Involvement of p53 and p21 in Cellular Defects and Tumorigenesis in Atm−/− Mice

Disruption of the mouse Atm gene, whose human counterpart is consistently mutated in ataxia-telangiectasia (A-T) patients, creates an A-T mouse model exhibiting most of the A-T-related systematic and cellular defects. While ATM plays a major role in signaling the p53 response to DNA strand break damage, Atm^−/− p53^−/− mice develop lymphomas earlier than Atm^−/− or p53^−/− mice, indicating that mutations in these two genes lead to synergy in tumorigenesis. The cell cycle G₁/S checkpoint is abolished in Atm^−/− p53^−/− mouse embryonic fibroblasts (MEFs) following γ-irradiation, suggesting that the partial G₁ cell cycle arrest in Atm^−/− cells following γ-irradiation is due to the residual p53 response in these cells. In addition, the Atm^−/− p21^−/− MEFs are more severely defective in their cell cycle G₁ arrest following γ-irradiation than Atm^−/− and p21^−/− MEFs. The Atm^−/− MEFs exhibit multiple cellular proliferative defects in culture, and an increased constitutive level of p21 in these cells might account for these cellular proliferation defects. Consistent with this notion, Atm^−/− p21^−/− MEFs proliferate similarly to wild-type MEFs and exhibit no premature senescence. These cellular proliferative defects are also rescued in Atm^−/− p53^−/− MEFs and little p21 can be detected in these cells, indicating that the abnormal p21 protein level in Atm^−/− cells is also p53 dependent and leads to the cellular proliferative defects in these cells. However, the p21 mRNA level in Atm^−/− MEFs is lower than that in Atm^+/+ MEFs, suggesting that the higher level of constitutive p21 protein in Atm^−/− MEFs is likely due to increased stability of the p21 protein.     

Epac‐2 ameliorates spontaneous colitis in Il‐10 −/− mice by protecting the intestinal barrier and suppressing NF‐κB/MAPK signalling

Intestinal barrier dysfunction and intestinal inflammation interact in the progression of Crohn''s disease (CD). A recent study indicated that Epac‐2 protected the intestinal barrier and had anti‐inflammatory effects. The present study examined the function of Epac‐2 in CD‐like colitis. Interleukin‐10 gene knockout (Il‐10 ^−/−) mice exhibit significant spontaneous enteritis and were used as the CD model. These mice were treated with Epac‐2 agonists (Me‐cAMP) or Epac‐2 antagonists (HJC‐0350) or were fed normally (control), and colitis and intestinal barrier structure and function were compared. A Caco‐2 and RAW 264.7 cell co‐culture system were used to analyse the effects of Epac‐2 on the cross‐talk between intestinal epithelial cells and inflammatory cells. Epac‐2 activation significantly ameliorated colitis in mice, which was indicated by reductions in the colitis inflammation score, the expression of inflammatory factors and intestinal permeability. Epac‐2 activation also decreased Caco‐2 cell permeability in an LPS‐induced cell co‐culture system. Epac‐2 activation significantly suppressed nuclear factor (NF)‐κB/mitogen‐activated protein kinase (MAPK) signalling in vivo and in vitro. Epac‐2 may be a therapeutic target for CD based on its anti‐inflammatory functions and protective effects on the intestinal barrier.     

Melanopsin as a Sleep Modulator: Circadian Gating of the Direct Effects of Light on Sleep and Altered Sleep Homeostasis in Opn4−/− Mice

Light influences sleep and alertness either indirectly through a well-characterized circadian pathway or directly through yet poorly understood mechanisms. Melanopsin (Opn4) is a retinal photopigment crucial for conveying nonvisual light information to the brain. Through extensive characterization of sleep and the electrocorticogram (ECoG) in melanopsin-deficient (Opn4^−/−) mice under various light–dark (LD) schedules, we assessed the role of melanopsin in mediating the effects of light on sleep and ECoG activity. In control mice, a light pulse given during the habitual dark period readily induced sleep, whereas a dark pulse given during the habitual light period induced waking with pronounced theta (7–10 Hz) and gamma (40–70 Hz) activity, the ECoG correlates of alertness. In contrast, light failed to induce sleep in Opn4^−/− mice, and the dark-pulse-induced increase in theta and gamma activity was delayed. A 24-h recording under a LD 1-h1-h schedule revealed that the failure to respond to light in Opn4^−/− mice was restricted to the subjective dark period. Light induced c-Fos immunoreactivity in the suprachiasmatic nuclei (SCN) and in sleep-active ventrolateral preoptic (VLPO) neurons was importantly reduced in Opn4^−/− mice, implicating both sleep-regulatory structures in the melanopsin-mediated effects of light. In addition to these acute light effects, Opn4^−/− mice slept 1 h less during the 12-h light period of a LD 1212 schedule owing to a lengthening of waking bouts. Despite this reduction in sleep time, ECoG delta power, a marker of sleep need, was decreased in Opn4^−/− mice for most of the (subjective) dark period. Delta power reached after a 6-h sleep deprivation was similarly reduced in Opn4^−/− mice. In mice, melanopsin's contribution to the direct effects of light on sleep is limited to the dark or active period, suggesting that at this circadian phase, melanopsin compensates for circadian variations in the photo sensitivity of other light-encoding pathways such as rod and cones. Our study, furthermore, demonstrates that lack of melanopsin alters sleep homeostasis. These findings call for a reevaluation of the role of light on mammalian physiology and behavior.     

Interleukin-17 deficiency reduced vascular inflammation and development of atherosclerosis in Western diet-induced apoE-deficient mice

ObjectiveSeveral reports describe the role of interleukin (IL)-17 in the development of atherosclerosis; however, its precise role remains controversial. We generated double-deficient mice for apolipoprotein E (apoE) and IL-17 (apoE^?/?IL-17^?/? mice) and investigated the effect of IL-17 deficiency on vascular inflammation and atherosclerosis.Methods and resultsAtherosclerotic plaque areas in apoE^?/?IL-17^?/? mice fed a Western diet (WD) were significantly reduced compared with those in apoE^?/? mice. No significant differences in plasma lipid profiles were observed between apoE^?/? and apoE^?/?IL-17^?/? mice. The number of infiltrated macrophages in the plaques was significantly decreased in WD-fed apoE^?/?IL-17^?/? mice compared with WD-fed apoE^?/? mice, whereas vascular smooth muscle cell content was not altered by IL-17 deficiency. Expression of inflammatory cytokines (MCP-1, IL-1β, IL-6, IFN-γ, and IL-12 p40) and scavenger receptors (Msr-1, Scarb1, and Olr1) in the plaques was inhibited in WD-fed apoE^?/?IL-17^?/? mice. Furthermore, expression of inducible nitric oxide (M1 marker) and arginase-1 (M2 marker) was inhibited in WD-fed apoE^?/?IL-17^?/? mice.ConclusionOur results indicate that IL-17 deficiency reduces vascular inflammation and atherosclerosis and that modulation of IL-17 could be a potential target for prevention and treatment of atherosclerosis.     

NRG1 / ERBB3 signaling in melanocyte development and melanoma: inhibition of differentiation and promotion of proliferation

Neuregulin (NRG) signaling through the receptor tyrosine kinase, ERBB3, is required for embryonic development, and dysregulated signaling has been associated with cancer progression. Here, we show that NRG1/ERBB3 signaling inhibits melanocyte (MC) maturation and promotes undifferentiated, migratory and proliferative cellular characteristics. Embryonic analyses demonstrated that initial MC specification and distribution were not dependent on ERBB3 signaling. However NRG1/ERBB3 signaling was both necessary and sufficient to inhibit differentiation of later stages of MC development in culture. Analysis of tissue arrays of human melanoma samples suggests that ERBB3 signaling may also contribute to metastatic progression of melanoma as ERBB3 was phosphorylated in primary tumors compared with nevi or metastatic lesions. Neuregulin 1‐treated MCs demonstrated increased proliferation and invasion and altered morphology concomitant with decreased levels of differentiation genes, increased levels of proliferation genes and altered levels of melanoma progression and metastases genes. ERBB3 activation in primary melanomas suggests that NRG1/ERBB3 signaling may contribute to the progression of melanoma from benign nevi to malignancies. We propose that targeting ERBB3 activation and downstream genes identified in this study may provide novel therapeutic interventions for malignant melanoma.     

Infection of B cell-deficient mice with CDC 1551, a clinical isolate of Mycobacterium tuberculosis: delay in dissemination and development of lung pathology

Long-term survival of mice infected with Mycobacterium tuberculosis is dependent upon IFN-gamma and T cells, but events in early phases of the immune response are not well understood. In this study, we describe a role for B cells during early immune responses to infection with a clinical isolate of M. tuberculosis (CDC 1551). Following a low-dose infection with M. tuberculosis CDC 1551, similar numbers of bacteria were detected in the lungs of both B cell knockout (IgH 6-, BKO) and C57BL/6J (wild-type) mice. However, despite comparable bacterial loads in the lungs, less severe pulmonary granuloma formation and delayed dissemination of bacteria from lungs to peripheral organs were observed in BKO mice. BKO mice reconstituted with naive B cells, but not those given M. tuberculosis-specific Abs, before infection developed pulmonary granulomas and dissemination patterns similar to wild-type animals. Further analysis of lung cell populations revealed greater numbers of lymphocytes, especially CD8+ T cells, macrophages, and neutrophils in wild-type and reconstituted mice than in BKO mice. Thus, less severe lesion formation and delayed dissemination of bacteria found in BKO mice were dependent on B cells, not Abs, and were associated with altered cellular infiltrate to the lungs. These observations demonstrate an important, previously unappreciated, role for B cells during early immune responses to M. tuberculosis infections.     

CD23<Superscript>+</Superscript>/CD21<Superscript>hi</Superscript> B-cell translocation and ipsilateral lymph node collapse is associated with asymmetric arthritic flare in TNF-Tg mice

Introduction  

Rheumatoid arthritis (RA) is a chronic autoimmune disease with episodic flares in affected joints. However, how arthritic flare occurs only in select joints during a systemic autoimmune disease remains an enigma. To better understand these observations, we developed longitudinal imaging outcomes of synovitis and lymphatic flow in mouse models of RA, and identified that asymmetric knee flare is associated with ipsilateral popliteal lymph node (PLN) collapse and the translocation of CD23⁺/CD21^hi B-cells (B-in) into the paracortical sinus space of the node. In order to understand the relationship between this B-in translocation and lymph drainage from flaring joints, we tested the hypothesis that asymmetric tumor necrosis factor (TNF)-induced knee arthritis is associated with ipsilateral PLN and iliac lymph node (ILN) collapse, B-in translocation, and decreased afferent lymphatic flow.     

Transmission of X-ray-induced reciprocal translocations in normal male mice and in male mice with a reduced sperm count due to translocation homozygosity

Normal (+/+) and translocation T(1; 11.13S)70H homozygous (T/T) male mice received 2 X 2.5 Gy X-rays with a 24-h interval. After 120 days, the frequency of late diplotene-metaphase I spermatocytes with translocation multivalents was 14.1% for +/+ and 13.7% for T/T males, respectively, in one group of animals of each type. The difference is not significant. A second group was allowed to sire progeny for 60 days with 2 normal females per week. Reciprocal translocations detectable at diakinesis/metaphase I were observed in 2.5% of the 395 male progeny from the irradiated +/+ fathers, and in 2.9% of the 489 male progeny from the irradiated T/T fathers. This leads to a pooled estimated transmission of 0.81 +/- 0.19. Translocations induced in the long 11.13 metacentric chromosome were not transmitted with a different frequency. The rate of heritable induced translocations in this study was 5.4 X 10(-5)/rad/gamete. On the basis of the data of Generoso et al. (1984) for the frequency of the heritable spontaneous translocations in male mice, it is concluded that, because of their low doubling dose (3.3-4.6 rad), the spontaneous translocations are probably of postmeiotic origin.     

Expression of LIF in transgenic mice results in altered thymic epithelium and apparent interconversion of thymic and lymph node morphologies.

Leukemia inhibitory factor (LIF) is a cytokine involved in embryonic and hematopoietic development. To investigate the effects of LIF on the lymphoid system, we generated a line of transgenic mice that expresses diffusible LIF protein specifically in T cells. These mice display two categories of phenotype that were not previously attributed to LIF overexpression. First, they display B cell hyperplasia, polyclonal hypergammaglobulinemia and mesangial proliferative glomerulonephritis, defects similar to those described for transgenic mice overexpressing the functionally related cytokine, interleukin-6. Secondly, the LIF transgenic mice display novel thymic and lymph node abnormalities. In the thymus, cortical CD4+CD8+ lymphocytes are lost, while numerous B cell follicles develop. Peripheral lymph nodes contain a vastly expanded CD4+CD8+ lymphocyte population. Furthermore, the thymic epithelium is profoundly disorganized, suggesting that disruption of stroma-lymphocyte interactions is responsible for many observed defects. Transplantation of transgenic bone marrow into wild type recipients transfers both the thymic and lymph node defects. However, transplantation of wild type marrow into transgenic recipients rescues the lymph node abnormality, but not the thymic defect, indicating the thymic epithelium is irreversibly altered. Our observations are consistent with a role for LIF in maintaining a functional thymic epithelium that will support proper T cell maturation.     

Estradiol deficiency during development modulates the expression of circadian and daily rhythms in male and female aromatase knockout mice

Gonadal steroids modify the phase, amplitude and period of circadian rhythms. To further resolve the role of estradiol, we examined daily patterns of activity, circadian free running period and behavioral responses to light pulses using aromatase deficient (ArKO) mice. These animals lack the enzyme necessary to produce estradiol. We hypothesized that circulating estrogens during development and adulthood modulate the amount of activity, the temporal relationship of activity patterns relative to a light:dark cycle, and the free running period. Intact and gonadectomized male and female ArKO and wildtype (WT) littermates were used. WT males, but not ArKO males, retained the ability to respond to steroid hormones; the time of activity onset, free running period in constant darkness, and total daily activity were significantly different in gonadectomized compared to intact males. In contrast, gonadectomy did not alter the expression of these variables in ArKO males. ArKO females had a longer free running period in constant darkness compared to WT females regardless of gonadal state. Ovariectomized ArKO females had a significantly delayed activity onset when compared to intact ArKO females and ovariectomized WT females, despite all 3 groups being estrogen deficient. Phase shifts in response to light pulses given at different times of the day revealed an interaction between genotype, sex, and circulating steroids. These results from ArKO animals strongly suggest an organizational effect of estradiol during a critical period of development on the expression of biological rhythms.     

Mutation of a novel gene results in abnormal development of spermatid flagella,loss of intermale aggression and reduced body fat in mice

ROSA22 male mice are sterile due to a recessive gene-trap mutation that affects development of the spermatid flagellum. The defect involves the flagellar axoneme, which becomes unstable around the time of its assembly. Despite a subsequent complete failure in flagellar assembly, development of the spermatid head appears normal and the spermatid head is released at the correct stage in spermatogenesis. The mutation is pleiotropic. Although ROSA22 homozygote males have normal levels of circulating testosterone and display normal mating behavior, they do not exhibit intermale aggressive behavior and have reduced body fat. The mutated gene (Gtrgeo22) maps to mouse chromosome 10 and is closely flanked by two known genes, Madcam1 and Cdc34. Ribonuclease protection analysis indicates that expression of the flanking genes is unaffected by the mutation. Gtrgeo22 is expressed at low levels in epithelial cells in several tissues, as well as in testis and brain. Analysis of the peptide coding sequence suggests that Gtrgeo22 encodes a novel transmembrane protein, which contains dileucine and tyrosine-based motifs involved in intracellular sorting of transmembrane proteins. Analysis of the Gtrgeo22 gene product should provide novel insight into the molecular basis for intermale aggression and sperm flagellar development.     

Immunohistochemical biomarkers and distribution of telocytes in ApoE−/− mice

Telocytes had been identified as a peculiar stromal cell type implicated in tissue homeostasis and the development and pathophysiology of diseases. Telocyte existed in most organs and tissues in humans and animals. However, few studies have examined telocytes in ApoE gene deficient mice. In our studies, we verified the existence, the morphology and immunohistochemical characteristics of telocytes in critical organs of the ApoE^?/? mice. Male adult ApoE^?/? mice were selected as an experimental model. Immunohistochemical bio‐markers, such as CD34, CD117, CD28, Vimentin and PDGFR‐α were utilized to determine the distribution and morphology of telocytes in the heart, liver and kidney. Telocyte expressed positively for CD34 and CD117, and partial telocyte and telopode expressed positively for PDGFR‐α in heart and liver, but negatively in kidney. Double immunofluorescence assays for CD28/Vimentin, CD34/CD117 and CD34/PDGFR‐α were used to demonstrate the biochemistry speciality of telocytes, respectively. The evidence of telocytes in the ApoE^‐/‐ mice is the first step of our sturdy, which aims to demonstrate changes in telocytes in atherosclerosis in this animal model.     

Correlation of sequential floral and male gametophyte development and preliminary results on anther culture in <Emphasis Type="Italic">Opuntia ficus-indica</Emphasis>

Before approaching anther culture as a tool to trigger an androgenic response in a new species, it is advisable to characterize and correlate flower and male gametophyte development to enable reproducible identification of the appropriate starting material. Buds and flowers of Opuntia ficus-indica cv. Gialla were classified in eight stages according to their total length at the earlier stages and the length of the corolla in flowers with emerging sepals. Due to the low condensation of chromatin in the microspore nucleus as well as in the vegetative nucleus of the bi- and tricellular pollen along with the high autofluorescence of the intricate exine, DAPI staining turned out not to be feasible in this species. Therefore an approach based on light-microscopy observation of semithin sections was used. These sections were stained with toluidine blue for general structure recognition and I₂KI to study starch deposition. Correlations were made between the sequential floral and male gametophyte development. Using this approach we determined the timing of pollen formation and observed that pollen development is impaired in plants producing seedless fruits. Furthermore, anther culture was carried out with anthers collected from flower buds at stages 2 and 3. Most of the anthers produced callus, however no regeneration was obtained.     

Loss of FBXO7 (PARK15) results in reduced proteasome activity and models a parkinsonism‐like phenotype in mice

Mutations in the FBXO7 (PARK15) gene have been implicated in a juvenile form of parkinsonism termed parkinsonian pyramidal syndrome (PPS), characterized by Parkinsonian symptoms and pyramidal tract signs. FBXO7 (F‐box protein only 7) is a subunit of the SCF (SKP1/cullin‐1/F‐box protein) E3 ubiquitin ligase complex, but its relevance and function in neurons remain to be elucidated. Here, we report that the E3 ligase FBXO7‐SCF binds to and ubiquitinates the proteasomal subunit PSMA2. In addition, we show that FBXO7 is a proteasome‐associated protein involved in proteasome assembly. In FBXO7 knockout mice, we find reduced proteasome activity and early‐onset motor deficits together with premature death. In addition, we demonstrate that NEX (neuronal helix–loop–helix protein‐1)‐Cre‐induced deletion of the FBXO7 gene in forebrain neurons or the loss of FBXO7 in tyrosine hydroxylase (TH)‐positive neurons results in motor defects, reminiscent of the phenotype in PARK15 patients. Taken together, our study establishes a vital role for FBXO7 in neurons, which is required for proper motor control and accentuates the importance of FBXO7 in proteasome function.