Segmented Gene Conversion as a Mechanism of Correction of 18S rRNA Pseudogene Located Outside of rDNA Cluster in D. melanogaster

The peculiarities of the sequences of 18S rDNA included in a 90-kb DNA segment cloned in YAC vector are described. This heterochromatic segment is situated on the X chromosome distal to the main rDNA cluster. The pseudo 18S rDNA sequence comprised undamaged stretches of rDNA interspersed with segments characterized by high density of nucleotide substitutions and insertions/deletions. The observed patchwork arrangement of unaltered rDNA sequences was considered as evidence of segmented gene conversion events between the normal and damaged genes which are thought to constitute one of the mechanisms of rDNA array homogenization. The 18S rDNA fragment (510 bp) located nearby, homologous to the internal, undamaged part of pseudo 18S rDNA, carries comparable density of randomly distributed nucleotide substitutions with no evidence of correction. Received: 8 August 1996 / Accepted: 7 December 1996     

Hypoxic initiation of pulmonary hypertension is mediated by serotonin secretion from neuroepithelial bodies in chemodenervated dogs

The purpose of this study was to investigate the stimulatory effect of hypoxia on the secretion of serotonin by neuroepithelial bodies (NEB) as well as to determine the relation between its level and changes in pulmonary arterial pressure (PAP) and also to determinate the effect of serotonin antagonists (pizotifen and methysergide) on the responses of pulmonary and systemic arterial pressures. The experiments were carried out in peripheral chemoreceptor-denervated dogs anesthetized with Na penthabarbital (30 mg/kg i.v.). On the breathing of normoxic and hypoxic (7% O2-93% N2) gas mixtures and on the injection of KCN (80 microg/kg i.v.), PAP, systemic arterial blood pressure (BP), tidal volume (VT), respiratory frequency (f/min), ventilation minute volume (VE) were determined. Also PAP and BP were recorded before and after the injection of pizotifen (0.5 mg/kg i.v.) and methysergide (1 mg/kg i.v.) during normoxic or hypoxic gas mixture breathing. At the end of each experimantal phase, serotonin level, PaO2, PaCO2 and pHa values in blood samples obtained from left ventricle and femoral artery were determined. On the breathing of the hypoxic gas mixture of the chemodenervated dogs, VT, VE and BP significantly decreased (P < 0.001, P < 0.001, P < 0.01). The mean value of PAP and serotonin levels (ventricular and femoral) were found significantly increased when compared with the corresponding normoxic values (P < 0.001, P < 0.05). On the other hand, injection of KCN produced no significant changes in PAP, serotonin levels, BP and respiratory parameters. After the injection of pizotifen, PAP was significantly increased in hypoxia (P < 0.01). After the injection of methysergide, the response of PAP was completely abolished during the breathing of hypoxic gas mixture. The finding of the abolition of response of PAP to hypoxia after the injection of methysergide indicates that serotonin release from NEB may be responsible for the elevation of PAP in hypoxic hypoxia.     

Downregulation of angiotensin converting enzyme by TNF-alpha in differentiating human macrophages

OBJECTIVE: To investigate regulation of angiotensin converting enzyme (ACE) by tumour necrosis factor alpha (TNF-alpha) in differentiating human peripheral blood monocytes (PBM). METHODS: Human PBM were allowed to differentiate to macrophages for 0-7 days and ACE amount was measured during differentiation. Experiments with TNF-alpha were performed after 2 days of differentiation. Cell cultures were incubated with TNF-alpha (0.5-10ng/ml) without or with SB 202190 (5microM), or PD 98059 (40microM). ACE amounts were measured by an inhibitor binding assay (IBA) and ACE mRNA levels by RNase protection assay (RPA). Activated p44/42 and p38 MAP kinases were measured by Western Blot analysis using phospho-p44/42 and -p38 MAPK antibodies. RESULTS: ACE amount increased by 40-fold along with macrophage differentiation. TNF-alpha caused dose dependent suppression of the amount of ACE and decreased levels of ACE mRNA. TNF-alpha activated p44/42 and p38 MAP kinases, which was inhibited by the specific inhibitors of these kinases, PD98059 or SB202190, respectively. Pretreatment of the cells with SB 202190, or PD 98059 both partly reversed TNF-alpha induced ACE suppression. CONCLUSIONS: TNF-alpha downregulated ACE, which effect was probably mediated by both p44/42 and p38 MAPK pathways. Local downregulation of ACE by TNF-alpha may be a counterbalancing mechanism in inflammatory processes.     

Functional reassembly of the Escherichia coli maltose transporter following purification of a MalF-MalG subassembly

Taking advantage of a chaperone-like function of MalK, a stable complex of MalF-MalG could be solubilized from the Escherichia coli membrane and purified in high yield in the absence of MalK. This MalF-MalG complex was competent for efficient reassembly of a functional MalFGK(2) maltose transporter complex both in detergent solution and in proteoliposomes.     

Cyclic AMP increases bradykinin receptor binding affinity in human endothelial cells

We demonstrated bradykinin receptors in human endothelial cells and studied whether bradykinin receptors might be regulated by cyclic AMP. Messenger RNA for bradykinin B(1) and B(2) receptors was detected with real-time PCR and B(2) receptor protein was confirmed by immunoblotting. Saturation binding experiments with increasing concentrations of (125)I-[Tyr(8)]-bradykinin (25-700 pM) were made to determine maximal binding capacity and dissociation constant. However, saturation binding experiments suggested one class of binding sites, maximal binding capacity of 39.3 +/- 1.3 fmol/mg protein and dissociation constant of 352 +/- 27 pM. Competition studies with bradykinin B(1) and B(2) receptor antagonists showed that binding was competed by a B(1) antagonist, and when internalization was inhibited with hypertonic buffer, by both B(1) and B(2) antagonists. Stimulating cells with dibutyryl-cAMP, cholera toxin and forskolin for 24 h increased (125)I-[Tyr(8)]-bradykinin (90 pM) binding with approximately 50%. Saturation binding experiments with dibutyryl-cAMP stimulated cells showed, that the dissociation constant was altered from 352 +/- 27 pM in non-stimulated cells, to 203 +/- 18 pM (P < 0.001) in stimulated cells, while maximal binding capacity remained unchanged. Binding was competed similarly by the B(1) antagonist in stimulated and control cells. These results suggest, that the dibutyryl-cAMP stimulated increase in (125)I-[Tyr(8)]-bradykinin binding is probably due to increased B(1) receptor affinity with no change in receptor capacity. In conclusion, bradykinin B(1) and B(2) receptor mRNA was shown in human endothelial cells. Binding studies suggest that bradykinin receptors are competable with bradykinin antagonists. Adenylate cyclase activators probably increase bradykinin B(1) receptor affinity, without changing capacity, and thus increase bradykinin binding.     

Atorvastatin completely inhibits VEGF-induced ACE upregulation in human endothelial cells

Angiotensin-converting enzyme (ACE) plays an important role in the pathophysiology of cardiovascular disease. We investigated whether atorvastatin, a powerful agent for the prevention and treatment of cardiovascular disease, influences ACE production in endothelial cells. Human umbilical cord vein endothelial cells were treated with VEGF (476 pM), which induced ACE upregulation. Cotreatment with atorvastatin (0.1-10 microM) dose dependently inhibited VEGF-induced ACE upregulation. In the presence of mevalonate (100 microM), atorvastatin failed to downregulate VEGF-induced ACE production. Cotreatment of the cells with either farnesylpyrophosphate (FPP; 5 microM) or geranylgeranylpyrophosphate (GGPP; 5 microM) partially inhibited the suppressive effect of atorvastatin. Pretreatment of the cells with Rho-associated protein kinase inhibitor, Y-27632 (10 microM), partially inhibited VEGF-induced ACE upregulation. VEGF (476 pM) caused PKC phosphorylation, which was inhibited by cotreatment of the cells with atorvastatin. Atorvastatin inhibited VEGF-induced ACE upregulation probably by inhibiting PKC phosphorylation. This effect was mediated via inhibition of the mevalonate pathway. ACE downregulation may be an additional beneficial effect of statins in the treatment of cardiovascular disease.