Alcohol-induced pancreatic oxidative stress: protection by phospholipid repletion

Oxidative stress is considered to be a forerunner of pancreatitis. Since we had found polyenylphosphatidylcholine, a mixture of polyunsaturated phosphatidylcholines extracted from soybeans, to protect against hepatic oxidative stress, we now tested its effects on the pancreas. Sprague-Dawley rats were pair-fed for two months nutritionally adequate liquid diet containing ethanol (36% of energy) or isocaloric carbohydrate, with either polyenylphosphatidylcholine (3 g/1000 kcal) or safflower oil, with or without 5 g/1000 kcal carbonyl iron. Parameters of oxidative stress (F2-isoprostanes, 4-hydroxynonenal, reduced glutathione), ubiquinol-10, ubiquinol-9 and vitamin E, as well as phosphatidylcholine species, were assessed by GC/MS and/or HPLC. Alcohol feeding increased pancreatic 4-hydroxynonenal three-fold, F2-isoprostanes and ubiquinol-9 by more than 70%, whereas it decreased total phospholipids, several phosphatidylcholine species, ubiquinol-10 and glutathione, especially in iron fed rats. Polyenylphosphatidylcholine prevented the rise in 4-hydroxynonenal and F2-isoprostanes, the decrease in dilinoleoylphosphatidylcholine and oleoyllinoleoylphosphatidylcholine and opposed the alcohol-induced decrease of glutathione; alpha-tocopherol remained unchanged. Iron had no significant effect except for decreasing ubiquinol-10 in the pancreas and increasing aminotransferases in the plasma. Thus, the alcohol-induced oxidative stress in the pancreas was shown to be prevented by polyenylphosphatidylcholine which may act, in part, by correcting the depletion of several phosphatidylcholine species.     

Dilinoleoylphosphatidylcholine decreases ethanol-induced cytochrome P4502E1.

Cytochrome P4502E1 (CYP2E1) induction by ethanol contributes to alcoholic liver disease and we found that a mixture of polyunsaturated phosphatidylcholines (PPC), which protects against alcohol-induced liver injury, also decreases CYP2E1. Since dilinoleoylphosphatidylcholine (DLPC) is the major component of PPC, we assessed here whether it is responsible for the protection of PPC by feeding rats for 8 weeks our liquid diet containing ethanol (36% of energy) or isocaloric carbohydrates, with either DLPC (1.5 g/1000 cal), PPC (3 g/1000 cal), or linoleate. CYP2E1 was assessed by Western blots and by two of its enzyme activities: the microsomal ethanol-oxidizing system (MEOS) and p-nitrophenolhydroxylase (PNP). With ethanol, CYP2E1 increased 10-fold, with corresponding rises in PNP and MEOS activities. Compared to linoleate, DLPC significantly decreased cytochrome b(5), total cytochromes P450, CYP2E1 content and its corresponding activities. DLPC decreases ethanol-induced CYP2E1 and should be considered for the prevention of alcoholic liver disease.     

A novel disorder caused by defective biosynthesis of N-linked oligosaccharides due to glucosidase I deficiency

Glucosidase I is an important enzyme in N-linked glycoprotein processing, removing specifically distal alpha-1,2-linked glucose from the Glc3Man9GlcNAc2 precursor after its en bloc transfer from dolichyl diphosphate to a nascent polypeptide chain in the endoplasmic reticulum. We have identified a glucosidase I defect in a neonate with severe generalized hypotonia and dysmorphic features. The clinical course was progressive and was characterized by the occurrence of hepatomegaly, hypoventilation, feeding problems, seizures, and fatal outcome at age 74 d. The accumulation of the tetrasaccharide Glc(alpha1-2)Glc(alpha1-3)Glc(alpha1-3)Man in the patient's urine indicated a glycosylation disorder. Enzymological studies on liver tissue and cultured skin fibroblasts revealed a severe glucosidase I deficiency. The residual activity was <3% of that of controls. Glucosidase I activities in cultured skin fibroblasts from both parents were found to be 50% of those of controls. Tissues from the patient subjected to SDS-PAGE followed by immunoblotting revealed strongly decreased amounts of glucosidase I protein in the homogenate of the liver, and a less-severe decrease in cultured skin fibroblasts. Molecular studies showed that the patient was a compound heterozygote for two missense mutations in the glucosidase I gene: (1) one allele harbored a G-->C transition at nucleotide (nt) 1587, resulting in the substitution of Arg at position 486 by Thr (R486T), and (2) on the other allele a T-->C transition at nt 2085 resulted in the substitution of Phe at position 652 by Leu (F652L). The mother was heterozygous for the G-->C transition, whereas the father was heterozygous for the T-->C transition. These base changes were not seen in 100 control DNA samples. A causal relationship between the alpha-glucosidase I deficiency and the disease is postulated.     

Effect of postnatal development on calcium currents and slow charge movement in mammalian skeletal muscle

Single- (whole-cell patch) and two-electrode voltage-clamp techniques were used to measure transient (Ifast) and sustained (Islow) calcium currents, linear capacitance, and slow, voltage-dependent charge movements in freshly dissociated fibers of the flexor digitorum brevis (FDB) muscle of rats of various postnatal ages. Peak Ifast was largest in FDB fibers of neonatal (1-5 d) rats, having a magnitude in 10 mM external Ca of 1.4 +/- 0.9 pA/pF (mean +/- SD; current normalized by linear fiber capacitance). Peak Ifast was smaller in FDB fibers of older animals, and by approximately 3 wk postnatal, it was so small as to be unmeasurable. By contrast, the magnitudes of Islow and charge movement increased substantially during postnatal development. Peak Islow was 3.6 +/- 2.5 pA/pF in FDB fibers of 1-5-d rats and increased to 16.4 +/- 6.5 pA/pF in 45-50-d-old rats; for these same two age groups, Qmax, the total mobile charge measurable as charge movement, was 6.0 +/- 1.7 and 23.8 +/- 4.0 nC/microF, respectively. As both Islow and charge movement are thought to arise in the transverse-tubular system, linear capacitance normalized by the area of fiber surface was determined as an indirect measure of the membrane area of the t-system relative to that of the fiber surface. This parameter increased from 1.5 +/- 0.2 microF/cm2 in 2-d fibers to 2.9 +/- 0.4 microF/cm2 in 44-d fibers. The increases in peak Islow, Qmax, and normalized linear capacitance all had similar time courses. Although the function of Islow is unknown, the substantial postnatal increase in its magnitude suggests that it plays an important role in the physiology of skeletal muscle.     

Desmoglein 2 is a receptor for adenovirus serotypes 3, 7, 11 and 14

We have identified desmoglein-2 (DSG-2) as the primary high-affinity receptor used by adenoviruses Ad3, Ad7, Ad11 and Ad14. These serotypes represent key human pathogens causing respiratory and urinary tract infections. In epithelial cells, adenovirus binding of DSG-2 triggers events reminiscent of epithelial-to-mesenchymal transition, leading to transient opening of intercellular junctions. This opening improves access to receptors, for example, CD46 and Her2/neu, that are trapped in intercellular junctions. In addition to complete virions, dodecahedral particles (PtDds), formed by excess amounts of viral capsid proteins, penton base and fiber during viral replication, can trigger DSG-2-mediated opening of intercellular junctions as shown by studies with recombinant Ad3 PtDds. Our findings shed light on adenovirus biology and pathogenesis and may have implications for cancer therapy.     

Modulation of social interactions by immune stimulation in honey bee, <Emphasis Type="Italic">Apis mellifera</Emphasis>, workers

Background  

Immune response pathways have been relatively well-conserved across animal species, with similar systems in both mammals and invertebrates. Interestingly, honey bees have substantially reduced numbers of genes associated with immune function compared with solitary insect species. However, social species such as honey bees provide an excellent environment for pathogen or parasite transmission with controlled environmental conditions in the hive, high population densities, and frequent interactions. This suggests that honey bees may have developed complementary mechanisms, such as behavioral modifications, to deal with disease.     

A theoretical entropy score as a single value to express inhibitor selectivity

Background  

Designing maximally selective ligands that act on individual targets is the dominant paradigm in drug discovery. Poor selectivity can underlie toxicity and side effects in the clinic, and for this reason compound selectivity is increasingly monitored from very early on in the drug discovery process. To make sense of large amounts of profiling data, and to determine when a compound is sufficiently selective, there is a need for a proper quantitative measure of selectivity.     

Genome-wide association and genomic prediction for host response to porcine reproductive and respiratory syndrome virus infection

Background

Host genetics has been shown to play a role in porcine reproductive and respiratory syndrome (PRRS), which is the most economically important disease in the swine industry. A region on Sus scrofa chromosome (SSC) 4 has been previously reported to have a strong association with serum viremia and weight gain in pigs experimentally infected with the PRRS virus (PRRSV). The objective here was to identify haplotypes associated with the favorable phenotype, investigate additional genomic regions associated with host response to PRRSV, and to determine the predictive ability of genomic estimated breeding values (GEBV) based on the SSC4 region and based on the rest of the genome. Phenotypic data and 60 K SNP genotypes from eight trials of ~200 pigs from different commercial crosses were used to address these objectives.

Results

Across the eight trials, heritability estimates were 0.44 and 0.29 for viral load (VL, area under the curve of log-transformed serum viremia from 0 to 21 days post infection) and weight gain to 42 days post infection (WG), respectively. Genomic regions associated with VL were identified on chromosomes 4, X, and 1. Genomic regions associated with WG were identified on chromosomes 4, 5, and 7. Apart from the SSC4 region, the regions associated with these two traits each explained less than 3% of the genetic variance. Due to the strong linkage disequilibrium in the SSC4 region, only 19 unique haplotypes were identified across all populations, of which four were associated with the favorable phenotype. Through cross-validation, accuracies of EBV based on the SSC4 region were high (0.55), while the rest of the genome had little predictive ability across populations (0.09).

Conclusions

Traits associated with response to PRRSV infection in growing pigs are largely controlled by genomic regions with relatively small effects, with the exception of SSC4. Accuracies of EBV based on the SSC4 region were high compared to the rest of the genome. These results show that selection for the SSC4 region could potentially reduce the effects of PRRS in growing pigs, ultimately reducing the economic impact of this disease.     

The effect of chronic alcohol feeding on lipid peroxidation in microsomes: lack of relationship to hydroxyl radical generation

Chronic alcohol feeding causes microsomal induction including increased generation of hydroxyl radicals. Ethanol induced liver injury may be mediated by lipid peroxidation for which hydroxyl radicals have been proposed as major mediators. Ethanol promotes lipid peroxidation when given acutely but also may serve as a hydroxyl radical scavenger. Therefore, we studied the acute and chronic effects of alcohol on microsomal lipid peroxidation and hydroxyl radical generation. Chronic alcohol feeding in rats increased microsomal generation of hydroxyl radicals but lipid peroxidation of endogenous lipid was inversely related to hydroxyl radical generation. Ethanol (50mM) had a slight inhibitory effect on hydroxyl radical production in peroxidizing microsomes, no effect on endogenous lipid peroxidation and enhanced the lysis of RBCs added as targets of peroxidation. Enhanced microsomal generation of hydroxyl radicals following chronic alcohol feeding is not an important mediator of lipid peroxidation.     

The characterization of a mammalian DNA structure-specific endonuclease.

The repair of some types of DNA double-strand breaks is thought to proceed through DNA flap structure intermediates. A DNA flap is a bifurcated structure composed of double-stranded DNA and a displaced single-strand. To identify DNA flap cleaving activities in mammalian nuclear extracts, we created an assay utilizing a synthetic DNA flap substrate. This assay has allowed the first purification of a mammalian DNA structure-specific nuclease. The enzyme described here, flap endonuclease-1 (FEN-1), cleaves DNA flap strands that terminate with a 5' single-stranded end. As expected for an enzyme which functions in double-strand break repair flap resolution, FEN-1 cleavage is flap strand-specific and independent of flap strand length. Furthermore, efficient flap cleavage requires the presence of the entire flap structure. Substrates missing one strand are not cleaved by FEN-1. Other branch structures, including Holliday junctions, are also not cleaved by FEN-1. In addition to endonuclease activity, FEN-1 has a 5'-3' exonuclease activity which is specific for double-stranded DNA. The endo- and exonuclease activities of FEN-1 are discussed in the context of DNA replication, recombination and repair.