primary resources in this effort.

Codes of Ethics↗
PubMed2001

Development of a new mobility scale for people living in the community after stroke: content validity.

Mobility inside and outside the home, and in the community, is important to independence of lifestyle after stroke. Existing measures lack content validity relating to these three environments. The aim of this study was to contribute to the content validity phase of developing a new scale for assessing mobility of people with stroke in these settings. An open-ended questionnaire was used to obtain responses from 15 physiotherapists with substantial experience in neurological physiotherapy. Responses were coded to provide a comprehensive list of tasks and destinations. A wide range of destinations was identified for i) inside the home: bedroom, bathroom/toilet, living room, kitchen; ii) outside the home: access to and from property, outside buildings, clothes line, garden and letterbox; iii) in the community: access to health and shopping facilities, leisure sites and public transport. Although a diverse range of tasks was identified, a common theme emerged to emphasise the impact of the environment on mobility. Responses were recorded in all dimensions of a recently published conceptual model of mobility that focused on environment. These dimensions included distance, time constraints, ambient conditions, terrain characteristics, external physical load, attentional demands, postural transitions and traffic level. Further work is required to quantify environmental dimensions which are relevant to the destinations identified in the three environmental settings of this study.

Activities of Daily Living↗
PubMed2003

Conducting international research in midwifery: a workshop held at the ICM Congress, Vienna, April 2002.

A workshop on international research in midwifery was held at the triennial International Confederation of Midwives (ICM) Congress in Vienna, April 2002. Thirty-five participants from 12 countries took part. The participants themselves defined the agenda, and subsequent discussion addressed the following issues: international research relationships and collaboration; ethical conduct in international research in midwifery; the role of the ICM in international research; and identifying topics for an international midwifery research agenda. Recommendations arising from this workshop were: that guidelines and a code of ethics for the conduct of international research in midwifery be developed; that the ICM and national midwifery organisations continue to actively support research, and further develop that support; that the ICM support education and capacity building for research at basic and continuing education levels; and that the priorities identified for collaborative international studies be updated on a regular basis.

Education↗
PubMed1998

Comparison of EGS4 and MCNP Monte Carlo codes when calculating radiotherapy depth doses.

The Monte Carlo codes EGS4 and MCNP have been compared when calculating radiotherapy depth doses in water. The aims of the work were to study (i) the differences between calculated depth doses in water for a range of monoenergetic photon energies and (ii) the relative efficiency of the two codes for different electron transport energy cut-offs. The depth doses from the two codes agree with each other within the statistical uncertainties of the calculations (1-2%). The relative depth doses also agree with data tabulated in the British Journal of Radiology Supplement 25. A discrepancy in the dose build-up region may by attributed to the different electron transport algorithims used by EGS4 and MCNP. This discrepancy is considerably reduced when the improved electron transport routines are used in the latest (4B) version of MCNP. Timing calculations show that EGS4 is at least 50% faster than MCNP for the geometries used in the simulations.

Calibration↗
PubMed2001

MedlineQBE (Query-by-Example).

Medline has the potential to significantly improve medical care but effective information retrieval remains difficult. Custom interfaces and relevance feedback are two approaches that have been successfully used to improve information retrieval. There are, however, many ways to implement these approaches. A system that facilitates rapid implementation and evaluation of novel algorithms has the potential to speed research progress. This paper describes MedlineQBE, a research workbench for implementing and evaluating information retrieval strategies. User interface, database access and display of results are abstracted leaving developers with the task of coding only the algorithm of interest. We implemented several custom interfaces, search-refinement strategies and a result-ordering algorithm using MedlineQBE. Preliminary evaluations of an oncology-patient interface and a relevance feedback algorithm that builds upon PubMed's "related articles" feature are promising. We conclude that custom interfaces and novel relevance feedback strategies have the potential to improve information retrieval from Medline.

Algorithms↗
PubMed2002

Integral algorithm and density matrix integration scheme for ab initio band structure calculations on polymeric systems.

A new program for band structure calculations of periodic one-dimensional systems has been constructed. It is distinguishable from other codes by the efficient two-electron integral evaluation and the integration schemes of the density matrix in the first Brillouin zone. The computation of polymeric two-electron integrals is based on the McMurchie Davidson algorithm and builds batches of the different cell indices included in the polymeric system. Consequently it presents efficient scaling with respect to the number of unit cells taken into account. Our algorithm takes into account fully the polymeric symmetry rather than the molecular symmetry. A semidirect procedure where only exchange integrals are computed at each SCF cycle is proposed in order to maintain balance between computation time and disk space. In addition, the integration of the density matrix over a large number of cell indices can be performed by different methods, such as Gauss-Legendre, Clenshaw-Curtis, Filon, and Alaylioglu-Evans-Hyslop. This last scheme is able to obtain an accuracy of 10(-13) a.u. on each individual density matrix element for all cell indices with only 48 k-points.

Journal Article↗
PubMed2004

SITEBLAST--rapid and sensitive local alignment of genomic sequences employing motif anchors.

MOTIVATION: Comparative sequence analysis is the essence of many approaches to genome annotation. Heuristic alignment algorithms utilize similar seed pairs to anchor an alignment. Some applications of local alignment algorithms (e.g. phylogenetic footprinting) would benefit from including prior knowledge (e.g. binding site motifs) in the alignment building process. RESULTS: We introduce predefined sequence patterns as anchor points into a heuristic local alignment strategy. We extended the BLASTZ program for this purpose. A set of seed patterns is either given as consensus sequences in IUPAC code or position-weight-matrices. Phylogenetic footprinting of promoter regions is one of many potential applications for the SITEBLAST software. AVAILABILITY: The source code is freely available to the academic community from http://corg.molgen.mpg.de/software

Algorithms↗
PubMed2003

RevTrans: Multiple alignment of coding DNA from aligned amino acid sequences.

The simple fact that proteins are built from 20 amino acids while DNA only contains four different bases, means that the 'signal-to-noise ratio' in protein sequence alignments is much better than in alignments of DNA. Besides this information-theoretical advantage, protein alignments also benefit from the information that is implicit in empirical substitution matrices such as BLOSUM-62. Taken together with the generally higher rate of synonymous mutations over non-synonymous ones, this means that the phylogenetic signal disappears much more rapidly from DNA sequences than from the encoded proteins. It is therefore preferable to align coding DNA at the amino acid level and it is for this purpose we have constructed the program RevTrans. RevTrans constructs a multiple DNA alignment by: (i) translating the DNA; (ii) aligning the resulting peptide sequences; and (iii) building a multiple DNA alignment by 'reverse translation' of the aligned protein sequences. In the resulting DNA alignment, gaps occur in groups of three corresponding to entire codons, and analogous codon positions are therefore always lined up. These features are useful when constructing multiple DNA alignments for phylogenetic analysis. RevTrans also accepts user-provided protein alignments for greater control of the alignment process. The RevTrans web server is freely available at http://www.cbs.dtu.dk/services/RevTrans/.

Amino Acid Substitution↗
PubMed2024

Taking advantage of reference-guided assembly in a slowly-evolving lineage: Application to Testudo graeca.

BACKGROUND: Obtaining de novo chromosome-level genome assemblies greatly enhances conservation and evolutionary biology studies. For many research teams, long-read sequencing technologies (that produce highly contiguous assemblies) remain unaffordable or unpractical. For the groups that display high synteny conservation, these limitations can be overcome by a reference-guided assembly using a close relative genome. Among chelonians, tortoises (Testudinidae) are considered one of the most endangered taxa, which calls for more genomic resources. Here we make the most of high synteny conservation in chelonians to produce the first chromosome-level genome assembly of the genus Testudo with one of the most iconic tortoise species in the Mediterranean basin: Testudo graeca. RESULTS: We used high-quality, paired-end Illumina sequences to build a reference-guided assembly with the chromosome-level reference of Gopherus evgoodei. We reconstructed a 2.29 Gb haploid genome with a scaffold N50 of 107.598 Mb and 5.37% gaps. We sequenced 25,998 protein-coding genes, and identified 41.2% of the assembly as repeats. Demographic history reconstruction based on the genome revealed two events (population decline and recovery) that were consistent with previously suggested phylogeographic patterns for the species. This outlines the value of such reference-guided assemblies for phylogeographic studies. CONCLUSIONS: Our results highlight the value of using close relatives to produce de novo draft assemblies in species where such resources are unavailable. Our annotated genome of T. graeca paves the way to delve deeper into the species' evolutionary history and provides a valuable resource to enhance direct conservation efforts on their threatened populations.

Animals↗
PubMed2005

The predicted impact of coding single nucleotide polymorphisms database.

Nonsynonymous single nucleotide polymorphisms (nsSNP) have the potential to affect the structure or function of expressed proteins and are, therefore, likely to represent modifiers of inherited susceptibility. We have classified and catalogued the predicted functionality of nsSNPs in genes relevant to the biology of cancer to facilitate sequence-based association studies. Candidate genes were identified using targeted search terms and pathways to interrogate the Gene Ontology Consortium database, Kyoto Encyclopedia of Genes and Genomes database, Iobion's Interaction Explorer PathwayAssist Program, National Center for Biotechnology Information Entrez Gene database, and CancerGene database. A total of 9,537 validated nsSNPs located within annotated genes were retrieved from National Center for Biotechnology Information dbSNP Build 123. Filtering this list and linking it to 7,080 candidate genes yielded 3,666 validated nsSNPs with minor allele frequencies > or =0.01 in Caucasian populations. The functional effect of nsSNPs in genes with a single mRNA transcript was predicted using three computational tools-Grantham matrix, Polymorphism Phenotyping, and Sorting Intolerant from Tolerant algorithms. The resultant pool of 3,009 fully annotated nsSNPs is accessible from the Predicted Impact of Coding SNPs database at http://www.icr.ac.uk/cancgen/molgen/MolPopGen_PICS_database.htm. Predicted Impact of Coding SNPs is an ongoing project that will continue to curate and release data on the putative functionality of coding SNPs.

Algorithms↗
PubMedDate not supplied

Photoreceptor dysplasia (pd) in miniature schnauzer dogs: evaluation of candidate genes by molecular genetic analysis.

Photoreceptor dysplasia (pd) is one of a group of at least six distinct autosomal and one X-linked retinal disorders identified in dogs which are collectively known as progressive retinal atrophy (PRA). It is an early onset retinal disease identified in miniature schnauzer dogs, and pedigree analysis and breeding studies have established autosomal recessive inheritance of the disease. Using a gene-based approach, a number of retina-expressed genes, including some members of the phototransduction pathway, have been causally implicated in retinal diseases of humans and other animals. Here we examined seven such potential candidate genes (opsin, RDS/peripherin, ROM1, rod cGMP-gated cation channel alpha-subunit, and three subunits of transducin) for their causal association with the pd locus by testing segregation of intragenic markers with the disease locus, or, in the absence of informative polymorphisms, sequencing of the coding regions of the genes. Based on these results, we have conclusively excluded four photoreceptor-specific genes as candidates for pd by linkage analysis. For three other photoreceptor-specific genes, we did not find any mutation in the coding sequences of the genes and have excluded them provisionally. Formal exclusion would require investigation of the levels of expression of the candidate genes in pd-affected dogs relative to age-matched controls. At present we are building suitable informative pedigrees for the disease locus with a sufficient number of meiosis to be useful for genomewide screening. This should identify markers linked to the disease locus and eventually permit progress toward the identification of the photoreceptor dysplasia gene and the disease-causing mutation.

Animals↗