Refining cage change routines: comparison of cardiovascular responses to three different ways of cage change in rats

Cage change is one of the unavoidable routines in laboratory rodent care. However, cage change disrupts the rodents' olfactory environment and can evoke stress reactions. In this study, the short-term cardiovascular responses to three different cage change procedures were compared with telemetric monitoring. These procedures were: placing the rats into a new, clean cage (NEW), transferring the old cage lid into the clean cage (LID) and transferring an enrichment object into the clean cage (ENR) with the animals. Seven outbred rats (four Hsd:Sprague-Dawley and three HsdBrlHan:WIST) were instrumented with telemetric transmitters. The reactions were recorded during the 24 h following the cage change procedures. All cage change procedures (and also simple handling) caused elevated heart rate and mean arterial pressure levels for up to 5 h after the procedure, with the largest effect seen during the first hour. The reactions observed after cage change were significantly (P < 0.05) greater than those observed after simple handling. The reactions after NEW were significantly higher than the reactions after ENR or LID, though the results were dependent on the stock. In Wistar rats the LID procedure resulted in smaller reactions than ENR. In Sprague-Dawley rats, the differences between ENR and LID were not so clear, but the transfer of scent-marked material into the new cage decreased the reactions compared with the NEW procedure also in this stock. Based on these results, using the old cage lid on the new cage could reduce the disturbance of cage change in rats.     

Epigenetic "bivalently marked" process of cancer stem cell-driven tumorigenesis

Silencing of tumor suppressor genes (TSGs), by DNA methylation, is well known in adult cancers. However, based on the "stem cell" theory of tumorigenesis, the early epigenetic events arising in malignant precursors remain unknown. A recent report demonstrates that, while pluripotent embryonic stem cells lack DNA methylation and possess a "bivalent" pattern of activating and repressive histone marks in numerous TSGs, analogous multipotent malignant cells derived from germ cell tumors (embryonic carcinoma cells) gain additional silencing modifications to those same genes. These results suggest a possible mechanism by which aberrant differentiation, mediated by histone and DNA methylation, instigates tumor progression.     

Clustered DNA methylation changes in polycomb target genes in early-stage liver cancer

Polycomb-group proteins mark specific chromatin conformations in embryonic and somatic stem cells that are critical for maintenance of their "stemness". These proteins also mark altered chromatin modifications identified in various cancers. In normal differentiated cells or advanced cancerous cells, these polycomb-associated loci are frequently associated with increased DNA methylation. It has thus been hypothesized that changes in DNA methylation status within polycomb-associated loci may dictate cell fate and that abnormal methylation within these loci may be associated with tumor development. To assess this, we examined the methylation states of four polycomb target loci -Trip10, Casp8AP2, ENSA, and ZNF484 - in liver cancer. These four targets were selected because their methylation levels are increased during mesenchymal stem cell-to-liver differentiation. We found that these four loci were hypomethylated in most early-stage liver cancer specimens. For comparison, two non-polycomb tumor suppressor genes, HIC1 and RassF1A, were also examined. Whereas the methylation level of HIC1 did not differ significantly between normal and tumor samples, RassF1A was significantly hypermethylated in liver tumor samples. Unsupervised clustering analysis classified the methylation changes within polycomb and non-polycomb targets to be independent, indicating independent epigenetic evolution. Thus, pre-deposited polycomb marks within somatic stem cells may contribute to the determination of methylation changes during hepatic tumorigenesis.     

Targeted Casp8AP2 methylation increases drug resistance in mesenchymal stem cells and cancer cells

Casp8AP2 contains a FLASH functional domain and is critical for the formation of death complex and the relay of death signal into the cells. Genetic defects in Casp8AP2 are associated with several diseases. A CpG island within the Casp8AP2 promoter is differentially regulated during somatic stem cell differentiation, and aberrant DNA methylation within the Casp8AP2 promoter has been reported in cancers. We hypothesized that abnormal DNA methylation of Casp8AP2 promoter might contribute to prolonged cellular survival or drug resistance in cancer. The epigenetic state within the Casp8AP2 promoter was then determined in different cancer cell lines and patient samples by methylation-specific PCR. Targeted Casp8AP2 methylation within normal and tumor cells was performed to see whether methylation promoted drug resistance. We found differential Casp8AP2 methylation among the normal and tumoral samples. Global demethylation in a platinum drug-resistant human gastric cancer cell line reversed Casp8AP2 methylation and diminished drug resistance. Targeted methylation of the Casp8AP2 promoter in somatic stem cells and cancer cells increased their resistance to drugs including platinum drugs. These data demonstrate that methylation within the Casp8AP2 promoter correlates with the development of drug resistance and might serve as a biomarker and treatment target for drug resistance in cancer cells.     

Role of cage material, working style and hearing sensitivity in perception of animal care noise

During daily care, laboratory animals are exposed to a variety of sounds which may have effects on welfare and also cause physiological and behavioural changes. So far, almost no attention has been paid to individual sounds or the sound level caused by animal care or the sound level inside the animal cage. In this study, sounds from selected rat care procedures were recorded: pulling cage out of the rack, placing it onto a table and replacing the cage back into the rack; with measurements made inside the rat cage and in the adjacent cage. Diet was poured into the food hopper and sounds were recorded inside the cage and also the adjacent cage. The work was repeated in a calm and also in a hurried style, using stainless steel and polycarbonate cages. Finally, the sounds produced by running tap water were recorded. Differences between rat and human hearing were compared using novel species-specific sound level weightings: R-weighting for rats dB(R) and H-weighting for human dB(H). Hurried work with steel caused sound exposure levels exceeding 90 dB(R) when the cages were placed into the rack and about 80 dB(R) when pulling them out of the rack or placing onto a table. With polycarbonate, the levels were 10-15 dB(R) lower. Unhurried calm working produced lower sound exposure levels than hurried working in many procedures. When the procedures were repeated with measurements in the adjacent cage, the sound exposure levels were lower, but the results were similar. Pouring food pellets into a hopper above the rat's head caused 15 dB(R) higher sound exposure levels than pouring food to an adjacent cage. In general, humans hear these sounds about 10-15 dB louder than rats. In conclusion, cage material, working style and hearing sensitivity all have an impact on the sound exposure level in the rodent cage. With correct working methods, high sound levels can be efficiently avoided in most cases.     

Signaling pathways from membrane lipid rafts to JNK1 activation in reactive nitrogen species-induced non-apoptotic cell death

At present, the signaling pathways controlling reactive nitrogen species (RNS)-induced non-apoptotic cell death are relatively less understood. In this work, various RNS donors are found to induce caspase-independent non-apoptotic cell death in mouse embryonic fibroblasts (MEF). In search of the molecular mechanisms, we first established the role of c-Jun N-terminal kinase (JNK) in RNS-induced non-apoptotic cell death. RNS readily activate JNK, and the jnk1-/- MEF are resistant to RNS-induced cell death. Moreover, the reconstitution of JNK1 effectively restores the sensitivity to RNS. Next, we identified tumor necrosis factor receptor-associated factor 2 (TRAF2) and apoptosis signal-regulating kinase 1 (ASK1) as the essential upstream molecules for RNS-induced JNK activation and cell death. RNS fail to activate JNK and induce cell death in traf2-/- MEF; and reconstitution of TRAF2 effectively restores the responsiveness of traf2-/- MEF to RNS. Moreover, RNS-induced ASK1 activation is impaired in traf2-/- cells and overexpression of a mutant ASK1 protein suppresses RNS-induced cell death in wild-type MEF cells. Last, we explored the signaling events upstream of TRAF2 and found that translocation of TRAF2 and JNK1 onto membrane lipid rafts is required for RNS-mediated JNK1 activation and cell death. Taken together, data from our study reveal a novel signaling pathway regulating RNS-induced JNK1 activation and non-apoptotic cell death.     

Interaction between Paranosema locustae and Metarhizium anisopliae var. acridum, two pathogens of the desert locust, Schistocerca gregaria under laboratory conditions

The interaction between two pathogens, the microsporidian Paranosema locustae Canning and the fungus Metarhizium anisopliae var. acridum Driver and Milner was studied under laboratory conditions in an attempt to develop an improved method of microbial control for the desert locust, Schistocerca gregaria Forsk?l. Fifth-instar locust nymphs, reared in the laboratory, were treated with various concentrations of one of the two pathogens or with both pathogens. The numbers of locusts killed were recorded each day and the production of pathogen spores within the dead locusts was assessed at the end (day 21) of each experiment. Locust nymphs treated with both P. locustae and M. anisopliae died sooner than nymphs infected with only one of the pathogens. At the lower concentrations of pathogen tested, the effects of the two pathogens were additive. At the higher concentrations the combined effects were synergistic. In terms of locust mortality, there was no evidence of any antagonistic effects between the two pathogens. However, the production of spores by P. locustae was reduced considerably when the host insects were infected also with M. anisopliae. For example, nymphs treated initially with P. locustae and then treated 3 and 10 days later with M. anisopliae produced 3-20 times and 2.5-8 times fewer spores, respectively, than nymphs treated only with P. locustae. Hence, in areas where M. anisopliae is applied, the natural persistence of P. locustae in the local grasshopper and locust populations may be diminished.     

Obesity and weight gain are associated with increased incidence of hyperinsulinemia in non-diabetic men.

We investigated the temporal relationships between obesity, weight change and hyperinsulinemia in a population-based 4-year follow-up study of 695 middle-aged, non-diabetic, and normoinsulinemic men. Thirty-eight men developed hyperinsulinemia during the follow-up (fasting serum insulin > or = 12.0 mU/l). In logistic regression analysis adjusting for other risk factors, men with body mass index of > or = 26.7 kg/m2 (highest third) had a 6.6-fold (p = 0.001) risk of developing hyperinsulinemia, compared with men with body mass index of < 24.4 kg/m2 (lowest third). Correspondingly, men with waist-to-hip ratio of > or = 0.95 (highest third) had a 3.5-fold (p = 0.028) incidence of hyperinsulinemia compared with men with waist-to-hip ratio of < 0.90 (lowest third). Weight gain in middle age and weight gain from the age of 20 years to middle age were also associated with increased risk of hyperinsulinemia. Hyperinsulinemia at baseline was not associated with weight gain during the follow-up. This prospective population-based study emphasizes the importance of avoiding obesity and weight gain during adulthood in preventing hyperinsulinemia.     

Two polymorphic microsatellites in a coding segment of the canine androgen receptor gene

A 0.6-kb segment of exon 1 of the canine androgen receptor gene contains two polymorphic CAG tandem repeats which encode strings of glutamine homopolymers. The number of CAGs in each tandem repeat was determined by (1) polymerase chain reaction (PCR) amplification of a gene segment containing both repeats, (2) cleavage between repeats with restriction enzyme EcoO109I and (3) fractionation of the restriction fragments containing individual CAG repeats by denaturing polyacrylamide gel electrophoresis (PAGE). Individual genomic DNA samples from 80 unrelated dogs (53 males plus 27 females for a total of 107 X chromosomes) contained 10–12 CAGs in the 5′ repeats and 10–13 CAGs in the 3′ repeats. Thirteen distinct androgen receptor genotypes were identified. Eleven (or 41%) of the 27 unrelated females were heterozygous in one or both repeat regions, whereas all male samples produced single bands as expected for X chromosome markers. A total of seven distinct haplotypes contributed to the 13 genotypes. The ‘polymorphism information content’ or PIC for this seven-allele X chromosome marker was 0.67.