Alternative rapamycin treatment regimens mitigate the impact of rapamycin on glucose homeostasis and the immune system

Inhibition of the mechanistic target of rapamycin (mTOR) signaling pathway by the FDA‐approved drug rapamycin has been shown to promote lifespan and delay age‐related diseases in model organisms including mice. Unfortunately, rapamycin has potentially serious side effects in humans, including glucose intolerance and immunosuppression, which may preclude the long‐term prophylactic use of rapamycin as a therapy for age‐related diseases. While the beneficial effects of rapamycin are largely mediated by the inhibition of mTOR complex 1 (mTORC1), which is acutely sensitive to rapamycin, many of the negative side effects are mediated by the inhibition of a second mTOR‐containing complex, mTORC2, which is much less sensitive to rapamycin. We hypothesized that different rapamycin dosing schedules or the use of FDA‐approved rapamycin analogs with different pharmacokinetics might expand the therapeutic window of rapamycin by more specifically targeting mTORC1. Here, we identified an intermittent rapamycin dosing schedule with minimal effects on glucose tolerance, and we find that this schedule has a reduced impact on pyruvate tolerance, fasting glucose and insulin levels, beta cell function, and the immune system compared to daily rapamycin treatment. Further, we find that the FDA‐approved rapamycin analogs everolimus and temsirolimus efficiently inhibit mTORC1 while having a reduced impact on glucose and pyruvate tolerance. Our results suggest that many of the negative side effects of rapamycin treatment can be mitigated through intermittent dosing or the use of rapamycin analogs.     

Adjustments of Protein Metabolism in Fasting Arctic Charr,Salvelinus alpinus

Protein metabolism, including the interrelated processes of synthesis and degradation, mediates the growth of an animal. In ectothermic animals, protein metabolism is responsive to changes in both biotic and abiotic conditions. This study aimed to characterise responses of protein metabolism to food deprivation that occur in the coldwater salmonid, Arctic charr, Salvelinus alpinus. We compared two groups of Arctic charr: one fed continuously and the other deprived of food for 36 days. We measured the fractional rate of protein synthesis (K_S) in individuals from the fed and fasted groups using a flooding dose technique modified for the use of deuterium-labelled phenylalanine. The enzyme activities of the three major protein degradation pathways (ubiquitin proteasome, lysosomal cathepsins and the calpain systems) were measured in the same fish. This study is the first to measure both K_S and the enzymatic activity of protein degradation in the same fish, allowing us to examine the apparent contribution of different protein degradation pathways to protein turnover in various tissues (red and white muscle, liver, heart and gills). K_S was lower in the white muscle and in liver of the fasted fish compared to the fed fish. There were no observable effects of food deprivation on the protease activities in any of the tissues with the exception of liver, where the ubiquitin proteasome pathway seemed to be activated during fasting conditions. Lysosomal proteolysis appears to be the primary degradation pathway for muscle protein, while the ubiquitin proteasome pathway seems to predominate in the liver. We speculate that Arctic charr regulate protein metabolism during food deprivation to conserve proteins.     

Implications of Harvest on the Boundaries of Protected Areas for Large Carnivore Viewing Opportunities

The desire to see free ranging large carnivores in their natural habitat is a driver of tourism in protected areas around the globe. However, large carnivores are wide-ranging and subject to human-caused mortality outside protected area boundaries. The impact of harvest (trapping or hunting) on wildlife viewing opportunities has been the subject of intense debate and speculation, but quantitative analyses have been lacking. We examined the effect of legal harvest of wolves (Canis lupus) along the boundaries of two North American National Parks, Denali (DNPP) and Yellowstone (YNP), on wolf viewing opportunities within the parks during peak tourist season. We used data on wolf sightings, pack sizes, den site locations, and harvest adjacent to DNPP from 1997–2013 and YNP from 2008–2013 to evaluate the relationship between harvest and wolf viewing opportunities. Although sightings were largely driven by wolf population size and proximity of den sites to roads, sightings in both parks were significantly reduced by harvest. Sightings in YNP increased by 45% following years with no harvest of a wolf from a pack, and sightings in DNPP were more than twice as likely during a period with a harvest buffer zone than in years without the buffer. These findings show that harvest of wolves adjacent to protected areas can reduce sightings within those areas despite minimal impacts on the size of protected wolf populations. Consumptive use of carnivores adjacent to protected areas may therefore reduce their potential for non-consumptive use, and these tradeoffs should be considered when developing regional wildlife management policies.     

A blind prometaphase study of Prader-Willi syndrome: frequency and consistency in interpretation of del 15q.

Controversy continues to exist concerning the proportion of individuals with Prader-Willi syndrome who have a chromosome 15 deletion and concerning the reliability with which a cytogenetic service laboratory can accurately perform the appropriate analysis. Blind prometaphase cytogenetic study of 13 individuals from a Prader-Willi syndrome clinic and seven controls has revealed that approximately 70% of accurately diagnosed clinically typical patients with this disorder have an evident deletion of at least 15q12. Blind analysis of panels of chromosome 15 pairs from all cases in this study by the directors of four independent cytogenetic service laboratories demonstrated substantial interobserver consistency in interpretation of results. The possibility of euploid mosaicism for del 15q was investigated, but remains unresolved.     

Hydration of stratum corneum

A model for the hydration behavior of human stratum corneum has been developed from measurements on in vitro samples isolated in a manner which minimized tissue treatment and trauma. Water sorption/desorption rate measurements as a function of water activity, temperature, and tissue integrity are reported. These data, together with thermodynamic data (infrared and nmr results reported earlier) are consistent with a model in which rapidly sorbed/desorbed water (ca. 0.5 mg water/mg stratum corneum) is associated with (“bound” by) the tissue, while slowly sorbed/desorbed “free” water (up to 12 mg water/mg stratum corneum) is kinetically restricted and probably intracellular in location. Both equilibrium water binding and desorption kinetic data suggest structural changes of this cellular water barrier upon hydration. Evidence for hysteresis in water sorption isotherms (reported by others) could not be reproduced.     

Trisomy 15 with loss of the paternal 15 as a cause of Prader-Willi syndrome due to maternal disomy.

Uniparental disomy has recently been recognized to cause human disorders, including Prader-Willi syndrome (PWS). We describe a particularly instructive case which raises important issues concerning the mechanisms producing uniparental disomy and whose evaluation provides evidence that trisomy may precede uniparental disomy in a fetus. Chorionic villus sampling performed for advanced maternal age revealed trisomy 15 in all direct and cultured cells, though the fetus appeared normal. Chromosome analysis of amniocytes obtained at 15 wk was normal in over 100 cells studied. The child was hypotonic at birth, and high-resolution banding failed to reveal the deletion of 15q11-13, a deletion which is found in 50%-70% of patients with PWS. Over time, typical features of PWS developed. Molecular genetic analysis using probes for chromosome 15 revealed maternal disomy. Maternal nondisjunction with fertilization of a disomic egg by a normal sperm, followed by loss of the paternal 15, is a likely cause of confined placental mosaicism and uniparental disomy in this case of PWS, and advanced maternal age may be a predisposing factor.     

Effect of pneumonectomy on the remaining lung in dogs

To determine the magnitude of functional compensation after pneumonectomy and whether compensation is related to maturity of the animal at the time of resection, we performed left pneumonectomy in either adult or 10-wk-old beagles. Studies were performed in adults 7-9 mo after surgery and in puppies 18-23 mo after surgery when the dogs reached full maturity. Results were compared with those in age- and sex-matched unoperated controls. Measurements included pressure-volume relationships, pulmonary hemodynamics, rebreathing studies of lung volume, diffusing capacity and its components, lung tissue volume, and pulmonary blood flow. Computerized-tomographic scans were performed in the puppy groups to determine changes in thoracic shape and size. Morphometric analysis of the lungs was performed under light microscopy. There was partial compensation for loss of one lung by functional improvement in the remaining lung. Compensation was greater in those pneumonectomized as puppies than as adults. Volume of the remaining lung was larger than predicted for a given transpulmonary pressure in both groups. Diffusing capacity, pulmonary capillary blood volume, and lung tissue volume were larger than expected for the normal right lung. After pneumonectomy, compliance of the rib cage was greater in puppies than in adults. Weight of the costal diaphragm was reduced in pneumonectomized puppies. Pulmonary hypertension at rest did not develop, and pulmonary vascular reactivity to hypoxia was unchanged after pneumonectomy in both groups. Significant correlations were obtained between physiological and morphometric measurements.     

Characterization of rat ribosomal DNA. The highly repetitive sequences that flank the ribosomal RNA transcription unit are homologous and contain RNA polymerase III transcription initiation sites

The non-transcribed spacers (NTS) of the ribosomal genes of a number of organisms have been studied and were found to contain repetitive sequences. In these studies with plasmid subclones of NTS, designated p3.4, p2.6 and p1.7, which come from both 5' and 3' flanking regions of the rat ribosomal genes, respectively, it has been determined that these sequences are found elsewhere within the genome. Southern hybridization analysis has demonstrated that the 5' and 3' NTS subclones cross-hybridize, and that the cross-hybridizing regions are synonymous with the highly repetitive regions. Sequences homologous to the rat NTS were specifically localized to both 5' and 3' flanking regions as well as to a number of the introns of cloned genes including rat serum albumin, rat alpha-fetoprotein, rat casein and human serum albumin. No hybridization was detected of the 5' NTS subclone to the human Alu sequence clone, Blur 8, or to the rodent equivalent, a clone containing Chinese hamster ovary type I and II Alu sequences. However, as reported for type II Alu sequences, the subcloned rat NTS sequences contain RNA polymerase III initiation sites and also hybridize to a number of small RNAs, but not 4.5 S or 7 S RNA. Sequence analysis of two distinct repetitive regions in p1.7 has revealed a region of alternating purine-pyrimidine nucleotides, potentially of Z DNA, and stretches of repetitive sequences. The possible roles for these repetitive sequences in recombination and in maintaining a hierarchical structure for the ribosomal genes are discussed.     

Occupational hydrocarbon exposure among fathers of Prader-Willi syndrome patients with and without deletions of 15q.

Prader-Willi syndrome (PWS) is a multiple-anomaly disorder in which 50%-70% of cases are associated with a de novo interstitial deletion [del 15(q11-13)] on prometaphase cytogenetic analysis, the remainder having apparently normal chromosomes. In most instances, the paternally derived chromosome has become deleted in the affected child, suggesting the possibility of a predisposing environmental factor. Strakowski and Butler found an increased incidence of paternal periconceptional employment in hydrocarbon-exposing occupations in this population. This observation may suggest a causal relationship to PWS. To determine whether this association may distinguish the cytogenetically different groups, we identified 81 patients with the disorder who were physically and cytogenetically examined in three centers, and we compared the frequency of possible periconceptional occupational hydrocarbon exposure between fathers of patients who demonstrate a 15q deletion and those who do not. There was no statistically significant difference between the cytogenetically different groups. In both groups, approximately half of the fathers had been employed in hydrocarbon-exposing jobs. These findings suggest lack of etiologic heterogeneity between the cytogenetically different groups for PWS and affirm the need to seek submicroscopic deletions through molecular genetic studies. These data also provide additional evidence that hydrocarbon exposure among fathers of children with PWS may be causally related to the disorder, and they also suggest the need for more accurate assessment of exposure via a large, controlled study.