Rapamycin-induced insulin resistance is mediated by mTORC2 loss and uncoupled from longevity

Rapamycin, an inhibitor of mechanistic target of rapamycin complex 1 (mTORC1), extends the life spans of yeast, flies, and mice. Calorie restriction, which increases life span and insulin sensitivity, is proposed to function by inhibition of mTORC1, yet paradoxically, chronic administration of rapamycin substantially impairs glucose tolerance and insulin action. We demonstrate that rapamycin disrupted a second mTOR complex, mTORC2, in vivo and that mTORC2 was required for the insulin-mediated suppression of hepatic gluconeogenesis. Further, decreased mTORC1 signaling was sufficient to extend life span independently from changes in glucose homeostasis, as female mice heterozygous for both mTOR and mLST8 exhibited decreased mTORC1 activity and extended life span but had normal glucose tolerance and insulin sensitivity. Thus, mTORC2 disruption is an important mediator of the effects of rapamycin in vivo.     

Evaluation of various compounds to inhibit activity of matrix metalloproteinases in the tear film of horses with ulcerative keratitis

OBJECTIVE: To examine in vitro effects of various antiproteolytic compounds on activity of matrix metalloproteinase (MMP)-2 and -9 in the tear film of horses with active corneal ulcers. SAMPLE POPULATION: Samples of tear film obtained from the eyes of 34 horses with active ulcerative keratitis. PROCEDURE: Horses were sedated, and tear samples were collected from the lower fornix of 34 ulcerated eyes by use of capillary tubes. The protease inhibitors 0.2% EDTA, 0.1% doxycycline, 10% N-acetylcysteine (NAC), 0.1% solution of a modified dipeptide that contains hydroxamic acid (ie, ilomostat), 0.1% alpha1-proteinase inhibitor (PI), 0.5% alpha1-PI, and 100% fresh equine serum (ES) were used to treat pooled samples. Amount of latent and active MMP-2 and -9 was measured by optical density scanning of gelatin zymograms of treated and untreated tear samples. RESULTS: Pooled tear samples obtained from ulcerated eyes contained the latent and active forms of MMP-2 and -9. Compared with MMP activity in untreated samples, total MMP activity (sum of all bands detected) observed on the gelatin zymogram gels was reduced by 99.4% by EDTA, 96.3% by doxycycline, 98.8% by NAC, 98.9% by ilomostat, 52.4% by 0.1% alpha1-PI, 93.6% by 0.5% alpha1-PI, and 90.0% by ES. CONCLUSIONS AND CLINICAL RELEVANCE: We documented that EDTA, doxycycline, NAC, ilomostat, alpha1PI, and ES inhibited MMP activity in vitro. Because these compounds use different mechanisms to inhibit various families of proteases in the tear film of horses, a combination of these protease inhibitors may be beneficial for treatment of corneal ulcers in horses.     

Molecular differentiation of Hypoderma bovis and Hypoderma lineatum (Diptera,Oestridae) by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP)

The most variable region of the cytochrome oxidase I (COI) gene of Hypoderma bovis(1) and Hypoderma lineatum(2) (Diptera, Oestridae) was amplified by PCR and the amplicons were sequenced and analysed. PCR products were digested with three restriction enzymes, namely BfaI, HinfI and TaqI, providing informative profiles. H. bovis and H. lineatum sequences revealed an inter-specific variation rate of 8.5%, and an intra-specific variation rate of 0.87 and 0.29%, respectively. The results showed that the COI gene region examined was useful for the differentiation of H. bovis and H. lineatum and that a PCR-RFLP assay is a practical tool for their identification, offering additional diagnostic and epidemiological instruments for the study of cattle grub infestation.     

Iron storage disease in captive Egyptian fruit bats (Rousettus aegyptiacus): relationship of blood iron parameters to hepatic iron concentrations and hepatic histopathology.

This study evaluated the relationship between blood iron parameters and hepatic iron concentrations, and correlation of histologic findings with hepatic iron concentrations in a captive population of Egyptian fruit bats (Rousettus aegyptiacus) and island flying foxes (Pteropus hypomelanus). Blood samples were collected for complete blood counts, plasma biochemical profiles, serum iron concentrations, total iron-binding capacity, whole-blood lead concentrations, and plasma ferritin assays. Liver samples obtained by laparotomy were divided, with one half processed for histologic examination and the other half frozen and submitted for tissue mineral analysis. The histologic sections were scored by two blinded observers for iron deposition, necrosis, and fibrosis. The Egyptian fruit bats had significantly higher liver iron (mean = 3,669 +/- 1,823 ppm) and lead (mean = 8.9 +/- 5.8 ppm) concentrations than the island flying foxes (mean [Fe] = 174 +/- 173 ppm, mean [Pb] = 1.9 +/- 0.5 ppm). Hepatic iron concentrations significantly correlated with tissue lead concentrations, histologic grading for iron and necrosis, serum iron, transferrin saturation, and plasma ferritin (P < 0.001). Blood lead concentrations negatively correlated with tissue lead concentrations (P < 0.001). When the product of transferrin saturation and serum iron was greater than 51, an individual animal had a high probability of having iron overload. When the product of these two variables was greater than 90, there was a high probability that the animal had hemochromatosis. On the basis of this study, it appears that evaluation of serum iron, transferrin saturation, and plasma ferritin are useful and noninvasive methods for diagnosis of hemochromatosis in Egyptian fruit bats.     

Distribution and properties of geographically distinct isolates of sugar beet yellowing viruses

From a total of 261 yellow sugarbeet leaves collected from 10 countries representing three continents, the incidence and distribution of strains of Beet mild yellowing virus (BMYV), Beet chlorosis virus (BChV) and Beet yellows virus (BYV) were analysed using serological and molecular methods. BMYV was found in all countries except Greece, and more frequently in the northern and western areas of Europe, whereas BYV predominated in Turkey, Spain, Greece, the USA and Chile. BChV, originally found in the USA and the UK in 1989, was identified in France, Spain, the Netherlands and Chile. Nine sugar beet poleroviruses, plus a reference isolate of Turnip yellows virus (TuYV, syn. Beet western yellows virus ), were further characterized and compared. Isolates obtained from sugar beet infected this species, but not oilseed rape or lettuce; all isolates except one infected Capsella bursa-pastoris . The coat-protein sequences of these isolates were highly similar, with the consensus sequence representing 89% of nucleotide residues. Within the coat-protein gene, two regions were identified that could represent specific epitopes to which monoclonal antibody BYDV-PAV-IL-1 could bind; this antibody is used to distinguish beet poleroviruses in ELISA. Comparison of the sequences at the 5' end showed that sequence homology existed only between isolates with the same host range. The first sequence data of polerovirus isolates from Chile are presented, showing that the coat protein and the 5' end of their genomes are highly similar to those of BMYV isolates found in Europe. Chilean polerovirus isolates may have been imported from the northern hemisphere in sugar beet breeding material.     

Application of nox-restriction fragment length polymorphism for the differentiation of Brachyspira intestinal spirochetes isolated from pigs and poultry in Australia.

Sixty-nine intestinal spirochetes isolated from pigs and poultry in eastern Australia were selected to evaluate the effectiveness of a species-specific PCR-based restriction fragment length polymorphism (RFLP) analysis of the Brachyspira nox gene. For comparative purposes, all isolates were subjected to species-specific PCRs for the pathogenic species Brachyspira hyodysenteriae and Brachyspira pilosicoli, and selected isolates were examined further by sequence analysis of the nox and 16S ribosomal RNA genes. Modifications to the original nox-RFLP method included direct inoculation of bacterial cells into the amplification mixture and purification of the PCR product, which further optimized the nox-RFLP for use in a veterinary diagnostic laboratory, producing sufficient product for both species identification and future comparisons. Although some novel profiles that prevented definitive identification were observed, the nox-RFLP method successfully classified 45 of 51 (88%) porcine and 15 of 18 (83%) avian isolates into 5 of the 6 recognized species of Brachyspira. This protocol represents a significant improvement over conventional methods currently used in veterinary diagnostic laboratories for rapid specific identification of Brachyspira spp. isolated from both pigs and poultry.     

Temperature effects on the action of acylurea insecticides against tobacco hornworm (Manduca sexta) larvae

The toxic effects of six acylurea insecticides on larvae of the tobacco hornworm were investigated at each of four environmental temperatures (20, 25, 30 and 35°C). This spans the range of temperatures which the insects can tolerate. For all the acylureas tested, mortality increased with temperature when either newly hatched or fourth-instar larvae were given insecticide in their food. Sub-lethal growth inhibition also became more pronounced at progressively higher environmental temperatures. This temperature dependence of acylurea action was not due to altered uptake of the insecticide, since there was no significant variation with temperature in the amount of [¹⁴C]flufenoxuron taken up by fifth-instar larvae when given a single meal containing labelled insecticide. Additionally, mortality of fourth-instar larvae given a single intra-haemocoelic injection of flufenoxuron was significantly greater at higher temperatures, implying that temperature affects a process that occurs after insecticide uptake. The intrinsic ability of acylureas to inhibit chitin synthesis is temperaturesensitive, since flufenoxuron inhibited the incorporation of [¹⁴C]N-acetylalucosamine into chitin by proleg epidermis in vitro significantly less well at 20°C than at the higher temperatures tested. However, there was no significant variation between the effectiveness of in-vitro chitin synthesis inhibition at 25, 30 and 35°C. These data show that the effectiveness of acylurea insecticides is subject to strong temperature effects in the range of temperatures likely to be experienced in the field.