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Regulation of protein phosphorylation in ocular ciliary epithelial cells by A, C and Ca2+/calmodulin-dependent protein kinases.

We have studied protein phosphorylation events in a cell line (8-SVHCE) derived from the human ocular ciliary epithelium after transformation within Simian Virus-40. We have investigated the time-course and identification of intracellular phosphorylated protein substrates in response to isoproterenol, phorbol-12-myristate-13-acetate (PMA), and ionophore A23187, which are activators of protein kinase A, C and calcium/calmodulin-dependent protein kinase, respectively. Five major endogenous phosphoproteins were readily identified by two-dimensional polyacrylamide gel electrophoresis with the following molecular weights: 80, 57, 24 and 19 kDa. Tryptic peptide analysis and phosphoaminoacid composition were utilized to aid the identification of the phosphoproteins. From these studies we have observed the following: (a) the most prominent phosphorylation of the 80-kDa protein occurs rapidly (1 min) in response to PMA treatment and is potentiated by isoproterenol, (b) the phosphorylation of the 57-kDa substrate (vimentin) occurs preferentially with isoproterenol treatment and increases gradually from 1 to 30 min, (c) late phosphorylation (60 min) of the 80-kDa protein by PMA is potentiated by isoproterenol, and (d) late phosphorylation of 19-kDa and 24-kDa substrates occurs with PMA treatment and is potentiated by A21387. The desensitization of adenylate cyclase activity by PMA or isoproterenol in 8-SVHCE cells results in altered adenylate cyclase activity, which appears to be correlated with similar alterations in the phosphorylation of the 57-kDa substrate (vimentin).(ABSTRACT TRUNCATED AT 250 WORDS)

Adenylyl Cyclases↗

Isolation of a cDNA encoding a glutathione S-transferase (GST) class-pi from the bovine ocular ciliary epithelium.

We have used a polyclonal antiserum to bovine ciliary epithelium, a secretory tissue involved in the formation of aqueous humor, to immunoscreen a directional lambda gt11 Sfi-Not cDNA expression library prepared from bovine ciliary epithelium poly(A)+ RNA. After immunoscreening 6 x 10(5) independent clones, 41 cDNA clones positive for ciliary epithelium were isolated and characterized. About one-third of the positive cDNA clones were found to be identical and to encode a glutathione S-transferase (GST) class-pi. The largest bovine GST cDNA clone isolated, pCN11, contains an open reading frame of 630 bases, encoding a protein of 210 amino acids with a calculated molecular weight of 23,335 Da. The corresponding amino acid sequence showed an overall identity of 85.6% with the human, and 85.2% with the rat and mouse GST class-pi. Northern analysis of bovine ocular tissues revealed that the GST class-pi gene encodes a 0.8-kilobase mRNA which is expressed most abundantly in cornea, ciliary epithelium and retina, and in lower levels in iris and lens. Cell lines derived from non-pigmented or pigmented bovine ciliary epithelium also showed high levels of GST-pi mRNA expression. These results provide additional evidence for differential gene expression of GST class-pi mRNA in various areas of the bovine eye.

Animals↗

Convexity meningioma and glioblastoma in collision.

An unusual case of benign convexity meningioma and glioblastoma in collision is presented. The preoperative diagnosis of this association is difficult to make based on symptomatology or computed tomography scans alone. This case supports the possibility of a malignant transformation within the gliosis surrounding a convexity meningioma.

Aged↗

Schwannomas of the falx.

A case of multiple schwannomas arising from the falx in a 17-year-old girl, previously operated on for an intracerebral schwannoma, is presented. The appearance of multiple schwannomas has to be considered in the followup of the rare cases of intracranial schwannomas not related to cranial nerves.

Adolescent↗

Crocin, safranal and picrocrocin from saffron (Crocus sativus L.) inhibit the growth of human cancer cells in vitro.

Extracts of saffron (Crocus sativus L.) have been reported to inhibit cell growth of human tumor cells. In order to study the cytotoxic effect of the characteristic compounds of saffron spice, we have isolated crocin, crocetin, picrocrocin and safranal. Doses inducing 50% cell growth inhibition (LD50) on HeLa cells were 2.3 mg/ml for an ethanolic extract of saffron dry stigmas, 3 mM for crocin, 0.8 mM for safranal and 3 mM for picrocrocin. Crocetin did not show cytotoxic effect. Cells treated with crocin exhibited wide cytoplasmic vacuole-like areas, reduced cytoplasm, cell shrinkage and pyknotic nuclei, suggesting apoptosis induction. Considering its water-solubility and high inhibitory growth effect, crocin is the more promising saffron compound to be assayed as a cancer therapeutic agent.

Antineoplastic Agents, Phytogenic↗

Intracellular increases of cAMP induce opposite effects in glutamic acid decarboxylase (GAD67) and glial fibrillary acidic protein immunoreactivities in C6 cells.

C6 is a cell line that expresses glial and neuronal markers. Treatments that increase intracellular cAMP levels induce the differentiation of these cells. We had previously demonstrated that forskolin, an agent that activates adenylate cyclase, produced changes in gene expression in C6 cells. As a consequence of this treatment, glutamic acid decarboxylase (GAD) activity and the mRNA for GAD67, one of the isoforms of the enzyme, decreased. In contrast, this treatment increased the transcription of the glial fibrillary acidic protein (GFAP) gene. We now show, by immunocytochemistry, that the changes in gene expression are phenotypically reflected by corresponding changes in the levels of the proteins encoded by the GAD67 and GFAP genes. Computer-assisted image analysis demonstrated that both the increase in GFAP immunofluorescence, and the decrease in GAD67 immunofluorescence are statistically significant. The changes in gene expression and in protein immunoreactivity are part of the differentiation process of the C6 cells towards a more mature glial phenotype.

Animals↗

The comparative phytochemistry of the genus Erythroxylon.

Recent research suggests that, in addition to the well-documented occurrence of significant quantities of cocaine and related alkaloids in a limited number of species, the genus Erythroxylon will prove to be a rich source of tropane alkaloids. These bases are esters of a range of acids with tropine, 3 alpha, 6 beta-dihydroxytropane and 3 alpha, 6 beta, 7 beta-trihydroxytropane; the numbers are further increased by nor-derivatives, and stereoisomerism involving the C-3 hydroxyl of the tropane skeleton. The diterpenes of the genus also appear worthy of further study.

Alkaloids↗

Urinary excretion of benzoylecgonine following ingestion of Health Inca Tea.

Four males ingested one cup of Health Inca Tea which contained 1.87 mg of cocaine. Urine specimens collected for 36 h post-ingestion were analyzed for benzoylecgonine (BE) by EMIT-d.a.u., TDx and gas chromatography/mass spectrometry (GC/MS). Positive immunoassay results were obtained for 21-26 h post tea ingestion. Discrepant immunoassay results occurred with only one specimen: EMIT positive; TDx negative, 0.25 mg/l; GC/MS, 0.273 mg/l. Quantitative TDx results were well correlated with GC/MS results, r2 = 0.963, n = 45. Maximum urinary BE concentrations ranged from 1.4-2.8 mg/l, occurring from 4-11 h, post ingestion. Total BE excretion in 36 h ranged from 1.05 to 1.45 mg, 59-90% of the ingested cocaine dose. Urinary excretion rate constant (Km) ranged from -0.073 to 0.111/h. Health Inca Tea ingestion should be considered when interpreting urinary BE concentrations.

Beverages↗

Effect of long-term antihypertensive therapy with angiotensin converting enzyme inhibitors on red cell sodium transport.

Several sodium transport abnormalities have been reported in erythrocytes from essential hypertensive patients. The possible modification of these parameters under antihypertensive treatment remains controversial. We have measured the maximal rates of the Na+/K+ pump, Na+/K+/Cl- cotransport, and Na+/Li+ countertransport and the rate constant of Na+ leak in erythrocytes from 22 essential hypertensive patient responders to angiotensin converting enzyme inhibitors quinapril or captopril, and from 17 patient nonresponders to these drugs. In the former group, sodium transport measurements were performed at the baseline placebo period and after 6 months of active treatment. The maximal rate of Na+/Li+ countertransport decreased significantly after 6 months of treatment, without differences between both groups of treatment. Angiotensin converting enzyme inhibitors did not significantly modify other sodium transport parameters. The basal activity of erythrocyte sodium transport was not different between patients who responded or not to antihypertensive treatment with those drugs, excluding a predictive value of these measurements concerning the response to angiotensin converting enzyme inhibitors.

Adult↗

Detection of a circulating inhibitor of the Na(+)-K(+)-Cl(-) cotransport system in plasma and urine after high salt intake.

The aim of the study was to detect the presence of an endogenous Na(+)-K(+)-Cl- cotransport inhibitor factor in plasma and urine from salt-loaded subjects. Twenty-six essential hypertensive patients (13 men, 13 women) and 6 normotensive controls (2 men, 4 women) received a diet containing 260 mmol/day of NaCl tablets for 7 days. Human erythrocytes from healthy blood donors (using bumetanide-stimulated Li+ efflux) were used to test the inhibition of Na(+)-K(+)-Cl- cotransport by different plasma and urine concentrations collected the last day of the salt-loaded period. The addition of plasma and urine from salt-loaded subjects significantly inhibited erythrocyte Na(+)-K(+)-Cl- cotransport (P < .001). No differences were observed between hypertensive patients and normotensive controls. These results provide evidence of an Na(+)-K(+)-Cl- cotransport inhibitor factor in plasma and urine from salt-loaded humans, in concordance with previous reports in salt-loaded rats.

Adult↗

Assessment of salt sensitivity in essential hypertension by 24-h ambulatory blood pressure monitoring.

We used ambulatory blood pressure monitoring (ABPM) in the assessment of salt sensitivity in 40 essential hypertensive patients, comparing 24-h mean blood pressure during 7 days of low salt (20 mmol NaCl/day) and high salt (260 mmol NaCl/day) intake. Salt sensitivity was diagnosed in 18 essential hypertensive patients (45%), each of them showing a significant increase in mean blood pressure (P < .05) from low to high salt diet. Salt-sensitive patients exhibited a high-salt-dependent increase in all blood pressure parameters including 24-h systolic, mean, diastolic blood pressure, blood pressure load, area under the curve, and awake and asleep blood pressure values. These patients exhibited a nondipper profile on both low-salt and high-salt diets. Salt-resistant patients (55%) showed a decrease in awake, and an increase in asleep blood pressure values after high salt intake, thus tending to flatten the circadian blood pressure profile. We conclude that ABPM is a useful method to assess salt sensitivity. In salt-resistant patients high salt intake induces a significant increase in asleep blood pressure with no significant changes in 24-h blood pressure, promoting a flattened blood pressure curve and tending to transform a dipper into a nondipper profile, which could have important implications in end-organ damage.

Adult↗

Finger heat flux/temperature as an indicator of thermal imbalance with application for extravehicular activity.

The designation of a simple, non-invasive, and highly precise method to monitor the thermal status of astronauts is important to enhance safety during extravehicular activities (EVA) and onboard emergencies. Finger temperature (Tfing), finger heat flux, and indices of core temperature (Tc) [rectal (Tre), ear canal (Tec)] were assessed in 3 studies involving different patterns of heat removal/insertion from/to the body by a multi-compartment liquid cooling/warming garment (LCWG). Under both uniform and nonuniform temperature conditions on the body surface, Tfing and finger heat flux were highly correlated with garment heat flux, and also highly correlated with each other. Tc responses did not adequately reflect changes in thermal balance during the ongoing process of heat insertion/removal from the body. Overall, Tfing/finger heat flux adequately reflected the initial destabilization of thermal balance, and therefore appears to have significant potential as a useful index for monitoring and maintaining thermal balance and comfort in extreme conditions in space as well as on Earth.

Adult↗

Erectile dysfunction in essential arterial hypertension and effects of sildenafil: results of a Spanish national study.

BACKGROUND: The aim of this study was to establish the prevalence of erectile dysfunction (ED) in hypertensive patients in specialized care hypertension units (SCHUs) and to assess the effectiveness and tolerability of sildenafil treatment. METHODS: This was a multicenter, prospective, open, observational pharmacoepidemiology study conducted in 25 Spanish SCHUs. A total of 2130 men with essential hypertension under treatment were recruited. In a second phase, 291 subjects with a score < or = 21 in the Sexual Health Inventory for Men (SHIM) received sildenafil (50 mg/day) as required 30 to 60 minutes before sexual activity, and were evaluated by the International Index of Erectile Function (IIEF). RESULTS: A total of 975 subjects (45.8%) had a score < or = 21 in the SHIM. In the second phase, sildenafil improved the score in the erectile function domain in 232 patients (83.2%). Severity of ED significantly improved (P <.001); severe (22.3% to 7.7%), moderate (23% to 5.6%), and mild impairment (36.3% to 44.8%). The IIEF was normalized in 39.1% of patients who completed post-treatment IIEF. In all, 33 subjects (11.8%) failed to complete the study: two (0.7%) because of lack of efficacy, two (0.7%) intercurrent disease, 10 (3.6%) failure to return to the visits, three (1.1%) fear of therapy, four (1.4%) adverse effects requiring treatment discontinuation, and 12 (4.3%) protocol violations. No statistically significant association was found between the prevalence of adverse effects and antihypertensive treatment with single drug or combination therapy. CONCLUSIONS: A high incidence of ED was found in hypertensive patients from Spanish SCHUs. Sildenafil showed an excellent response and safety profile.

3',5'-Cyclic-GMP Phosphodiesterases↗

Silent cerebral white matter lesions and cognitive function in middle-aged essential hypertensive patients.

BACKGROUND: An association between midlife blood pressure levels and late-life cognitive impairment has been reported. Hypertension is one of the most important factors related to the presence of cerebral white matter lesions, which is a prognostic factor for the development of cognitive impairment. Studies have shown a relationship between white matter lesions and cognitive decline in elderly hypertensive patients. The aim of the present study was to evaluate cognitive function in asymptomatic middle-aged hypertensive patients according to the presence or absence of white matter lesions. METHODS: Sixty never-treated essential hypertensive patients (38 men, 22 women), aged 50 to 60 years (mean age, 54.4 +/- 3.8 years), without clinical evidence of target organ damage, were studied. All patients underwent brain magnetic resonance imaging to establish the presence or absence of white matter lesions, using the Rotterdam criteria. Cognitive function was evaluated by a neuropsychologic test battery measuring attention, memory, intelligence, anxiety, and depression. RESULTS: Twenty-three hypertensive patients (38%) were found to have white matter lesions on brain resonance. These patients exhibited a significantly worse performance on digit span forward, a standardized measure of attention than hypertensives without white matter lesions (4.86 +/- 1.14 v 5.51 +/- 0.97; P =.027). Hypertensive patients with white matter lesions showed no differences on both visual and logical memory tests when compared with patients without lesions. CONCLUSIONS: We conclude that the presence of silent cerebral white matter lesions in middle-aged hypertensive patients is associated with a mild decline in basic attention.

Blood Pressure↗

Blood pressure variability and silent cerebral damage in essential hypertension.

BACKGROUND: It is recognized that blood pressure (BP) variability has prognostic significance in determining target organ damage and cardiovascular mortality and morbidity. The aim of this study was to analyze the association between blood pressure variability and the presence of silent cerebral white matter lesions in middle-aged asymptomatic essential hypertensives. METHODS: We studied 43 middle-aged untreated hypertensive patients. Blood pressure variabilities (short-term and long-term) were evaluated by using both non-invasive, beat-to-beat, continuous finger 24-hour monitoring (Portapres) and oscillometric automated discontinuous ambulatory blood pressure monitoring. All patients underwent cerebral magnetic resonance imaging to detect the presence or not of white matter lesions. RESULTS: Hypertensive patients with cerebral white matter lesions exhibited significantly higher values of long-term systolic blood pressure variability (standard deviation of 24-hour blood pressure) measured both by continuous beat-to-beat monitoring (16.2 +/- 3.7 v 13.7 +/- 3.6 mm Hg; P = 0.047) and by ambulatory blood pressure monitoring (15.2 +/- 3.8 v 12.8 +/- 2.7 mm Hg; P = 0.022). However, these differences were not independent on blood pressure elevation and did not maintain their significance after adjusting for 24-hour systolic blood pressure. Neither short-term systolic blood pressure variability, nor short-term or long-term diastolic blood pressure variabilities showed differences between patients with and without white matter lesions. CONCLUSION: The present study indicates that long-term systolic blood pressure variability is significantly related to the presence of silent cerebral white matter lesions in essential hypertensive patients, although this relationship is partially dependent on absolute blood pressure elevation.

Biomarkers↗