Multiple roles of mouse Numb in tuning developmental cell fates.

BACKGROUND: Notch signaling regulates multiple differentiation processes and cell fate decisions during both invertebrate and vertebrate development. Numb encodes an intracellular protein that was shown in Drosophila to antagonize Notch signaling at binary cell fate decisions of certain cell lineages. Although overexpression experiments suggested that Numb might also antagonize some Notch activity in vertebrates, the developmental processes in which Numb is involved remained elusive. RESULTS: We generated mice with a homozygous inactivation of Numb. These mice died before embryonic day E11.5, probably because of defects in angiogenic remodeling and placental dysfunction. Mutant embryos had an open anterior neural tube and impaired neuronal differentiation within the developing cranial central nervous system (CNS). In the developing spinal cord, the number of differentiated motoneurons was reduced. Within the peripheral nervous system (PNS), ganglia of cranial sensory neurons were formed. Trunk neural crest cells migrated and differentiated into sympathetic neurons. In contrast, a selective differentiation anomaly was observed in dorsal root ganglia, where neural crest--derived progenitor cells had migrated normally to form ganglionic structures, but failed to differentiate into sensory neurons. CONCLUSIONS: Mouse Numb is involved in multiple developmental processes and required for cell fate tuning in a variety of lineages. In the nervous system, Numb is required for the generation of a large subset of neuronal lineages. The restricted requirement of Numb during neural development in the mouse suggests that in some neuronal lineages, Notch signaling may be regulated independently of Numb.

Modulation of the human homeobox genes PRX-2 and HOXB13 in scarless fetal wounds.

Scarless healing of cutaneous wounds occurs in humans during the first two trimesters of development, but by birth all wounds are repaired with scar formation. To search for transcriptional regulatory genes that might mediate fetal tissue regeneration, we surveyed homeobox gene expression in proliferating fetal fibroblasts and in wounded and unwounded skin. Two novel human homeobox genes, PRX-2 and HOXB13, were identified that were differentially expressed during fetal versus adult wound healing. Both genes were predominantly expressed in proliferating fetal fibroblasts and developing dermis, and PRX-2 was downregulated in adult skin. In a model of scarless fetal skin regeneration, PRX-2 expression was strongly increased compared with unwounded skin and the signal was localized to the wounded dermis, the site of scarless repair. Conversely, in adult skin weak epidermal PRX-2 expression was observed, mRNA levels were not increased by wounding, and no dermal expression was detected. HOXB13 expression was decreased in wounded fetal tissue relative to unwounded fetal controls or wounded adult skin. Thus both HOXB13 and PRX-2 are expressed in patterns consistent with roles in fetal skin development and cutaneous regeneration.

Metabolism of promutagens catalyzed by Drosophila melanogaster CYP6A2 enzyme in Saccharomyces cerevisiae.

The somatic mutation and recombination test (SMART) in Drosophila melanogaster allows screening of chemicals for genotoxicity in a multicellular organism. In order to correlate data obtained in the SMART with those from genotoxicity tests in rodents, it is important to learn more on the variety of drug-metabolizing enzymes present in this insect and to identify their substrate specificities. In this study we have concentrated on the phase I enzyme cytochrome P450 6A2, which is the first cytochrome P450 cloned from Drosophila. A genomic CYP6A2 DNA fragment and its corresponding cDNA were cloned and sequenced, revealing a previously unidentified intron with an inframe stop codon. This intron is invariantly present in an insecticide resistant [OR(R)] and a sensitive (flr3) strain. Developmental Northern analysis of CYP6A2 mRNA demonstrated a peak of expression in the third larval and pupal stage. CYP6A2 mRNA was found to be present in the insecticide-resistant strain at higher levels than in the insecticide-sensitive strain. Therefore, insecticide resistance might be correlated with enhanced CYP6A2 expression. The substrate specificity of CYP6A2 enzyme was investigated by coexpressing CYP6A2 cDNA with the cDNA for human NADPH-cytochrome P450 reductase in the yeast Saccharomyces cerevisiae. The transformed strain activated the mycotoxin aflatoxin B1 to a product that induced gene conversion, scored at the trp5 locus. Two other compounds, 7,12-dimethylbenz[a]anthracene (DMBA) and 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2), were metabolized in the transformed strain to cytotoxic products.

Normalization of coronary vasomotion after percutaneous transluminal coronary angioplasty?

BACKGROUND: Coronary vasomotion was evaluated at rest and during bicycle exercise in 33 patients (age, 53 +/- 7 years) with coronary artery disease. In a first group of patients (n = 15), vasomotion was studied before and 4.3 +/- 2.3 months (early) after percutaneous transluminal coronary angioplasty (PTCA), whereas in a second group (n = 18), exercise coronary arteriography was performed 30 +/- 11 months (late) after successful PTCA. Patients with restenosis (percent area stenosis greater than or equal to 75% or percent diameter stenosis greater than or equal to 50%) were excluded. METHODS AND RESULTS: Luminal areas of a normal segment and the stenotic segment were determined at rest, during supine bicycle exercise, and 5 minutes after sublingual nitrate administration by using biplane quantitative coronary arteriography. Work loads before and early after PTCA were identical in group 1 and similar late after PTCA in group 2. Percent area stenosis decreased from 86% to 36% (p less than 0.001) in group 1 and from 93% to 46% (p less than 0.001) in group 2. Normal coronary arteries showed mild vasodilation during exercise before (+3%, NS versus rest), early (+7%, NS versus rest), and late after (+10%, p less than 0.05 versus rest) PTCA. Administration of sublingual nitrate was associated with significant vasodilation of the normal vessel segment before (+27%, p less than 0.001 versus rest), early (+31%, p less than 0.001 versus rest), and late (+21%, p less than 0.001 versus rest) after PTCA. In contrast, the stenotic vessel segments showed coronary vasoconstriction during exercise before PTCA (-25%, p less than 0.001 versus rest), whereas minimal vasomotion was observed early (+2%; NS versus rest) as well as late (+5%; NS versus rest) after PTCA. Individual post-PTCA (early and late) exercise data elicited vasodilation in 19, no vasomotion in four, and vasoconstriction in 10 instances. Sublingual administration of nitrate was associated with a significant increase in minimal luminal area before (+18%, p less than 0.05 versus rest), early (+24%, p less than 0.01 versus rest), and late (+16%, p less than 0.001 versus rest) after PTCA. An inverse linear correlation was found between the percent change in minimal luminal area during peak exercise and percent area stenosis at rest (r = 0.77, p less than 0.001). CONCLUSIONS: Exercise-induced stenosis narrowing is observed before PTCA but normal vasomotion is reestablished in two thirds of all patients early and late after PTCA. In one third, an abnormal reaction to exercise (i.e., vasoconstriction) persisted after PTCA, mainly in those patients with a residual area stenosis of 50% (percent diameter stenosis of 30%) or more. Thus, PTCA appears to have a salutary effect on coronary vasomotion during exercise, which, however, remains dependent on the severity of the residual stenosis.

Oxprenolol and the circulation during anaesthesia in the dog: influence of intrinsic sympathomimetic activity.

Oxprenolol is a non-selective adrenergic beta-receptor antagonist displaying beta-mimetic activity. To test the hypothesis that beta-mimetic activity could minimize the response of the circulation to adrenergic beta-receptor blockade, cumulative dose-response curves to oxprenolol 0.1-1.6 mg kg-1 were obtained in seven anaesthetized dogs. Anaesthesia was maintained with 0.5% halothane supplementing nitrous oxide 66% in oxygen, under moderately hypocapnic IPPV. Oxprenolol, up to 0.4 mg kg-1 i.v., caused modest increases in heart rate, LV dP/dt max and cardiac output. With the largest dose (1.6 mg kg-1), significant increases in heart rate (+19%), LV dP/dt max (+13%) and cardiac output (+27%) were observed while arterial pressure remained unchanged and systemic vascular resistance decreased (-18%).

Circulatory responses of the dog to acute isovolumic anaemia in the presence of high-grade adrenergic beta-receptor blockade.

The haemodynamic responses to isovolumic anaemia (dextran-for-blood exchange) were studied in seven dogs pretreated with propranolol 20 mg/kg body weight for 3 weeks. Adrenergic beta-receptor blockade did not prevent significant increases in heart rate (up to 19%). Cardiac output increased by about 20% while systemic vascular resistance decreased by the same proportion. Oxygen extraction increased from 24% to 35% and this was reflected by a decrease in mixed venous PO2 by 1.2 kPa. The modest hyperdynamic response of the circulation to haemodilution in the presence of high-grade adrenergic beta-receptor blockade is in sharp contrast with the large hyperdynamic response that has been reported in its absence and indicates that normal compensatory mechanisms are considerably impaired.

Haemodynamic interactions of high-dose propranolol pretreatment and anaesthesia in the dog. III: The effects of haemorrhage during halothane and trichloroethylene anaesthesia.

We have examined the haemodynamic effects of 0.8% trichloroethylene and 1% halothane anaesthesia in a control group of five dogs, chronically implanted with cardiovascular flow- and pressure-measuring apparatus and compared them with a similar group of six dogs pretreated for 3 weeks with oral propranolol (20 mg/kg/day). The effects of graded haemorrhage of 25% of the estimated blood volume and re-transfusion were studied. Cardiovascular function was satisfactory at all stages of the study except during trichloroethylene anaesthesia in the beta-blocked dogs when the response to blood loss was impaired severely. Therefore the use of trichloroethylene in the presence of propranolol may not be advisable in clinical practice.

[CO2 "wash-out" : a factor contributing to increase myocardial performance in the face of hypoxia (author's transl)]. / Le "lavage" du CO2 myocardique : un facteur d'augmentation de la performance cardiaque en cas d'hypoxie

The cardiovascular system of the anaesthetized dog has been used as an experimental model for studying the mechanisms of the haemodynamic responses to hypoxia. Together with aortic and left circumflex coronary artery blood flow (electromagnetic flow transducers), aortic and left ventricular pressures have been recorded and blood has been sampled from the aorta and the coronary sinus (PO2, PCO2, pH). During short periods of hypoxia an improvement of myocardial performance has been observed both before and after administration of a beta-adrenergic receptor blocker and a marked reduction of coronary sinus PCO2 has been noted. When hypoxia was caused by a mixture of nitrogen (95%) and CO2 (5%) an improvement of performance was observed only before administration of the beta-blocker. The slope of the relationship between PCSCO2 and cardiac performance was found to be similar before and after administration of the beta-blocker and also similar to that observed in studies of the response of the isolated heart muscle to acute hypocapnia. Besides beta-adrenergic receptor stimulation, a reduction of coronary sinus PCO2 (CO2 wash-out due to an increase of coronary blood flow) could be a factor contributing to the maintenance of myocardial performance in the face of hypoxia.