Analysis of a Drosophila cyclin E hypomorphic mutation suggests a novel role for cyclin E in cell proliferation control during eye imaginal disc development.

We have generated and characterized a Drosophila cyclin E hypomorphic mutation, DmcycEJP, that is homozygous viable and fertile, but results in adults with rough eyes. The mutation arose from an internal deletion of an existing P[w+lacZ] element inserted 14 kb upstream of the transcription start site of the DmcycE zygotic mRNA. The presence of this deleted P element, but not the P[w+lacZ] element from which it was derived, leads to a decreased level of DmcycE expression during eye imaginal disc development. Eye imaginal discs from DmcycEJP larvae contain fewer S phase cells, both anterior and posterior to the morphogenetic furrow. This results in adults with small rough eyes, largely due to insufficient numbers of pigment cells. Altering the dosage of the Drosophila cdk2 homolog, cdc2c, retinoblastoma, or p21(CIP1) homolog dacapo, which encode proteins known to physically interact with Cyclin E, modified the DmcycEJP rough eye phenotype as expected. Decreasing the dosage of the S phase transcription factor gene, dE2F, enhanced the DmcycEJP rough eye phenotype. Surprisingly, mutations in G2/M phase regulators cyclin A and string (cdc25), but not cyclin B1, B3, or cdc2, enhanced the DmcycEJP phenotype without affecting the number of cells entering S phase, but by decreasing the number of cells entering mitosis. Our analysis establishes the DmcycEJP allele as an excellent resource for searching for novel cyclin E genetic interactors. In addition, this analysis has identified cyclin A and string as DmcycEJP interactors, suggesting a novel role for cyclin E in the regulation of Cyclin A and String function during eye development.

Suppression of noradrenaline spillover by the dopamine prodrug gamma-L-glutamyl-L-dopa: a central effect?

The DA prodrug gamma-L-glutamyl-L-dopa (gludopa) has a high degree of renal selectivity with 2-step conversion to DA in the kidney. The effects of gludopa, with and without DA-2 receptor blockade, on renal and total noradrenaline (NA) spillover, were studied in two groups of rabbits. Eight rabbits received gludopa infusion (25 and 100 micrograms/kg/min and 8 received an infusion of gludopa and DA-2 receptor antagonist, YM-09151 (50 micrograms/kg i.v.). Renal and total NA spillover rates were measured by 3H-NA tracer method before and after gludopa infusion. Brain NA, DA, gludopa and L-dopa content were measured after gludopa infusion in 5 rabbits; control values for tissue catecholamine and drug levels were obtained in 5 untreated rabbits. Gludopa infusion markedly increased kidney DA content (300-fold) and DA excretion (6000-fold) but had little effect on plasma DA. It produced a dose-related fall in mean (+/- SEM) renal NA spillover (21.6 +/- 3.7 to 10.6 +/- 2.7, 7.2 +/- 2.7 ng/min, p < 0.01). Even greater falls were observed in total NA spillover after gludopa (43.1 +/- 10.2 to 19.7 +/- 3.4, 9.4 +/- 1.8 ng/min, p < 0.01). DA-2 receptor antagonism had no influence on the effects of gludopa on either renal or total NA spillover. Significant amounts of gludopa were detected in the brain after drug infusion (0.28 +/- 13 nmol/g brain tissue). Gludopa, a putative renal selective dopamine prodrug with effects mediated via DA-1 receptors also significantly inhibits both renal and extra-renal NA spillover. This effect is not a DA-2 effect but may be mediated centrally.

Complement levels and exposure in the hypobaric chamber.

Decompression sickness sometimes occurs in routine hypobaric chamber runs. Susceptibility to decompression sickness has been correlated with increases in C3a and C5a metabolites of complement activation by air bubbles. If personnel susceptible to decompression sickness show changes in C3a and C5a after decompression, then measurement of these metabolites may be useful in assisting diagnosis. C3a and C5a metabolites of complement activation were measured in 11 male and 2 female volunteers 1 h before (A), 1 h after (B), and 24 h after (C) exposure in routine hypobaric chamber runs to 25,000 ft equivalent altitude. The results were compared with 4 controls (3 males and 1 female). A one-way analysis of variance showed no significant difference between the experimental and control groups overall (p = 0.308; p > 0.05; N = 17). T-tests at each of the results further suggest non-significant differences between the experimental and control groups (p = 0.473; p = 0.341; p = 1.051; p > 0.05; N = 17) at results A, B, and C, respectively. The C5a assay was less than 10 micrograms.L-1 in all individuals, and the particular kit used was not sensitive below 10 micrograms.L-1. As none of the participants developed decompression sickness, it would be useful to determine levels of C3a and C5a in individuals who subsequently develop decompression sickness. One member of the experimental group who had undergone many chamber runs had much higher pre- and post-chamber run levels of C3a. This raises the question of the effect of repeated exposure in low pressure chambers on complement activation, and requires further study.

The dopamine prodrug, gludopa, decreases both renal and extrarenal noradrenaline spillover in conscious rabbits.

1. Renal and total noradrenaline (NA) spillover rates were examined under control conditions and during graded infusions of gludopa (gamma-L-glutamyl-L-dopa) in conscious rabbits. 2. Gludopa infusion at 25 and 100 micrograms/kg per min did not alter mean arterial pressure (MAP) and heart rate (HR), but had significant dose-related effects on the renal dopamine (DA) system. At the high dose there were pronounced increases in urinary DA excretion (> 6000-fold) and renal DA content (> 100-fold); renal NA content doubled. 3. Renal venous DA increased after gludopa infusion, but arterial plasma DA concentrations were not significantly changed. Mean arterial plasma gludopa and L-dopa concentrations reached 890, 3190 ng/mL and 3, 10 ng/mL at low and high doses, respectively. 4. Gludopa resulted in a pronounced dose-dependent fall in renal NA spillover, which at 100 micrograms/kg per min accounted for almost half of the reduction in overall NA spillover rate. 5. The significant falls in renal and extrarenal NA spillover rate during gludopa infusion are consistent with suppression of renal and overall sympathetic activity. Gludopa-induced inhibition of renal NA spillover is likely to be due to the actions of DA generated in the kidney on presynaptic DA-2 and alpha-2 receptors. A central sympathoinhibitory mechanism may explain the reduced total NA spillover.

Sympatho-inhibitory effects of r-l-glutamyl-l-dopa in conscious rabbits.

Renal and total norepinephrine (NE) spillover rates were studied with [3H] NE kinetic method during graded r-l-glutamyl-l-dopa (gludopa) i.v. infusion in conscious rabbits. Mean arterial pressure (MAP) and heart rate (HR) remained constant during the experiment. Gludopa i.v. infusion at 25 and 100 micrograms.kg-1.min-1 produced marked increases in urinary dopamine (DA) excretion and renal DA content. Although renal venous DA rose after gludopa infusion, the arterial DA was not significantly altered. Arterial plasma gludopa and levodopa levels reached 0.9 +/- 0.5, 3.2 +/- 0.8 micrograms.ml-1 and 3.0 +/- 1.8, 10.1 +/- 5.1 ng.ml-1 at the lower and higher gludopa doses, respectively. Gludopa elicited a pronounced dose-related fall in renal NE spillover, which only accounted for about one half of the reduction in overall NE spillover rate. Renal NE content was doubled. These results indicated that gludopa decreased the renal and extrarenal NE spillover to plasma. This reduction may be mediated by intrarenally synthesized DA via presynaptic DA-2 and alpha-2 receptors, but could also be explained by some central sympatho-inhibitory mechanism.

Bronchoalveolar lavage fluid urea as a measure of pulmonary permeability in healthy smokers.

The effects of cigarette smoking on blood to airway pulmonary permeability to the low-molecular-weight solute urea were investigated, in an attempt to evaluate its use as a dilution marker for bronchoalveolar lavage (BAL) studies. Five healthy normal smokers who smoked a cigarette 10 min prior to undergoing a 3 x 60 mL bronchoalveolar lavage (BAL), and five nonsmokers who also underwent BAL but without cigarette smoke exposure were studied. Five minutes before bronchoscopy, 4 MBq 3H-water and 1 MBq 14C-urea were injected intravenously and biochemical urea assays and an indirect radiotracer method were used to evaluate permeability. It was shown that the smoking group had less urea in their BAL supernatants compared to nonsmokers the results using the radiotracer method being significant (p<0.005). Using both methods, it was shown that levels of urea increased in sequentially aspirated aliquots in both groups. The median directly assayed levels of urea in the smokers rose as follows: aliquot 1 0.05 micromol x mL(-1), (range 0.03-0.14), aliquot 2 0.10 micromol x mL(-1) (0.07-0.17), aliquot 3 0.12 micromol x mL(-1) (0.06-0.23) (p<0.05). This led to significantly increased calculated levels of epithelial lining fluid in the sequential aliquots (p<0.05). In addition, there were large but variable amounts of labelled water detected in both subject groups indicating a complex interaction between the BAL procedure and the circulation. Changing urea measurements during the bronchoalveolar lavage procedure confound the use of the urea (epithelial lining fluid) method for normalizing dilution factors. The use of epithelial lining fluid determinations in smokers ignores the additional and probably complex permeability changes. The present data suggest that acute exposure to cigarette smoke in smokers may decrease blood to airway permeability.

Sympatho-inhibitory effects of gamma-l-glutamyl-l-dopa are not mediated by activation of dopamine-2 receptors in conscious rabbits.

AIM: To define the role of dopamine-2 receptors in the sympatho-inhibitory effects of gamma-l-glutamyl-l-dopa in conscious rabbits. METHOD: gamma-l-glutamyl-l-dopa (gludopa) was infused iv at 25 and 100 micrograms.kg-1.min-1 with and without prior dopamine-2 receptor blockade by YM-09151-2 (50 micrograms.kg-1 iv) in conscious rabbits. RESULTS: Mean arterial pressure and heart rate remained unchanged while renal plasma flow increased. Arterial norepinephrine (NE) concentration, total and renal NE spillover rate were markedly decreased in a dose-related manner, which were not affected by prior dopamine-2 receptor blockade. Gludopa was detected in the whole brain (92 +/- 112 ng/g wet brain tissue) at the end of experiment although brain tissue levodopa, NE, and dopamine contents were not much different from those in the control group. CONCLUSION: Gludopa decreased dose-dependently plasma NE concentration, and total and renal NE overflow to plasma, which were not mediated by activation of dopamine D2 receptors.

decapentaplegic is required for arrest in G1 phase during Drosophila eye development.

During eye development in Drosophila, cell cycle progression is coordinated with differentiation. Prior to differentiation, cells arrest in G1 phase anterior to and within the morphogenetic furrow. We show that Decapentaplegic (Dpp), a TGF-&bgr; family member, is required to establish this G1 arrest, since Dpp-unresponsive cells located in the anterior half of the morphogenetic furrow show ectopic S phases and ectopic expression of the cell cycle regulators Cyclins A, E and B. Conversely, ubiquitous over-expression of Dpp in the eye imaginal disc transiently inhibits S phase without affecting Cyclin E or Cyclin A abundance. This Dpp-mediated inhibition of S phase occurs independently of the Cyclin A inhibitor Roughex and of the expression of Dacapo, a Cyclin E-Cdk2 inhibitor. Furthermore, Dpp-signaling genes interact genetically with a hypomorphic cyclin E allele. Taken together our results suggest that Dpp acts to induce G1 arrest in the anterior part of the morphogenetic furrow by a novel inhibitory mechanism. In addition, our results provide evidence for a Dpp-independent mechanism that acts in the posterior part of the morphogenetic furrow to maintain G1 arrest.