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Biomedical subjects

J Martinez

Publications and source records attributed to J Martinez.

At least 127 records · Page 7Linked to original sources
PubMed2000

Neurotensin modulates the electrical activity of frog pituitary melanotropes via activation of a G-protein-coupled receptor pharmacologically related to both the NTS1 and nts2 receptors of mammals.

The primary structure of frog neurotensin (fNT) has recently been determined and it has been shown that fNT is a potent stimulator of alpha-MSH secretion by frog pituitary melanotropes. In the present study, we have investigated the effects of fNT on the electrical activity of cultured frog melanotropes by using the patch-clamp technique and we have determined the pharmacological profile of the receptors mediating the effect of fNT. In the cell-attached configuration, fNT (10(-7) M) provoked an increase in the action current discharge followed by an arrest of spike firing. In the gramicidin-perforated patch configuration, fNT (10(-7) M) induced a depolarization accompanied by an increase in action potential frequency and a decrease in membrane resistance. Administration of graded concentrations (10(-10) to 10(-6) M) of fNT or the C-terminal hexapeptide NT(8-13) caused a dose-dependent increase in the frequency of action potentials with EC(50) of 2 x 10(-8) and 5 x 10(-9) M, respectively. The stimulatory effect of fNT was mimicked by various pseudopeptide analogs, with the following order of potency: Boc-[Trp(11)]NT(8-13) > Boc-[D-Trp(11)]NT(8-13) > Boc-[Lys(8,9), Nal(11)]NT(8-13) > Boc-[Psi11,12]NT(8-13). In contrast, the cyclic pseudopeptide analogs of NT(8-13), Lys-Lys-Pro-D-Trp-Ile-Leu and Lys-Lys-Pro-D-Trp-Glu-Leu-OH, did not affect the electrical activity. The NTS1 receptor antagonist and nts2 receptor agonist SR 48692 (10(-5) M) stimulated the spike discharge but did not block the response to fNT. In contrast, SR 142948A (10(-5) M), another NTS1 receptor antagonist and nts2 receptor agonist, inhibited the excitatory effect of fNT. The specific nts2 receptor ligand levocabastine (10(-6) M) had no effect on the basal electrical activity and the response of melanotropes to fNT. In cells which were dialyzed with guanosine-5'-O-(3-thiotriphosphate) (10(-4) M), fNT caused an irreversible stimulation of the action potential discharge. Conversely, dialysis of melanotropes with guanosine-5'-O-(2-thiodiphosphate) (10(-4) M) completely blocked the effect of fNT. Pretreatment of cells with cholera toxin (1 microg/ml) or pertussis toxin (0.2 microg/ml) did not affect the electrical response to fNT. Intracellular application of the G(o/i/s) protein antagonist GPAnt-1 (3 x 10(-5) M) had no effect on the fNT-evoked stimulation. In contrast, dialysis of melanotropes with the G(q/11) protein antagonist GPAnt-2A (3 x 10(-5) M) abrogated the response to fNT. The present data demonstrate that fNT is a potent stimulator of the electrical activity of frog pituitary melanotropes. These results also reveal that the electrophysiological response evoked by fNT can be accounted for by activation of a G(q/11)-protein-coupled receptor subtype whose pharmacological profile shares similarities with those of mammalian NTS1 and nts2 receptors.

Adamantane↗
PubMed2000

Positive association between blood brain barrier disruption and osmotically-induced demyelination.

Rapid correction of chronic hyponatremia can cause osmotic brain demyelination in animals and humans. Why demyelination develops is unknown, but blood brain-barrier disruption might expose oligodendrocytes to substances normally excluded from the brain. To test this hypothesis, chronic hyponatremia was induced and corrected using a new, reproducible rat model for producing osmotic brain demyelination. Blood brain barrier integrity was assessed by NMR imaging at either 3, 16 or 24 h during the first day of correction. Demyelination was determined histopathologically 5 - 6 days later. Of 96 rats studied, demyelination developed 5 - 6 days later in 37 rats, 89% of whom showed barrier disruption. In the 59 rats who did not develop demyelination, 45 (76%) had no barrier disruption. Thus, blood-brain barrier disruption during the first 24 h of correction was associated with a 70% risk of developing demyelination. By contrast, the risk of developing subsequent demyelination was only 8% when the barrier was intact. This strong association between barrier disruption and subsequent demyelination provides new insights into the role of blood brain barrier function in demyelinative disorders such as the osmotic demyelination syndrome and by extension to other demyelinative disorders such as multiple sclerosis.

Animals↗
PubMed2000

Detection of heat shock protein-70 from Trichinella spiralis larvae using a modification of the routine western blotting procedure.

This is the first study that establishes a standardized western blotting method for the detection of heat shock protein (HSP) 70 from Trichinella spiralis using (selected) monoclonal antibodies (mAbs). Enhancement of HSP transfer onto the supportive membrane and increased retention of protein by the membrane are prominent features of the procedure. The reactivity of T. spiralis HSP70 on western blots was substantially increased by the use of a 10% acrylamide gel, the optimization of conditions during electrotransfer, and transfer onto Immobilon membranes. These data indicate that mAbs actually capable of detecting the agent of interest may be discarded because of nonoptimal testing conditions. We suggest that this method will aid in understanding the role and function of T. spiralis HSP70 in host-parasite (inter)relationship(s).

Animals↗
PubMed2000

Mutagenesis in the switch IV of the helical domain of the human Gsalpha reduces its GDP/GTP exchange rate.

The Galpha subunits of heterotrimeric G proteins are constituted by a conserved GTPase "Ras-like" domain (RasD) and by a unique alpha-helical domain (HD). Upon GTP binding, four regions, called switch I, II, III, and IV, have been identified as undergoing structural changes. Switch I, II, and III are located in RasD and switch IV in HD. All Galpha known functions, such as GTPase activity and receptor, effector, and Gbetagamma interaction sites have been found to be localized in RasD, but little is known about the role of HD and its switch IV region. Through the construction of chimeras between human and Xenopus Gsalpha we have previously identified a HD region, encompassing helices alphaA, alphaB, and alphaC, that was responsible for the observed functional differences in their capacity to activate adenylyl cyclase (Antonelli et al. [1994]: FEBS Lett 340:249-254). Since switch IV is located within this region and contains most of the nonconservative amino acid differences between both Gsalpha proteins, in the present work we constructed two human Gsalpha mutant proteins in which we have changed four and five switch IV residues for the ones present in the Xenopus protein. Mutants M15 (hGsalphaalphaS133N, M135P, P138K, P143S) and M17 (hGsalphaalphaS133N, M135P, V137Y, P138K, P143S) were expressed in Escherichia coli, purified, and characterized by their ability to bind GTPgammaS, dissociate GDP, hydrolyze GTP, and activate adenylyl cyclase. A decreased rate of GDP release, GTPgammaS binding, and GTP hydrolysis was observed for both mutants, M17 having considerably slower kinetics than M15 for all functions tested. Reconstituted adenylyl cyclase activity with both mutants showed normal activation in the presence of AlF(4)(-), but a decreased activation with GTPgammaS, which is consistent with the lower GDP dissociating rate they displayed. These data provide new evidence on the role that HD is playing in modulating the GDP/GTP exchange of the Gsalpha subunit.

Adenylyl Cyclases↗
PubMed2000

Adherence to antiviral drug regimens in HIV-infected adolescent patients engaged in care in a comprehensive adolescent and young adult clinic.

Inconsistent use of antiviral medications for the treatment of HIV may lead to the emergence of resistant strains in HIV-infected adults. Patterns of adherence with these drug regimens in adolescents remains unknown. Identifying nonadherence in HIV-infected patients to antiviral regimens and developing corrective measures could improve patient outcomes. This study was undertaken to understand adherence in HIV-infected youths engaged in care and to reduce patterns of nonadherence. A retrospective analysis of 25 charts (78%) of HIV-infected youths (n = 32, age 13 to 21 years) were consecutively reviewed from January 1993 to May 1998. Charts were reviewed for documentation of factors previously documented to be associated with adherence: housing stability, social support, prior sexually transmitted diseases (STDs) and/or pregnancy, HIV exposure category, number of clinic visits, number of pills per day, number of medications per day, knowledge of medication schedule, age, gender, race/ethnicity, health status as revealed by CD4 count and viral load, and recorded patterns of adherence to medications and clinic appointments. Thirteen of the 18 (72%) patients who were receiving antiretroviral medication were nonadherent. Sixty-seven percent of the females and 80% of the males reported missing doses. Housing instability (p = 0.031) and/or length of treatment with antiviral medications (months of treatment) (p = 0.043) were significantly correlated with nonadherence. The stability of the adolescents' living situations was the most significant correlate of medication adherence for this population of HIV-infected youth.

Adolescent↗
PubMed2000

[New derivatives of pantolactonyl N-phthalimido-2-arylpropanoate].

We present the crystal and molecular structures of two new N-phthalyl-3-amino-2-arylpropionic acid pantolactonyl ester derivatives with 4-fluorophenyl and 3,4-dimethoxyphenyl as the aryl group, 2,3,4,5-tetrahydro-4,4-dimethyl-2-oxofuran-3-yl 3-phthalimido-2-(4-fluorophenyl)propanoate, C23H20FNO6, and 2,3,4,5-tetrahydro-4,4-dimethyl-2-oxofuran-3-yl 3-phthalimido-2-(3,4-dimethoxyphenyl)propanoate ethyl acetate hemisolvate, C25H25NO8.0.5C4H8O2. This structural study confirms the S configuration of the C2 and validates the stereospecificity of our synthesis strategy.

4-Butyrolactone↗
PubMed2000

FGFR3 gene mutation (Gly380Arg) with achondroplasia and i(21q) Down syndrome: phenotype-genotype correlation.

We report the case of a boy with achondroplasia and i(21q) Down syndrome. Besides craniofacial features typical in Down syndrome, the skeletal findings of achondroplasia dominate the clinical picture. The diagnosis of Down syndrome was based on clinical features and the cytogenetic finding of i(21q) trisomy 21. The diagnosis of achondroplasia was based on the presence of clinical and radiographic findings and confirmed by the presence of a common FGFR3 gene mutation (Gly380Arg) detected by restriction enzyme analysis and sequencing of the polymerase chain reaction products. This is the first report of achondroplasia associated with i(21q) Down syndrome.

Achondroplasia↗
PubMed1999

Unilateral eye abnormalities in reared Mediterranean gilthead sea bream.

Eye abnormalities in reared gilthead sea bream (Sparus aurata) were investigated clinically and by histological techniques. A significant number of fish had a cataract in one eye and ocular inflammation including gross exophthalmos. In a small number of fish gas bubbles were observed in the anterior chamber of the eye. Histological examination provided no evidence of an infectious process and a possible behavioural aetiology is discussed.

Animals↗
PubMed1999

Design and synthesis of potent bradykinin agonists containing a benzothiazepine moiety.

A bradykinin analogue (H-Arg-Pro-Pro-Gly-Phe-Ser-D-BT-Arg-OH, 3) in which the Pro-Phe dipeptide was replaced by the (3S)[amino]-5-(carbonylmethyl)-2,3-dihydro-1, 5-benzothiazepin-4(5H)-one (D-BT) moiety has been synthesized. The same modification was performed on the potent bradykinin B(2) receptor antagonist HOE 140 (H-D-Arg-Arg-Pro-Hyp-Gly-Thi-Ser-D-Tic-Oic-Arg-OH), in which the -D-Tic-Oic- moiety was replaced by D-BT to yield H-D-Arg-Arg-Pro-Hyp-Gly-Thi-Ser-D-BT-Arg-OH, 1 (JMV1116). These compounds were examined in vitro for their binding affinity toward bradykinin B(1) and B(2) receptors as well as for their ability to interfere with bradykinin-induced contraction of both human umbilical vein and rat uterus. The two compounds 3 and 1 competed with [(3)H]bradykinin binding to the human cloned B(2) receptor giving K(i) values of 13 +/- 2 and 0.7 +/- 0.1 nM, respectively. Unexpectedly, both compounds were full bradykinin B(2) receptor agonists on the human umbilical vein (pD(2) = 6.60 +/- 0.07 for 3 and 6.80 +/- 0.08 for 1) and rat uterus (pD(2) = 7.20 +/- 0.09 for 3 and 7.50 +/- 0.09 for 1) preparations with the same efficacy as bradykinin. In addition 1 induced a concentration-dependent phosphoinositide production in CHO cells expressing the human cloned B(2) receptor. These data provide evidence for a bioactive conformation of bradykinin constrained at the dipeptide Pro-Phe.

Angiotensin-Converting Enzyme Inhibitors↗
PubMed1999

Synthesis and characterization of bradykinin B(2) receptor agonists containing constrained dipeptide mimics.

We have previously shown that substitution of the D-Tic-Oic dipeptide by a (3S)-[amino]-5-(carbonylmethyl)-2,3-dihydro-1, 5-benzothiazepin-4(5H)-one (D-BT) moiety in the bradykinin B(2) receptor antagonist HOE 140 resulted in a full potent and selective bradykinin B(2) receptor agonist (H-DArg-Arg-Pro-Hyp-Gly-Thi-Ser-D-BT-Arg-OH, JMV1116) exhibiting a high affinity for the human receptor (K(i) 0.7 nM). In the present study, we have investigated the effects of replacement of the D-Tic-Oic moiety by various constrained dipeptide mimetics. The resulting compounds were tested for their binding affinity toward the cloned human B(2) receptor and for their functional interaction with the bradykinin-induced contraction of isolated human umbilical vein. Subsequently, we have designed novel bradykinin B(2) receptor agonists which are likely to be resistant to enzymatic cleavage by endopeptidases and which might represent interesting new pharmacological tools. In an attempt to increase the potency of compound JMV1116, both its N-terminal part and the D-BT moiety were modified. Substitution of the D-arginine residue by a L-lysine residue led to a 10-fold more potent bradykinin B(2) ligand [compound 22 (JMV1465) (K(i) 0.07 nM)], retaining full agonist activity on human umbilical vein. Substitution of the D-BT moiety by a (3S)-[amino]-5-(carbonylmethyl)-2,3-dihydro-8-methyl-1, 5-benzothiazepin-4(5H)-one [D-BT(Me)] moiety led to compound 23 (JMV1609) which exhibited a higher agonist activity (pD(2) = 7.4) than JMV1116 (pD(2) = 6.8).

Animals↗
PubMed1999

Using heat shock proteins as indicators of the immune function in wistar rats during a secondary Trichinella spiralis infection.

The muscle, liver, brain and spleen tissues from Wistar rats with either a primary Trichinella spiralis infection alone, or reinfected 45 days after primary infection, were collected at Days 1, 7, 14, 20 and 27 post reinfection. They were then assayed for levels of four heat shock proteins (HSPs), i.e. hsp90, hsp70, hsp60 and hsp25. Detection and quantitation of the separate HSPs in tissue specimens were achieved using Western blot and image analysis technique, respectively. The results show that the elements affecting altered expression of rat organs' HSP were 'neutralized' by resistance-related events in immunized rats. Thus, while rat organs exhibited varying HSP levels in primary infections [Martinez, J., Perez-Serrano, J., Bernadina, W., Rodriguez-Caabeiro, F., 1999. Parasitology 118, 202-209], there was, in reinfected versus primary-infected rats, no difference in test HSPs levels in any organ, and at any time within the time course of this study. We have interpreted the results by using the model that involves induction of anti-T.spiralis immunity during primary infection and (almost) complete removal of effectors of tissue injury (infective T.spiralis larvae and newborn larvae) during reinfection.

Animals↗
PubMed1999

Evidence for a direct interaction between the penultimate aspartic acid of cholecystokinin and histidine 207, located in the second extracellular loop of the cholecystokinin B receptor.

Recently, we reported that the mutation of His(207) to Phe located in the second extracellular loop of the cholecystokinin B receptor strongly affected cholecystokinin (CCK) binding (Silvente-Poirot, S., Escrieut, C., and Wank, S. A. (1998) Mol. Pharmacol. 54, 364-371). To characterize the functional group in CCK that interacts with His(207), we first substituted His(207) to Ala. This mutation decreased the affinity and the potency of CCK to produce total inositol phosphates 302-fold and 456-fold without affecting the expression of the mutant receptor. The screening of L-alanine-modified CCK peptides to bind and activate the wild type and mutant receptors allowed the identification of the interaction of the C-terminal Asp(8) of CCK with His(207). The H207A-CCKBR mutant, unlike the wild type receptor, was insensitive to substitution of Asp(8) of CCK to other amino acid residues. This interaction was further confirmed by mutating His(207) to Asp. The affinity of CCK for the H207D-CCKBR mutant was 100-fold lower than for the H207A-CCKBR mutant, consistent with an electrostatic repulsion between the negative charges of the two interacting aspartic acids. Peptides with neutral amino acids in position eight of CCK reversed this effect and displayed a gain of affinity for the H207D mutant compared with CCK. To date, this is the first report concerning the identification of a direct contact point between the CCKB receptor and CCK.

Amino Acid Sequence↗
PubMed1999

Arginine 336 and asparagine 333 of the human cholecystokinin-A receptor binding site interact with the penultimate aspartic acid and the C-terminal amide of cholecystokinin.

The cholecystokinin-A receptor (CCK-AR) is a G protein-coupled receptor that mediates important central and peripheral cholecystokinin actions. Residues of the CCK-AR binding site that interact with the C-terminal part of CCK that is endowed with biological activity are still unknown. Here we report on the identification of Arg-336 and Asn-333 of CCK-AR, which interact with the Asp-8 carboxylate and the C-terminal amide of CCK-9, respectively. Identification of the two amino acids was achieved by dynamics-based docking of CCK in a refined three-dimensional model of CCK-AR using, as constraints, previous results that demonstrated that Trp-39/Gln-40 and Met-195/Arg-197 interact with the N terminus and the sulfated tyrosine of CCK, respectively. Arg-336-Asp-8 and Asn-333-amide interactions were pharmacologically assessed by mutational exchange of Arg-336 and Asn-333 in the receptor or reciprocal elimination of the partner chemical functions in CCK. This study also allowed us to demonstrate that (i) the identified interactions are crucial for stabilizing the high affinity phospholipase C-coupled state of the CCK-AR.CCK complex, (ii) Arg-336 and Asn-333 are directly involved in interactions with nonpeptide antagonists SR-27,897 and L-364,718, and (iii) Arg-336 but not Asn-333 is directly involved in the binding of the peptide antagonist JMV 179 and the peptide partial agonist JMV 180. These data will be used to obtain an integrated dynamic view of the molecular processes that link agonist binding to receptor activation.

Amides↗
PubMed1999

The interleukin-1 type 2 receptor gene displays immediate early gene responsiveness in glucocorticoid-stimulated human epidermal keratinocytes.

Human epidermal keratinocytes (HEKs) in primary culture (P2-P4) were used to study glucocorticoid (GC)-mediated transcription of the genes encoding the constitutively expressed interleukin-1 type 1 receptor (IL-1R1) and the inducible interleukin-1 type 2 receptor (IL-1R2). Utilizing Northern dot blot analysis and a quantitative reverse transcription-polymerase chain reaction protocol for IL-1R1 and IL-1R2, dexamethasone and, in particular, the budesonide epimer R were shown to effectively and rapidly induce transcription from the IL-IR2 gene when compared with IL-1R1 or beta-actin RNA message levels in the same sample. Southern blot analysis of newly generated IL-1R2 reverse transcription-polymerase chain reaction products using end-labeled IL-1R2 intron probes suggested that GC enhancement of IL-1R2 expression was regulated primarily at the level of de novo transcription. GC-induced IL-1R2 gene transcription displayed features characteristic of a classical immediate early gene response, including a signal transduction function, a relatively low basal abundance, a rapid, transient induction, cycloheximide superinduction, actinomycin D suppression, and a rapid decay of IL-1R2 RNA message. Parallel time course kinetic analysis of IL-1R2 RNA message levels with Western immunoblotting revealed tight coupling of de novo IL-IR2 gene transcription with translation of the IL-1R2 RNA message; a newly synthesized ( approximately 46-kDa) IL-1R2 protein was detected in the HEK growth medium as early as 1 h after budesonide epimer R treatment. These data indicate that different GC compounds can variably up-regulate the IL-1R2 response in HEKs through transcription-mediated mechanisms and, for the first time, suggest that a gene encoding a soluble cytokine receptor can respond like an immediate early gene.

Budesonide↗
PubMed1999

Identification of the receptor subtype involved in the analgesic effect of neurotensin.

The neuropeptide neurotensin (NT) elicits hypothermic and naloxone-insensitive analgesic responses after brain injection. Recent pharmacological evidence obtained with NT agonists and antagonists suggests that these effects are mediated by a receptor distinct from the initially cloned high-affinity NT receptor (NTR1). The recent cloning of a second NT receptor (NTR2) prompted us to evaluate its role in NT-induced analgesia. Intracerebroventricular injections in mice of two different antisense oligodeoxynucleotides from the NTR2 markedly decreased NTR2 mRNA and protein and reduced NT-induced analgesia. This effect was specific, because NTR1 levels were unaffected, and sense or scramble oligodeoxynucleotides had no effect. Structure-activity studies revealed a close correlation between the analgesic potency of NT analogs and their affinity for the NTR2 and disclosed potent and selective agonists of this receptor. These data confirm that NTR1 is involved in the NT-elicited turning behavior and demonstrate that the NTR2 mediates NT-induced analgesia.

Analgesics↗
PubMed1999

Pulmonary hypertension in patients with myelofibrosis secondary to myeloproliferative diseases.

We examined the clinical characteristics of six patients with myelofibrosis secondary to myeloproliferative diseases whose clinical courses were complicated by pulmonary hypertension to determine possible causal links between the two disorders. Six patients (four male, two female), with diagnoses of myeloproliferative disease, myelofibrosis (one with polycythemia vera, three with agnogenic myeloid metaplasia, one with unclassified myeloproliferative syndrome, one with essential thrombocytosis), and pulmonary hypertension are presented. Measurement of the pulmonary artery pressure was performed by Doppler echocardiography in all patients and by right sided heart catheterization in four patients. The range of resting pulmonary artery systolic pressure was 35 to 47 mmHg above the mean right atrium by echocardiography. One patient had autopsy evidence of pulmonary myeloid metaplasia and interstitial fibrosis; another had acute leukemic infiltration of the lung parenchyma. All patients had thrombocytosis; symptomatology in one patient with marked thrombocytosis improved with plateletpheresis. Two patients suffered systemic thrombosis. All patients had severe hepatomegaly. Two patients had evidence of left ventricular dysfunction. The interval between the development of dyspnea and death was less than seven months in five of the patients. A causal link between pulmonary hypertension and myelofibrosis secondary to myeloproliferative diseases is suggested for each patient. Hematopoietic infiltration of the pulmonary parenchyma, portal hypertension, thrombocytosis, hypercoagulability, and left ventricular failure may account in part for the development of pulmonary hypertension in these patients. Patients with myelofibrosis and dyspnea should have Doppler echocardiography to evaluate pulmonary artery pressures.

Adult↗
PubMed1999

Imaging time-of-flight secondary ion mass spectrometry of solid-phase peptide syntheses.

Imaging time-of-flight secondary ion mass spectrometry (TOF-SIMS) of solid-phase peptide syntheses carried out by the Merrifield and Sheppard strategies is described. Mixtures of resin beads mixed at random from batch syntheses or obtained in combinatorial chemistry by the mix and split technique, where each bead is functionalized by a unique peptide, were analyzed directly without any chemical cleavage of the growing chains to assess the nature of the growing structure on any bead of the mixture without its isolation.

Amino Acids↗