Activation of particulate guanylyl cyclase by Vibrio vulnificus hemolysin

Recently we reported that Vibrio vulnificus hemolysin, an exotoxin produced by V. vulnificus, dilates rat thoracic aorta via elevated cGMP levels without affecting nitric oxide synthase. We investigated the mechanism further by observing the guanylyl cyclase activities in cytosolic, membrane, unfractionated, or reconstituted preparations. Hemolysin did not activate guanylyl cyclase in the membrane or cytosolic fraction, while it activated guanylyl cyclase in unfractionated or reconstituted preparation. The increased activity was not inhibited by the HS-142-1, a microbial polysaccharide which antagonizes atrial natriuretic peptide receptor, or 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), a soluble guanylyl cyclase inhibitor. However, it was attenuated by 6-(phenylamino)-5,8-quinolinedione (LY 83.583), which inhibits the catalytic domain of both guanylyl cyclases, and by cholesterol, which blocks hemolysin-incorporation into the membrane. Removing ATP, a cofactor of particulate guanylyl cyclase, attenuated the activation and ATPgammaS, a non-phosphorylating analog, restored it. These results suggest that V. vulnificus hemolysin activates particulate guanylyl cyclase via hemolysin incorporation into the vascular smooth muscle cell membrane in cooperation with certain unidentified cytosolic component(s).     

Outcome of moderate carotid artery stenosis in patients who are asymptomatic

PURPOSE: The incidence rate of disease progression and stroke after the diagnosis of a moderate (50% to 79%) carotid stenosis was determined by means of color-flow duplex scanning. METHODS: During a 4-year period, 344 male veterans with moderate internal carotid artery stenoses, on one or both sides, were examined at regular intervals for a mean period of 25 months. Carotid color-flow scans were obtained semiannually. Clinical follow-up was performed to determine the incidence rate of amaurosis fugax, transient ischemic attacks, nonhemispheric symptoms, and strokes. RESULTS: New neurologic symptoms developed in 75 patients (21.8%). Fifty-one (14.8%) had ipsilateral symptoms during follow-up: 18 amaurosis fugax (5.2%), 14 transient ischemic attacks (4%), 5 nonhemispheric symptoms (1.4%), and 14 strokes (4%). Twenty-four patients (6.9%) had contralateral symptoms: 20 strokes (5.8%) and 4 transient ischemic attacks (1.2%). Life-table analysis showed that the annual rate of ipsilateral neurologic events was 8.1%, and the annual rate of stroke was 2.1%. Seventy-five patients (22%) died in the follow-up period. Disease progression to 80% to 99% stenosis or occlusion occurred in 71 of 458 vessels (15.5%). The internal carotid arteries that showed evidence of disease progression had a significantly higher initial peak systolic velocity (251 vs 190 cm/s; P <.0001) and end diastolic velocity (74 vs 52 cm/s; P < 0.0001). Black patients and patients with ischemic heart disease were at a higher risk for disease progression. We could not identify any atherosclerotic risk factors that reliably predicted patients in whom future ipsilateral neurologic symptoms were more likely to develop. However, there was an increased risk of stroke associated with progression of disease. CONCLUSION: Patients who are asymptomatic and who have moderate carotid stenoses are at significant risk for neurologic symptoms and death, but have a relatively low incidence rate of ipsilateral events. The initial flow characteristics in the stenotic vessel are predictive of future disease progression, but they are not helpful in identifying patients in whom symptoms will develop.     

High-resolution crystal structures of delta5-3-ketosteroid isomerase with and without a reaction intermediate analogue

Bacterial Delta5-3-ketosteroid isomerase (KSI) catalyzes a stereospecific isomerization of steroid substrates at an extremely fast rate, overcoming a large disparity of pKa values between a catalytic residue and its target. The crystal structures of KSI from Pseudomonas putida and of the enzyme in complex with equilenin, an analogue of the reaction intermediate, have been determined at 1.9 and 2.5 A resolution, respectively. The structures reveal that the side chains of Tyr14 and Asp99 (a newly identified catalytic residue) form hydrogen bonds directly with the oxyanion of the bound inhibitor in a completely apolar milieu of the active site. No water molecule is found at the active site, and the access of bulk solvent is blocked by a layer of apolar residues. Asp99 is surrounded by six apolar residues, and consequently, its pKa appears to be elevated as high as 9.5 to be consistent with early studies. No interaction was found between the bound inhibitor and the residue 101 (phenylalanine in Pseudomonas testosteroni and methionine in P. putida KSI) which was suggested to contribute significantly to the rate enhancement based on mutational analysis. This observation excludes the residue 101 as a potential catalytic residue and requires that the rate enhancement should be explained solely by Tyr14 and Asp99. Kinetic analyses of Y14F and D99L mutant enzymes demonstrate that Tyr14 contributes much more significantly to the rate enhancement than Asp99. Previous studies and the structural analysis strongly suggest that the low-barrier hydrogen bond of Tyr14 (>7.1 kcal/mol), along with a moderate strength hydrogen bond of Asp99 ( approximately 4 kcal/mol), accounts for the required energy of 11 kcal/mol for the transition-state stabilization.     

Expression of differential nitric oxide synthase isoforms in human normal gastric mucosa and gastric cancer tissue

The present study investigated the expression and distribution of three isoforms of nitric oxide synthase (NOS) in different anatomical regions of the human stomach and in gastric neoplastic tissues by immunohistochemistry using specific antibodies. Intracellular localization of individual isoenzymes of NOS was detected in normal gastric mucosa. Gastric cancer tissues had a marked reduction of all three NOS isoforms expression. The expression of the endothelial NOS, neuronal NOS and inducible NOS in the tumor tissue was significantly lower than in normal gastric mucosa (P = 0.01, P = 0.02, P < 0.01, respectively). In the tumor tissue the expression of inducible NOS was significantly lower than the expression of both constitutive forms of NOS (P < 0.01). There was a tendency to higher expression of both constitutive forms of NOS in earlier stages T2 of the tumor compared to advanced T4 tumor. In contrast, the expression of inducible NOS was higher than in the advanced T4 tumor than in the earlier stages T2 of the tumor. The mapping of the expression of endothelial NOS, neuronal NOS and inducible NOS in human stomach showed higher expression of NOS isoforms in the distal third than in the proximal third of the stomach (P = 0.03, P = 0.04, P = 0.01, respectively). We conclude that there is greater expression of NOS in the stomach corpus and in antrum than in the proximal third of the normal human stomach mirroring the anatomical predilection of common pathological changes in this part of the human stomach. Furthermore, there was loss of the expression of individual isoenzymes in gastric neoplasms.     

Coexpression of Bcl-2, c-Myc, and p53 oncoproteins as prognostic discriminants in patients with colorectal carcinoma

Lanepitant is a high-affinity, selective neurokinin-1 receptor (NK-1) and is effective in the dural inflammation model of acute migraine. Lanepitant 30, 80, and 240 mg given orally was evaluated in a double-blind, placebo-controlled crossover study to determine its effect in reducing migraine pain and severity of associated symptoms. Outpatients treated four migraine headaches of moderate or severe pain intensity with study drug according to a randomization schedule. They recorded their pain intensity and severity of migraine-associated symptoms at 30, 60, 90, and 120 min. Although 53 patients were randomly allocated to a treatment sequence, only 40 patients completed all treatments. There was no statistically significant difference in improvement in migraine pain at any time for any of the treatments. Additionally, there was no change in severity of migraine-associated symptoms associated with lanepitant therapy. No adverse events could be attributed to lanepitant. Lanepitant was ineffective orally in treating acute migraine in this trial. This may be due to poor bioavailability during a migraine attack. Alternatively, the neurogenic inflammation hypothesis may not apply to migraine.     

Primary pulmonary hypertension

Justification of early treatment of nocturnal enuresis is founded in the negative psychological impact on the child. In fact nocturnal enuresis delays early autonomy and socialisation by decreasing in self-esteem and self-confidence. Nocturnal enuresis classification is the preliminary step to correct therapy. Enuresis must be classified as primary (never acquired nocturnal control) or secondary (at least 6 months of dry nights). A child is also classified as having monosymptomatic enuresis if she/he experienced only night wetting and symptomatic enuresis if she/he experienced night wetting associated with diurnal voiding symptoms (urinated > or = 7 times a day, urgency, damp pants, squatting, holding the perineum, sitting on one heel). Monosymptomatic patients must be treated with desmopressin nasal spray at the daily dose of 20 micrograms at bed time. If the reduction of at least the 50% of the basal number of the wet nights is not achieved, the dosage must be increased until 40 micrograms. For patients affected by rhinitis or asthma, desmopressin is now available in tablets. In symptomatic patients desmopressin therapy must be associated to oxybutinin (5 mg x 2). Therapy interruption must be gradual with desmopressin reduction of 10 micrograms every 30 days. In symptomatic patients oxybutinin must be introduced only at bed time. The efficacy of the drugs depends on the therapy length. The highest percentage of success is obtained if the treatment is protracted for at least six months. Antidepressants are also used for nocturnal enuresis especially imipramine. The dosage varies between 0.5-1.5 mg/ kg/daily. As plasmatic levels are achieved only in 30% of treated patients, a 3-5 fold increase in suggested. Nevertheless these levels result in near toxic threshold concentration. Sporadic treatment purposes include amytriptiline, diclofenac sodicum, viloxsazine and methilphenidate if giggle incontinence is present. Non responders may be treated with alarm. If after 16 weeks of treatment no success is obtained alarm use must be interrupted.     

Limb development: marginal fringe benefits

Recent results show that in the developing Drosophila wing, the secreted pioneer protein Fringe regulates the sensitivity of the Notch signaling pathway to different ligands. This provides a likely mechanism by which Fringe-like molecules may control patterning in both Drosophila and vertebrates.     

Prognostic significance of tumour markers in endometrial cancer

BACKGROUND: Steroid 5 alpha-reductase is implicated in the pathogenesis of benign prostatic hyperplasia (BPH). We studied the in vitro and in vivo effects of FR146687, a new inhibitor of 5 alpha-reductase. METHODS: Two isozymes of rat and human 5 alpha-reductases were expressed in 293 cells. In vivo effects of drugs were evaluated on rat and dog prostates. Castrated immature rats were injected with testosterone propionate (TP) or 5 alpha-dihydrotestosterone propionate (DHTP) to induce growth of the ventral prostates. Testosterone and 5 alpha-dihydrotestosterone (DHT) contents in rat and dog prostates were measured by gas chromatography-mass spectrophotometry (GC-MS). RESULTS: FR146687 showed noncompetitive inhibition in both isozymes and no inhibitory effects on other steroid oxidoreductases. In mature rats and castrated immature rats treated with TP, FR146687 dose-dependently reduced ventral prostate and seminal vesicle weight at doses above 0.1 mg/kg, while castrated immature rats treated with DHTP were not affected by FR146687. FR146687 showed more potent reduction of rat prostates than finasteride. DHT concentration in the prostates was significantly reduced when FR146687 was administered to rats and beagles. CONCLUSIONS: FR146687 is a dual inhibitor for 5 alpha-reductase isozymes and significantly reduced the growth and DHT content in the prostate.