The calcineurin-NFAT pathway negatively regulates megakaryopoiesis.

The calcium regulated calcineurin-nuclear factor of activated T cells (NFAT) pathway modulates the physiology of numerous cell types, including hematopoietic. Upon activation, calcineurin dephosphorylates NFAT family transcription factors, triggering their nuclear entry and activation or repression of target genes. NFATc1 and c2 isoforms are expressed in megakaryocytes. Moreover, human chromosome 21 (Hsa21) encodes several negative regulators of calcineurin-NFAT, candidates in the pathogenesis of Down syndrome (trisomy 21)-associated transient myeloproliferative disorder and acute megakaryoblastic leukemia. To investigate the role of calcineurin-NFAT in megakaryopoiesis, we examined wild-type mice treated with the calcineurin inhibitor cyclosporin A and transgenic mice expressing a targeted single extra copy of Dscr1, an Hsa21-encoded calcineurin inhibitor. Both murine models exhibited thrombocytosis with increased megakaryocytes and megakaryocyte progenitors. Pharmacological or genetic inhibition of calcineurin in mice caused accumulation of megakaryocytes exhibiting enhanced 5-bromo-2'-deoxyuridine uptake and increased expression of messenger RNAs encoding CDK4 and G1 cyclins, which promote cell division. Additionally, human megakaryocytes with trisomy 21 show increased proliferation and decreased NFAT activation compared with euploid controls. Our data indicate that inhibition of calcineurin-NFAT drives proliferation of megakaryocyte precursors by de-repressing genes that drive cell division, providing insights into mechanisms of normal megakaryopoiesis and megakaryocytic abnormalities that accompany Down syndrome.

Overcoming evasive resistance from vascular endothelial growth factor a inhibition in sarcomas by genetic or pharmacologic targeting of hypoxia-inducible factor 1α.

Increased levels of hypoxia and hypoxia-inducible factor 1α (HIF-1α) in human sarcomas correlate with tumor progression and radiation resistance. Prolonged antiangiogenic therapy of tumors not only delays tumor growth but may also increase hypoxia and HIF-1α activity. In our recent clinical trial, treatment with the vascular endothelial growth factor A (VEGF-A) antibody, bevacizumab, followed by a combination of bevacizumab and radiation led to near complete necrosis in nearly half of sarcomas. Gene Set Enrichment Analysis of microarrays from pretreatment biopsies found that the Gene Ontology category "Response to hypoxia" was upregulated in poor responders and that the hierarchical clustering based on 140 hypoxia-responsive genes reliably separated poor responders from good responders. The most commonly used chemotherapeutic drug for sarcomas, doxorubicin (Dox), was recently found to block HIF-1α binding to DNA at low metronomic doses. In four sarcoma cell lines, HIF-1α shRNA or Dox at low concentrations blocked HIF-1α induction of VEGF-A by 84-97% and carbonic anhydrase 9 by 83-93%. HT1080 sarcoma xenografts had increased hypoxia and/or HIF-1α activity with increasing tumor size and with anti-VEGF receptor antibody (DC101) treatment. Combining DC101 with HIF-1α shRNA or metronomic Dox had a synergistic effect in suppressing growth of HT1080 xenografts, at least in part via induction of tumor endothelial cell apoptosis. In conclusion, sarcomas respond to increased hypoxia by expressing HIF-1α target genes that may promote resistance to antiangiogenic and other therapies. HIF-1α inhibition blocks this evasive resistance and augments destruction of the tumor vasculature.

Hydrogen peroxide induces hyphal differentiation in Candida albicans.

In this study, we demonstrate that hyphal differentiation is induced by the subtoxic concentration of exogenous H(2)O(2) in Candida albicans. This finding is confirmed by the changing intracellular concentration of H(2)O(2). In order to induce the same level of differentiation, low concentrations of exogenous H(2)O(2) are required for the null mutants of the thiol-specific antioxidant and catalase, while higher concentrations are needed for cells treated with ascorbic acid, an antioxidant chemical.

Does delayed facial involvement implicate a pattern of "descending reversible paralysis" in Fisher syndrome?

OBJECTIVE: Delayed facial palsy (DFP) has often been described during the recovery stage of Fisher syndrome (FS), but the implications of DFP in FS pathophysiology have not been reported previously. The aim of this study was to identify the incidence and clinical course of DFP in FS, and to determine its clinical/pathophysiological implications in FS. METHODS: About 71 FS patients were enrolled from seven university-based hospitals in Korea. DFP was defined with respect to new development of unilateral or bilateral facial palsies with delayed onset after either the nadir or improvement of initial neurological signs of FS. RESULTS: Eleven of the 71 patients (16%) satisfied the definition of DFP. No other cranial palsies developed as a delayed pattern. With the exception of two patients with bulbar involvement, DFP developed after a latent period of upper-cranial neuropathies. Comparison of FS patients without and with DFP revealed no significant clinical, serological, or electrophysiological differences. All except one patient with DFP exhibited a good outcome within 1 month of follow-up. CONCLUSION: DFP was identified as a common and specific phenomenon in FS. Nearly all cases of DFP were developed in a descending manner and were associated with a good outcome, while other cranial neuropathies developed or followed as a sequential pattern. These findings suggest the involvement of so-called "descending reversible paralysis" in the pathophysiology of FS.

Inhibition of vascular endothelial growth factor A and hypoxia-inducible factor 1α maximizes the effects of radiation in sarcoma mouse models through destruction of tumor vasculature.

PURPOSE: To examine the addition of genetic or pharmacologic inhibition of hypoxia-inducible factor 1α (HIF-1α) to radiation therapy (RT) and vascular endothelial growth factor A (VEGF-A) inhibition (ie trimodality therapy) for soft-tissue sarcoma. METHODS AND MATERIALS: Hypoxia-inducible factor 1α was inhibited using short hairpin RNA or low metronomic doses of doxorubicin, which blocks HIF-1α binding to DNA. Trimodality therapy was examined in a mouse xenograft model and a genetically engineered mouse model of sarcoma, as well as in vitro in tumor endothelial cells (ECs) and 4 sarcoma cell lines. RESULTS: In both mouse models, any monotherapy or bimodality therapy resulted in tumor growth beyond 250 mm(3) within the 12-day treatment period, but trimodality therapy with RT, VEGF-A inhibition, and HIF-1α inhibition kept tumors at <250 mm(3) for up to 30 days. Trimodality therapy on tumors reduced HIF-1α activity as measured by expression of nuclear HIF-1α by 87% to 95% compared with RT alone, and cytoplasmic carbonic anhydrase 9 by 79% to 82%. Trimodality therapy also increased EC-specific apoptosis 2- to 4-fold more than RT alone and reduced microvessel density by 75% to 82%. When tumor ECs were treated in vitro with trimodality therapy under hypoxia, there were significant decreases in proliferation and colony formation and increases in DNA damage (as measured by Comet assay and γH2AX expression) and apoptosis (as measured by cleaved caspase 3 expression). Trimodality therapy had much less pronounced effects when 4 sarcoma cell lines were examined in these same assays. CONCLUSIONS: Inhibition of HIF-1α is highly effective when combined with RT and VEGF-A inhibition in blocking sarcoma growth by maximizing DNA damage and apoptosis in tumor ECs, leading to loss of tumor vasculature.

Development of DNA chip for the simultaneous detection of various beta-lactam antibiotic-resistant genes.

A robust and fast DNA chip method was developed in order to detect the various beta-lactam antibiotic-resistance genes in one slide. These genes included PSE, OXA, FOX, MEN, CMY, TEM, SHV, OXY, and AmpC. beta-lactam antibiotic-resistance genes were labeled with a fluorescent nucleotide by a multiplex polymerase chain reaction using a mixture of specific primer sets for each gene. This labeled target was hybridized with a DNA chip that contained the spots of the specific probe DNAs for each beta-lactam antibiotic-resistance gene. This technique made it possible to detect the specific resistance gene, even in a single bacterium.

Dopa responsive slow orthostatic tremor in Parkinson's disease.

Slow orthostatic tremor (OT) occurred to longer and lower frequency regular rhythmic bursts in leg muscle upon standing. The slow OT was often able to clinically confused with orthostatic myoclonus. We described a Parkinson's disease patient with levodopa responsive slow OT. She showed abnormal movements of more regular rhythms and stable frequency on both legs on standing. These symptoms were aggravated at off state and improved by increasing levodopa.

Rupture of external iliac artery during microscopic lumbar disc surgery -A case report-.

Iatrogenic vascular injury during lumbar disc surgery is a rare but serious complication. This paper reports a patient who sustained an injury to the iliac artery while undergoing intervertebral disc surgery at the lumbar region. He suffered from massive bleeding and shock. An urgent laparatomy was performed under cardiopulmonary bypass, and the vascular injuries were repaired successfully. This case shows that a rapid diagnosis and immediate intervention can result in a favorable outcome.

Characterization of thiol-specific antioxidant 1 (TSA1) of Candida albicans.

We previously identified several proteins that are differentially expressed in pathogenic hyphae by comparing protein profiles of yeast and hyphae of Candida albicans. One of these, thiol-specific antioxidant 1 (TSA1), attracted our attention because it may play some roles in surviving an unfavourable oxidative environment created by host cells. Two alleles of the C. albicans TSA1 (CaTSA1) gene are located in opposite orientation on the same chromosome. Using PCR-directed disruption cassettes and URA-Blaster, a series of deletion mutants that lack one to four copies were constructed to examine the functions of CaTSA1. Northern and Western analyses showed that both the transcript and protein products of CaTSA1 decreased proportionally to the disrupted copy number and were completely absent in the null mutant, indicating that all four TSA1 copies are equally functional at the transcriptional level. Intracellular H2O2 increased by an order of magnitude in deletion mutants lacking three to four copies, suggesting that CaTsa1p is not a redundant H2O2 scavenger. CaTsa1p was indispensable for yeast-to-hyphal transition when C. albicans was cultured under oxidative stress. The level of its oxidized form increased approximately five-fold in hyphal cells, whereas that of the reduced form increased two-fold compared to yeast cells. The ratio of oxidized to reduced form was increased three-fold in hyphal cells. This overall increase was found to be controlled at the post-transcriptional level. Interestingly, CaTsa1p is translocated to the nucleus of hyphal cells. These findings may be of biological significance for differentiation and pathogenicity.