Lamin A/C-dependent localization of Nesprin-2, a giant scaffolder at the nuclear envelope

The vertebrate proteins Nesprin-1 and Nesprin-2 (also referred to as Enaptin and NUANCE) together with ANC-1 of Caenorhabditis elegans and MSP-300 of Drosophila melanogaster belong to a novel family of alpha-actinin type actin-binding proteins residing at the nuclear membrane. Using biochemical techniques, we demonstrate that Nesprin-2 binds directly to emerin and the C-terminal common region of lamin A/C. Selective disruption of the lamin A/C network in COS7 cells, using a dominant negative lamin B mutant, resulted in the redistribution of Nesprin-2. Furthermore, using lamin A/C knockout fibroblasts we show that lamin A/C is necessary for the nuclear envelope localization of Nesprin-2. In normal skin where lamin A/C is differentially expressed, strong Nesprin-2 expression was found in all epidermal layers, including the basal layer where only lamin C is present. This indicates that lamin C is sufficient for proper Nesprin-2 localization at the nuclear envelope. Expression of dominant negative Nesprin-2 constructs and knockdown studies in COS7 cells revealed that the presence of Nesprin-2 at the nuclear envelope is necessary for the proper localization of emerin. Our data imply a scaffolding function of Nesprin-2 at the nuclear membrane and suggest a potential involvement of this multi-isomeric protein in human disease.     

Declines of zoonotic agents in liquid livestock wastes stored in batches on-farm

AIM: To measure the decline rates of zoonotic agents introduced into liquid livestock wastes in on-farm storage tanks. METHODS AND RESULTS: Salmonella spp., Escherichia coli O157, Campylobacter jejuni, Listeria monocytogenes and Cryptosporidium parvum, propagated in laboratory-controlled conditions, were inoculated into 35,000-l volumes of fresh livestock wastes (pig slurries, cattle slurries and dirty waters). D-values for bacteria were six to 44 days, and for C. parvum were 133 to 345 days. Campylobacter jejuni declined significantly more rapidly than the other bacterial pathogens, while E. coli O157 declined significantly more slowly. On average, bacterial declines were not affected by the season of waste deposition and storage or by the dry matter content of the wastes, but were more rapid in dirty waters than in pig slurries. The physiciochemical composition of wastes in each category varied significantly. CONCLUSIONS: Zoonotic agents can survive for several months during storage of liquid livestock wastes. Livestock wastes should be batch-stored and not subjected to continuous additions. SIGNIFICANCE AND IMPACT OF THE STUDY: This study indicates that batches of liquid livestock waste, if contaminated with bacterial pathogens, should be stored for 6 months to reduce contamination levels. Alternative strategies for reducing C. parvum levels in liquid livestock wastes should be explored.     

Decline of zoonotic agents in livestock waste and bedding heaps

AIMS: To measure the rates of decline of zoonotic agents introduced into heaps of spent bedding and faecal wastes generated by commercially farmed livestock and managed in a similar way to that of a working farm. METHODS AND RESULTS: Livestock isolates of Salmonella, pathogenic Listeria, Campylobacter and Escherichia coli O157 were laboratory cultured and used to inoculate 5 m3 heaps of cattle, sheep or pig wastes mixed with bedding materials. Decline of each of the infectious agents was monitored with time as was the temperature inside each heap. Temperatures of >50 degrees C were typically achieved at the core of the heaps. Pathogen decline was rapid, typically <3 days for a 1-log reduction in levels. The longest time that zoonotic agents were isolated from the heaps was 93 days. CONCLUSIONS: Movement of heaps of livestock bedding waste from animal pens to a secondary store, and storing them under conditions conducive for increased temperature is a simple and cost-effective treatment for rapidly lowering levels of zoonotic agents in solid farm wastes. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrates a simple and cheap treatment that can be used to help prevent the spread of zoonotic agents through agricultural environments.     

Cold hardiness of Habrobracon hebetor (Say) (Hymenoptera: Braconidae), a parasitoid of pyralid moths

The ectoparasitoid Habrobracon hebetor (Say) attacks stored-product infesting pyralid moths that are able to overwinter under extremely cold conditions. The extent to which H. hebetor can withstand these conditions is not known, but has important implications for the ability of H. hebetor to provide long-term suppression of these pests in temperate climates. We investigated basic cold hardiness aspects of a mutant eye-color strain of H. hebetor. Feeding larvae and adults of H. hebetor had supercooling points (SCPs) at temperatures higher than those of eggs and pupae. Mean SCPs of females and males were equivalent, as were those of naked and silk-encased pupae. Feeding on honey prior to being subjected to low temperatures significantly increased the SCP of adult females by approximately 8 degrees C. Mortality of pupae and adults increased significantly whenever the temperature dropped below the mean SCP, indicating that H. hebetor does not tolerate freezing. For pupae and adults exposed to -12 and -5 degrees C, the hourly mortality rate increased with time of exposure. Pupae and adults exposed to -12 degrees C for different time intervals showed high mortality after only 1d of exposure. At -5 degrees C, none survived 12d of exposure. A better understanding of how well this parasitoid tolerates low temperatures will be useful in evaluating its potential as a biological control agent of stored-product moths in temperate regions.     

A 50-kDa cytosolic protein complexed with the 90-kDa heat shock protein (hsp90) is the same protein complexed with pp60v-src hsp90 in cells transformed by the Rous sarcoma virus.

We have previously shown that a 50-kDa protein is one component of a heteromeric complex immunoprecipitated by the 90-kDa heat shock protein (hsp90) monoclonal antibodies 8D3 and 3G3 (Perdew, G. H., and Whitelaw, M. L. (1991) J. Biol. Chem. 266, 6708-6713). In this report, we compare the 50-kDa protein with that found in pp60v-src-hsp90-p50 complexes immunoprecipitated from Rous sarcoma virus-transformed cells with antibodies to pp60v-src. 35S- and 32P-labeled p50 proteins from each system were identical in their mobilities by sodium dodecyl sulfate-polyacryl-amide gel electrophoresis. The profile of N-chlorosuccinimide cleavage products derived from each 32P-labeled p50 protein were also identical when resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. We have developed a mouse monoclonal antibody, 3M/1B5p50, capable of detecting p50 on Western blots. This antibody detected the 50-kDa protein which co-purified with the pa104 pp60v-src mutant of the avian sarcoma virus oncoprotein in 44A rat fibroblasts. We did not detect p50 in association with native glucocorticoid receptor in L cells or with the overexpressed glucocorticoid receptor in Chinese hamster ovary cells. Two experiments utilizing immunochemical staining implied that essentially all cytosolic p50 is associated with hsp90. Firstly, immunoprecipitating hsp90 from Hepa 1 cytosol with monoclonal antibody 3G3 left the cytosol depleted of p50. Secondly, cytosol fractionated by sucrose gradient revealed that p50 cosedimented with hsp90, confirming the existence of p50 only in association with hsp90.     

Effects of lipopolysaccharide and acclimation temperature on induced behavioral fever in juvenile Iguana iguana

We examined the effects of acclimation temperature and two doses (2.5 and 25mgkg(-1)) of a pyrogen (lipopolysaccharide, LPS) on behavioral thermoregulation in juvenile green iguanas. Overall means of body temperatures for the three-day trial periods were compared among three groups of animals acclimated at 15, 25, and 34 degrees C. The responses of each group of animals to the two dosages of LPS and a control saline injection were examined. Within each treatment block, animals either chose high body temperatures characteristic of a fever response or chose low body temperatures characteristic of a hypothermic response. Thermoregulation was influenced by interaction effects between and among, and independent effects of, acclimation temperature, dose of LPS, and day. In some treatment blocks, individual lizard mass positively correlated with mean individual body temperature. Mean mass of lizards that chose higher body temperatures within a treatment block was higher than the mean mass of lizards that chose lower body temperatures. From these results, we concluded that LPS may induce two different behavioral thermoregulatory responses: fever or hypothermia. The actual amplitude and direction of body temperature change appears to be affected by acclimation temperature and possibly by mass or energy reserves of the animal. If the energy reserves are not sufficient to sustain the higher rate of metabolism associated with the higher body temperatures of a hyperthermic or feverish state, the animal may resort to hypothermia.     

Pigment Epithelium‐Derived Factor,Insulin Sensitivity,and Adiposity in Polycystic Ovary Syndrome: Impact of Exercise Training

Pigment epithelium‐derived factor (PEDF) is upregulated in obese rodents and is involved in the development of insulin resistance (IR). We aim to explore the relationships between PEDF, adiposity, insulin sensitivity, and cardiovascular risk factors in obese women with polycystic ovary syndrome (PCOS) and weight‐matched controls and to examine the impact of endurance exercise training on PEDF. This prospective cohort intervention study was based at a tertiary medical center. Twenty obese PCOS women and 14 non‐PCOS weight‐matched women were studied at baseline. PEDF, cardiometabolic markers, detailed body composition, and euglycemic—hyperinsulinemic clamps were performed and measures were repeated in 10 PCOS and 8 non‐PCOS women following 12 weeks of intensified aerobic exercise. Mean glucose infusion rate (GIR) was 31.7% lower (P = 0.02) in PCOS compared to controls (175.6 ± 96.3 and 257.2 ± 64.3 mg.m^?2.min^?1) at baseline, yet both PEDF and BMI were similar between groups. PEDF negatively correlated to GIR (r = ?0.41, P = 0.03) and high‐density lipoprotein (HDL) (r = ?0.46, P = 0.01), and positively to cardiovascular risk factors, systolic (r = 0.41, P = 0.02) and diastolic blood pressure (r = 0.47, P = 0.01) and triglycerides (r = 0.49, P = 0.004). The correlation with GIR was not significant after adjusting for fat mass (P = 0.07). Exercise training maintained BMI and increased GIR in both groups; however, plasma PEDF was unchanged. In summary, PEDF is not elevated in PCOS, is not associated with IR when adjusted for fat mass, and is not reduced by endurance exercise training despite improved insulin sensitivity. PEDF was associated with cardiovascular risk factors, suggesting PEDF may be a marker of cardiovascular risk status.     

Elevated levels of oncogenic protein kinase Pim-1 induce the p53 pathway in cultured cells and correlate with increased Mdm2 in mantle cell lymphoma

Mutation of the p53 gene is a common event during tumor pathogenesis. Other mechanisms, such as mdm2 amplification, provide alternative routes through which dysfunction of the p53 pathway is promoted. Here, we address the hypothesis that elevated expression of pim oncogenes might suppress p53 by regulating Mdm2. At a physiological level, we show that endogenous Pim-1 and Pim-2 interact with endogenous Mdm2. Additionally, the Pim kinases phosphorylate Mdm2 in vitro and in cultured cells at Ser(166) and Ser(186), two previously identified targets of other signaling pathways, including Akt. Surprisingly, at high levels of Pim expression, as would occur in tumors, active, but not inactive, Pim-1 or Pim-2 blocks the degradation of both p53 and Mdm2 in a manner that is independent of Mdm2 phosphorylation, leading to increased p53 levels and, proportionately, p53-dependent transactivation. Additionally, Pim-1 induces endogenous ARF, p53, Mdm2, and p21 in primary murine embryo fibroblasts and stimulates senescence-associated beta-galactosidase levels, consistent with the induction of senescence. Immunohistochemical analysis of a cohort of 33 human mantle cell lymphomas shows that elevated expression of Pim-1 occurs in 42% of cases, with elevated Pim-2 occurring in 9% of cases, all of which also express Pim-1. Notably, elevated Pim-1 correlates with elevated Mdm2 in MCL with a p value of 0.003. Taken together, our data are consistent with the idea that Pim normally interacts with the p53 pathway but, when expressed at pathological levels, behaves as a classic dominant oncogene that stimulates a protective response through induction of the p53 pathway.