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PubMed1995

Transfer of yeast artificial chromosomes into mammalian cells and comparative study of their integrity.

Yeast artificial chromosomes (YACs) from the CEPH MegaYAC library (Paris, France) ranging in size from 350 to 1600 kb and mapping to the q22.1 and q22.2 regions of human chromosome 21 were transferred into mammalian cells by spheroplast fusion. The integrity of the YACs from two adjacent parts of the region was compared after retrofitting and stable transfer into mammalian cells. We found that large YACs could easily be manipulated to allow transfer of the YAC material into mammalian cells and that the size of the YAC did not appear to be limiting for fusion. However, we show that there was great variability in the integrity of the YACs from the two regions, which was not related to the size of the YACs. Four YACs in region I from sequence-tagged site (STS) G51E05 up to STS LL103 showed, in general, no loss of material and correct gene transfer into mammalian cells. In contrast, the three YACs in the more centromeric region II (from STS G51B09 up to G51E05) frequently showed a loss of human material during handling, retrofitting and transfer. As a YAC from another library covering region II was also found to be unstable, we propose that the integrity of the YACs is highly dependent on the incorporated human chromosomal DNA.

Animals↗
PubMed2006

Gene profiling of cattle blastocysts derived from nuclear transfer, in vitro fertilization and in vivo development based on cDNA library.

Gene expression analysis of cloned embryos would enable us to better understand the early biological events during preimplantation after NT (nuclear transfer). Routine RT-PCR and Northern-blot were limited because it could not analyze tens of thousands of genes at one time and were impeded by minimum material. Based on the developed RT-PCR methodology, we previously constructed cDNA libraries with equivalent to single embryo from the pooled AI-blastocysts (artificial insemination and in vivo developed blastocysts) of cattle. To identify gene expression profiles in NT- and IVF (in vitro fertilized)-blastocysts, and search for new candidate genes involved during this period, here we created cDNA sources from three types of blastocysts (AI-, IVF- and NT-blastocysts). The expressions of 60 genes previously identified from cDNA library were compared in three types of blastocyst. Results showed that the gene expression profile of NT-blastocysts was more similar to that of AI-blastocysts than that of created from IVF-blastocysts. Several important genes, such as Oct-4 and IFN-iota, only detected in the early embryonic development, were highly expressed in three types of blastocysts and showed no significant difference, it indicated that the donor nuclear undergone efficient reprogramming by the blastocyst stage and gained totipotential after nuclear transfer. The gene expression profiles in three types of blastocysts suggested that nuclear transfer and in vitro culture environments impaired the viability of embryos in different ways.

Animals↗
PubMed2001

Subtraction-hybridization method for the identification of imprinted genes.

Imprinted genes show monoallelic expression from either the paternal or maternal genome (1,2), and their regulated expression is usually associated with the existence of parentally differentially methylated regions on genomic DNAs (3,4). Because of this, essentially two different approaches, using either cDNA or genomic DNA as starting material (5) have been developed for systematic isolation of imprinted genes. In this chapter, we describe a subtraction-hybridization method (6-8) as an example of the former approach. Both parthenogenetic embryos and androgenetic embryos (9,10) are the most suitable biological materials for the subtraction or detection of imprinted genes. However, it is difficult to obtain a large amount of such special materials because only a small number of these embryos develop to the d 10 stage (9,10). Thus, polymerase chain reaction (PCR)-based techniques, such as the differential display (11-13) and subtraction-hybridization methods, are necessary to accomplish this experiment. The subtraction-hybridization method has been successfully applied for isolation of both paternally expressed genes (Pegs) (6,14,15) and maternally expressed genes (Megs) (7), and it allows cDNA libraries to be made from a very small amount of biological material. We are convinced that this method can be applied in many fields of biological science.

Animals↗
PubMed1990

Cloning of PCR-amplified total cDNA: construction of a mouse oocyte cDNA library.

We describe a general method for the synthesis and cloning of cDNA, applicable to cases in which the availability of biological material for mRNA extraction is extremely limited. A protocol allowing amplification of a heterogeneous mixture of cDNAs by the polymerase chain reaction has been devised and applied successfully to the construction of an apparently representative cDNA library, using as a model of a scarce RNA source 50 mouse ovulated eggs that can yield a maximum of 1.75 ng of poly(A)+ RNA. However, about 5% of the material obtained after amplification was adequate for cloning. Using the cloned sequences, we have derived a preliminary indirect measurement of the sequence complexity of the maternal poly(A)+ RNA in this mammalian oocyte.

Animals↗
PubMed1994

Current diagnosis and treatment of gastroesophageal reflux disease.

OBJECTIVE: To review recent advances in the diagnosis and treatment of gastroesophageal reflux disease (GERD). MATERIAL AND METHODS: Original English language reports were obtained through a Medline search of the National Library of Medicine up to and including 1993. The reference lists of all original reports and review articles were searched to locate any further material. In the evaluation of therapeutic efficacy, randomized studies were preferentially considered; greatest priority was given to double-blind, placebo-controlled trials. Abstracts, nonrandomized trials, and non-English language publications were considered only when other data were unavailable. RESULTS: Information obtained from histories and physical examinations suggests that GERD occurs in many patients. Evaluation of mucosal injury with use of either endoscopy or air contrast barium radiography is an important early step in the diagnosis of GERD. Endoscopy obtains tissue for histologic study, especially in Barrett's esophagus. Prolonged esophageal pH monitoring is the most useful determinant of the presence and amount of reflux of acid. Patients with GERD should be counseled on lifestyle modification and the use of antacids and antirefluxants. Histamine type 2 receptor antagonists provide symptomatic relief in 32 to 82% of patients with GERD and resolution of verified esophagitis in 0 to 82%. Responses with omeprazole therapy are higher; symptomatic responses were noted in 62 to 94% of patients, and healing of esophagitis occurred in 71 to 96%. Promotility agents and surgical therapy have a role in selected patients. CONCLUSION: GERD is a chronic disorder that often necessitates individualized lifelong therapy. Many questions remain to be answered about the cost-effectiveness of both diagnostic tests and therapy for GERD.

Gastroesophageal Reflux↗
PubMed1994

Systematic analytic procedure for identification of unknown medicaments in biological material.

The presented method has been used in toxicological practice for identification of unknown medicaments in biological material longer than fifteen years with good results. This method described as TLC/CR-screening is a combination of thin layer chromatography (TLC) with a system of colour reactions (CR-screening), the principle being evaluation of characteristic features (qualities) of each detected unknown substance (parent medicament form or its metabolite) from three points of view: the type of ionization during the extraction, the mobility in basic TLC-system in comparison with five reference standards, the reactivity with system reagents for detection. The properties of each detected substance expressed by adequate symbols are put together into three component codes, under which corresponding standards of medicaments and/or their metabolites can be found in the library of code system.

Biocompatible Materials↗
PubMed1998

Protein F, a fibronectin-binding protein of Streptococcus pyogenes, also binds human fibrinogen: isolation of the protein and mapping of the binding region.

During screening of a gene library of Streptococcus pyogenes type M15 for fibrinogen-binding material, a protein of approximately 100 kDa, encoded outside the vir region, was found. DNA sequencing revealed this component to be identical to protein F, a fibronectin-binding protein. Isolation of the recombinant protein, termed F15, was performed by the use of fibrinogen affinity chromatography. The affinity constant (Ka) of protein F15 for fibrinogen, 1.25 x 10(7) mol-1, was lower than that for fibronectin, 1.8 x 10(8) mol-1. The fibrinogen-binding domain was located in the N-terminal part of the molecule, while the fibronectin-binding domains, as previously determined, were in the C-terminal portion of protein F. To examine the amino acid sequence heterogeneity of protein F, the 5' part of the prtF gene, corresponding to the N-terminal variable region of the protein, was amplified by PCR from 12 strains of S. pyogenes belonging to six different M-types. Alignment of these nucleotide sequences indicated that the 5' portion of the prtF gene had probably undergone a number of intragenic recombination and horizontal gene transfer events, allowing a pattern of structural diversity of protein F observed earlier for some other streptococcal virulence factors. There was no strict correlation between M-type and nucleotide sequence of the variable region of the prtF gene and, compared to streptococcal M protein, the overall variation observed for protein F appeared more limited.

Adhesins, Bacterial↗
PubMed1970

Hospital administrators' views of medical libraries.

Ways in which the library can become involved in hospital educational programs are discussed. The librarian can set out materials on specific topics for discussion among nurses, and can encourage continuing education among physicians. The need for this is stressed, together with the necessity for adequate hospital library standards to meet this demand.

Hospital Administration↗
PubMed1990

How to select Plasmodium falciparum pre-erythrocytic antigens in an expression library without defined probe.

The restricted access to Plasmodium falciparum liver stages has greatly limited the analysis of the antigenic content of that stage. Due to the lack of material to perform immunochemical studies, of access to mRNA, and of monoclonal probes, we decided to screen a genomic library with stage-restricted human antibodies. This strategy led to the identification of a large number of DNA fragments encoding both sporozoite specific as well as liver-stage specific epitopes. Following the initial characterization of one liver-stage antigen, further screening was performed by using additional selective human antibodies. These were defined as having a high degree of reactivity with native antigens on either of the two stages while being negative with the already known molecules of the two stages. From this second screening and the study of cross-reactions, several subsets of DNA clones expressing antigens present on the surface of sporozoites, or in liver stages, or in both, could be identified. In exposed individuals a high prevalence of antibodies to several of these antigens was found.

Animals↗
PubMed1983

Establishing a psychiatric library.

The amount of information in the psychiatric field has increased to such an extent that a specialized library is an essential part of the mental health facility. Guidelines are presented for establishing a psychiatric library, including staffing, budgeting, physical layout, and selection and handling of books, reference materials, journals and annuals, microfilm and microfiche, and audiovisuals. The expertise of the librarian is the single most important factor in determining the quality of the collection and the services provided, the author believes. Because of escalating costs, developing a cooperative library network of small, specialized mental health collections may be the most productive course for the future.

Humans↗
PubMed2000

The search for an ideal method of abdominal fascial closure: a meta-analysis.

BACKGROUND AND OBJECTIVE: The ideal suture for abdominal fascial closure has yet to be determined. Surgical practice continues to rely largely on tradition rather than high-quality level I evidence. The authors conducted a systematic review and meta-analysis of randomized controlled trials to determine which suture material and technique reduces the odds of incisional hernia. METHODS: MEDLINE and Cochrane Library databases were searched for articles in English published from 1966 to 1998 using the keywords "suture", "abdomen/surgery", and "randomized controlled trials". Randomized controlled trials, trials of adult patients, and trials with a Jadad Quality Score of more than 3, comparing suture materials, technique, or both, were included. Two independent reviewers critically appraised study quality and extracted data. The reviewers were masked to the study site, authors, journal, and date to minimize bias. The primary outcome was postoperative incisional hernia. Secondary outcomes included wound dehiscence, infection, wound pain, and suture sinus formation. RESULTS: The occurrence of incisional hernia was significantly lower when nonabsorbable sutures were used. Suture technique favored nonabsorbable continuous closure. Suture sinuses and wound pain were significantly lower when absorbable sutures were used. There were no differences in the incidence of wound dehiscence or wound infection with respect to suture material or method of closure. Subgroup analyses of individual sutures showed no significant difference in incisional hernia rates between polydioxanone and polypropylene. Polyglactin showed an increased wound failure rate. CONCLUSIONS: Abdominal fascial closure with a continuous nonabsorbable suture had a significantly lower rate of incisional hernia. The ideal suture is nonabsorbable, and the ideal technique is continuous.

Abdominal Muscles↗
PubMed1991

Partial characterization of 2-beta-mercaptoethanol-soluble surface-associated antigens of Onchocerca volvulus.

Antigens were extracted from the epicuticle/cuticle of intact female Onchocerca volvulus using 2% 2-beta-mercaptoethanol and 1% SDS. In Western blot analysis a human infection serum selected for its high antibody titre against whole worm homogenates did not recognize any component solubilized by 1% SDS. However, the same serum did bind at least 7 antigens among the material extracted with 2-beta-mercaptoethanol. These antigens have apparent molecular weights (Mr) of: 15,000, 18,000, 28,000, 78,000, 98,000, 120,000 and 200,000. In ELISA using this preparation as target antigen, 151 out of 153 human infection sera gave positive results. An Onchocerca-specific IgG1 monoclonal antibody, designated Cam1, recognized the 28,000 Mr antigen, which is the most prominent antigen detected by Western blot analysis using human infection sera. In ELISA, using material affinity-purified with Cam1 as target antigen, 149 out of 153 human infection sera gave a positive IgG response. From a cDNA library three expressing clones were isolated with a rabbit serum raised against 2-beta-mercaptoethanol solubilized material. One of these clones was recognized by the monoclonal antibody Cam1.

Animals↗
PubMed1991

National Library of Medicine programs--PHS. Final rule.

These regulations govern the programs of the National Library of Medicine (NLM). The regulations permit the Regional Medical Libraries to recover part or all of the costs of providing photocopies of biomedical materials; improve readability of the regulations; and update references to statutory authorities and uniform administrative requirements.

Copying Processes↗
PubMed2004

Sol-gel polycondensation of tetraethyl orthosilicate (TEOS) in sugar-based porphyrin organogels: inorganic conversion of a sugar-directed porphyrinic fiber library through sol-gel transcription processes.

Sugar-appended porphyrins (1 a-e) with monosaccharide groups at their periphery have been rationally designed for a new class of gelating reagents. A few of these compounds have the tendancy to form one-dimensional aggregates stable enough to show successful gelation ability for DMF-alcohol mixed solvents. The aggregation mode in the specific columnar super structures has been evaluated in detail by UV-visible spectrometry (UV/Vis), circular dichroism (CD), scanning electron microscopy (SEM), and transmission electron microscopy (TEM). All UV-visible spectra of sugar-appended porphyrinic gels obtained from 1 a-c exhibit Soret band absorptions, which shift to lower wavelength and are significantly broadened. This phenomenon indicates that these porphyrin cores strongly interact with each other in an H-aggregate fashion, which drives the generation of a one-dimensional porphyrin-stacking array. The CD spectra of the organogels from 1 a and 1 b, which are in anomers, exhibit an almost symmetric pattern, whereas the gel from 1 c gives a completely different pattern. This implies that the gel fibrils wind themselves in a right- or left-handed fashion; this reflects chirality in the specific molecular structure of the gelators. The results from SEM for the gel fibrils are in good agreement with the CD patterns. The gel fibrils in 1 a possess left-handed helicity, whereas those in 1 b wind themselves right-handedly. Macroscopic helical morphology reflects the microscopic structure well at a molecular level, which gives structural variety of the gel fibrils, which can be defined by the sugar library. Inorganic conversion of the organic helical fibrils by a sol-gel transcription process successfully gives the helical-silica structures, which finely inherit the organic morphology. A striking observation is that a unimolecular porphyrin-stacking array is also transcribed into silica fibers when the optimized sol-gel reaction conditions are selected. A sugar-based organic-fiber library in porphyrinic gels thus provides a variety of inorganic materials through the sol-gel transcription process.

Journal Article↗
PubMed1998

Gene structures and properties of enzymes of the plasmid-encoded nicotine catabolism of Arthrobacter nicotinovorans.

Arthrobacter nicotinovorans is a Gram-positive aerobic soil bacterium able to grow on nicotine as its sole source of carbon and nitrogen. The initial steps of nicotine catabolism are catalyzed by nicotine dehydrogenase, the l- and d-specific 6-hydroxynicotine oxidases, and ketone dehydrogenase. The genes encoding these enzymes reside on a 160 kb plasmid, pAO1. The cccDNA of this plasmid was isolated in high purity and reasonable yield. It served as template material for the construction of a lambda-phage DNA library of the plasmid. The genes coding for 6-hydroxy-l-nicotine oxidase and for the subunits of the heterotrimeric ketone dehydrogenase were identified, subcloned and sequenced. The 6-hlno gene was identified as a 1278 bp open reading frame; its regulatory elements were also recognized. The derived primary structure of the monomer of apo-6-hydroxy-l-nicotine oxidase (46,264.5 Da) agrees with the data obtained by partial amino acid sequencing. 6-Hydroxy-l-nicotine oxidase and 6-hydroxy-d-nicotine oxidase were expressed in Escherichia coli and obtained in a state of high purity and crystallized. Ketone dehydrogenase (KDH) was found to be a heterotrimer with subunits of molecular mass 89,021.71, 26,778.65 and 17,638.88. The genes of KDH-A and KDH-B are juxtaposed; the A of the stop codon of KDH-A is used in the start codon of KDH-B, eliciting a frame shift. KDH-C is separated from KDH-A by 281 bp.

Arthrobacter↗
PubMed2005

Fluorous parallel synthesis of a hydantoin/thiohydantoin library.

Fluorous tagging strategy is applied to solution-phase parallel synthesis of a library containing hydantoin and thiohydantoin analogs. Two perfluoroalkyl (Rf)-tagged alpha-amino esters each react with six aromatic aldehydes under reductive amination conditions. Twelve amino esters then each react with 10 isocyanates and isothiocyanates in parallel. The resulting 120 ureas and thioureas undergo spontaneous cyclization to form the corresponding hydantoins and thiohydantoins. The intermediate and final product purifications are performed with solid-phase extraction (SPE) over FluoroFlash cartridges, no chromatography is required. Using standard instruments and straightforward SPE technique, one chemist accomplished the 120-member library synthesis in less than five working days, including starting material synthesis and product analysis.

Aldehydes↗
PubMed2004

Synthetic molecules as antibody replacements.

Antibodies are by far the most versatile, valuable, and widely used protein-binding agents. They are essential tools in biological research and are increasingly being developed as therapeutic reagents. However, antibodies have a number of practical limitations, and it would be desirable in many applications to replace them with simpler, more robust synthetic molecules. Unfortunately, synthetic protein-binding agents rarely exhibit the high affinity and specificity typical of a good antibody. This article reviews efforts to overcome these limitations and to develop a facile, high-throughput methodology for the isolation of synthetic protein ligands with antibody-like binding characteristics.

Antibodies, Bispecific↗
PubMedDate not supplied

Lipase-mediated purification of methyl nonactate, an important natural product building block for diversity-oriented synthesis.

Methyl nonactate is a valuable starting material for the production of natural product-like combinatorial libraries and ketide amino acids, a series of novel conformationally constrained amino acid analogues. Fermentation of Streptomyces griseus generates high titers of macrotetrolide antibiotics from which methyl nonactate can be generated by methanolysis. Unfortunately, this approach generates mixtures of homologues that are not economically separable. We report a specific lipase-mediated hydrolysis of nonactate derivatives that discriminates between homologues and is the foundation of an economically tractable preparation of methyl nonactate in scale.

Amino Acids↗