Severe Painful Vaso-Occlusive Crises and Mortality in a Contemporary Adult Sickle Cell Anemia Cohort Study

Background

Frequent painful vaso-occlusive crises (VOCs) were associated with mortality in the Cooperative Study of Sickle Cell Disease (CSSCD) over twenty years ago. Modern therapies for sickle cell anemia (SCA) like hydroxyurea are believed to have improved overall patient survival. The current study sought to determine the relevance of the association between more frequent VOCs and death and its relative impact upon overall mortality compared to other known risk factors in a contemporary adult SCA cohort.

Methods

Two hundred sixty four SCA adults were assigned into two groups based on patient reported outcomes for emergency department (ED) visits or hospitalizations for painful VOC treatment during the 12 months prior to evaluation.

Results

Higher baseline hematocrit (p = 0.0008), ferritin (p = 0.005), and HDL cholesterol (p = 0.01) were independently associated with 1 or more painful VOCs requiring an ED visit or hospitalization for acute pain. During a median follow-up of 5 years, mortality was higher in the ED visit/hospitalization group (relative risk [RR] 2.68, 95% CI 1.1-6.5, p = 0.03). Higher tricuspid regurgitatant jet velocity (TRV) (RR 2.41, 95% CI 1.5-3.9, p < 0.0001), elevated ferritin (RR 4.00, 95% CI 1.8-9.0, p = 0.001) and lower glomerular filtration rate (RR=2.73, 95% CI 1.6-4.6, p < 0.0001) were also independent risk factors for mortality.

Conclusions

Severe painful VOCs remain a marker for SCA disease severity and premature mortality in a modern cohort along with other known risk factors for death including high TRV, high ferritin and lower renal function. The number of patient reported pain crises requiring healthcare utilization is an easily obtained outcome that could help to identify high risk patients for disease modifying therapies.

Trial Registration

ClinicalTrials.gov NCT00011648 http://clinicaltrials.gov/     

Internalization and processing of human angiogenin by cultured aortic smooth muscle cells

Human angiogenin is a 14-kDa plasma protein with angiogenic and ribonucleolytic activities. Angiogenin binds specifically to aortic smooth muscle cells, activates second messenger pathways, and inhibits their proliferation. Human and bovine aortic smooth muscle cells were used to study the internalization and intracellular fate of human angiogenin at 37 degrees C. Using a specific antibody against angiogenin, we found that the internalized native protein was localized in the perinuclear region at 30 min and then dispersed throughout the cytoplasm. In conditions favoring receptor-mediated endocytosis, internalization of iodinated angiogenin showed a first peak at 5 min and then further increased for up to 24 h. The half-life of the molecule, calculated as 12 h in chase experiments, could contribute to its intracellular accumulation. In cell extracts, in addition to the 14-kDa protein, a 8.7-kDa fragment was observed at 24 h, and three fragments with molecular mass of 10.5, 8.7, and 6. 1 kDa were detected at 48 h. Our data point to a specific internalization and processing of human angiogenin by aortic smooth muscle cells.     

Effects of medium viscosity on kinetics of the enzymatic reaction catalyzed by bacterial RNase

Effects of medium viscosity on kinetic parameters of poly(U) hydrolysis catalyzed by RNase from Bac. intermedius 7P (binase) were studied in solutions of sucrose (4-50 wt. %) and glycerol (35-62 wt. %) in Tris--sodium acetate buffer (pH 7.5) at 25 degreesC. The rate constant of reaction kcat was practically unchanged over a wide range of viscosities (1-15 cP for sucrose and 2.5-3 cP for glycerol). In glycerol solutions, kcat slightly increased with viscosity increase from 4 to 10 cP. Addition of NaCl to the buffer medium resulted in an inhibitory effect of Na+ on kcat, prevented by 50% sucrose or 60% glycerol. It is concluded that binase-catalyzed poly(U) cleavage occurs through a "tense"-substrate mechanism, similarly to reactions catalyzed by alpha-chymotrypsin, trypsin, and laccase.     

A web server to locate periodicities in a sequence

Summary : FT is a tool written in C++, which implements the Fourier analysis method to locate periodicities in aminoacid or DNA sequences. It is provided for free public use on a WWW server with a Java interface. Availability : The server address is http://o2.db. uoa.gr/FT Contact : shamodr@atlas.uoa.gr      

Affinity-based proteomics reveal cancer-specific networks coordinated by Hsp90

Most cancers are characterized by multiple molecular alterations, but identification of the key proteins involved in these signaling pathways is currently beyond reach. We show that the inhibitor PU-H71 preferentially targets tumor-enriched Hsp90 complexes and affinity captures Hsp90-dependent oncogenic client proteins. We have used PU-H71 affinity capture to design a proteomic approach that, when combined with bioinformatic pathway analysis, identifies dysregulated signaling networks and key oncoproteins in chronic myeloid leukemia. The identified interactome overlaps with the well-characterized altered proteome in this cancer, indicating that this method can provide global insights into the biology of individual tumors, including primary patient specimens. In addition, we show that this approach can be used to identify previously uncharacterized oncoproteins and mechanisms, potentially leading to new targeted therapies. We further show that the abundance of the PU-H71-enriched Hsp90 species, which is not dictated by Hsp90 expression alone, is predictive of the cell's sensitivity to Hsp90 inhibition.     

BRCA1 role in the mitigation of radiotoxicity and chromosomal instability through repair of clustered DNA lesions

Oxidatively-induced clustered DNA lesions are considered the signature of any ionizing radiation like the ones human beings are exposed daily from various environmental sources (medical X-rays, radon, etc.). To evaluate the role of BRCA1 deficiencies in the mitigation of radiation-induced toxicity and chromosomal instability we have used two human breast cancer cell lines, the BRCA1 deficient HCC1937 cells and as a control the BRCA1 wild-type MCF-7 cells. As an additional control for the DNA damage repair measurements, the HCC1937 cells with partially reconstituted BRCA1 expression were used. Since clustered DNA damage is considered the signature of ionizing radiation, we have measured the repair of double strand breaks (DSBs), non-DSB bistranded oxidative clustered DNA lesions (OCDLs) as well as single strand breaks (SSBs) in cells exposed to radiotherapy-relevant γ-ray doses. Parallel measurements were performed in the accumulation of chromatid and isochromatid breaks. For the measurement of OCDL repair, we have used a novel adaptation of the denaturing single cell gel electrophoresis (Comet assay) and pulsed field gel electrophoresis with Escherichia coli repair enzymes as DNA damage probes. Independent monitoring of the γ-H2AX foci was also performed while metaphase chromatid lesions were measured as an indicator of chromosomal instability. HCC1937 cells showed a significant accumulation of all types of DNA damage and chromatid breaks compared to MCF-7 while BRCA1 partial expression contributed significantly in the overall repair of OCDLs. These results further support the biological significance of repair resistant clustered DNA damage leading to chromosomal instability. The current results combined with previous findings on the minimized ability of base clusters to induce cell death (mainly induced by DSBs), enhance the potential association of OCDLs with breast cancer development especially in the case of a BRCA1 deficiency leading to the survival of breast cells carrying a high load of unrepaired DNA damage clusters.     

The radiosensitizing potential of glutaraldehyde on MCF7 breast cancer cells as quantified by means of the G2-chromosomal radiosensitivity assay

Glutaraldehyde (GA) is a high production volume chemical that is very reactive with a wide spectrum of medical, scientific and industrial applications. Concerning the genotoxic and carcinogenic effect of GA, controversial results have been reported, while in humans no studies with positive carcinogenic results for GA have been published. However, our previous study concerning the combined effects of exposure to both GA and ionising radiation (IR) in peripheral blood lymphocytes of healthy donors has shown that non-genotoxic doses of the chemical induces a statistically significant increase in chromosomal radiosensitivity. The lack of information concerning the radiosensitizing potential of GA on cancerous cells triggered us to test the radiosensitizing effect of GA on breast cancer cells (MCF7). For this purpose the G2-chromosomal radiosensitivity assay (G2-assay) was used. The assay involves G2-phase irradiation and quantitation of the chromosomal fragility in the subsequent metaphase. The experimental data show that 48 h exposure to GA, at doses that are not clastogenic to MCF7 breast cancer cells enhances G2-chromosomal radiosensitivity of this cell line. In an effort to evaluate whether the observed increase in GAs-induced G2-chromosomal radiosensitization is linked to GA-induced alterations in the cell cycle and feedback control mechanism, Mitotic Index analysis was performed. The results have shown that such a mechanism cannot be directly related to the observed GA-induced increase in G2-chromosomal radiosensitivity. Since increased G2-chromosomal radiosensitivity has been linked with cancer proneness, the radiosensitizing effect of GA at non-clastogenic doses highlights its potential carcinogenic profile.     

Comparison of the stepwise and convergent approaches in the solid-phase synthesis of rat Tyr0-atriopeptin II

Summary Tyr^o-Atriopeptin II was synthesized on a 2-chlorotrityl resin by both, the stepwise and the convergent approach. For both methods an Fmoc/tBu(Trt)-based protection scheme was used. The convergent methodology utilizes the sequential condensation of four protected peptide fragments. These were chosen so that after every condensation reaction, the amino-terminal region of the newly formed resin-bound peptide did not contain a β-turn. This ‘designed’ convergent synthesis gave the target peptide in much higher yield and purity than the conventional stepby-step synthesis. HOAc, acetic acid; Boc,tert-butyloxycarbonyl; DCC, dicyclohexylcarbodiimide: DCM, dichloromethane; DIC, diisopropylcarbodiimide; DIEA,N,N-diisopropylethylamine; DMFN,N-dimethylformamide; DMSO, dimethylsulfoxide; EDT. ethanedithiol; FAB-MS, fast atom bombardment mass spectrometry; Fmoc, 9-luorenylmethoxycarbonyl; HOBt, 1-hydroxybenzotriazole; HPLC, high-performance liquid chromatography; i-PrOH, isopropanol; Mmt, 4-methoxytrityl; PEG-PS, polvethyleneglycol grafted polystyrene; Pme, 2,2,5,7,8-pentamethylchroman-6-sulfonyl; RP, reversed phase; rt, room temperature; SPPS, solid phase peptide synthesis;tBu,tert-butyl; TFA, trifluoroacetic acid; TFE, trifluoroethanol; TLC, thin layer chromatography; Trt, triphenylmethyl, trityl. Abbreviations used for amino acids follow the rules of the IUPAC-IUB Commission of Biochemical Nomenclature [J. Biol. Chem. 247 (1972), 977]. All amino acids are of the L-configuration.     

N-myc oncogene overexpression down-regulates leukemia inhibitory factor in neuroblastoma.

Amplification of N-myc oncogene is a frequent event in advanced stages of human neuroblastoma and correlates with poor prognosis and enhanced neovascularization. Angiogenesis is an indispensable prerequisite for the progression and metastasis of solid malignancies, which is modulated by tumor suppressors and oncogenes. We have addressed the possibility that N-myc oncogene might regulate angiogenesis in neuroblastoma. Here, we report that experimental N-Myc overexpression results in down-regulation of leukemia inhibitory factor (LIF), a modulator of endothelial cell proliferation. Reporter assays using the LIF promoter and a series of N-Myc mutants clearly demonstrated that down-regulation of the LIF promoter was independent of Myc/Max interaction and required a contiguous N-terminal N-Myc domain. STAT3, a downstream signal transducer, was essential for LIF activity as infection with adenoviruses expressing a phosphorylation-deficient STAT3 mutant rendered endothelial cells insensitive to the antiproliferative action of LIF. LIF did not influence neuroblastoma cell proliferation suggesting that, at least in the context of neuroblastoma, LIF is involved in paracrine rather than autocrine interactions. Our data shed light on the mechanisms by which N-myc oncogene amplification enhances the malignant phenotype in neuroblastoma.