Distinct mechanisms of splicing regulation in vivo by the Drosophila protein Sex-lethal

The protein Sex-lethal (SXL) controls pre-mRNA splicing of two genes involved in Drosophila sex determination: transformer (tra) and the Sxl gene itself. Previous in vitro results indicated that SXL antagonizes the general splicing factor U2AF⁶⁵ to regulate splicing of tra. In this report, we have used transgenic flies expressing chimeric proteins between SXL and the effector domain of U2AF⁶⁵ to study the mechanisms of splicing regulation by SXL in vivo. Conferring U2AF activity to SXL relieves its inhibitory activity on tra splicing but not on Sxl splicing. Therefore, antagonizing U2AF⁶⁵ can explain tra splicing regulation both in vitro and in vivo, but this mechanism cannot explain splicing regulation of Sxl pre-mRNA. These results are a direct proof that Sxl, the master regulatory gene in sex determination, has multiple and separable activities in the regulation of pre-mRNA splicing.     

T-wave alternans: predicting the unpredictable?

Sudden cardiac death (SCD) due to ventricular arrhythmias hasa high morbidity and mortality. It is no wonder that cardiologists,rather than patching up survivors, are dedicated to findingpredictors of cardiac electrical instability to prevent SCD.The implantable cardiac defibrillator (ICD) has evolved as theultimate strategy for sudden arrhythmic death. In secondaryprevention, individuals are recognized by an aborted arrhythmicevent. By stratifying according to left ventricular ejectionfraction (LVEF) below 35%, SCD may also be prevented by an ICDin individuals without any prior arrhythmic event.¹ However,the relative SCD risk based     

Phenotype traits associated with different alleles at the RPS5 locus in Saccharomyces cerevisiae

Summary The RPS5 gene has been characterised through its ability to reduce invertase production by the SUC5 gene. In this paper we show that RPS5 acts by maintaining low levels of SUC5 mRNA. We also show that RPS5 acts on the SUC1 and SUC4 genes but not on SUC2 and SUC3, which are members of the SUC family. RPS5 also shows a pleiotropic effect on the amount of mitochondrial cytochromes.     

Comparing the validity of clinician-generated diagnosis of conduct disorder to the diagnostic interview schedule for children.

Clinician diagnoses of conduct disorder (CD) were compared to the diagnoses of CD generated by a structured interview against an observed criterion. Participants were 534 youth from a large residential program in the Midwest for delinquent youth. Rates of in-program CD behaviors were gathered from staff observations of the youth over a 9-month time period. Youth diagnosed with CD by the Diagnostic Interview Schedule for Children (DISC) displayed significantly more CD behaviors in the first 6 months of treatment compared to both youth without an externalizing disorder and youth diagnosed with CD by a clinician. Youth diagnosed with CD by a clinician had rates of CD identical to youth without an externalizing disorder. Clinicians may have weighted contextual information more heavily, as this group was significantly more likely to have an arrest record. Results support the use of structured interviews and provide evidence that typical clinician diagnoses may lack adequate validity.     

A 94-kDa protein, identified as neutral endopeptidase-24.11, can inactivate atrial natriuretic peptide in the vascular endothelium.

Neutral endopeptidase 24.11 (EC 3.4.24.11) inactivates atrial natriuretic peptide by cleaving the hormone between Cys7 and Phe8, and inhibitors of the enzyme have consequent natriuretic and diuretic properties. The in vivo sites of degradation of this peptide by the zinc-metallopeptidase, however, remain to be established. Because an endopeptidase-24.11-like activity has recently been reported in the rat mesenteric artery, we have further investigated the degradation of atrial natriuretic peptide in vascular tissue. Endopeptidase-24.11 activity was detected in solubilized membrane preparations from rat and rabbit vascular tissue, using [3H]D-Ala2-leucine enkephalin as substrate, and both rabbit and rat aorta preparations were also found to cleave atrial natriuretic peptide between Cys7 and Phe8. In both cases, hydrolysis was inhibited by neutral endopeptidase inhibitors, with Ki values close to their Ki values for the pure enzyme. In preparations of rabbit aorta denuded of endothelium by saponin treatment, the hydrolysis of the Gly3-Phe4 bond of [3H]D-Ala2-leucine enkephalin and the Cys7-Phe8 bond of atrial natriuretic peptide was reduced by greater than 90%. The high performance liquid chromatography method used to follow the degradation of atrial natriuretic peptide differed from previously published procedures, in that samples to be injected were first treated with excess dithiothreitol to reduce the Cys7-Cys23 disulfide bridge. This facilitated the separation of the intact peptide and its metabolites. The presence of the 94-kDa neutral endopeptidase in rabbit aortic tissue was definitively established using a new potent 125I-labeled inhibitor, [125I]RB104 [2-[(3-[125I]iodo-4-hydroxy)phenylmethyl]-4-N-[3- hydroxyamino-3-oxo-1-phenylmethyl propyl]amino-4-oxobutanoic acid] (Ki, 30 pM), which selectively labeled the enzyme after sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the membrane preparations. Therefore, despite its low concentrations in the vasculature, the presence of endopeptidase-24.11 almost exclusively in endothelial tissue suggests that the enzyme is ideally localized to inactivate circulating atrial natriuretic peptide.     

Prospective Evaluation of the Chemiluminescence Test for the Detection of Granulocyte Antibodies: Comparison with the Granulocyte Immunofluorescence Test

A series of 213 neutropenic patients were tested for the presence of granulocyte antibodies using the granulocyte chemiluminescence test (GCLT) and the granulocyte immunofluorescence test (GIFT). Sera containing lymphocyte (HLA) antibodies were excluded from the study. A direct GIFT was performed on granulocytes from 56 patients. Samples were obtained from patients with a range of clinical conditions including primary adult autoimmune neutropenia, autoimmune neutropenia of infancy, autoimmune neutropenia secondary to Felty's syndrome, rheumatoid arthritis, idiopathic thrombocytopenic purpura, systemic lupus erythematosus, proliferative disorders, bone marrow transplantation and patients with documented febrile or pulmonary transfusion reactions. Overall, granulocyte antibodies were detected in 52.1% of patient sera. Results for the GCLT and GIFT (IgG) were strongly correlated (plt;0.001) for both primary and secondary immune neutropenias. The results confirm the applicability of the GCLT in the granulocyte serology laboratory.     

Phase IB study of doxorubicin in combination with the multidrug resistance reversing agent S9788 in advanced colorectal and renal cell cancer.

S9788 is a new triazineaminopiperidine derivate capable of reversing multidrug resistance (MDR) in cells resistant to chemotherapeutic agents such as doxorubicin. It does not belong to a known class of MDR revertants, but its action involves the binding of P-glycoprotein. Thirty-eight evaluable patients with advanced colorectal or renal cell cancer were treated with doxorubicin alone (16 patients) followed after disease progression with combination treatment of doxorubicin plus S9788 (12 patients) or upfront with the combination of doxorubicin plus S9788 (22 patients). S9788 was given i.v. as a loading dose of 56 mg m-2 over 30 min followed by doxorubicin given at 50 mg m-2 as a bolus infusion. Thereafter, a 2-h infusion of S9788 was administered at escalating doses ranging from 24 to 120 mg m-2 in subsequent cohorts of 4-10 patients. Pharmacokinetic analysis demonstrated that concentrations of S9788 that are known to reverse MDR in vitro were achieved in patients at non-toxic doses. Compared with treatment with doxorubicin alone, treatment with the combination of doxorubicin and S9788 produced a significant increase in the occurrence of WHO grade 3-4 granulocytopenia. Treatment with S9788 was cardiotoxic as it caused a dose-dependent and reversible increase in corrected QT intervals as well as clinically non-significant arrhythmias on 24- or 48-h Holter recordings. Although clinically relevant cardiac toxicities did not occur, the study was terminated as higher doses of S9788 may increase the risk of severe cardiac arrhythmias. Twenty-nine patients treated with S9788 plus doxorubicin were evaluable for response, and one patient, who progressed after treatment with doxorubicin alone, achieved a partial response. We conclude that S9788 administered at the doses and schedule used in this study results in relevant plasma concentrations in humans and can safely be administered in combination with doxorubicin.     

MR-Guided percutaneous ethanol ablation of liver tissue in a .2-T open MR system: Preliminary study in porcine model

The purpose of this study was to test the feasibility of MR-guided percutaneous ethanol ablation of liver tissue on a .2-T open MR scanner. Needles were placed by MR guidance first into an ex vivo sheep liver and then into livers of three anesthetized pigs, and injection of 10 ml of 96% alcohol was performed. T1 fast low-angle shot (FLASH), T2 turbo spin echo (TSE), and T1 spin echo (SE) images were obtained after incremental volumes of injection. In one pig, simultaneous injection of saline into normal liver was also performed with subsequent pathological correlation. Ethanol-infiltrated liver was hypointense to liver on all sequences, whereas saline caused no tissue signal changes on T1 SE and either isointense or hyperintense changes on T2 TSE images. Pathological examination confirmed ethanol-induced acute liver changes as compared with the control. MR guidance of needle placement and monitoring of ethanol effects on liver tissue is feasible. This may have implications for potential MR-guided hepatic tumor ablation.