Phosphorylation of tobacco eukaryotic translation initiation factor 4A upon pollen tube germination

Eukaryotic translation initiation factor eIF-4A is a member of the DEAD box family of RNA helicases and RNA-dependent ATPases. In tobacco, eIF-4A is encoded by a gene family with one isoform, eIF-4A8, being exclusively expressed in pollen. This pollen-specific isoform is a candidate for mediating translational control in the developing gametophyte. Here we show that eIF-4A is barely phosphorylated in mature pollen, but during pollen tube germination, two isoforms of eIF-4A become phosphorylated. Phosphoamino acid analysis indicated phosphorylation of threonine. In order to determine whether pollen-specific eIF-4A8 is among the phosphorylated isoforms, we raised transgenic tobacco plants overexpressing eIF-4A8 containing a histidine tag. Hereby, we could show that indeed eIF-4A8 is modified through phosphorylation. The biological relevance of the phosphorylation of eIF-4A is discussed.     

Adenovirus-mediated gene transfer of inhibitors of apoptosis protein delays apoptosis in cerebellar granule neurons

The inhibitor of apoptosis (IAP) family of antiapoptotic genes, originally discovered in baculovirus, exists in animals ranging from insects to humans. Here, we investigated the ability of IAPs to suppress cell death in both a neuronal model of apoptosis and excitotoxicity. Cerebellar granule neurons undergo apoptosis when switched from 25 to 5 mM potassium, and excitotoxic cell death in response to glutamate. We examined the endogenous expression of four members of the IAP family, X chromosome-linked IAP (XIAP), rat IAP1 (RIAP1), RIAP2, and neuronal apoptosis inhibitory protein (NAIP), by semiquantitative reverse PCR and immunoblot analysis in cultured cerebellar granule neurons. Cerebellar granule neurons express significant levels of RIAP2 mRNA and protein, but expression of RIAP1, NAIP, and XIAP was not detected. RIAP2 mRNA content and protein levels did not change when cells were switched from 25 to 5 mM potassium. To determine whether ectopic expression of IAP influenced neuronal survival after potassium withdrawal or glutamate exposure, we used recombinant adenoviral vectors to target XIAP, human IAP1 (HIAP1), HIAP2, and NAIP into cerebellar granule neurons. We demonstrate that forced expression of IAPs efficiently blocked potassium withdrawal-induced N-acetyl-Asp-Glu-Val-Asp-specific caspase activity and reduced DNA fragmentation. However, neurons were only protected from apoptosis up to 24 h after potassium withdrawal, but not at later time points, suggesting that IAPs delay but do not block apoptosis in cerebellar granule neurons. In contrast, treatment with 100 microM or 1 mM glutamate did not induce caspase activity and adenoviral-mediated expression of IAPs had no influence on subsequent excitotoxic cell death.     

Endotoxin impairs the engraftment of rat islets transplanted beneath the kidney capsule of C57BL/6-mice

The primary objective of this investigation was to determine the effect of endotoxin on islet xenograft survival within the first three days after transplantation. Pancreatic islets from Lewis rats were prepared under endotoxin-free conditions with Liberase (Boehringer) and purified by centrifugation on endotoxin-free Ficoll/Histopaque. After overnight incubation, with or without 10 microg/ml endotoxin, the islets were transplanted beneath the kidney capsule of normoglycemic C57Bl/6-mice. Three days later, kidneys were removed and their insulin content were measured. We could demonstrate significant differences (P<0.01) in insulin recovery between lipopolysaccharide-free and lipopolysaccharide-containing grafts. In case of endotoxin contaminated islets, we found only 13+/-2% (n=9) of the original insulin content, in contrast to 53+/-7% (n=9) when endotoxin-free islets where grafted. In experiments with islets isolated by use of conventional (lipopolysaccharide-containing) collagenase, and then cultured in endotoxin-free medium, insulin recovery three days after transplantation was 36+/-1% (n=13).     

Differences in prostate size between patients from University and Veterans Affairs Medical Center populations

We have previously determined that Nippostrongylus brasiliensis secretes three monomeric nonamphiphilic (G1na) variants of acetylcholinesterase (AChE) with broadly similar properties. In this study we have examined AChE expression in somatic extracts of N. brasiliensis and report the identification of an additional enzyme which is not secreted. The enzyme was resolved by sucrose density gradient centrifugation with a sedimentation coefficient of 10.2 S which was shifted to 9.4 S in the presence of Triton X-100, identifying the enzyme as a tetrameric amphiphilic (G4a) form. The amphiphilic properties of this enzyme were confirmed by charge-shift electrophoresis, in which migration was accelerated by interaction with sodium deoxycholate. The enzyme showed low activity with butyrylthiocholine, and a Michaelis constant of 91 +/- 13 microM for acetylthiocholine was determined. It was highly sensitive to the AChE-specific inhibitor bis (4-allyldimethylammoniumphenyl)pentan-3-one dibromide, with an IC50 of 6.5 +/- 0.4 microM, but was also inhibited by the butyrylcholinesterase-specific inhibitor tetramonoisopropylpyrophosphortetramide, albeit with a higher IC50 of 46.5 +/- 6.1 microM. This enzyme can therefore be distinguished from the secreted AChEs by its amphiphilic properties, sedimentation in sucrose gradients, and sensitivity to cholinesterase inhibitors.     

Monitoring of impurities using high-performance liquid chromatography with diode-array detection: eigenvalue plots of partially overlapping tailing peaks

The chromatogram of ropinirole in the presence of about 5% of a closely eluting impurity, obtained by HPLC with diode-array detection, was analysed by chemometric procedures. Log eigenvalue plots were used to determine the relative composition of regions of the chromatogram. It is shown that since the peaks exhibit tailing, unusual behaviour is found in the plots. This is verified by performing simulations, in which it is demonstrated that peak asymmetry has a pronounced influence on this chemometric approach. In many cases of liquid chromatographic analysis, asymmetric peak shapes are encountered and methods for peak purity assessment should be re-evaluated in the light of these asymmetries.     

Of sick turkeys, kwashiorkor, malaria, perinatal mortality, heroin addicts and food poisoning: research on the influence of aflatoxins on child health in the tropics

Similarities between the geographical and climatic prevalences of kwashiorkor and of exposure to dietary aflatoxins, and between the biochemical, metabolic and immunological derangements in kwashiorkor and those in animals exposed to aflatoxins, prompted investigation of the associations between kwashiorkor and aflatoxins. Studies in Africa in the 1980s indicated a role for these toxins in the pathogenesis of the disease. Paediatric cases of kwashiorkor are less prone to severe Plasmodium falciparum malaria than normal children. In mice infected with P. berghei, aflatoxin exposure inhibits parasite growth and ameliorates morbidity. Aflatoxins occur in < or = 40% of samples of breast milk from tropical Africa, usually as low concentrations of the relatively non-toxic derivatives of aflatoxin B1 (AFB1) but sometimes as high concentrations of the very toxic AFB1. This could explain kwashiorkor in breast-fed babies. Aflatoxin exposure occurs in > or = 30% of pregnancies in tropical Africa and the toxins are often in cord blood, sometimes at extremely high concentrations. Aflatoxins are now incriminated in neonatal jaundice and there is circumstantial evidence that they cause perinatal death and reduced birthweight. Aflatoxin-induced immunosuppresion may explain the aggressive behaviour of HIV infection in Africa. There are similarities between observations on HIV cases in Africa and those on heroin addicts in Europe, where 'street' heroin is frequently contaminated with aflatoxin. Aflatoxins were found in 20% of random urine samples from heroin addicts in the U.K. and the Netherlands. Aflatoxins have also been incriminated in episodes of food poisoning which have been associated with serious morbidity and mortality, particularly among young children.     

Danger ideation reduction therapy (DIRT): preliminary findings with three obsessive-compulsive washers

Three obsessive-compulsive patients received Danger Ideation Reduction Therapy (DIRT) in an initial treatment trial. All three subjects presented with contamination/washing concerns but refused to participate in exposure and response prevention. DIRT is solely directed at decreasing danger-related expectancies concerning contamination. DIRT procedures do not attempt to address inflated personal responsibility. In addition, DIRT does not involve direct or filmed exposure to contamination-related stimuli, or behavioural experiments. Components of DIRT include corrective information cognitive restructuring, filmed interviews, microbiological experiments, attentional focusing and Hoekstra's (1989) probability of catastrophe estimation task. Treatment consisted of between six and ten 1-hr weekly sessions. At post-treatment, substantial reductions in scores on the Padua Inventory, Maudsley Obsessional-Compulsive Inventory and two global rating scales were apparent for all subjects. These improvements were maintained at a 3-month follow-up. The theoretical and clinical implications of these preliminary findings are discussed.     

Nationwide survey of fluoroscopy: radiation dose and image quality

PURPOSE: To determine the average abdominal entrance air kerma, low-contrast sensitivity, and spatial resolution in upper gastrointestinal tract fluoroscopy in the United States. MATERIALS AND METHODS: A random sample of fluoroscopic facilities was selected to be surveyed for the Nationwide Evaluation of X-ray Trends program. Measurements were performed by using a newly developed fluoroscopic phantom. The surveys were conducted by state radiation control personnel. RESULTS: Average air kerma rates 1 cm above the tabletop, free in air, were 43 mGy/min (n = 340). The rate increased to 64 mGy/min when a 1.6-mm-thick copper filter, which simulated the use of barium contrast medium, was added to increase attenuation. The average entrance air kerma, free in air, for radiographs was 3.4 mGy, and an average of 12 radiographs were obtained per examination. Of 352 facilities surveyed, 306 (87%) were able to resolve wire mesh with 20 or more lines per inch. Of 339 facilities for which percentage contrast could be calculated, 192 (57%) had minimum percentage contrast values of 4% or more. CONCLUSION: Spatial resolution for fluoroscopy is adequate for most of the facilities surveyed, but a substantial proportion of facilities could not visualize low-contrast test objects, which strongly suggests image quality problems.