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Biomedical subjects

J Alvarez

Publications and source records attributed to J Alvarez.

At least 127 records · Page 7Linked to original sources
PubMed1997

The discovery, characterization and crystallographically determined binding mode of an FMOC-containing inhibitor of HIV-1 protease.

A pharmacophore derived from the structure of the dithiolane derivative of haloperidol bound in the active site of the HIV-1 protease (HIV-1 PR) has been used to search a three-dimensional database for new inhibitory frameworks. This search identified an FMOC-protected N-tosyl arginine as a lead candidate. A derivative in which the arginine carboxyl has been converted to an amide has been crystallized with HIV-1 PR and the structure has been determined to a resolution of 2.5 A with a final R-factor of 18.5%. The inhibitor binds in an extended conformation that results in occupancy of the S2, S1', and S3' subsites of the active site. Initial structure-activity studies indicate that: (1) the FMOC fluorenyl moiety interacts closely with active site residues and is important for binding; (2) the N(G)-tosyl group is necessary to suppress protonation of the arginine guanidinyl terminus; and (3) the arginine carboxamide function is involved in interactions with the water coordinated to the catalytic aspartyl groups. FMOC-protected arginine derivatives, which appear to be relatively specific and nontoxic, offer promise for the development of useful HIV-1 protease inhibitors.

Amino Acids↗
PubMed1997

Dissociation of the effects of the antitumour ether lipid ET-18-OCH3 on cytosolic calcium and on apoptosis.

1. We have compared the effects of 1-O-octadecyl-2-O-methyl-sn-glycero-3-phosphocholine (ET-18-OCH3) on the cytosolic free calcium concentration ([Ca2+]i) and on apoptosis in several normal and leukaemia cells, including human polymorphonuclear neutrophils (PMNs), U937 cells, and undifferentiated as well as dimethylsulphoxide-differentiated HL60 cells (uHL60 and dHL60, respectively). 2. ET-18-OCH3 produced apoptosis, as evidenced by DNA degradation into oligonucleosome-size fragments, in U937 and uHL60 cells, but not in dHL60 cells or PMNs. 3. ET-18-OCH3 induced an increase in [Ca2+]i mediated through the platelet-activating factor (PAF) receptor in U937, dHL60 cells and PMNs, as shown by cross-desensitization experiments and by prevention of the [Ca2+]i changes by the PAF antagonist WEB-2170. The EC50 values for the increase in [Ca2+]i induced by PAF and ET-18-OCH3 were 5 x 10(-11) and 2.5 x 10(-7) M, respectively. In uHL60 cells the effect of ET-18-OCH3 on [Ca2+]i was very small and was not affected by WEB-2170. 4. PAF did not produce apoptosis in any of the cell types tested. WEB-2170 did not prevent the apoptosis induced by ET-18-OCH3. 5. The uptake of [3H]-ET-18-OCH3 was much larger in U937 and uHL60 cells than in dHL60 cells and PMNs. 6. Our results indicate that the apoptotic effect of ET-18-OCH3 is not related to the changes in [Ca2+]i, effected by interaction with plasma membrane PAF receptors, but to other actions which are associated with the uptake of this drug into the cells.

Antineoplastic Agents↗
PubMed1997

Urinary cyclic GMP, endothelin, and prostaglandin E2 in normal pregnancy and preeclampsia.

Cyclic GMP, endothelin and prostaglandin E2 (PGE2) all have systemic vasoactive properties (with cyclic GMP acting as a second messenger of nitric oxide). Intrarenally they act as natriuretics and urinary levels reflect intrarenal production. Cyclic GMP and PGE2 also act as important inhibitors of platelet activation and thrombosis. The purpose of this study was to determine if urinary levels of cyclic GMP, endothelin, and PGE2 differ in preeclamptic as compared to normal pregnancies. Parameters were compared in 13 normotensive, nonpreeclamptic pregnancies, and 32 preeclamptic pregnancies. Preeclamptic women had significantly lower levels of urinary cyclic GMP (0.67 +/- 0.12 vs. 2.1 +/- 0.5 nmol/g creatinine), endothelin (0.88 +/- 0.09 vs. 3.75 +/- 1.4 ng/g creatinine), and PGE2 (26 +/- 4 vs. 9 ng/g creatinine) as compared to normals (p < 0.05). Intrarenal production of cyclic GMP, endothelin, and PGE2 are all disturbed in preeclampsia and may have implications in the sodium retention, hypertension, and intrarenal thrombosis and vasospasm of preeclamptic pregnancy.

Adult↗
PubMed1997

[Ca2+] microdomains control agonist-induced Ca2+ release in intact HeLa cells.

We have monitored specifically the [Ca2+] in the lumen of the endoplasmic reticulum (ER) of intact HeLa cells using an ER-targeted low-Ca2+-affinity aequorin. The steady-state [Ca2+] in the ER was around 600 microM. Histamine induced a concentration-dependent decrease in lumenal [Ca2+], whose rate increased near one order of magnitude and became "quantal" when cytosolic [Ca2+] ([Ca2+]c) was clamped with the Ca2+ chelator BAPTA. This effect was not due to decreased Ca2+ pumping because simultaneous addition of a SERCA inhibitor produced only additive effects. Given that inhibition by [Ca2+]c of the inositol 1,4,5-trisphosphate-gated channels requires a [Ca2+]c much higher than that observed in the bulk cytosol after histamine addition, we conclude that local [Ca2+]c microdomains at the site of release strongly inhibit agonist-induced Ca2+ mobilization in intact cells. This effect should play a key role in the mechanism controlling cytosolic [Ca2+] oscillations and waves, and therefore in the generation of spatio-temporal Ca2+ patterns.

Calcium↗
PubMed1997

A family with a milder form of adult dominant polycystic kidney disease not linked to the PKD1 (16p) or PKD2 (4q) genes.

Autosomal dominant polycystic kidney disease (ADPKD) is a genetically heterogeneous disease. Most families show positive linkage to polymorphic markers around the PKD1 (16p13.3) or PKD2 (4q21-23) loci. The PKD1 and PKD2 genes have been cloned and mutations defined in a number of patients. Several clinical studies have described a milder phenotype for PKD2 patients. More recently, evidence for a third genetic locus has been found in one Portuguese, one French-Canadian, and one Italian family. We identified a Spanish family with negative linkage to the PKD1 and the PKD2 loci. This family showed a very mild clinical phenotype compared to the other forms of ADPKD, including the non-PKD1/non-PKD2 families previously described.

Adult↗
PubMed1997

Anaerobic threshold determination with analysis of salivary amylase.

The purpose of this study was to determine the anaerobic threshold from analysis of amylase concentration in total saliva during a laboratory exercise test. Each of 20 healthy young men performed both a submaximal and a maximal test on a treadmill. During the submaximal test, capillary blood and total saliva samples were collected for determination of anaerobic threshold (AT) and saliva threshold (Tsa), respectively. Tsa was defined as the point at which the first continuous increase in amylase concentration occurred during exercise. The results showed no significant difference between values of AT and Tsa when both were expressed either as running velocity or as heart rate. In addition, there existed a high correlation between AT and Tsa (r = .93, p < .001). It was therefore concluded that the analysis of amylase concentration in total saliva during exercise might be used as a valid new method for determining AT.

Adult↗
PubMed1997

Gene expression patterns of murine dentin matrix protein 1 (Dmp1) and dentin sialophosphoprotein (DSPP) suggest distinct developmental functions in vivo.

Although the precise mechanisms of the conversion of predentin to dentin are not well understood, several lines of evidence implicate the noncollagenous proteins (NCPs) as important regulators of dentin biomineralization. Here we compared the in vivo temporospatial expression patterns of two dentin NCP genes, dentin matrix protein 1 (Dmp1), and dentin sialophosphoprotein (DSPP) in developing molars. Reverse transcription-polymerase chain reaction was performed on embryonic day 13 to 1-day-old first molars using Dmp1- and DSPP-specific primer sets. Dmp1 transcripts appeared at the late bud stage, while DSPP mRNA was seen at the cap stage. Expression of both genes was sustained throughout odontogenesis. In situ hybridization analysis revealed interesting differences in the expression patterns of these genes. While Dmp1 and DSPP showed coexpression in young odontoblasts before the start of mineralization, the expression of these genes was notably distinct at later stages. Dmp1 expression decreased in secretory odontoblasts after the appearance of mineral, while high levels of DSPP were sustained in odontoblasts. In early secretory ameloblasts, DSPP expression was transient and down-regulated with the appearance ofdentin matrix. Interestingly, Dmp1 expression became evident in ameloblasts during the maturative phase of amelogenesis. In contrast to Dspp expression that was tooth-specific, Dmp1 was expressed by osteoblasts throughout ossification in the skeleton. Probes directed to the "DSP" and "DPP" regions of the DSPP gene showed identical patterns of mRNA expression. These data show that the developmental expression patterns of Dmp1 and DSPP are distinct, implying that these molecules serve different biological functions in vivo.

Animals↗
PubMed1997

Effects of heavy water on hepatic intracellular pH and phosphatidylcholine turnover. A 31P NMR study.

The short term effect of heavy water (2H2O) in intracellular pH (pHi) and phosphatidylcholine (PtdCho) turnover have been studied by 31P NMR spectroscopy in the perfused mouse liver metabolizing alanine. Hepatic pHi decreased from 7.19 +/- 0.01 (n = 10) to 7.01 +/- 0.03 (n = 4) after the addition of 6 mM alanine to Krebs Ringer bicarbonate (KRB) perfusion medium. Replacement of 50% of the KRB water with 2H2O during alanine perfusion inhibited the intracellular acidification induced by alanine and caused i) a decrease in the hepatic content of PtdCho, and ii) increases in phosphocholine and glycerophosphocholine, respectively. Amiloride (1 mM) of 5-(N-ethyl-N-isopropyl)-amiloride (10 microM), two previously reported inhibitors of the Na+/H+ exchangers, mimicked the effects produced by 2H2O on pHi and PtdCho turnover. Replacement of 50% of the KRB water with 2H2O or the addition of 1mM amiloride to KRB only, did not modify pHi nor increase the levels of phosphocholine of glycerophosphocholine. Thus, the observed increases are the result of alanine perfusion in the presence of 2H2O or amiloride. These results suggest that 2H2O behaves similarly to previously reported inhibitors of Na+/H+ exchange, disclosing also a novel role for PtdCho metabolism in the regulation on hepatic pHi.

Alanine↗
PubMed1997

[Biopharmaceutical studies of similar theophylline products in the national market].

BACKGROUND: Theophylline has a narrow therapeutic margin, therefore it should be formulated as a sustained release pharmaceutical form. In Chile there are several brands of sustained release theophylline forms that have not been tested. AIM: To perform in vitro release-dissolution studies of sustained release theophylline forms commercialized in Chile. MATERIALS AND METHODS: Five sustained release products were studied according to the specifications of the seventh supplement of The United States Pharmacopoeia Convention. Release and dissolution rates were compared with a rapid release aminophylline form. The identity, validation and active principle content of each product was also studied. RESULTS: All the studied forms complied with the quality requirements of the USP XII. The dissolution rate constants of the products at pH 6 ranged from 0.0583 to 0.2130, compared with 7.1 for the rapid release form. The dissolution of all products, except one, was pH dependent. CONCLUSIONS: These results underscore the need to systematically study the quality of pharmaceutical products, considered similar, whose differences could have potential risks for patients.

Bronchodilator Agents↗
PubMed1996

Hormonal regulation of FHG22 mRNA in Syrian hamster harderian glands: role of estradiol.

The regulation of the FHG22 gene by sex steroids has been studied in Syrian hamster Harderian gland, an organ with sexual dimorphism in which the FHG22 mRNA is female-specific. Testosterone treatment of females caused irregular inhibitory effects on the FHG22 mRNA levels, whereas male castration originated transitory increases during less than 2 weeks. Treatment of 15 day-castrated males for 1 or 2 days with beta-estradiol-3-benzoate caused a marked stimulation in the FHG22 mRNA levels. The results found in vivo may be explained considering those found in female Harderian gland serum-free primary cell cultures. In the absence of hormones, the FHG22 mRNA levels decreased along the time and neither progesterone, testosterone, or 5 alpha-dihydrotestosterone affected the expression. However, estradiol stimulated the FHG22 mRNA expression in a time and dose-dependent manner: increasing effects were detected between 8-96 h of treatment and the EC50 was about 10(-9) M. The estradiol effect was reverted by the antiestrogen ICI 164,384 or by cycloheximide. We conclude that estradiol stimulates FHG22 mRNA expression in Harderian gland, although other agents may also control the expression in vivo.

Animals↗
PubMed1996

Segmental demyelination induced by cerebrospinal fluid of progressive spastic paraparesis: correlation with altered proteolytic parameters.

Progressive spastic paraparesis (PSP) is a demyelinating disease of the central nervous system. We studied the ability of the cerebrospinal fluid (CSF) of patients to induce alterations in rat peroneal nerves, and to modify the proteolytic activity of trypsin in vitro. Subperineurial injection of native or heated CSF of patients induced segmental demyelination and other cytological alterations 5-7 days later, in the infiltrated zone, while proximal and distal regions were normal. The CSF of normal subjects did not induce demyelination, but upon heating, it did so. Trypsin was strongly inhibited by the normal CSF but upon heating, its inhibitory activity was replaced by a strong potentiation. In contrast, native and heated CSF of patients potentiated trypsin. Our findings indicate that (1) the normal CSF contains a thermostable factor that potentiates trypsin whose function is overruled by thermolabile protease inhibitors; (2) the CSF of PSP patients has a reduced inhibitory activity and a conserved ability to potentiate trypsin; and (3) the CSF is endowed with a pathogenic power that correlates with an unchecked potentiating activity. We propose that the imbalance of a protease system may play a role in the pathogenesis of PSP lesions.

Animals↗
PubMed1996

Sprouting and abnormal contacts of nonmedullated axons, and deposition of extracellular material induced by the amyloid precursor protein (APP) and other protease inhibitors.

We have reported that the local administration of serine protease inhibitors (amyloid precursor protein with the Kunitz insert (APP K+), aprotinin, and leupeptin) to the rat sciatic nerve determines a sprouting response of myelinated axons, proliferation of Schwann cells, and demyelination, 5 to 7 days later. Further study of these nerves with the electron microscope revealed (i) a sprouting response of nonmedullated axons, (ii) the appearance of fine axons with a few turns of compact myclin, (iii) abnormal contracts of axons with basal laminae, with fibroblast-like cells, and between them, (iv) the occurrence of hemidesmosome- and desmosome-like junctions between Schwann cell processes, and between Schwann cells and axons, and (v) the appearance of amorphous and fibrillary extracellular deposits alongside the axolemma. The adjacent proximal and distal segments were normal, i.e., axons remained continuous, and the alterations were confined to the segment exposed to the protease inhibitors. Heated APP Kappa +, APP without the Kunitz insert (APP K-), bovine serum albumin, and saline, did not elicit cytological alterations. Our results suggest that these inhibitors of serine proteases (i) set free a sprouting drive of axons by disrupting an ongoing repressive mechanism: (ii) modify the adhesive properties of axons and Schwann cells, and (iii) alter the natural history of an extracellular material. The imbalance of an extracellular protease system may participate in the pathogenesis of Alzheimer's disease.

Amyloid beta-Protein Precursor↗
PubMed1996

Molecular genetics of ICL2, encoding a non-functional isocitrate lyase in Saccharomyces cerevisiae.

In this work, we identified an open reading frame 5' to the yeast HALI gene, that shares a 38% identity in the deduced amino acid sequence with gluconeogenic enzyme isocitrate lyase, encoded by ICL1. We therefore termed the new gene ICL2. The latter is not capable of complementing an icl1 deletion for growth on ethanol neither in its original context, nor when expressed under the control of the glycolytic PFK2 promoter. Nevertheless, fusions of the 5'-non-coding region of ICL2 to lacZ reporter gene revealed that the gene is transcribed and that the transcriptional regulation is similar to that of other gluconeogenic genes, i.e. high-level expression on ethanol that is drastically reduced on glucose media. Therefore, we attribute the lack of complementation to a lack of function of the encoded protein as an isocitrate lyase. The deduced amino acid sequences of Icl1 and Icl2 differ in a conserved motif used to identify isocitrate lyases, the hexapeptide KKCGHM, where the second lysine residue of Icl1 is replaced by an arginine in Icl2. However, we here demonstrated by in vitro mutagenesis of ICL1 that such an exchange, even though it affects Icl activity to some degree, does not lead to a complete lack of function. Thus, the results presented in this work argue for ICL2 encoding a non-functional isocitrate lyase and provide evidence that lysine 216 of Icl1 is not essential for catalysis.

Amino Acid Sequence↗