Targeted disruption of p53 attenuates doxorubicin-induced cardiac toxicity in mice

Use of the chemotherapeutic agent doxorubicin (Dox) is limited by dose-dependent cardiotoxic effects. The molecular mechanism underlying these toxicities are incompletely understood, but previous results have demonstrated that Dox induces p53 expression. Because p53 is an important regulator of the cell birth and death we hypothesized that targeted disruption of the p53 gene would attenuate Dox-induced cardiotoxicity. To test this, female 6–8 wk old C57BL wild-type (WT) or p53 knockout (p53 KO) mice were randomized to either saline or Dox 20 mg/kg via intraperitoneal injection. Animals were serially imaged with high-frequency (14 MHz) two-dimensional echocardiography. Measurements of left ventricle (LV) systolic function as assessed by fractional shortening (FS) demonstrated a decline in WT mice as early as 4 days after Dox injection and by 2 wk demonstrated a reduction of 31± 16% (P < 0.05) from the baseline. In contrast, in p53 KO mice, LV FS was unchanged over the 2 wk period following Dox injection. Apoptosis of cardiac myocytes as measured by the TUNEL and ligase reactions were significantly increased at 24 h after Dox treatment in WT mice but not in p53 KO mice. After Dox injection, levels of myocardial glutathione and Cu/Zn superoxide dismutase were preserved in p53 KO mice, but not in WT animals. These observations suggest that p53 mediated signals are likely to play a significant role in Dox-induced cardiac toxicity and that they may modulate Dox-induced oxidative stress.These two authors equally contributed to this study.     

Bone quality and healing in a swine vascularized bone allotransplantation model using cyclosporine-based immunosuppression therapy

Although vascularized bone and joint allotransplantation is a promising new treatment option for reconstructing large bone defects, the need for immunosuppressive agents to prevent rejection in these procedures poses a major problem. This problem stems from the fact that several of these agents can cause harmful side effects, such as alterations in bone quality and healing. Therefore, the purpose of this study was to determine what effect the commonly used immunosuppressant regimen cyclosporine A-based combination therapy has on bone quality and healing. In 10 pigs, vascularized bone allografts with skin and muscle components (osteomyocutaneous free flaps) were transplanted from size-matched donor animals. Recipient animals received oral cyclosporine A/mycophenolate mofetil/prednisone therapy for 90 days. Bone quality was studied before and after transplantation by measuring the bone's acoustic velocity and density and calculating the bone's elastic coefficient. Bone healing was assessed using radiographic analysis. Four animals were lost as a result of graft rejection or immunosuppression-related complications before the 90-day endpoint of the study. Although bone specimens taken from the six animals that completed the 90-day protocol had histological signs of rejection, they all seemed to have normal bone healing. Posttransplant bone density values were significantly decreased (p < 0.05) (1544.7 +/- 47.5 kg/m3) as compared with pretransplant values (1722.7 +/- 44.1 kg/m3). Results of the acoustic velocity and elastic coefficients measurements showed a significant decrease (p < 0.05) in posttransplant values (from 3503.0 +/- 165.1 meters/sec to 2963.0 +/- 54.6 meters/sec and from 21.6 +/- 2.2 GPa to 13.6 +/- 0.5 GPa, respectively), indicating diminished bone quality. The findings indicate that cyclosporine A/mycophenolate mofetil/prednisone combination therapy is ineffective in preventing bone rejection, that it decreases bone quality, and that it is associated with systemic toxicity, suggesting that this immunosuppressive regimen at the doses used in this study is not ideal for vascularized bone allotransplantation procedures.     

Detection of Brazilian spotted fever infection by polymerase chain reaction in a patient from the state of São Paulo

Brazilian spotted fever (BSF) cases have been increasing in the state of S?o Paulo but no genomic information about local rickettsia isolated from humans has been well documented. We recovered spotted-fever group rickettsiae from a sample of patient blood cultured in Vero cells using the shell vial technique. Rickettsial DNA fragments (gltA, ompA, and, ompB genes) were detected, and analysis of the ompB gene base sequences showed identity with the Rickettsia rickettsii ompB sequence available in the GenBank.     

On the influence of mechanical conditions in osteochondral defect healing

Despite the introduction of new surgical techniques, the treatment of cartilage defects remains challenging. Delay or complete failure of cartilage healing is associated with problems in biological regeneration. The influence of mechanical conditions on this process, however, remains unevaluated. Osteochondral defects were generated on the left femoral condyle in 18 Yucatan minipigs. After 4, 6 and 12 weeks the defect filling, trabecular orientation and bone density were compared to the intact contralateral side. The mechanical straining during this period was then analyzed using an adaptive finite element technique. Histologically, the osteochondral defects showed bone resorption at the base and bone formation from the circumference. At 12 weeks, the macroscopically healed specimens showed fibrous cartilage formation, a minimally organized trabecular structure and increased trabecular volume fraction compared to the controls (p < 0.002). The amount of cancellous, cartilagineous, and fibrous tissue and the defect size as measured in histomorphometric analysis for the three time points (4, 6 and 12 weeks) was comparable in magnitude to that predicted by finite element analysis. The simulated osteochondral healing process was not fully capable of re-establishing a hyaline-like cartilage layer. The correlation between simulation and histology allows identification of mechanical factors that appear to have a larger impact on the healing of osteochondral defects than previously considered.     

Estrogen preferentially promotes the differentiation of CD11c+ CD11b(intermediate) dendritic cells from bone marrow precursors

Sex biases in autoimmunity and infection suggest that steroid sex hormones directly modulate immune cells. We show in this study that 17-beta-estradiol (E2) promotes the differentiation of functional dendritic cells (DC) from murine bone marrow precursor cells. Remarkably, ex vivo DC differentiation was inhibited in steroid hormone-deficient medium, and was restored by addition of physiological amounts of E2, but not dihydrotestosterone. DC differentiation was inhibited by the estrogen receptor (ER) antagonists ICI 182,780 and tamoxifen, and from ERalpha(-/-) bone marrow cells, indicating that E2 acted via ERs. E2 addition was most effective in promoting DC differentiation immediately ex vivo, but did not increase DC proliferation. E2 treatment specifically promoted differentiation of a CD11c(+) CD11b(int) DC population that displayed high levels of cell surface MHC class II and CD86, suggesting that E2 could augment numbers of potent APC. DC that differentiated in E2-supplemented medium were fully functional in their capability to mediate presentation of self and foreign Ags and stimulate the proliferation of naive CD4(+) T cells. The requirement for estrogen during DC differentiation suggests a mechanism by which E2 levels in peripheral tissues might modulate both the number and functional capabilities of DC in vivo, thereby influencing immune responses.     

Phylogeography and Pleistocene demographic history of the endangered marsh deer (Blastocerus dichotomus) from the Río de la Plata Basin

The marsh deer is the largest neotropical cervid with morphological and ecological adaptations to wetlands and riparian habitats. Historically, this now endangered species occupied habitats along the major river basins in South America, ranging from southern Amazonia into northern Argentina to the Paraná river delta. This particularly close association with wetlands makes marsh deer an excellent species for studying the effects of Pleistocene climatic changes on their demographic and phylogeographic patterns. We examined mitochondrial DNA variation in 127 marsh deer from 4 areas distributed throughout the Río de la Plata basin. We found 17 haplotypes in marsh deer from Brazil, Bolivia and Argentina that differed by 1–8 substitutions in a 601 bp fragment of mitochondrial control region sequence, and 486 bp of cytochrome b revealed only 3 variable sites that defined 4 haplotypes. Phylogeny and distribution of control region haplotypes suggest that populations close to the Pantanal area in central Brazil underwent a rapid population expansion and that this occurred approximately 28,000–25,000 years BP. Paleoclimatic data from this period suggests that there was a dramatic increase for precipitation in the medium latitudes in South America and these conditions may have fostered marsh deer’s population growth.     

The zebrafish mutant vps18 as a model for vesicle‐traffic related hypopigmentation diseases

Hypopigmentation is a characteristic of several diseases associated with vesicle traffic defects, like the Hermansky–Pudlak, Chediak–Higashi, and Griscelli syndromes. Hypopigmentation is also a characteristic of the zebrafish mutant vps18^hi2499A, which is affected in the gene vps18, a component of the homotypic fusion and protein sorting complex that is involved in tethering during vesicular traffic. Vps18, as part of this complex, participates in the formation of early endosomes, late endosomes, and lysosomes. Here, we show that Vps18 is also involved in the formation of melanosomes. In the zebrafish mutant vps18^hi2499A the retroviral insertion located at exon 4 of vps18, leads to the formation of two abnormal splicing variants lacking the coding sequence for the clathrin repeat and the RING finger conserved domains. A deficiency of Vps18 in zebrafish larvae results in hepatomegaly and skin hypopigmentation. We also observed a drastic reduction in the number of melanosomes in the eye's retinal pigmented epithelium along with the accumulation of immature melanosomes. A significant reduction in the vps18^hi2499A larvae visual system capacity was found using the optokinetic response assay. We propose that the insertional mutant vps18^hi2499A can be used as a model for studying hypopigmentation diseases in which vesicle traffic problems exist.     

A field model of learning: 1. Short-term memory in the crab Chasmagnathus granulatus

Learning and memory studies have been performed for more than two decades using the crab Chasmagnathus in our laboratory. Here, our research was aimed at disclosing some instances of learning in field conditions. Three experiments were performed non-simultaneously, all with a 22.5-min pre-training preceding the first visual-danger-stimulus, an opaque rectangle passing overhead. In Experiment 1, crabs received a single stimulus followed by 22.5-min testing without stimulation, where the re-emerging latency was considered the basic latency response. In Experiment 2, training consisted of 15 stimulus 3-min apart, followed by 22.5-min testing without stimulation. Throughout training crabs were underground but re-emerged at testing with latencies longer than the basic latency response. Both at pre-training and testing the usual strategy of exploring was the short-near excursions. In Experiment 3, training included three stimulus 22.5-min apart, followed by 22.5-min testing. Crabs left their burrows before the end of each inter-trial, showing a mean latency like the basic latency response, but a sensitization to the stimulus and a preponderance of the fast-far excursions over the usual slow-near. In brief: through 15-3 training, crabs learn that the stimulus is iteratively presented; through 3-22.5 training, crabs acquire sensitization to the stimulus and a different strategy of exploration.     

E6AP ubiquitin ligase mediates ubiquitin‐dependent degradation of peroxiredoxin 1

E6‐associated protein (E6AP) is a cellular ubiquitin protein ligase that mediates ubiquitylation and degradation of tumor suppressor p53 in conjunction with the high‐risk human papillomavirus E6 protein. We previously reported that E6AP targets annexin A1 protein for ubiquitin‐dependent proteasomal degradation. To gain a better understanding of the physiological function of E6AP, we have been seeking to identify novel substrates of E6AP. Here, we identified peroxiredoxin 1 (Prx1) as a novel E6AP‐binding protein using a tandem affinity purification procedure coupled with mass spectrometry. Prx1 is a 25‐kDa member of the Prx family, a ubiquitous family of antioxidant peroxidases that regulate many cellular processes through intracellular oxidative signal transduction pathways. Immunoprecipitation analysis showed that E6AP binds Prx1 in vivo. Pull‐down experiments showed that E6AP binds Prx1 in vitro. Ectopic expression of E6AP enhanced the degradation of Prx1 in vivo. In vivo and in vitro ubiquitylation assays revealed that E6AP promoted polyubiquitylation of Prx1. RNAi‐mediated downregulation of endogenous E6AP increased the level of endogenous Prx1 protein. Taken together, our data suggest that E6AP mediates the ubiquitin‐dependent proteasomal degradation of Prx1. Our findings raise a possibility that E6AP may play a role in regulating Prx1‐dependent intracellular oxidative signal transduction pathways. J. Cell. Biochem. 111: 676–685, 2010. © 2010 Wiley‐Liss, Inc.