Growth and body composition changes in mice selected for high post-weaning weight gain on two levels of feeding

Summary Two lines of mice were selected for high post-weaning weight gain (3 to 6 weeks) adjusted for 3 week weight. One line (F) was grown on freely available food and the other (S) on a feeding scale set at the same level for all mice. Food intake of the S line averaged 80% of the F line. The realised heritabilities after 6 generations of selection were 0.38±0.06 and 0.33±0.07 for the F and S lines, respectively. In generation 7, mice from the F and S lines and from an unselected control line (C) were compared on both free and set levels of feeding from 3 weeks to 9 weeks of age. Measurements taken were growth rate, appetite, food conversion efficiency (weight gain/food intake) and body composition (fat, protein, ash, water). The F and S lines grew more rapidly and efficiently than the C line on both levels of feeding, each line performing best on the level of feeding on which it was selected. The average genetic correlation between growth rates of the same line on the two feeding levels was 0.54±0.10. The F line grew 19% faster and was 9% more efficient than the S line on free feeding but the S line grew 15% faster and was 15% more efficient than the F line on set feeding. Relative to the C line, food intake per day on free feeding was 4% higher in the F line and 6% lower in the S line. There was no difference between the lines in food intake/g body weight. The rate of deposition of all body components increased in both selection lines. In the F, S and C lines respectively, efficiencies of gains in body components (10²x gain/food) were 1.79, 1.31 and 1.06 for fat, 1.53, 1.63 and 1.22 for protein and 5.88, 6.45 and 4.98 for protein + water. Apparently energy lost as heat was reduced in both the F and S lines. The partitioning of energy retained was altered in favour of more fat in the F line and more protein in the S line.     

How house officers cope with their mistakes.

We examined how house officers coped with serious medical mistakes to gain insight into how medical educators should handle these situations. An anonymous questionnaire was mailed to 254 house officers in internal medicine asking them to describe their most important mistake and their response to it; 45% (N = 114) reported a mistake and completed the questionnaire. House officers experienced considerable emotional distress in response to their mistakes and used a variety of strategies to cope. In multivariate analysis, those who coped by accepting responsibility were more likely to make constructive changes in practice, but to experience more emotional distress. House officers who coped by escape-avoidance were more likely to report defensive changes in practice. For house officers who have made a mistake, we suggest that medical educators provide specific advice about preventing a recurrence of the mistake, provide emotional support, and help them understand that distress is an expected concomitant of learning from the experience.     

The lepidopteran mitochondrial control region: structure and evolution

For several species of lepidoptera, most of the approximately 350-bp mitochondrial control-region sequences were determined. Six of these species are in one genus, Jalmenus; are closely related; and are believed to have undergone recent rapid speciation. Recent speciation was supported by the observation of low interspecific sequence divergence. Thus, no useful phylogeny could be constructed for the genus. Despite a surprising conservation of control-region length, there was little conservation of primary sequences either among the three lepidopteran genera or between lepidoptera and Drosophila. Analysis of secondary structure indicated only one possible feature in common--inferred stem loops with higher-than-random folding energies-- although the positions of the structures in different species were unrelated to regions of primary sequence similarity. We suggest that the conserved, short length of control regions is related to the observed lack of heteroplasmy in lepidopteran mitochondrial genomes. In addition, determination of flanking sequences for one Jalmenus species indicated (i) only weak support for the available model of insect 12S rRNA structure and (ii) that tRNA translocation is a frequent event in the evolution of insect mitochondrial genomes.      

Inhibition of human immunodeficiency virus replication by the herpes simplex virus virion host shutoff protein.

The herpes simplex virus (HSV) virion host shutoff gene (vhs) encodes a protein which nonspecifically accelerates the degradation of mRNA molecules, leading to inhibition of protein synthesis. This ability to inhibit a critical cellular function suggested that vhs could be used as a suicide gene in certain gene therapy applications. To investigate whether vhs might be useful for treatment of AIDS, we tested the ability of both HSV type 1 (HSV-1) and HSV-2 vhs to inhibit replication of human immunodeficiency virus (HIV). Replication of HIV was substantially inhibited when an infectious HIV proviral clone was cotransfected into HeLa cells together with vhs under the control of the cytomegalovirus (CMV) immediate-early promoter. HSV-2 vhs was more active than HSV-1 vhs in these experiments, consistent with previously published studies on these genes. Since expression of vhs from the CMV promoter is essentially unregulated, we also tested the ability of vhs expressed from the HIV long terminal repeat (LTR) promoter to inhibit HIV replication. Wild-type HSV-1 vhs inhibited HIV replication more than 44,000-fold in comparison to a mutant vhs gene encoding a nonfunctional form of the Vhs protein. Production of Vhs in transfected cells was verified by Western blot assays. A larger amount of Vhs was observed in cells transfected with plasmids expressing vhs from the HIV LTR than from the CMV promoter, consistent with the greater inhibition of HIV replication observed with these constructs. Mutant forms of Vhs were expressed at higher levels than wild-type Vhs, most likely due to the ability of wild-type Vhs to degrade its own mRNA. The strong inhibitory activity of the vhs gene and its unique biological properties make vhs an interesting candidate for use as a suicide gene for HIV gene therapy.     

The effects of antidiuretic hormone on urine flow and composition in the chronically-cannulated ovine fetus.

The fetuses of nine pregnant ewes were chronically cannulated between 86 and 130 days with cannulae in one carotid artery, one jugular vein, the fetal bladder and the amniotic cavity. The effects of infused AVP on fetal urine flow rate and composition were studied. A dose of 35 pmol. h-1 always caused an increase in urine osmolality and a decrease in flow rate and free water clearance without change in blood pressure. Higher doses (140 pmol.h-1) were significantly pressor and caused increased excretion of sodium, chloride, urea and creatinine. Although this dose always caused an increase in urine osmolality, in five experiments the flow rate also rose. It is concluded that the ovine fetus has the ability to control its own urine flow and composition, and possibly also amniotic fluid volume and composition.     

Identification of factors that function in Drosophila salivary gland cell death during development using proteomics

Proteasome inhibitors induce cell death and are used in cancer therapy, but little is known about the relationship between proteasome impairment and cell death under normal physiological conditions. Here, we investigate the relationship between proteasome function and larval salivary gland cell death during development in Drosophila. Drosophila larval salivary gland cells undergo synchronized programmed cell death requiring both caspases and autophagy (Atg) genes during development. Here, we show that ubiquitin proteasome system (UPS) function is reduced during normal salivary gland cell death, and that ectopic proteasome impairment in salivary gland cells leads to early DNA fragmentation and salivary gland condensation in vivo. Shotgun proteomic analyses of purified dying salivary glands identified the UPS as the top category of proteins enriched, suggesting a possible compensatory induction of these factors to maintain proteolysis during cell death. We compared the proteome following ectopic proteasome impairment to the proteome during developmental cell death in salivary gland cells. Proteins that were enriched in both populations of cells were screened for their function in salivary gland degradation using RNAi knockdown. We identified several factors, including trol, a novel gene CG11880, and the cop9 signalsome component cop9 signalsome 6, as required for Drosophila larval salivary gland degradation.     

Population genetic structure of Bryde’s whales (<Emphasis Type="Italic">Balaenoptera brydei</Emphasis>) at the inter-oceanic and trans-equatorial levels

Bryde’s whales (Balaenoptera brydei) differ from other typical baleen whale species because they are restricted to tropical and warm temperate waters in major oceans, and frequent trans-equatorial movement has been suggested for the species. We tested this hypothesis by analyzing genetic variation at 17 microsatellite loci (N = 508) and 299 bp of mitochondrial DNA (mtDNA) control region sequences (N = 472) in individuals obtained from the western North Pacific, South Pacific, and eastern Indian Ocean. Combined use of microsatellite and mtDNA markers allowed us to distinguish between contemporary gene flow and ancestral polymorphism and to describe sex-specific philopatry. A high level of genetic diversity was found within the samples. Both nuclear and mtDNA markers displayed similar population structure, indicating a lack of sex-specific philopatry. Spatial structuring was detected using both frequency-based population parameters and individual-based Bayesian approaches. Whales in the samples from different oceanic regions came from genetically distinct populations with evidence of limited gene flow. We observed low mtDNA sequence divergence among populations and a lack of concordance between geographic and phylogenetic position of mtDNA haplotypes, suggesting recent separation of populations rather than frequent trans-equatorial and inter-oceanic movement. We conclude that current gene flow between Bryde’s whale populations is low and that effective management actions should treat them as separate entities to ensure continued existence of the species.     

Adenosine 3',5'-cyclic monophosphate (cAMP)-dependent inhibition of IL-5 from human T lymphocytes is not mediated by the cAMP-dependent protein kinase A

IL-5 is implicated in the pathogenesis of asthma and is predominantly released from T lymphocytes of the Th2 phenotype. In anti-CD3 plus anti-CD28-stimulated PBMC, albuterol, isoproterenol, rolipram, PGE2, forskolin, cholera toxin, and the cAMP analog, 8-bromoadenosine cAMP (8-Br-cAMP) all inhibited the release of IL-5 and lymphocyte proliferation. Although all of the above compounds share the ability to increase intracellular cAMP levels and activate protein kinase (PK) A, the PKA inhibitor H-89 failed to ablate the inhibition of IL-5 production mediated by 8-Br-cAMP, rolipram, forskolin, or PGE2. Similarly, H-89 had no effect on the cAMP-mediated inhibition of lymphocyte proliferation. Significantly, these observations occurred at a concentration of H-89 (3 microM) that inhibited both PKA activity and CREB phosphorylation in intact cells. Additional studies showed that the PKA inhibitors H-8, 8-(4-chlorophenylthio) adenosine-3',5'-cyclic monophosphorothioate Rp isomer, and a myristolated PKA inhibitor peptide also failed to block the 8-Br-cAMP-mediated inhibition of IL-5 release from PBMC. Likewise, a role for PKG was considered unlikely because both activators and inhibitors of this enzyme had no effect on IL-5 release. Western blotting identified Rap1, a downstream target of the cAMP-binding proteins, exchange protein directly activated by cAMP/cAMP-guanine nucleotide exchange factors 1 and 2, in PBMC. However, Rap1 activation assays revealed that this pathway is also unlikely to be involved in the cAMP-mediated inhibition of IL-5. Taken together, these results indicate that cAMP-elevating agents inhibit IL-5 release from PBMC by a novel cAMP-dependent mechanism that does not involve the activation of PKA.     

Role of amino acid residues in transmembrane segments IS6 and IIS6 of the Na+ channel alpha subunit in voltage-dependent gating and drug block

Alanine-scanning mutagenesis of transmembrane segments IS6 and IIS6 of the rat brain Na(v)1.2 channel alpha subunit identified mutations N418A in IS6 and L975A in IIS6 as causing strong positive shifts in the voltage dependence of activation. In contrast, mutations V424A in IS6 and L983A in IIS6 caused strong negative shifts. Most IS6 mutations opposed inactivation from closed states, but most IIS6 mutations favored such inactivation. Mutations L421C and L983A near the intracellular ends of IS6 and IIS6, respectively, exhibited significant sustained Na(+) currents at the end of 30-ms depolarizations, indicating a role for these residues in Na(+) channel fast inactivation. These residues, in combination with residues at the intracellular end of IVS6, are well situated to form an inactivation gate receptor. Mutation I409A in IS6 reduced the affinity of the local anesthetic etidocaine for the inactivated state by 6-fold, and mutations I409A and N418A reduced use-dependent block by etidocaine. No IS6 or IIS6 mutations studied affected inactivated-state affinity or use-dependent block by the neuroprotective drug sipatrigine (compound 619C89). These results suggest that the local anesthetic receptor site is formed primarily by residues in segments IIIS6 and IVS6 with the contribution of a single amino acid in segment IS6.