Three-day regimen improves faecal tagging for minimal preparation CT examination of the colon.

This study set out to determine whether extending the length of oral contrast administration in minimal preparation CT of the colon improves faecal tagging. Two cohorts of 50 patients each were compared, one with a 2-day the other with a 3-day faecal tagging regimen. The degree of faecal tagging was graded by two blinded observers. The 3-day regimen showed significantly better tagging in the rectum and sigmoid colon (p = 0.006 and p = 0.009, respectively, using the Mann-Whitney test). The percentage of patients who had faecal tagging in the sigmoid colon graded as "complete" was 64% for the 3-day regimen as opposed to 34% for the 2-day regimen. The corresponding percentages for the rectum were 64% for the 3-day regimen and 36% for the 2-day regimen. Extending the length of oral contrast administration from 2 to 3 days significantly improves the quality of faecal tagging in the rectum and sigmoid colon.

Multi-detector CT: review of its use in acute GI haemorrhage.

The advent of multi-section computed tomography (CT) technology allows the non-invasive assessment of the arterial tree. Using current software, it is now possible to produce high-quality, angiographic-like images that can be used to plan and guide therapeutic procedures. One such clinical situation is the assessment of patients with acute gastrointestinal (GI) haemorrhage. Multi-section CT has a number of advantages over conventional angiography in this situation. The simplicity and non-invasive nature of the technique compared with conventional angiography make CT angiography possible in situations where conventional angiography is not available. Movement artefact from respiration and peristalsis is a common problem in the interpretation of conventional angiography; this is essentially abolished with rapid acquisition times and the use of multi-planar images to remove overlying bowel loops. Cross-sectional imaging with the ability for multi-planar reconstruction allows the accurate anatomical localization of the bleeding site, as well as an assessment of the underlying pathology: this can be used to plan therapy (embolization or surgery). The aim of this paper is to review the current use of CT in the investigation of patients with GI haemorrhage, illustrated with images from our Institution. For patients in whom GI endoscopy has failed to establish a diagnosis, we propose multi-section CT angiography as the initial imaging investigation in acute GI haemorrhage.

Effects of dopamine beta-hydroxylase genotype and disulfiram inhibition on catecholamine homeostasis in mice.

RATIONALE: Dopamine beta-hydroxylase (DBH) converts dopamine (DA) to norepinephrine (NE), thus playing a critical role in catecholamine metabolism. OBJECTIVES/METHODS: We examined the effects of Dbh gene dosage and the DBH inhibitor disulfiram in mice with zero, one, or two null Dbh alleles (+/+, +/-, and-/- mice). RESULTS: DBH protein levels in adrenal and prefrontal cortex (PFC) and adrenal DBH activity were proportional to number of wild-type alleles. Adrenal DA was slightly increased in+/- mice and markedly increased (80-fold) in -/- mice compared to wild-type animals. While adrenal NE and epinephrine (EPI) were undetectable in -/- mice, adrenal concentrations of NE and EPI were similar in +/+ and +/- mice, suggesting that the increase in DA maintains the normal rate of beta-hydroxylation in Dbh +/- mice. Disulfiram had little effect on adrenal catecholamine levels, regardless of genotype or dose. NE was absent in the PFC of -/- mice, but only slightly reduced in +/- animals compared to wild-type animals. PFC DA was increased twofold in +/- mice and fivefold in -/- mice, and the NE to DA ratio was reduced ( approximately 35%) in +/- mice, compared to wild-type mice. Disulfiram significantly decreased PFC NE and increased DA in +/+ and +/- animals, with the disulfiram and genotype effects on the PFC NE to DA ratio apparently additive. CONCLUSIONS: The data reveal potentially important and apparently additive effects of Dbh genotype and disulfiram administration on PFC catecholamine metabolism. These effects may have implications for genetic control of DBH activity in humans and for understanding therapeutic effects of disulfiram.

pTRIPLEscript: a novel cloning vector for generating in vitro transcripts from tandem promoters for SP6, T7 and T3 RNA polymerase.

We have constructed a family of novel in vitro transcription vectors in which functional T3, T7 and SP6 RNA polymerase promoters are arranged in tandem and directed towards a multiple cloning site. This prototype vector, named pTRIPLEscript, permits the transcription of one strand of a DNA insert by any of the three commonly used bacteriophage RNA polymerases with no apparent cross talk, i.e., use of the wrong promoter sequence. The vector has two main uses: (i) to clone probe sequences that will be distributed to many laboratories, allowing the use of the most convenient RNA polymerase; and (ii) to circumvent the problem of RNA polymerase-dependent premature termination.

Cloning and expression of eukaryotic initiation factor 4B cDNA: sequence determination identifies a common RNA recognition motif.

Eukaryotic protein synthesis initiation factor 4B (eIF-4B) is an 80,000 dalton polypeptide which is essential for the binding of mRNA to ribosomes. A highly purified preparation of eIF-4B from HeLa cells was subjected to enzymatic cleavage and amino-terminal amino acid sequence analysis. Degenerate oligonucleotide probes were used to isolate a 3851 bp cDNA encoding eIF-4B from a human cDNA library. The DNA encodes a protein comprising 611 residues with a mass of 69,843 daltons. The amino-terminal domain of eIF-4B contains a consensus RNA binding domain present in a number of other RNA binding proteins. Expression of eIF-4B in transfected COS-1 cells yielded a polypeptide which reacted with anti-eIF-4B antiserum and comigrated with purified eIF-4B. Expression of eIF-4B in COS-1 cells resulted in a general inhibition of translation, possibly due to a 50-fold eIF-4B overproduction.

Selection of the 5'-proximal translation initiation site is influenced by mRNA and eIF-2 concentrations.

A cDNA clone of the influenza virus NS (non-structural protein) gene in a vector carrying a bacteriophage T7 RNA polymerase promoter was manipulated so as to reiterate the initiation site to give two in-frame AUG codons 57 nucleotide residues apart. Each initiation site was in either a preferred context (...AUAAUGG...) or a less favourable context (...UUUAUGG...) and the four possible permutations were constructed. When capped mRNA transcripts of these clones were translated in the rabbit reticulocyte lysate system, products from initiation at both AUG codons were observed. At low RNA concentrations the frequency of initiation at the 5'-proximal AUG codon rather than the second was higher when the first AUG codon was in the preferred context, in qualitative agreement with the scanning ribosome model. However, a completely unexpected finding was that the ratio of initiation at the first AUG codon to initiation at the second decreased with increasing mRNA concentration, irrespective of the particular context involved. Several lines of evidence indicated that the increased frequency of initiation at the second AUG codon was not due solely to the lower density of ribosome loading per mRNA at high RNA concentrations, and may therefore be the result of high RNA concentrations out-titring the capacity of endogenous reticulocyte factors responsible for preferential initiation at the 5'-proximal AUG codon. The effect of supplementing the system with purified initiation factors was examined. Only eIF-2 was capable of decreasing the frequency of initiation at the second AUG codon and promoting use of the first AUG at high mRNA concentrations; eIF-3, 4A, 4B, 4C + 4D, 4F and 5 were inactive.

Immunoblot analysis of the structure of protein synthesis initiation factor eIF3 from HeLa cells.

Translational initiation factor eIF3 is a large, multisubunit protein complex involved in early steps of the initiation pathway. Affinity-purified polyclonal antibodies were used to analyze by immunoblotting the mass and charge characteristics of the subunits in HeLa cell lysates and in purified eIF3 preparations. The evidence indicates that eIF3 comprises at least seven antigenically distinct subunits: p170, p115, p66, p47, p44, p40, and p36. During purification, p170, p115, and p66 are partially degraded to smaller forms, which appear to be the major cause of variable subunit composition among preparations of eIF3.

Identification of the 80-kDa protein that crosslinks to the cap structure of eukaryotic mRNAs as initiation factor eIF-4B.

Preparations of either crude or purified protein synthesis initiation factors, when tested by crosslinking to the m7G-cap structure of mRNAs, exhibit specific crosslinking to an 80-kDa protein. Polyclonal antibodies specific for eIF-4B precipitate the 80-kDa cap-radiolabeled protein, thereby demonstrating that eIF-4B binds mRNA near its 5'-terminus.

Separation of protein synthesis initiation factor eIF4A from a p220-associated cap binding complex activity.

A cap binding complex activity was purified from HeLa cells by a procedure which does not depend on the use of cap-affinity chromatography. The activity co-purified with a Mr 220,000 polypeptide (p220), but not with eIF4A. The active complex therefore differs from eIF4F, the complex purified by cap analog-affinity chromatography, in that it lacks the Mr 50,000 subunit which is antigenically identical to eIF4A. The activities of eIF4F, CBP I and the eIF4A-free complex purified here were compared in a fractionated system translating capped globin mRNA. Results indicate that the two complexes have similar activities and that they perform a function which cannot be provided by CBP I alone. Cap binding complex activity can be partly separated from eIF4A activity on sucrose gradients, thus eIF4A provides a function that is distinct from cap binding complex activity. The results indicate that eIF4A can be physically separated from the cap binding complex without affecting the ability of the remaining structure to function in an in vitro translation system. They suggest that the eIF4A-free complex may provide a function that is not a property of either CBP I or of eIF4A, but may be a property of p220.

Regulated phosphorylation and low abundance of HeLa cell initiation factor eIF-4F suggest a role in translational control. Heat shock effects on eIF-4F.

Initiation factor eIF-4F, a multiprotein cap binding protein complex, was purified from HeLa cells by m7G affinity chromatography and independently by phosphocellulose column chromatography. The m7G affinity-purified sample contains three major proteins, p220, eIF-4A, and p28 (also known as CBP-I or eIF-4E). The abundancies of these proteins are roughly 2, 10, and 0.8 X 10(6) molecules/cell, respectively. Two-dimensional isoelectric focusing/sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the eIF-4F samples shows that p28 comprises two isoelectric variants, one of which labels with phosphate and disappears when samples are treated with alkaline phosphatase. The 45,000-dalton protein in eIF-4F appears to be identical to eIF-4A. The p220 subunit rarely produces discrete spots on two-dimensional gel electrophoresis; in purified samples it usually forms 3 closely spaced streaks. eIF-4F fractionated by phosphocellulose chromatography separates into forms containing either phosphorylated or unphosphorylated p28. However, both fractions possess similar specific activities in in vitro translation assays for eIF-4F activity. The phosphorylation of p28 decreases upon heat shock when protein synthesis is repressed. The correlation of dephosphorylation of p28 with the inhibition of protein synthesis and the relatively low abundance of the eIF-4F complex suggest that eIF-4F plays a role in the translational control of mRNA binding. Limitations of the in vitro assay system may account for the failure to detect phosphorylation-dependent activity differences.

Demonstration in vitro that eucaryotic initiation factor 3 is active but that a cap-binding protein complex is inactive in poliovirus-infected HeLa cells.

Protein synthesis initiation factor preparations from poliovirus-infected HeLa cells have reduced ability to initiate translation on capped mRNA. The defect in initiation factors has been variously attributed to inactivation of eucaryotic initiation factor 3 (eIF3), eIF4B, or a cap-binding protein (CBP) complex. We have developed a series of in vitro protein synthesis assays to show that eIF3 is active but a CBP complex activity is inactivated after poliovirus infection. eIF3 activity, when determined in the presence of purified CBP complex, is present in sucrose gradients of factors from both infected and uninfected cells. CBP complex activity, determined in the presence of eIF3 from poliovirus-infected cells, is present in uninfected cells only and comigrates on sucrose gradient with an activity which restores the ability of crude initiation factors from infected cells to translate capped globin mRNA. This is the first demonstration by a fractionated translation system that an activity which is attributable to CBP complex is inactivated in poliovirus-infected cells. The results also indicate that eIF3 is undetectable or has greatly reduced activity in the absence of CBP complex.

Involvement of eukaryotic initiation factor 4A in the cap recognition process.

Antibodies against eukaryotic initiation factor 4A (eIF-4A) were used to study the involvement of this factor in recognizing the 5' cap structure of eukaryotic mRNA. We demonstrate that an approximately 50-kilodalton polypeptide present in rabbit reticulocyte ribosomal high salt wash which can be specifically cross-linked to the 5' oxidized cap structure of reovirus mRNA (Sonenberg, N. (1981) Nucleic Acids Res. 9, 1643) reacts with an anti-eIF-4A monoclonal antibody. We also show that antibodies against eIF-4A react with a 50-kilodalton polypeptide present in a cap-binding protein complex obtained by elution from a m7GTP-agarose affinity column. Comparative peptide analysis of eIF-4A and the 50-kilodalton component of the cap-binding protein complex indicates a very strong similarity between the two polypeptides.

A comparison of the training responses to aerobic dance and jogging in college females.

The purpose of this study was to compare the physiological alterations that occur in college females as a result of a 7-wk jogging and aerobic dance-training program. Forty-six subjects (18-29 yr) volunteered to participate and included 15 dancers, 19 joggers, and 12 controls. All subjects were given a pre- and post-VO2max treadmill test. The joggers and dancers trained 4 d/wk, 30 min/d for 7 wk at an intensity that represented approximately 83 and 84% of their initial maximal heart rates, respectively. Both experimental groups significantly (P less than 0.05) increased their VO2max, VEmax, and maximal treadmill running times and significantly (P less than 0.05) decreased their maximal heart rates as a result of the training. The control group showed no significant (P greater than 0.05) changes in any of the variables measured. It was concluded that both aerobic dance and jogging were equally effective (P less than 0.05) exercise modalities for improving cardiorespiratory endurance when performed at similar intensities, frequencies, and durations.

Inhibition of HeLa cell protein synthesis following poliovirus infection correlates with the proteolysis of a 220,000-dalton polypeptide associated with eucaryotic initiation factor 3 and a cap binding protein complex.

Following poliovirus infection of HeLa cells, the synthesis of cellular proteins is inhibited but translation of poliovirus mRNA proceeds. The defect in the recognition of host cell mRNA may be due to a change in a cap recognition complex which, when added to an infected cell lysate, restores the ability to translate capped mRNAs. We employed immunoblotting techniques to examine initiation factors in crude lysates from uninfected and poliovirus-infected HeLa cells. Using an antiserum against eucaryotic initiation factor 3, we detected an antigen of approximate molecular weight 220,000 in uninfected cell lysates but not in infected cell lysates. Antigenically related polypeptides of 100,000 to 130,000 daltons, presumably degradation products, were detected in the infected cell lysate. The time course for degradation of the 220,000-dalton polypeptide correlates with that for inhibition of cellular protein synthesis in vivo. A portion of the population of 220,000-dalton polypeptides apparently associates with initiation factor eIF3 but is readily dissociated in buffers containing high salt. Affinity-purified antibodies against the polypeptide recognize a protein of the same size in a purified preparation of a cap binding protein complex obtained by cap-affinity chromatography. We postulate that the 220,000-dalton polypeptide is an essential component of the cap recognition complex and that its degradation in poliovirus-infected cells results in the inhibition of host cell translation. These results are in the first demonstration of a specific structural defect in an initiation factor resulting from poliovirus infection.

Protein synthesis initiation factors from human HeLa cells and rabbit reticulocytes are similar: comparison of protein structure, activities, and immunochemical properties.

Five initiation factors, eIF-2, eIF-3, eIF-4A, eIF-4B, and eIF-5, were purified from human HeLa cells. Methods of protein fractionation and assays for initiation factors which had been developed for rabbit reticulocytes were found to be suitable for HeLa factors. The initiation factors from HeLa cells are similar to or indistinguishable from the corresponding rabbit reticulocyte factors with respect to specific activities, molecular weights as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and subunit structure. The molecular weight of eIF-3 particles from both species is about 410000 as determined by equilibrium sedimentation analytical centrifugation. The partial protease fragmentation patterns of corresponding proteins also are similar and indicate that the primary sequences of the factors are related in the two species. Antisera raised in goats against rabbit eIF-3 and human eIF-2, eIF-4A, and eIF-4B cross-react with the cognate factors from both species. On the basis of immunoblotting techniques, eIF-4A is highly conserved, eIF-2 alpha, eIF-3, and eIF-4B are somewhat less conserved, and eIF-2 beta is the least conserved of the proteins examined. The functional, structural, and immunological results are all consistent with the view that initiation factors from different mammalian cells are very similar.

Mood changes as reported by women taking the oral contraceptive pill.

PIP: In an effort to clarify the nature of emotional side effects frequently reported by women using oral contraceptives (OCs), an exploratory study was undertaken of the verbal descriptions of side effects provided by a sample of young, healthy, sexually active women attending the Family Planning Association of Queensland clinics in Brisbane; they had been using OCs for at least 6 months. The adjectives used in unstructured interviews by 20 women happy with pills and 20 complaining of side effects formed the basis for a questionnaire comparing occurrence of emotions before and after pill use. 3 consecutive sets of adjectives were tested using Principle Factor Analysis and orthogonal rotation. The final questionnaire along with questions concerning social variables were given to 100 women and the scores from different subgroups were compared using the t test. 4 distinct, specific, and uncorrelated factors containing 53 adjectives were identified, of which "annoyed" and "tiredness" suggest negative emotional effects and "passionate" and "reassured" suggest positive effects. The distribution of factor scores in subgroups with statistically significant differences indicate that increases in "annoyed" and "tiredness" scores are found mainly in women who complain of emotional side effects, who were bothered by remembering to take the pill, worried about pill-related dangers, and gained excessive weight. Increases in "passionate" scores were found in noncohabiting women with steady sexual relationships, and increases in "reassured" were universal. The results demonstrate the multidimensional nature of reported emotional side effects from the pill and indicate that highly specific and unique mood changes which do not fit into existing notions of depression or anxiety are reported by women taking the pill.^ieng