Biomedical subjects

L Hernandez

Publications and source records attributed to L Hernandez.

At least 73 records · Page 4Linked to original sources

PubMed1997

Atropine decreases drinking but not feeding and induces less hypothalamic acetylcholine release in diabetic rats.

Drinking, feeding and hypothalamic extracellular acetylcholine (ACh) release was measured before and after the administration of several doses of atropine sulfate in streptozotocin (STZ)-diabetic and normal rats. Drinking but not feeding was dose-relatedly decreased by i.p. or intrahypothalamic injections of atropine in STZ-diabetic rats. Hypothalamic ACh release, as measured by microdialysis, increased less (dose-related) in diabetic than normal rats following an i.p. administration of atropine. Ach basal levels were the same in both groups. These results are discussed in terms of a hyperactive hypothalamic cholinergic (muscarinic) system involved in the diabetic polydipsia.

Acetylcholine↗

PubMed1997

Potent and selective non-benzodioxole-containing endothelin-A receptor antagonists.

The benzodioxole ((methylenedioxy)benzene) group is present in a number of endothelin (ET) receptor antagonists thus far reported. As part of our own endothelin antagonist program we have developed (2R*,3R*,4S*)-1-(N,N-dibutylacetamido)-4-(1,3-benzodioxol-5- yl)-2-(4-methoxyphenyl)pyrrolidine-3-carboxylic acid (A-127722). This is a potent antagonist, binding to the ETA and ETB receptor subtypes with affinities (IC50) of 0.4 and 520 nM, respectively, and also contains the aforementioned benzodioxole. While this compound was seemingly optimized at its N-terminus, no effort had been directed toward understanding the contributions to binding affinity or receptor subtype selectivity conferred by the benzodioxole. Substitution by 1- or 2-naphthyl yielded weak antagonists. Oxygenated benzenes, such as p-anisyl, were potent compounds with IC50s in the low-nanomolar range. Simple deletion of either of the two oxygen atoms (dihydrobenzofurans) yielded extremely potent agents, possessing subnanomolar affinity for the ETA receptor. Additionally, the compounds showed enhanced selectivity, binding to the ETB receptor subtype in the micromolar range. This paper describes the development of this novel class of compounds.

Acetamides↗

PubMed1997

Deletions and loss of expression of p16INK4a and p21Waf1 genes are associated with aggressive variants of mantle cell lymphomas.

Mantle cell lymphoma (MCL) is molecularly characterized by bcl-1 rearrangement and cyclin D1 gene overexpression. Some aggressive variants of MCL have been described with blastic or large cell morphology, higher proliferative activity, and shorter survival. The cyclin-dependent kinase inhibitors (CDKIs) p21Waf1 and p16INK4a have been suggested as candidates for tumor-suppressor genes. To determine the role of p21Waf1 and p16INK4a gene alterations in MCLs, we examined the expression, deletions, and mutations of these genes in a series of 24 MCLs, 18 typical, and 6 aggressive variants. Loss of expression and/or deletions of p21Waf1 and p16INK4a genes were detected in 4 (67%) aggressive MCLs but in none of the typical variants. Two aggressive MCLs showed a loss of p16INK4a expression. These cases showed homozygous deletions of p16INK4a gene by Southern blot analysis. An additional aggressive MCL in which expression could not be examined showed a hemizygous 9p12 deletion. Loss of p21Waf1 expression at both protein and mRNA levels was detected in an additional aggressive MCL. No p21Waf1 gene deletions or mutations were found in this case. The p21Waf1 expression in MCLs was independent of p53 mutations. The two cases with p53 mutations showed p21Waf1 and p16INK4a expression whereas the 4 aggressive MCLs with p16INK4a and p21Waf1 gene alterations had a wild-type p53. p21Waf1 and p16INK4a were expressed at mRNA and protein levels in all typical MCLs examined. No gene deletions or point mutations were found in typical variants. Two typical MCLs showed an anomalous single-stranded conformation polymorphism corresponding to the known polymorphisms at codon 148 of p16INK4a gene and codon 31 of p21Waf1 gene. These findings indicate that p21Waf1 and p16INK4a alterations are rare in typical MCLs but the loss of p21Waf1 and p16INK4a expression, and deletions of p16INK4a gene are associated with aggressive variants of MCLs, and they occur in a subset of tumors with a wild-type p53 gene.

Blotting, Southern↗

PubMed1997

p21WAF1/Cip1 expression is associated with cell differentiation but not with p53 mutations in squamous cell carcinomas of the larynx.

p21WAF1/Cip1 is a recently identified gene involved in cell cycle regulation through cyclin-CDK-complex inhibition. The expression of this gene in several cell lines seems to be induced by wild-type, but not mutant, p53. p21WAF1/Cip1 expression has been studied at both mRNA and protein levels in a series of 49 normal mucosae and squamous cell carcinomas of the larynx. A significant association was found between mRNA and protein expression in tumours (P < 0.0001). p21WAF1/Cip1 expression was strongly associated with squamous cell differentiation of carcinomas, because six of seven (86 per cent) undifferentiated carcinomas (grade 4) showed very low levels of p21WAF1/Cip1 expression, whereas 41 out of 42 (98 per cent) carcinomas with squamous cell differentiation (grades 1-3) had normal or high levels of p21WAF1/Cip1 expression (P < 0.0001). In addition, p21WAF1/Cip1 expression was topologically related to the squamous differentiation of tumour cells with a distribution similar to that seen in normal squamous epithelium. No correlation was found between p21WAF1/Cip1 expression and the global S-phase of the carcinomas. p53 mutations (exons 5-9) were found in ten carcinomas with p21WAF1/Cip1 expression, but no p53 mutations were detected in three p21WAF1/Cip1-negative tumours. In conclusion, p21WAF1/Cip1 expression is frequently upregulated in squamous cell carcinomas of the larynx and is associated with tumour cell differentiation. p21WAF1/Cip1 expression in these tumours is independent of p53 gene mutations.

Adult↗

PubMed1997

Blood microdialysis in humans: a new method for monitoring plasma compounds.

A new venous microdialysis probe for humans was developed. The active area was made with cellulose membrane (220 mm outside diameter, length 20 mm, and 6000 molecular weight cut off). Glucose measurements were used to test it. The relative recovery was 50% in dialysates obtained in vitro, the step response was 91.8% in the first 2 min. The in vivo recovery was 65.8% obtained by the no net flux method, with the probe placed in the cubital vein of the subjects arms. Corrected glucose in dialysates was non significantly different from plasma glucose simultaneously collected. In healthy male volunteers from 18 to 37 years of age, mean 22.1 years, the glucose levels in the blood dialysates were measured every 30 min, 60 min before and 150 min after an oral load of 75 g of glucose or vehicle. Glucose increased significantly after the load. The differences with the control group were significant at 60 and 90 min after the treatment. The results showed the easy and safe application of this technique to monitor endogenous and exogenous compounds in the extracellular compartment of blood in out patients.

Administration, Oral↗

PubMed1997

Selective action of acute systemic clozapine on acetylcholine release in the rat prefrontal cortex by reference to the nucleus accumbens and striatum.

The effects of i.p. clozapine [0 (n = 6), 5 (n = 5), 10 (n = 5), 20 (n = 9) and 40 (n = 5) mg/kg] on acetylcholine (ACh) release in the prefrontal cortex (PFC), nucleus accumbens (NAC) and striatum (STR) were studied by simultaneous triple microdialysis in freely moving rats. Clozapine dose-responsively increased extracellular ACh in the studied areas. The effect was larger in the PFC. Comparisons of the slopes of the regression equations showed differences between the effects in PFC and nucleus accumbens (t = 4.29; df = 56; P < .001) and PFC and STR (t = 4.56), but not between nucleus accumbens and STR. These differential actions were not artifacts of the simultaneous perfusion because clozapine (20 mg/kg) increased ACh levels during single microdialysis of the PFC (353 +/- 72%; n = 5) or STR (168 +/- 24%; n = 5), in the same proportion as the respective increases in those areas during the simultaneous triple microdialysis (PFC = 330 +/- 41%; STR = 144 +/- 18%; n = 9). Local infusion of tetrodotoxin (10 microM) reduced ACh in the areas studied to about 30% of the mean baselines, confirming the neuronal origin of this neurotransmitter. Extrapolation of these results to humans suggests that adequate levels of cholinergic activity in the PFC are required for mental health, and that a similar ACh release in the human PFC by clozapine could be therapeutic. The low impact on striatal ACh could explain the lack of extrapyramidal symptoms by clozapine.

Acetylcholine↗

PubMed1996

Acute effects of digitalis and enalapril on the neurohormonal profile of chagasic patients with severe congestive heart failure.

Chagasic patients with congestive heart failure are usually treated with digitalis and converting enzyme inhibitors. According to the neurogenic and dysautonomic theories, chagasic patients would not benefit from these drugs. To clarify this controversial issue, we have studied patients with congestive heart failure and suspected Chagas' heart disease. All patients received intravenous methyl-digoxin for 24 h and oral enalapril for 96 h. Blood samples for plasma norepinephrine, aldosterone and renin were taken at baseline, after acute digitalization and following enalapril. Based on the serology for Chagas' disease, the patients were divided into non-chagasic and chagasic patients. In the chagasic group three patients were in functional class III and 3 were in functional class IV. In the non-chagasic group five patients were in functional class III and 2 were in functional class IV. Both groups had a marked and quantitatively similar degree of neurohormonal activation. All patients improved at least one functional class and lost more than 5 kg of body weight with treatment. The chagasic patients had a statistically significant reduction in plasma norepinephrine (2262 +/- 1407 to 865 +/- 390, P < 0.008, pg/ml, M +/- S.D.), plasma aldosterone (330 +/- 168 to 155 +/- 75, P < 0.01, pg/ml, M +/- S.D.) and plasma renin activity (14 +/- 13 to 2 +/- 1.6 ng/ml per h, M +/- S.D., P < 0.05), with digitalis. Following enalapril, norepinephrine and aldosterone there was a further but non-significant reduction, when compared to postdigitalis values. These results indicated that chagasic patients do benefit from digitalis and enalapril. Furthermore, the prominent and significant reduction in all three neurohormones suggest that the parasympathetic and sympathetic systems of these chagasic and non-chagasic patients, are responding to the neuromodulatory effects of digitalis and enalapril.

Aldosterone↗

PubMed1996

p53 gene mutations and protein overexpression are associated with aggressive variants of mantle cell lymphomas.

Mantle cell lymphoma (MCL) is molecularly characterized by bcl-1 rearrangement and cyclin D1/PRAD-1 gene overexpression. Some aggressive variants have been recognized with a blastic or large cell morphology, higher proliferative activity, and shorter survival. p53 gene mutations in lymphoid neoplasms have been detected mainly in high grade lymphomas and have been associated with tumor progression in follicular and small lymphocytic lymphomas. To determine the role of p53 alterations in MCL, we examined 35 typical and 8 aggressive variants (5 blastic and 3 large cell) of MCLs by a combination of immunohistochemistry, single-strand conformational polymorphism analysis of genomic DNA and/or cDNA obtained by reverse transcriptase-polymerase chain reaction, denaturing gradient gel electrophoresis, and sequencing. Of the 8 aggressive MCLs, 3 (38%) contained missense point mutations in axon 8 codon 278 (Pro --> Leu), exon 8 codon 273 Arg --> His), and exon 5 codon 151 (Pro --> Ser), respectively. A diffuse p53 protein overexpression was observed in more than 50% of the tumor cells in these 3 cases. A fourth blastic MCL also showed strong p53 immunoreactivity. However, no mutations were detected in exons 5-9 in this case. p53 expression was also detected in 10% of the cells in an additional large cell type of MCL and in less than 1% of the cells in 6 typical cases. No mutations were detected in any of these cases or in the remaining cases with no expression of the protein. Four nucleotide changes were observed by single-strand conformational polymorphism analysis in 4 typical MCLs with no overexpression of the protein. Direct sequencing showed that these nucleotide changes were located at exon 6 (1 case), intron 7 (2 cases), and intron 8 (1 case). The changes in exon 6 and intron 7 were known polymorphisms. The nucleotide change in intron 8 was outside splicing sites of the neighboring exons. The overall survival of the 3 patients with p53 mutations (median, 18.3 months) was significantly shorter than that of patients with the nonmutated MCLs (median, 49 months; P < .01). These findings indicate that p53 gene mutations are an infrequent phenomenon in MCLs and are associated with a subset of aggressive variants.

Base Sequence↗

PubMed1996

2,4-Diarylpyrrolidine-3-carboxylic acids--potent ETA selective endothelin receptor antagonists. 1. Discovery of A-127722.

We have discovered a novel class of endothelin (ET) receptor antagonists through pharmacophore analysis of the existing non-peptide ET antagonists. On the basis of this analysis, we determined that a pyrrolidine ring might replace the indian ring in SB 209670. The resultant compounds were readily prepared and amenable to extensive SAR studies. Thus a series of N-substituted trans,trans-2-(4-methoxyphenyl)-4-(1,3-benzodioxol-5-yl)pyrroli din e-3- carboxylic acids (8) have been synthesized and evaluated for binding at ET(A) and ET(B) receptors. Compounds with N-acyl and simple N-alkyl substituents had weak activity. Compounds with N-alkyl substituents containing ethers, sulfoxides, or sulfones showed increased activity. Much improved activity resulted from compounds where the N-substituents were acetamides. Compound 17u (A-127722) with the N,N-dibutylacetamide substituent is the best of the series. It has an IC(50)=0.36 nM for inhibition of ET-1 radioligand binding at the ET(A) receptor, with a 1000-fold selectivity for the ET(A) vs the ET(B) receptor. It is also a potent inhibitor (IC(50)=0.16 nM) of phosphoinositol hydrolysis stimulated by ET-1, and it antagonized the ET-1-induced contraction of the rabbit aorta with a pA(2)=9.20. The compound has 70% oral bioavailability in rats.

Animals↗

PubMed1996

Azole endothelin antagonists. 3. Using delta log P as a tool to improve absorption.

The oral absorption profile of a family of azole-based ET(A)-selective antagonists has been improved through a rational series of structural modifications which were suggested by analysis of the physicochemical parameter delta log P. Comparison of urea 2 with a series of well-absorbed compounds using delta log P analysis suggested that 2 has an excess capacity for forming hydrogen bonds with solvent. A series of urea modifications were explored as a means of reducing H-bonding capacity while maintaining affinity for the ET(A)-receptor. The correlation between delta log P values and absorption in an intraduodenal (id) bioavailability model was good; this strategy uncovered replacements for each of the urea NH groups which simultaneously improve both potency and drug absorption. A combination of these optimized modifications produces carbamate 16h, a highly-selective ET(A) antagonist with a potency/bioavailability profile consistent with an oral route of administration.

Administration, Oral↗

PubMed1996

Diabetes decreases limbic extracellular dopamine in rats.

Mesolimbic dopamine (DA) was measured by ventral striatum (including nucleus accumbens) microdialysis in freely moving streptozotocin (STZ)-diabetic male rats. DA and dihydroxyphenylacetic acid (DOPAC) basal levels and amphetamine-induced DA increase were lower in diabetic than in normal rats. These results are discussed in terms of decreased DA neuron activity and DA receptor hypersensitivity in the mesolimbic system of STZ-diabetic rats.

3,4-Dihydroxyphenylacetic Acid↗

PubMed1996

In vivo monitoring of brain neurotransmitter release for the assessment of neuroendocrine interactions.

1. The neurotransmitter mechanisms regulating neuroendocrine processes have been traditionally inferred from the effects of drugs purportedly acting through specific transmitter systems. The direct appraisal of changes in endogenous neuromediators had to rely initially on analyses of brain samples obtained post-morten. 2. Currently, a more physiological assessment is available through the monitoring ot the extracellular levels of neurotransmitters and their metabolites in discrete brain areas of living animals. Two methodologies, namely in vivo voltammetry and microdialysis, are being increasingly used for this purpose. This article summarizes their principles, relative merits, and limitations and presents some relevant applications. 3. Thus, microdialysis data show a differential response in the amphetamine-induced dopamine release in the nucleus accumbens in adult male and female rats castrated prepuberally. Given their high time-resolution, in vivo electrochemistry techniques seem especially suited for studying the fast, non-genomic effects of steroid hormones. This is illustrated by the voltammetric detection of a rapid release of dopamine in the corpus striatum induced by progesterone in males. 4. These methodologies should be regarded as complementary tools for the assessment of the neurochemical correlates of neuroendocrine interactions.

Animals↗

PubMed1996

Ethanol anapyrexia in rats.

In the present series of experiments we tested whether ethanol decreases body temperature by impairing thermal regulation (poikilothermia) or by shifting the set point downwards. The central temperature of rats kept in a thermocline and the selected ambient temperature were recorded by telemetry. After an IP injection of 2 g/kg of ethanol the rats selected an ambient temperature 7 degrees C lower than the one they selected before the ethanol injection and 8 degrees C lower than the one selected by the same rats after saline injection. At the same time the central temperature decreased by 2.5 degrees C. After about 40 min the rats preferred warmer ambient temperatures and 10 min later the central temperature began to rise. When, after ethanol, the rats were kept at 30 degrees C the central temperature remained at the normal level. At 35-36 degrees C the central temperature of normal rats without ethanol rose, in 1 h, from 37 degrees C to 39.75 degrees C. The results suggest that ethanol hypothermia is due to a downward shift of the set point and, in fact, is an anapyrexia, a condition inverse to fever.

Animals↗

PubMed1996

Systemic and local cocaine increase extracellular serotonin in the nucleus accumbens.

The effect of systemic or intra-accumbens injections of cocaine on serotonin (5-HT) overflow was studied by nucleus accumbens microdialysis in freely moving rats. In Experiment 1, cocaine was injected intraperitoneally at 0, 10, 20, and 30 mg/kg. In Experiment 2, cocaine (3.6, 7.2, and 14.4 mM), lidocaine (7.2 mM), or saline was infused through the probe by reverse microdialysis. Extracellular serotonin (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) were measured by high-pressure liquid chromatography and electrochemical detection. Systemic administration of cocaine induced a dose-related increase in 5-HT overflow and a decrease of 5-HIAA. Intra-accumbens cocaine infusion also caused a dose-related increase in 5-HT, but no effect on 5-HIAA. As a control for local anesthesia, equimolar lidocaine did not increase 5-HT. The difference between lidocaine and cocaine was not due to unequal diffusion out of the probe, because previous in vivo calibration of the probe showed that more lidocaine than cocaine diffused out of the probe when equimolar solutions were infused. These experiments suggest that systemic cocaine acts on the nucleus accumbens to increase synaptic 5-HT.

Animals↗

PubMed1996

Pharmacological characterization of A-127722: an orally active and highly potent ETA-selective receptor antagonist.

Endothelins (ET) are potent vasoactive peptides implicated in the pathogenesis of a number of vascular diseases. The effects of ET on mammalian organs and cells are initiated by binding to ETA or ETB receptors. In this report, we document the pharmacology of A-127722, a novel ETA-selective receptor antagonist. A-127722 inhibits [125I]ET-1 binding to cloned human ETA and ETB receptors competitively with Ki values of 69 pM and 139 nM, respectively. A-127722 exhibits a dose-dependent inhibition of ET-1-induced arachidonic acid release in human pericardium smooth muscle cells with a pA2 value of 10.5 and inhibits ET-1-induced vasoconstriction in isolated rat aorta with a pA2 value of 9.2. In vivo, A-127722 dose-dependently blocks the pressor response to ET-1 (0.3 nmol/kg i.v.) in conscious rats. Statistically significant (P < .05) antagonism is seen at doses greater than 0.1 mg/kg p.o. Maximal inhibition, at 10 mg/kg, remains constant for at least 8 hr after dosing. No effect is seen on the ETB-mediated transient vasodepressor effect of exogenous ET-1. In conclusion, A-127722 is ETA-selective, orally bioavailable and efficacious for inhibiting the effects of ET in the rat, and A-127722 is the most potent ET receptor antagonist yet reported.

Administration, Oral↗

PubMed1995

Plasma norepinephrine, myocardial damage and left ventricular systolic function in Chagas' heart disease.

The functional status of the sympathetic nervous system in Chagas' heart disease is still the subject of intense controversy. To determine the nature of the abnormalities of the sympathetic nervous system, we measured the plasma norepinephrine concentration of chagasic patients with varying degrees of myocardial damage. Thirty-six patients with positive serology for Chagas' disease were studied. Twenty patients were in Functional Class I (New York Heart Association), 10 were in Functional Class II and six were in Functional Classes III-IV. Cardiac catheterization was performed in 24 patients. The asymptomatic patients had a plasma norepinephrine concentration (121 +/- 37 pg/ml, mean +/- S.D.) not different from normal controls (103 +/- 59 pg/ml). The symptomatic patients, however, had a significantly elevated plasma norepinephrine concentration (665 +/- 354 pg/ml, P < 0.001). The baseline heart rate of the asymptomatic and symptomatic patients directly correlated with the plasma norepinephrine concentration (r = 0.69, P < 0.0001). The symptomatic patients had larger ventricular volumes, higher left ventricular end-diastolic pressures and lower ejection fractions than the asymptomatic patients and normal controls. The plasma norepinephrine concentration correlated linearly with the left ventricular end-diastolic volume (r = 0.77, P < 0.0001), and non-linearly with the ejection fraction (r = -0.70, P < 0.0001) and the left ventricular end-diastolic pressure (r = 0.53, P < 0.007). These results indicate that, in Chagas' heart disease as in most other cardiac diseases, sympathetic nervous system activation is a late and compensatory phenomenon. In other words, sympathetic activation is very likely related to the progressive impairment of left ventricular function.

Adult↗

PubMed1995

Bisimidazoacridones and related compounds: new antineoplastic agents with high selectivity against colon tumors.

A new class of potent and highly selective antitumor agents has been synthesized. Bisimidazoacridones, where the tetracyclic ring systems are held together by either a N2-methyldiethylenetriamine or 3,3'-diamino-N-methyldipropylamine linker, and related asymmetrical compounds, where one of the imidazoacridone ring system was replaced by a triazoloacridone ring system, were found to be cytostatic and cytotoxic in vitro. Some of these compounds, such as 5,5'-[(methylimino)bis(3,1-propanediylimino)]bis[6H-imidazo[ 4,5,1-de]acridin-6-one] (4b) showed remarkably high activity and selectivity for colon cancer in the National Cancer Institute screen. This antitumor effect was also apparent in colony survival assays utilizing the colon cancer line, HCT-116, and in in vivo assays involving xenografts of tumor derived from HCT-116 in nude mice. The tested compounds exhibited relatively low acute toxicity and were well-tolerated by the treated animals. The bisimidazoacridones interact with nucleic acids in vitro but preliminary experimental and modeling data indicate that in spite of their structure, they may not be bis-intercalators. While the precise mode of action of these compounds is not yet understood, they appear to be excellent candidates for clinical development.

Aminoacridines↗

PubMed1995

Isolation and enzymic properties of levansucrase secreted by Acetobacter diazotrophicus SRT4, a bacterium associated with sugar cane.

Acetobacter diazotrophicus, a nitrogen-fixing bacterium associated with sugar cane, secretes a levansucrase (sucrose-2,6-beta-D-fructan 6-beta-D-fructosyltransferase; EC 2.4.1.10). This enzyme is constitutively expressed and represents more than 70% of the total proteins secreted by strain SRT4. The purified protein consists of a single 58 kDa polypeptide with an isoelectric point of 5.5. Its activity is optimal at pH 5.0. It catalyses transfructosylation from sucrose to a variety of acceptors including water (sucrose hydrolysis), glucose (exchange reaction), fructan (polymerase reaction) and sucrose (oligofructoside synthesis). In vivo the polymerase activity leads to synthesis of a high-molecular-mass fructan of the levan type. A. diazotrophicus levansucrase catalyses transfructosylation via a Ping Pong mechanism involving the formation of a transient fructosyl-enzyme intermediate. The catalytic mechanism is very similar to that of Bacillus subtilis levansucrase. The kinetic parameters of the two enzymes are of the same order of magnitude. The main difference between the two enzyme specificities is the high yield of oligofructoside, particularly 1-kestotriose and kestotetraose, accumulated by A. diazotrophicus levansucrase during sucrose transformation. We discuss the hypothesis that these catalytic features may serve the different biological functions of each enzyme.

Acetobacter↗