Endoscopic retrograde cholangiopancreatography after a liquid fatty meal: effect on deep common bile duct cannulation time.

BACKGROUND AND STUDY AIM: Endoscopic retrograde cholangiopancreatography (ERCP) is an important gastrointestinal endoscopic procedure in the study and treatment of pancreaticobiliary diseases. The critical step of the procedure is cannulation of the common bile duct (CBD) and/or the pancreatic duct. Cannulation can be a technical challenge at times. Fat is a natural stimulator for bile secretion and relaxation of the sphincter of Oddi. The objective of this study was to determine the effect of a liquid fatty meal on deep CBD cannulation during ERCP. PATIENTS AND METHODS: We performed a randomized double-blind study in 84 patients to examine the effect of a liquid fatty meal on deep CBD cannulation during ERCP, in a teaching medical center. In the study group, each patient had a liquid fatty meal orally about 1 hour before the procedure. In the control group, each patient had the same volume of a non-fat meal. The appearance of the major papilla, the cannulation rate, the cannulation time, and the fluoroscopy time during cannulation were compared for the two groups. RESULTS: The orifice of the CBD/pancreatic duct was much more easily identified in the group who ingested the fatty meal. Compared with the non-fat meal group, in the fatty meal group the mean and the median deep CBD cannulation times were shorter, at 8.0 minutes vs. 14.7 minutes ( P = 0.005) and 8.0 minutes vs. 11.5 minutes ( P = 0.008), respectively. Additionally, in the fatty meal group, the mean and the median fluoroscopy times during deep CBD cannulation were lower, at 3.3 minutes vs. 6.1 minutes ( P = 0.040) and 2.5 minutes vs. 3.9 minutes ( P = 0.013), respectively. There were no complications, such as aspiration, associated with the liquid meals given shortly before the ERCP procedure. CONCLUSIONS: To avoid prolonged cannulation and unnecessary radiation exposure, patients should have a liquid fatty meal before ERCP procedures.

Continuous affinity-based selection: rapid screening and simultaneous amplification of bacterial surface-display libraries.

A new method for continuous biopanning has been developed. We have combined the power of affinity chromatography with the fecundity of bacteria in a unique process that mimics clonal selection. Mixed populations of bacteria were applied to a fermenter containing the immobilized ligand of interest. Bacteria retained in this affinity fermenter were allowed to grow under continuous washout conditions, such that weakly bound organisms were selectively lost. Those initially rare founder bacteria expressing a receptor for the immobilized ligand (R+ve) were thus enriched and amplified simultaneously. From an initial culture containing 1 x 10(10) R-ve cells spiked with fewer than 30 R+ve bacteria (<1 in 10(8)), final ratios of R+ve/R-ve bacteria as high as 1 in 12 were observed, representing an enrichment factor of 55 million-fold. This technology has considerable potential for rapid screening of bacterial surface-display libraries and in facilitating directed-evolution studies.

Evidence for heterotypic interaction between the receptor tyrosine kinases TIE-1 and TIE-2.

The orphan receptor tyrosine kinase Tie-1 is expressed in endothelial cells and is essential for vascular development. Nothing is known about the signaling pathways utilized by this receptor. In this study we have used chimeric receptors composed of the TrkA ectodomain fused to the transmembrane and intracellular domains of Tie-1, or the related receptor Tie-2, to examine Tie-1 signaling capacity. In contrast to TrkA/Tie-2, the Tie-1 chimera was unable to phosphorylate cellular proteins or undergo autophosphorylation. Consistent with this Tie-1 exhibited negligible kinase activity. Co-immunoprecipitation analysis revealed Tie-1 was present in endothelial cells bound to Tie-2. Full-length Tie-1 and truncated receptor, formed by regulated endoproteolytic cleavage, were found to complex with Tie-2. Association was mediated by the intracellular domains of the receptors and did not require Tie-1 to be membrane-localized. Tie-1 bound to Tie-2 was not tyrosine-phosphorylated under basal conditions or following Tie-2 stimulation. This study provides the first evidence for the existence of a pre-formed complex of Tie-1 and Tie-2 in endothelial cells. The data suggest Tie-1 does not signal via ligand-induced kinase activation involving homo-oligomerization. The physical association between Tie-1 and Tie-2 is consistent with Tie-1 having a role in modulating Tie-2 signaling.

Community-acquired bacteremia in HIV-positive patients: protective benefit of co-trimoxazole.

OBJECTIVE: To evaluate the effect of the type of Pneumocystis carinii pneumonia (PCP) prophylaxis on the development of community-acquired bacteremia. DESIGN: Case-control study using all cases of community-acquired bacteremia identified prospectively during a longitudinal study of all infections in a cohort of HIV-infected persons. SETTING: University-affiliated Department of Veterans Affairs Medical Center HIV program. PATIENTS: All patients with community-acquired bacteremia seen at the facility between January 1990 and December 1995 were included. Controls, seen at the same facility and matched by date and CD4 count, were used to assess risk factors. A total of 57 cases and 114 controls were analysed. MAIN OUTCOME MEASURES: Risk of development of bacteremia, distribution of organisms, and effect of specific prophylactic regimens for PCP. RESULTS: Bacteremia was caused by Staphylococcus aureus (23%), Pseudomonas aeruginosa (18%), Escherichia coli (16%), Streptococcus pneumoniae (14%) and others (31%). Groups were similar by age, race, HIV risk factors and CD4 count. The presence of an intravenous catheter was mildly predictive of the development of bacteremia [odds ratio (OR), 2.67; P = 0.024]. Type of PCP prophylaxis in cases and controls with CD4 < 200 x 10(6)/l included co-trimoxazole (trimethoprim-sulfamethoxazole, TMP-SMX; 31 and 60%, respectively), dapsone (33 and 24%, respectively) and aerosolized pentamidine (27 and 13%, respectively). Use of TMP-SMX (but not dapsone or aerosolized pentamidine) was associated with the absence of bacteremia (OR, 0.28; P = 0.001). A similar protective effect was found when controlling for the presence of an intravenous catheter. CONCLUSION: PCP prophylaxis with TMP-SMX apparently protects against community-acquired bacteremia in HIV-infected persons.

The production of PCR products with 5' single-stranded tails using primers that incorporate novel phosphoramidite intermediates.

We have prepared several novel phosphoramidites and have synthesised oligonucleotides incorporating them internally. The presence of these residues in an oligonucleotide template presents an impossible barrier to primed synthesis by Taq DNA polymerase. When extended as polymerase chain reaction products, these oligonucleotides no longer serve as templates for the polymerase beyond the insertion sites of the modified intermediates, thereby producing single-stranded tails on amplification products. These tails can then be used for solid phase capture and colorimetric detection of PCR products.

Lipocortin 1 fragment modifies pyrogenic actions of cytokines in rats.

Lipocortins form a group of proteins that have been proposed as mediators of the anti-inflammatory actions of glucocorticoids. Intracerebroventricular injection of a recombinant fragment of lipocortin 1 (NH2-terminal 1-188) caused dose-dependent (0.4-1.2 micrograms) reductions in the acute increases in colonic temperature and oxygen consumption, which occurred in response to central injections of recombinant interleukin 1 beta and gamma-interferon in conscious rats. In contrast the lipocortin fragment did not affect the response to prostaglandin E2, and its activity was prevented by heat treatment or by pretreatment of animals with polyclonal antiserum raised to the fragment. Central injection of antiserum significantly enhanced the thermogenic responses to interleukin 1 beta in rats treated with dexamethasone without affecting the responses in normal animals. These results support a physiological role for lipocortin in the central effects of glucocorticoids.

Analysis of A-MuLV transformed fibroblast lines for amplification of the c-myc, p53 and c-fos nuclear proto-oncogenes.

Aberrations in nuclear proto-oncogene organisation and/or gene expression have been implicated in cell transformation mediated by the v-abl gene. For example, it has been suggested that amplification of the c-myc proto-oncogene is a co-operative event in v-abl induced fibroblast transformation. We have investigated amplification of the c-myc, p53 and c-fos nuclear proto-oncogenes in several Abelson murine leukaemia virus (A-MuLV) transformed fibroblast lines. None of these proto-oncogenes were detectably rearranged or amplified in v-abl transformed Swiss 3T3 lines. In contrast, NIH3T3 fibroblasts transformed by the v-abl gene consistently showed a 4 to 16-fold amplification of the c-myc gene. These data show that c-myc gene amplification is not an obligatory event associated with A-MuLV transformation, but may be restricted to cell lines derived from NIH3T3. c-myc gene amplification also did not correlate with a reduced latency period for tumour induction in nude mice. In addition, c-myc amplification was not selected during tumourigenesis, indicating that this event is not required for A-MuLV transformed Swiss 3T3 cells to display a full tumourigenic phenotype.

Human complement factor I: analysis of cDNA-derived primary structure and assignment of its gene to chromosome 4.

Factor I is a serine proteinase of complement which together with one of several specific cofactors cleaves activation products of the third and fourth components of complement (C3b and C4b) and modulates the activity of C3 convertase. A heterodimer glycoprotein (Mr = 88,000), factor I is synthesized as a single-chain precursor, prepro-I, which undergoes intracellular proteolytic processing. The human hepatoma line HepG2, however, secretes predominantly the single-chain precursor pro-I. In order to determine the molecular basis for this apparent processing defect, factor I cDNA clones were isolated from a HepG2 mRNA-derived library. Sequencing of the largest insert, HI1971, revealed that it contains 14 base pairs of 5' untranslated region, the complete coding sequence for the 583-residue prepro-I (NH2-signal peptide-heavy chain-linking peptide-light chain-COOH), two polyadenylation signals within the 200-base pair 3' untranslated region, and a portion of poly(A) tail. Analysis of the derived protein structure 1) reveals a mosaic multidomain structure of the heavy chain; 2) demonstrates structural similarity between intracellular conversion of pro-I and activation of other serine proteinase zymogens; and 3) indicates that the light chain of factor I resembles most closely the active subunit of tissue plasminogen activator among all serine proteinases and factor D among complement proteinases. Furthermore, this protein sequence was compared to the sequences of factor I cDNA clones isolated from normal human liver libraries and found to be identical. By exclusion, this defines as cellular the basis for the inefficient processing of pro-I by the HepG2 line. Chromosomal localization by the somatic cell hybrid method maps the factor I gene to chromosome 4.

Extensive mutagenesis of the nuclear location signal of simian virus 40 large-T antigen.

Site-directed mutagenesis was used to change Lys-128 of the simian virus 40 large-T nuclear location signal to Met, Ile, Arg, Gln, Asn, Leu, or His. Except for the large-T antigen of the Arg mutation, which was present in cytoplasmic and nuclear compartments, the resultant proteins were unable to enter the nucleus. By contrast, mutations at other sites within the signal were generally less severe in their effect. In some cases (Lys-128 to Gln, Asn, and His), the apparently cytoplasmic variants were able to support limited plasmid DNA replication, suggesting that low levels of large-T antigen undetectable by immunofluorescence were present in the nucleus. Such mutants did not support viral DNA replication. We conclude that there is a strong requirement for a basic residue at position 128 in the large-T nuclear location signal, with Lys the preferred residue.

Functionally important conserved amino-acids in interferon-alpha 2 identified with analogues produced from synthetic genes.

A gene was chemically synthesised and expressed in Escherichia coli to produce [Ala30,32,33]IFN-alpha 2, an analogue of human alpha 2-interferon (IFN-alpha 2) which is devoid of activity on human cells. Eight additional analogues provided single changes in IFN-alpha 2 at each of these three conserved positions. No one residue is essential for activity, but both antiviral and anti-proliferative activity are particularly sensitive to changes in the side-chain of Arg33.

Human serum amyloid P component. cDNA isolation, complete sequence of pre-serum amyloid P component, and localization of the gene to chromosome 1.

Complementary DNA clones corresponding to the human serum amyloid P component (SAP) were isolated, and the complete nucleotide and derived amino acid sequence of preSAP was determined. PreSAP biosynthesis is directed by a 1.1-kilobase mRNA. Synthesis and postsynthetic processing of preSAP in Xenopus oocytes result in secretion of a protein with mobility similar to native purified SAP when analyzed by sodium dodecyl sulfate gel electrophoresis. The human SAP gene is on chromosome 1, probably closely linked to the gene for C-reactive protein which encodes the related acute phase reactant found in human plasma.

Human serum amyloid A (SAA): biosynthesis and postsynthetic processing of preSAA and structural variants defined by complementary DNA.

To study structural variants of human serum amyloid A (SAA), an apoprotein of high-density lipoprotein, complementary DNA clones were isolated from a human liver library with the use of two synthetic oligonucleotide mixtures containing sequences that could code for residues 33-38 and 90-95 of the protein sequence. The SAA-specific cDNA clone (pA1) contains the nucleotide sequence coding for the mature SAA and 10 amino acids of the 18-residue signal peptide. It also includes a 70 nucleotide long 3'-untranslated region and approximately 120 bases of the poly(A) tail. The derived amino acid sequence of pA1 is identical with the alpha form of apoSAA1. A fragment of pA1 containing the conserved (residues 33-38) region of SAA also hybridized with RNA from human acute phase liver and acute phase stimulated, but not unstimulated, mouse and rabbit liver. In contrast, a fragment corresponding to the variable region hybridized to a much greater extent with human than with rabbit or murine RNA. Human acute phase liver SAA mRNA (approximately 600 nucleotides in length) directs synthesis of preSAA (Mr 14 000) in a cell-free translating system. In a Xenopus oocyte translation system preSAA is synthesized and processed to the mature Mr 12 000 product. The complete 18 amino acid signal peptide sequence of preSAA was derived from sequencing cDNA synthesized by "primer extension" from the region of SAA mRNA corresponding to the amino terminus of the mature product. Two other SAA-specific cDNA clones (pA6 and pA10) differed from pA1 in that they lack the internal PstI restriction enzyme site spanning residues 54-56 of pA1.(ABSTRACT TRUNCATED AT 250 WORDS)

Cloning of human prealbumin complementary DNA. Localization of the gene to chromosome 18 and detection of a variant prealbumin allele in a family with familial amyloid polyneuropathy.

Prealbumin, a 55,000 Mr protein, is a normal constituent of human serum. In patients with familial amyloid polyneuropathy (FAP), an autosomal dominant disease, variant prealbumin molecules are found in association with systemic amyloid deposits. One variant prealbumin has a methionine for valine substitution at amino acid 30 and has been implicated in the pathogenesis of type 1 FAP. A prealbumin-specific complementary DNA clone has been isolated from an adult human liver library and used in Southern blot hybridization experiments to identify a unique NsiI restriction endonuclease site in the variant allele carried by type 1 FAP patients with the methionine for valine substitution. The complementary DNA clone has been used to analyse a panel of human-mouse and human--hamster somatic cell hybrid DNAs and localize the prealbumin gene to chromosome 18.

Isolation of a complementary DNA clone for the human complement protein C2 and its use in the identification of a restriction fragment length polymorphism.

Complementary DNA (cDNA) clones corresponding to the major histocompatibility (MHC) class III antigen, complement protein C2, have been isolated from human liver cDNA libraries with the use of a complex mixture of synthetic oligonucleotides (17 mer) that contains 576 different oligonucleotide sequences. The C2 cDNA were used to identify a DNA restriction enzyme fragment length polymorphism that provides a genetic marker within the MHC that was not detectable at the protein level. An extensive search for genomic polymorphisms using a cDNA clone for another MHC class III gene, factor B, failed to reveal any DNA variants. The genomic variants detected with the C2 cDNA probe provide an additional genetic marker for analysis of MHC-linked diseases.

The role of the beta-lactamase signal sequence in the secretion of proteins by Escherichia coli.

A derivative of pBR322 has been constructed that contains both a unique EcoRI restriction site right at the beginning of the signal codons of the beta-lactamase (bla) gene and a unique BstEII site just at the end of the bla signal codons. Although the signal peptide encoded by the new plasmid differs from the wild type (pBR322) by 2 amino acid residues (Ser 2 to Arg 2 and Ala 23 to Gly 23), the synthesis, transport, and processing of the beta-lactamase remain unchanged in Escherichia coli. Two deletion mutants, in which the bla signal codons have been almost completely excised, have also been constructed. Bacteria containing either of these plasmids produce, but do not secrete, an active beta-lactamase. Last, the bla signal codons have been precisely joined to the cDNA version of the triose phosphate isomerase (tpi) gene from chicken. Expression of this fusion gene in E. coli gives a hybrid protein that is neither secreted into the periplasm nor proteolytically processed. This result supports the view that there are characteristics of the mature protein that are necessary for the secretion across the inner membrane of E. coli.