Two structural domains of initiation factor eIF-4B are involved in binding to RNA

Translation initiation factor eIF-4B promotes the binding of mRNA to 40 S preinitiation complexes and together with eIF-4A possesses RNA helicase activity. To elucidate structural features involved in its function, a series of internal and C-terminal deletions, as well as point mutations, were constructed in the eIF-4B cDNA. The mutated cDNAs were expressed in transiently transfected COS-1 cells, and mutant forms of the factor were overproduced up to about 25-fold over endogenous eIF-4B levels. Inhibition of dihydrofolate reductase (DHFR) synthesis by high levels of eIF-4B variants was determined in vivo, and the binding of the eIF-4B forms to biotinylated RNA was measured in vitro. The results indicate that the N-terminal region containing the RNA binding motif with its RNP1 and RNP2 consensus elements is sufficient for inhibition of DHFR synthesis. Deletion of the RNP1 sequence abrogates RNA binding, but amino acid substitutions at conserved residues do not always inhibit RNA binding. Deletion of the DRYG domain near the middle of eIF-4B results in inhibition of RNA binding, but not of DHFR synthesis. Up to 164 residues of the C terminus are not required for RNA binding, but removal of 226 or more residues completely inhibits RNA binding, perhaps by the loss of two arginine-rich regions. The results suggest that both the RNA recognition motif and the arginine-rich region are required for stable RNA binding but that both are not necessary for in vivo inhibition of protein synthesis.     

Permanent neuronal cell loss in the inferior olive of adult rats exposed to alcohol during the brain growth spurt: a stereological investigation

The purpose of this study was to examine whether exposure of rat pups to alcohol postnatally over a period of brain development similar to that of the human 3rd trimester results in a permanent loss of cells in the inferior olivary nucleus. It was hypothesized that a deficit of neurons in the inferior olive, the sole source of climbing fibers, may contribute to the cerebellar dysfunction observed following exposure to alcohol during development. Sprague-Dawley rat pups were artificially reared and administered alcohol over postnatal days 4-9. One artificially reared group received a daily alcohol dose of 4.5 g/kg, administered as a 10.2% solution in 2 of 12 daily feedings (10.2% group). This pattern of alcohol administration resulted in high peak blood alcohol concentrations with near total clearance. The other artificially reared group was fed a diet made isocaloric to the alcohol-containing diet (gastrostomy control group). Pups were allowed to grow to adulthood and killed on postnatal day 115. The total number of neurons in the inferior olivary nucleus was estimated using unbiased stereological methods. Exposure to alcohol resulted in a significant deficit in the number of neurons in the inferior olive at 115 days of age. The total number of neurons in the alcohol-exposed group was 40.12 +/- 8.7 x 10(3), compared with 53.37 +/- 3.7 x 10(3) in the artificially reared controls. These results indicate that there is a permanent deficit of neurons in the inferior olive after postnatal exposure to alcohol.(ABSTRACT TRUNCATED AT 250 WORDS)     

The detection and documentation of trace wound patterns by use of an alternative light source

This article is a discussion of the use of narrow-band light sources coupled with cameras equipped with band-pass filters to document patterned injuries on human skin. Several case reports are included.     

Outcome of patients with haemorrhagic shock: an indicator of performance in a trauma centre

The survival rate of patients admitted to the Royal Prince Alfred Hospital (RPAH) from the central Sydney area with a major injury has improved since regionalization of trauma services in early 1992. This improvement has been attributed to education, better hospital care and changing trauma epidemiology. This study was conducted to assess whether the outcome of patients admitted with haemorrhagic shock had improved. This is proposed as a more subtle indicator of hospital performance than overall survival rates. A prospective before and after study was carried out comparing outcome in the 18 months preceding 1 January 1992 with that in the subsequent 18 months. Entry criteria to the study included all primary retrievals from the central Sydney area to RPAH with injury severity scores (ISS) > 15. Outcomes were compared generally and in those who were in a state of haemorrhagic shock (systolic blood pressure < or = 90 mmHg) on arrival at the emergency department. Three hundred and eight patients were entered into the study. Stratification showed similar numbers, demographic features and mechanisms of injury in the two groups. Forty patients were in a state of haemorrhagic shock on admission. The overall mortality was reduced from 31% to 11% (P < 0.001) over the two phases of the study. The mortality from blood loss in the 40 shocked patients fell from 10/25 in the first period to 2/15 (P = 0.07) in the latter. These improved survival rates were felt to reflect the value of the educational and organizational initiatives introduced following designation of the hospital as a trauma centre.     

Effects of amount of whole cottonseed on intake, digestibility, and physiological responses of dairy cows

In Experiment 1, 27 lactating cows were fed complete rations ad libitum of 0, 15, and 30% whole cottonseed to examine the effects on intake, digestibility, blood gases, and blood metabolites. Dry matter intake declined linearly with increased cottonseed, but because of greater energy density, calculated net energy for lactation intake was not depressed significantly. Ether extract, crude protein, and calcium digestibility increased with cottonseed in the diet. Respiration rates declined with dry matter intake and increased cottonseed feeding; some blood gases were influenced by cottonseed feeding but not in a detrimental way. No data among 13 blood metabolites indicated effects of gossypol toxicity with 30% whole cottonseed in the diet. In Experiment 2, 24 dry, nonpregnant Holstein cows were offered complete rations ad libitum of 15, 35, and 55% whole cottonseed to measure responses in intake, respiration rates, and blood metabolites. The highest cottonseed diet significantly depressed intake of both dry matter and calculated net energy for lactation. Maximum dry matter from cottonseed eaten by an individual was 8.4 kg/d (average for 1 wk). Respiration rates declined parallel to intake. No evidence for gossypol toxicity was seen among data of 11 metabolites in blood.     

MIM capacitor fabrication and assessment for GaAs MMICs

Metal-insulator-metal, MIM, capacitors have been fabricated using plasma deposited silicon nitride, SiN_x, films deposited under varying deposition conditions. The electrical properties of the MIM capacitors and the corresponding physical properties of the SiN_x films have been determined. The breakdown field strength of the films, which varied between 0.4–3.0 MVcm⁻¹, has been related to the amount of hydrogen incorporated in the SiN_x layers during deposition. Frenkel-Poole conduction through the silicon nitride has been observed at room temperature and this conduction mechanism is shown to be predominant and independent of the breakdown field strength, for the films investigated.     

Neuropathology of bovine herpesvirus type 5 (BHV-5) meningo-encephalitis in a rabbit seizure model

CI-994 (acetyldinaline) is an orally active anticancer drug currently in Phase 1 clinical trials. To assess its preclinical toxicity, CI-994 was administered orally as suspensions to Wistar rats (10/sex/dose) and in capsules to beagle dogs (3/sex/dose) once daily for two weeks. Doses were 1.5, 5, and 15 mg/kg for rats (9, 30, and 90 mg/m2, respectively), and 0.5, 2, and 5 mg/kg for dogs (10, 40, and 100 mg/m2, respectively). Systemic exposure was dose-proportional based on toxicokinetic analysis in dogs. Severe clinical signs and mortality occurred at the highest dose in both species beginning on Day 10. Neutropenia, lymphocytopenia, thrombocytopenia, lymphoid depletion, bone marrow hypocellularity, and testicular degeneration were observed in both species, primarily at the mid- and high-doses. Despite continued treatment, neutrophil counts in dogs returned to control levels in Week 2. Other microscopic findings in rats included splenic hematopoietic depletion at all doses and epithelial cell necrosis in various tissues at 15 mg/kg. Additional bone marrow changes in dogs involved myeloid and megakaryocyte hyperplasia at 2 mg/kg and abnormal myeloid and megakaryocyte maturation at 2 and 5 mg/kg. Except for the testicular effects in both species, all changes were reversible within a 4-week (rat) or 9-week (dog) recovery period. The results of these studies show that target organ effects of CI-994 principally involve tissues with rapidly dividing cell populations and that bone marrow suppression is the dose-limiting toxicity. CI-994 also seems to interfere with the release and/or maturation of cells in the bone marrow.     

Expression and circular dichroism studies of the extracellular domain of the alpha subunit of the nicotinic acetylcholine receptor

To provide material suitable for structural studies of the nicotinic acetylcholine receptor, we have expressed and purified the NH2-terminal extracellular domain of the mouse muscle alpha subunit. Several constructs were initially investigated using Xenopus oocytes as a convenient small scale expression system. A fusion protein (alpha210GPI) consisting of the 210 NH2-terminal amino acids of the alpha subunit and a glycosylphosphatidylinositol anchorage sequence conferred surface alpha-bungarotoxin binding in oocytes. Coexpression of alpha210GPI with an analogous construct made from the delta subunit showed no evidence of heterodimer formation. The alpha210GPI protein was chosen for large scale expression in transfected Chinese hamster ovary cells. The alpha210GPI protein was cleaved from these cells and purified on an immunoaffinity column. Gel and column chromatography show that the purified protein is processed as expected and exists as a monomer. The purified protein also retains the two distinct, conformation-specific binding sites expected for the correctly folded alpha subunit. Circular dichroism studies of alpha210GPI suggest that this region of the receptor includes considerable beta-sheet secondary structure, with a small proportion of alpha-helix.