Concurrent reductions in blood pressure and metabolic rate during fasting in the unrestrained SHR

Arctic ground squirrels (Spermophilus parryii) overwinter in hibernaculum conditions that are substantially below freezing. During torpor, captive arctic ground squirrels displayed ambient temperature (T(a))-dependent patterns of core body temperature (T(b)), metabolic rate (TMR), and metabolic fuel use, as determined by respiratory quotient (RQ). At T(a) 0 to -16 degrees C, T(b) remained relatively constant, and TMR rose proportionally with the expanding gradient between T(b) and T(a), increasing >15-fold from a minimum of 0.0115 +/- 0.0012 ml O(2). g(-1). h(-1). At T(a) 0-20 degrees C, T(b) increased with T(a); however, TMR did not change significantly from T(b) 0 to 12 degrees C, indicating temperature-independent inhibition of metabolic rate. The overall change in TMR from T(b) 4 to 20 degrees equates to a Q(10) of 2.4, but within this range of T(b), Q(10) changed from 1.0 to 14.1. During steady-state torpor at T(a) 4 and 8 degrees C, RQ averaged 0.70 +/- 0.013, indicating exclusive lipid catabolism. At T(a) -16 and 20 degrees C, RQ increased significantly to >0.85, consistent with recruitment of nonlipid fuels. RQ was negatively correlated with maximum torpor bout length. For T(a) values <0 degrees C, this relationship supports the hypothesis that availability of nonlipid metabolic fuels limits torpor duration in hibernating mammals; for T(a) values >0 degrees C, hypotheses linked to body temperature are supported. Because anterior body temperatures differ from core, overall, the duration torpor can be extended in hibernating mammals may be dependent on brain temperature.     

Divergence of Mammalian Higher Order Chromatin Structure Is Associated with Developmental Loci

Several recent studies have examined different aspects of mammalian higher order chromatin structure – replication timing, lamina association and Hi-C inter-locus interactions — and have suggested that most of these features of genome organisation are conserved over evolution. However, the extent of evolutionary divergence in higher order structure has not been rigorously measured across the mammalian genome, and until now little has been known about the characteristics of any divergent loci present. Here, we generate a dataset combining multiple measurements of chromatin structure and organisation over many embryonic cell types for both human and mouse that, for the first time, allows a comprehensive assessment of the extent of structural divergence between mammalian genomes. Comparison of orthologous regions confirms that all measurable facets of higher order structure are conserved between human and mouse, across the vast majority of the detectably orthologous genome. This broad similarity is observed in spite of many loci possessing cell type specific structures. However, we also identify hundreds of regions (from 100 Kb to 2.7 Mb in size) showing consistent evidence of divergence between these species, constituting at least 10% of the orthologous mammalian genome and encompassing many hundreds of human and mouse genes. These regions show unusual shifts in human GC content, are unevenly distributed across both genomes, and are enriched in human subtelomeric regions. Divergent regions are also relatively enriched for genes showing divergent expression patterns between human and mouse ES cells, implying these regions cause divergent regulation. Particular divergent loci are strikingly enriched in genes implicated in vertebrate development, suggesting important roles for structural divergence in the evolution of mammalian developmental programmes. These data suggest that, though relatively rare in the mammalian genome, divergence in higher order chromatin structure has played important roles during evolution.     

The endoscopic diagnosis of gastroesophageal malignancy. A cytologic review

Cytologic reports were compared to final diagnoses for 1,157 gastroesophageal samples from an eight-year period in order to evaluate the diagnostic accuracy of endoscopic cytology and to determine the significance of a suspicious cytologic report. In the subgroup of patients with adenocarcinoma evaluated by paired endoscopic biopsy and cytology, the relative and combined sensitivities of the sampling methods were studied. Cytologic examination was reported as positive or suspicious in 85% of 229 cases of malignancy. There were three false-positive diagnoses of squamous-cell carcinoma of the esophagus, representing 0.3% of all submitted samples. Suspicious cytologic reports were issued in 5% of all cases. The majority (63%) of patients with a suspicious cytologic report had a final diagnosis of malignancy, with gastric adenocarcinoma present in almost half of the cases. Adenocarcinoma was diagnosed in 168 of the patients. Combined endoscopic biopsy and cytology was more sensitive (96%) than biopsy alone (90%) in making the initial diagnosis. Cytology may be of particular value in the diagnosis of gastroesophageal malignancy when the lesions are small and superficial or where stricture precludes adequate biopsy. Regardless of the biopsy findings, patients with suspicious cytologic reports require careful reevaluation since a high percentage of those cases in our series were subsequently verified as having malignancy.     

Severe defect in thymic development in an insertional mutant mouse model

Transgenic mice were generated expressing NK1.1, an NK cell-associated receptor, under control of the human CD2 promoter. Unexpectedly, one of the founder lines, Tg66, showed a marked defect in thymic development characterized by disorganized architecture and small size. Mapping of the transgene insertion by fluorescence in situ hybridization revealed integration in chromosome 2, band G. Already from postnatal day 3, the thymic architecture was disturbed with a preferential loss of cortical thymic epithelial cells, a feature that became more pronounced over time. Compared with wild-type mice, total thymic cell numbers decreased dramatically between 10 and 20 days of age. Thymocytes isolated from adult Tg66 mice were predominantly immature double-negative cells, indicating a block in thymic development at an early stage of differentiation. Consequently, Tg66 mice had reduced numbers of peripheral CD4(+) and CD8(+) T cells. Bone marrow from Tg66 mice readily reconstituted thymi of irradiated wild-type as well as RAG-deficient mice. This indicates that the primary defect in Tg66 mice resided in nonhemopoietic stromal cells of the thymus. The phenotype is observed in mice heterozygous for the insertion and does not resemble any known mutations affecting thymic development. Preliminary studies in mice homozygous for transgene insertion reveal a more accelerated and pronounced phenotype suggesting a semidominant effect. The Tg66 mice may serve as a useful model to identify genes regulating thymic epithelial cell differentiation, thymic development, and function.     

Repair of Nitric Oxide-damaged DNA in ��-Cells Requires JNK-dependent GADD45�� Expression

Proinflammatory cytokines induce nitric oxide-dependent DNA damage and ultimately β-cell death. Not only does nitric oxide cause β-cell damage, it also activates a functional repair process. In this study, the mechanisms activated by nitric oxide that facilitate the repair of damaged β-cell DNA are examined. JNK plays a central regulatory role because inhibition of this kinase attenuates the repair of nitric oxide-induced DNA damage. p53 is a logical target of JNK-dependent DNA repair; however, nitric oxide does not stimulate p53 activation or accumulation in β-cells. Further, knockdown of basal p53 levels does not affect DNA repair. In contrast, expression of growth arrest and DNA damage (GADD) 45α, a DNA repair gene that can be regulated by p53-dependent and p53-independent pathways, is stimulated by nitric oxide in a JNK-dependent manner, and knockdown of GADD45α expression attenuates the repair of nitric oxide-induced β-cell DNA damage. These findings show that β-cells have the ability to repair nitric oxide-damaged DNA and that JNK and GADD45α mediate the p53-independent repair of this DNA damage.Insulin-dependent diabetes mellitus is an autoimmune disease characterized by the selective destruction of insulin-secreting pancreatic β-cells found in the islets of Langerhans (1). Cytokines, released from invading leukocytes during insulitis, are believed to participate in the initial destruction of β-cells, precipitating the autoimmune response (2, 3). Treatment of rat islets with the macrophage-derived cytokine interleukin-1 (IL-1)² results in the inhibition of glucose-stimulated insulin secretion and oxidative metabolism and in the induction of DNA damage that ultimately results in β-cell death (4–6). Nitric oxide, produced in micromolar levels following enhanced expression of the inducible nitric-oxide synthase in β-cells, mediates the damaging actions of cytokines on β-cell function (7–9). Nitric oxide inhibits insulin secretion by attenuating the oxidation of glucose to CO₂, reducing cellular levels of ATP and, thereby, attenuating ATP-inhibited K⁺ channel activity (10, 11). The net effect is the inhibition of β-cell depolarization, calcium entry, and calcium-dependent exocytosis. In addition to the inhibition of β-cell function, nitric oxide induces DNA damage in β-cells (4, 12, 13). Nitric oxide or the oxidation products N₂O₃ and ONOO⁻ induce DNA damage through direct strand breaks and base modification (14–16) and by inhibition of DNA repair enzymes, thereby enhancing the damaging actions of nitric oxide (17, 18).Recent studies have shown that β-cells maintain a limited ability to recover from cytokine-mediated damage (19, 20). The addition of a nitric-oxide synthase inhibitor to islets treated for 24 h with cytokine and continued culture with the nitric-oxide synthase inhibitor and cytokine results in a time-dependent restoration of insulin secretion, mitochondrial aconitase activity, and the repair of nitric oxide-damaged DNA (20, 21). Nitric oxide plays a dual role in modifying β-cell responses to cytokines. Nitric oxide induces β-cell damage and also activates a JNK-dependent recovery response that requires new gene expression (22). The ability of β-cells to recover from cytokine-mediated damage is temporally limited because cytokine-induced β-cell damage becomes irreversible following a 36-h incubation, and islets at this point are committed to degeneration (19).The purpose of this study was to determine the mechanisms by which β-cells repair nitric oxide-damaged DNA. Previous reports have shown that DNA damage induced by oxidizing agents, such as nitric oxide, is repaired through the base excision repair pathway (23), but how this pathway is activated in response to nitric oxide is unknown. Similar to the recovery of metabolic function, we now show that the activation of JNK by nitric oxide is required for repair of cytokine-induced DNA damage in β-cells. p53 is a logical candidate to mediate this repair because it plays a central role in DNA repair, is a target of JNK, and is activated by nitric oxide (24–27). However, we show that cytokines do not stimulate p53 phosphorylation, and nitric oxide fails to stimulate p53 accumulation and phosphorylation. Growth arrest and DNA damage (GADD) 45α is a DNA damage-inducible gene that can be regulated by both p53-dependent and p53-independent mechanisms (28–31). In contrast to p53, we show that cytokines stimulate GADD45α expression in a nitric oxide- and JNK-dependent manner and that siRNA-mediated knockdown of GADD45α results in an attenuation in the repair of nitric oxide-mediated DNA damage. These findings support a role for JNK in the regulation of GADD45α-dependent and p53-independent repair of nitric oxide-damaged β-cell DNA.