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G Gonzalez

Publications and source records attributed to G Gonzalez.

At least 19 recordsLinked to original sources
PubMed2000

Dos, a heme-binding PAS protein from Escherichia coli, is a direct oxygen sensor.

A direct sensor of O(2), the Dos protein, has been found in Escherichia coli. Previously, the only biological sensors known to respond to O(2) by direct and reversible binding were the FixL proteins of Rhizobia. A heme-binding region in Dos is 60% homologous to the O(2)-sensing PAS domain of the FixL protein, but the remainder of Dos does not resemble FixL. Specifically, the C-terminal domain of Dos, presumed to be a regulatory partner that couples to its heme-binding domain, is not a histidine kinase but more closely resembles a phosphodiesterase. The absorption spectra of Dos indicate that both axial positions of the heme iron are coordinated to side chains of the protein. Nevertheless, O(2) and CO bind to Dos with K(d) values of 13 and 10 microM, respectively, indicating a strong discrimination against CO binding. Association rate constants for binding of O(2) (3 mM(-)(1) s(-)(1)), CO (1 mM(-)(1) s(-)(1)) and even NO (2 mM(-)(1) s(-)(1)) are extraordinarily low and very similar. Displacement of an endogenous ligand, probably Met 95, from the heme iron in Dos triggers a conformational change that alters the activity of the enzymatic domain. This sensing mechanism differs from that of FixL but resembles that of the CO sensor CooA of Rhodospirillum rubrum. Overall the results provide evidence for a heme-binding subgroup of PAS-domain proteins whose working range, signaling mechanisms, and regulatory partners can vary considerably.

Amino Acid Sequence↗
PubMed2000

Pharmaco-topology of sulfonylurea receptors. Separate domains of the regulatory subunits of K(ATP) channel isoforms are required for selective interaction with K(+) channel openers.

The differential responsiveness of (SUR1/K(IR)6.2)(4) pancreatic beta-cell versus (SUR2A/K(IR)6.2)(4) sarcolemmal or (SUR2B/K(IR)6. 0)(4) smooth muscle cell K(ATP) channels to K(+) channel openers (KCOs) is the basis for the selective prevention of hyperinsulinemia, myocardial infarction, and acute hypertension. KCO-stimulation of K(ATP) channels is a unique example of functional coupling between a transport ATPase and a K(+) inward rectifier. KCO binding to SUR is Mg-ATP-dependent and antagonizes the inhibition of (K(IR)6.0)(4) pore opening by nucleotides. Patch-clamping of matched chimeric human SUR1-SUR2A/K(IR)6.2 channels was used to identify the SUR regions that specify the selective response of sarcolemmal versus beta-cell channels to cromakalim or pinacidil versus diazoxide. The SUR2 segment containing the 12th through 17th predicted transmembrane domains, TMD12-17, confers sensitivity to the benzopyran, cromakalim, and the pyridine, pinacidil, whereas an SUR1 segment which includes TMD6-11 and the first nucleotide-binding fold, NBF1, controls responsiveness to the benzothiadiazine, diazoxide. These data are incorporated into a functional topology model for the regulatory SUR subunits of K(ATP) channels.

ATP-Binding Cassette Transporters↗
PubMed1999

The tolbutamide site of SUR1 and a mechanism for its functional coupling to K(ATP) channel closure.

Micromolar concentrations of tolbutamide will inhibit (SUR1/K(IR)6. 2)(4) channels in pancreatic beta-cells, but not (SUR2A/K(IR)6.2)(4) channels in cardiomyocytes. Inhibition does not require Mg(2+) or nucleotides and is enhanced by intracellular nucleotides. Using chimeras between SUR1 and SUR2A, we show that transmembrane domains 12-17 (TMD12-17) are required for high-affinity tolbutamide inhibition of K(ATP) channels. Deletions demonstrate involvement of the cytoplasmic N-terminus of K(IR)6.2 in coupling sulfonylurea-binding with SUR1 to the stabilization of an interburst closed configuration of the channel. The increased efficacy of tolbutamide by nucleotides results from an impairment of their stimulatory action on SUR1 which unmasks their inhibitory effects. The mechanism of inhibition of beta-cell K(ATP) channels by sulfonylureas during treatment of non-insulin-dependent diabetes mellitus thus involves two components, drug-binding and conformational changes within SUR1 which are coupled to the pore subunit through its N-terminus and the disruption of nucleotide-dependent stimulatory effects of the regulatory subunit on the pore. These findings uncover a molecular basis for an inhibitory influence of SUR1, an ATP-binding cassette (ABC) protein, on K(IR)6.2, a ion channel subunit.

ATP-Binding Cassette Transporters↗
PubMed1999

The C terminus of SUR1 is required for trafficking of KATP channels.

In beta cells from the pancreas, ATP-sensitive potassium channels, or KATP channels, are composed of two subunits, SUR1 and KIR6.2, assembled in a (SUR1/KIR6.2)4 stoichiometry. The correct stoichiometry of channels at the cell surface is tightly regulated by the presence of novel endoplasmic reticulum (ER) retention signals in SUR1 and KIR6.2; incompletely assembled KATP channels fail to exit the ER/cis-Golgi compartments. In addition to these retrograde signals, we show that the C terminus of SUR1 has an anterograde signal, composed in part of a dileucine motif and downstream phenylalanine, which is required for KATP channels to exit the ER/cis-Golgi compartments and transit to the cell surface. Deletion of as few as seven amino acids, including the phenylalanine, from SUR1 markedly reduces surface expression of KATP channels. Mutations leading to truncation of the C terminus of SUR1 are one cause of a severe, recessive form of persistent hyperinsulinemic hypoglycemia of infancy. We propose that the complete loss of beta cell KATP channel activity seen in this form of hyperinsulinism is a failure of KATP channels to traffic to the plasma membrane.

ATP-Binding Cassette Transporters↗
PubMed1999

Two regions of sulfonylurea receptor specify the spontaneous bursting and ATP inhibition of KATP channel isoforms.

KATP channels are heteromultimers of KIR6.2 and a sulfonylurea receptor, SUR, an ATP binding cassette (ABC) protein with several isoforms. KIR6.2 forms a channel pore whose spontaneous activity and ATP sensitivity are modulated by the receptor via an unknown interaction(s). Side by side comparison of single-channel kinetics and steady-state ATP inhibition of human beta-cell, SUR1/KIR6.2, versus cardiac, SUR2A/KIR6.2 channels demonstrate that the latter have a greater mean burst duration and open probability in the absence of nucleotides and approximately 4-fold higher IC50(ATP). We have used matched chimeras of SUR1 and SUR2A to show that the kinetics, which determine the maximal open probability (Pomax), and the ATP sensitivity are functionally separable and to identify the two segments of SUR responsible for these isoform differences. A region within the first five transmembrane domains specifies the interburst kinetics, whereas a C-terminal segment determines the sensitivity to inhibitory ATP. The separable effects of SUR on ATP inhibition and channel kinetics implies that the cytoplasmic C terminus of SUR either directly modulates the affinity of a weak ATP binding site on the inward rectifier or affects linkage between the binding site and the gate. This is the first identification of parts of an ABC protein that interact with an ion channel subunit to modulate the spontaneous activity and ATP sensitivity of the heteromeric channel.

ATP-Binding Cassette Transporters↗
PubMed1999

Sulfonylurea receptors set the maximal open probability, ATP sensitivity and plasma membrane density of KATP channels.

KATP channels are heteromultimers of SUR and KIR6.2. C-terminal truncation of KIR6.2 allows surface expression of the pore. KIR6.2deltaC35 channels display approximately 7-fold lower maximal open probability, approximately 35-fold reduced ATP sensitivity, reduced mean open time, a markedly increased transition rate from a burst into a long-lived closed state, and have no counterpart in vivo. SUR1 and SUR2A restore wild-type bursting, ATP sensitivity and increase channel density in the plasma membrane. The high IC50(ATP) of approximately 4 mM for KIR6.2deltaCK185Q channels results from the additive effects of SUR removal and KIR6.2 modification. The results demonstrate allosteric interaction(s) are essential for normal intrinsic activity, ATP inhibition, and trafficking of KATP channels.

ATP-Binding Cassette Transporters↗
PubMed1999

The N-terminus of KIR6.2 limits spontaneous bursting and modulates the ATP-inhibition of KATP channels.

KATP channels are heteromultimers of a sulfonylurea receptor SUR and KIR6.2 with the inward rectifier forming the pore which is regulated by SUR. We have examined the contributions of the cytoplasmic domains of KIR6.2 to control of spontaneous bursting and ATP-inhibition in human SUR1/KIR6.2 KATP channels. Truncations of the N-terminus of KIR6.2 nearly eliminate transitions to interburst closed states without affecting the open or intraburst closed states, thus producing SUR1/DeltaNKIR6.2 channels with an extremely high open probability in the absence of nucleotides. These channels have a decrease apparent ATP-sensitivity which is consistent with the involvement of the N-terminus in a transition to an interburst closed state that preferentially binds inhibitory ATP. Mutations in both the N- and proximal C-termini of KIR6.2 can synergistically attenuate the ATP-inhibition. The results identify the N-terminus of KIR6.2 as a determinant of the interburst kinetics of KATP channels and suggest that the two cytoplasmic domains of KIR6.2 participate in ATP-inhibitory gating through distinct mechanisms.

ATP-Binding Cassette Transporters↗
PubMed1999

Influence of age and sex in serum osteocalcin levels in thoroughbred horses.

In this study, we assessed the potential value of free serum osteocalcin or bone gla protein (BGP), the most abundant non collagenous matrix protein found in bone and dentin, to reflect changes of bone turnover in thoroughbred horses. Levels of osteocalcin were analyzed in serum samples of 54 clinically normal animals divided into three groups (A, B, C) according to age: 8, 16-18 and 24-36 months, in order to determine the standard for young horses of different age and sex. Serum BGP was measured by an in-house developed double antibody radioimmunoassay using bovine antigen. The mean BGP levels (ng/ml) were 45.65 +/- 11.69; 33.65 +/- 16.65; 15.08 +/- 6.70 respectively for groups A, B and C; statistically significant differences were found between groups (A vs B and C; Bvs C). Difference between males and females was found significant in group C with higher values in the females: 18.75 +/- 5.00 against 14.43 +/- 10.47 i n the males. This can be considered a sex related effect on BGP serum levels after the onset of puberty. Correlation coefficient between age and serum BGP for females and males were r 5 20.598 ( P < 0.001) and r 5 200.807 (P < 0.001) respectively. A significant negative linear relationship could be established between these two parameters in males during the growth period. The regression equation between serum BGP and age for males was (month of age = 65.14-1.68. BGP). In the female group the gestation and lactation are variables that lower the correlation coefficient between age and serum BGP levels. These results suggest that serum BGP decreases in thoroughbred horses during the growth period, and significant differences between sexes were found only after the onset of puberty.

Aging↗
PubMed1999

Effect of varying doses of potassium-magnesium citrate on thiazide-induced hypokalemia and magnesium loss.

The purpose of this study was to compare the efficacy of three dosages of potassium-magnesium citrate in overcoming thiazide-induced hypokalemia and magnesium loss and increasing urinary pH and citrate. Sixty-one normal subjects first took hydrochlorothiazide at 50 mg/d. After 3 weeks of thiazide treatment or earlier if hypokalemia developed, the subjects were randomized to take one of three dosages of potassium-magnesium citrate (K ( 4 ) MgCit ( 2 ) ) for 3 weeks while continuing on the thiazide: 4 tablets per day (24 mEq potassium, 12 mEq magnesium, and 36 mEq citrate per day), 7 tablets per day (49 mEq potassium, 24.5 mEq magnesium, and 73.5 mEq citrate per day), or 10 tablets per day (70 mEq potassium, 35 mEq magnesium, and 105 mEq citrate per day). Outcome measures were changes in serum potassium and magnesium and urinary potassium, magnesium, pH, and citrate. All three dosages of potassium-magnesium citrate significantly increased serum potassium concentration, with >80% of subjects regaining normal values despite continued thiazide therapy. The two higher dosages, but not the lowest dosage, caused a small but significant increase in serum magnesium concentration, while substantially increasing urinary magnesium. All three dosages significantly increased urinary pH and citrate in a dose-dependent manner. The lowest dosage produced increases sufficient to prevent stone recurrence. Side effects of thiazide therapy were ameliorated by the highest dosage but not by the two lower dosages. Potassium-magnesium citrate at a dosage of 4 tablets per day is adequate to correct thiazide-induced hypokalemia and to increase urinary pH and citrate sufficiently for stone prevention. Higher dosages are probably required for the prevention of magnesium loss and adverse symptoms of thiazide therapy.

Adult↗
PubMed1999

Diffusion- and perfusion-weighted imaging in vasospasm after subarachnoid hemorrhage.

BACKGROUND AND PURPOSE: Better measures of cerebral tissue perfusion and earlier detection of ischemic injury are needed to guide therapy in subarachnoid hemorrhage (SAH) patients with vasospasm. We sought to identify tissue ischemia and early ischemic injury with combined diffusion-weighted (DW) and hemodynamically weighted (HW) MRI in patients with vasospasm after SAH. METHODS: Combined DW and HW imaging was used to study 6 patients with clinical and angiographic vasospasm, 1 patient without clinical signs of vasospasm but with severe angiographic vasospasm, and 1 patient without angiographic spasm. Analysis of the passage of an intravenous contrast bolus through brain was used to construct multislice maps of relative cerebral blood volume (rCBV), relative cerebral blood flow (rCBF), and tissue mean transit time (tMTT). We hypothesize that large HW imaging (HWI) abnormalities would be present in treated patients at the time they develop neurological deficit due to vasospasm without matching DW imaging (DWI) abnormalities. RESULTS: Small, sometimes multiple, ischemic lesions on DWI were seen encircled by a large area of decreased rCBF and increased tMTT in all patients with symptomatic vasospasm. Decreases in rCBV were not prominent. MRI hemodynamic abnormalities occurred in regions supplied by vessels with angiographic vasospasm or in their watershed territories. All patients with neurological deficit showed an area of abnormal tMTT much larger than the area of DWI abnormality. MRI images were normal in the asymptomatic patient with angiographic vasospasm and the patient with normal angiogram and no clinical signs of vasospasm. CONCLUSIONS: We conclude that DW/HW MRI in symptomatic vasospasm can detect widespread changes in tissue hemodynamics that encircle early foci of ischemic injury. With additional study, the technique could become a useful tool in the clinical management of patients with SAH.

Adult↗
PubMed1999

Phase II trial of weekly irinotecan plus cisplatin in advanced esophageal cancer.

PURPOSE: To evaluate the response, toxicity, survival, and quality of life in patients with unresectable or metastatic esophageal cancer treated with weekly irinotecan and cisplatin. PATIENTS AND METHODS: Thirty-five patients with metastatic or unresectable esophageal adenocarcinoma (23 patients) or squamous cell carcinoma (12 patients) were treated. No prior chemotherapy was allowed. The majority of patients had metastatic and bidimensionally measurable disease (34 patients each [97%]). Patients were treated with cisplatin 30 mg/m(2) and irinotecan 65 mg/m(2), repeated weekly for 4 weeks, followed by a 2-week rest period. Treatment was recycled every 6 weeks. Degree of dysphagia relief was monitored, and quality of life was measured prospectively using the European Organization for Research and Treatment of Cancer Quality of Life Questionnaire C-30 and Functional Assessment of Cancer Therapy-General instruments. RESULTS: Thirty-five patients were assessable for response and toxicity. Major objective responses were observed in 20 patients (57%; 95% confidence interval, 41% to 73%), including two complete responses (6%). Similar response rates were observed for adenocarcinoma (12 of 23 patients; 52%) and squamous carcinoma (eight of 12 patients; 66%). The median duration of response was 4.2 months (range, 1 to 8.8+ months). Median actuarial survival was 14.6 months (range, 1 to 15.2+ months). In 20 patients with dysphagia assessable at baseline, 18 (90%) noted either improvement or resolution of dysphagia on chemotherapy. Global quality of life improved in responding patients, primarily because of improvements in pain, emotional state, and relationships with family and friends. Toxicity was relatively mild and included only three patients (9%) with grade 4 neutropenia and four (11%) with grade 3 diarrhea. There were no treatment-related deaths. CONCLUSION: The combination of weekly cisplatin plus irinotecan had significant activity in metastatic esophageal carcinoma and resulted in significant relief of dysphagia. The regimen was well tolerated, with acceptable myelosuppression and rare treatment-related diarrhea. Further evaluation of the combination of weekly irinotecan and cisplatin, including the addition of other agents to this regimen, is indicated.

Adenocarcinoma↗
PubMed1998

Structure of a biological oxygen sensor: a new mechanism for heme-driven signal transduction.

The FixL proteins are biological oxygen sensors that restrict the expression of specific genes to hypoxic conditions. FixL's oxygen-detecting domain is a heme binding region that controls the activity of an attached histidine kinase. The FixL switch is regulated by binding of oxygen and other strong-field ligands. In the absence of bound ligand, the heme domain permits kinase activity. In the presence of bound ligand, this domain turns off kinase activity. Comparison of the structures of two forms of the Bradyrhizobium japonicum FixL heme domain, one in the "on" state without bound ligand and one in the "off" state with bound cyanide, reveals a mechanism of regulation by a heme that is distinct from the classical hemoglobin models. The close structural resemblance of the FixL heme domain to the photoactive yellow protein confirms the existence of a PAS structural motif but reveals the presence of an alternative regulatory gateway.

Amino Acid Sequence↗
PubMed1998

Reconstituted human cardiac KATP channels: functional identity with the native channels from the sarcolemma of human ventricular cells.

ATP-sensitive potassium (KATP) channels in striated myocytes are heteromultimers of KIR6.2, a weak potassium inward rectifier, plus SUR2A, a low-affinity sulfonylurea receptor. We have cloned human KIR6.2 (huKIR6.2) and a huSUR2A that corresponds to the major, full-length splice variant identified by polymerase chain reaction analysis of human cardiac poly A+ mRNA. ATP- and glibenclamide-sensitive K+ channels were produced when both subunits were coexpressed in COSm6 and Chinese hamster ovary cells lacking endogenous KATP channels, but not when huSUR2A or huKIR6.2 were transfected alone. Recombinant channels activated by metabolic inhibition in cell-attached configuration or in inside-out patches with ATP-free internal solution were compared with sarcolemmal KATP channels in human ventricular cells. The single-channel conductance of approximately 80 pS measured at -40 mV in quasi-symmetrical approximately 150 mmol/L K+ solutions, the intraburst kinetics that were dependent on K+ driving force, and the weak inward rectification were indistinguishable for both channels. Similar to the native channels, huSUR2A/huKIR6.2 recombinant channels were inhibited by ATP at quasi-physiological free Mg2+ ( approximately 0. 7 mmol/L) or in the absence of Mg2+, with an apparent IC50 of approximately 20 micromol/L and a pseudo-Hill coefficient of approximately 1. They were "refreshed" by MgATP and stimulated by ADP in the presence of Mg2+ when inhibited by ATP. The huSUR2A/huKIR6.2 channels were stimulated by cromakalim and pinacidil in the presence of ATP and Mg2+ but were insensitive to diazoxide. The results suggest that reconstituted huSUR2A/huKIR6.2 channels represent KATP channels in sarcolemma of human cardiomyocytes and are an adequate experimental model with which to examine structure-function relationships, molecular physiology, and pharmacology of these channels from human heart.

ATP-Binding Cassette Transporters↗
PubMed1998

Imidazole is a sensitive probe of steric hindrance in the distal pockets of oxygen-binding heme proteins.

The FixL heme-based sensor, despite its low affinity for oxygen, is much more reactive than myoglobin toward the large polar ligand imidazole. To determine which features of a myoglobin heme pocket favor binding of imidazole, we have measured binding of this ligand to the FixL heme domain, elephant myoglobin, wild-type sperm whale myoglobin, and sperm whale myoglobins having alanine, valine, threonine, glutamine, leucine, phenylalanine, or tryptophan substitutions of the distal (E7) histidine residue. Except for histidine, the association rate constants dropped more than 3000-fold as the volume of the E7 side chain, at position 64, was expanded from alanine (10(6) M-1 s-1) to phenylalanine (10(3) M-1 s-1). There was inhibition of imidazole binding due to displacement of coordinated water from H64 and H64Q sperm whale myoglobins, where the E7 side chain hydrogen bonds directly to the bound ligand. The imidazole dissociation rate constants varied less dramatically and less consistently with any single factor, though they were measurably decreased by hydrogen bonding to an E7 glutamine or histidine. On the whole, the results for the sperm whale myoglobin E7 substitutions show that the rate constants for imidazole binding are useful and sensitive indicators of steric hindrance and polar interactions in the distal pockets of myoglobins. The combined effects of the glutamine 64 and phenylalanine 29 in elephant myoglobin largely account for its increased imidazole association and dissociation rate constants, respectively, compared to those of sperm whale myoglobin. An unhindered distal pocket not competent to stabilize positive poles is indicated by the large imidazole association (>/=10(4) M-1 s-1) and dissociation (>/=50 s-1) rate constants, parameters that are characteristic of FixL.

Amino Acid Substitution↗
PubMed1998

Mechanisms of autoxidation of the oxygen sensor FixL and Aplysia myoglobin: implications for oxygen-binding heme proteins.

On exposure to oxygen, ferrous heme is thought to autoxidize via three distinct mechanisms: (i) dissociation of protonated superoxide from oxyheme; (ii) reaction between a noncoordinated oxygen molecule and pentacoordinate deoxyheme, and (iii) reaction between a noncoordinated oxygen molecule and an intermediate having water coordinated to the ferrous heme iron. The formation of a hexacoordinate aquomet (H2O.Fe3+) species has been proposed to drive mechanism (iii); consequently, heme proteins with a pentacoordinate met (Fe3+) form might be expected to lack this pathway. We have measured the dependence of autoxidation rate on oxygen concentration for Rhizobium meliloti FixL and Aplysia kurodai myoglobin, which have pentacoordinate met forms. For both proteins, the bell shape of this dependence shows that they autoxidize primarily by mechanism (iii), indicating that a hexacoordinate aquomet species is not required for this mechanism. A novel presentation of the oxygen dependence of autoxidation rates that uses heme saturation, rather than oxygen concentration, more clearly reveals the relative contributions of autoxidation pathways.

Animals↗
PubMed1998

Presence of integrons in isolates of different biotypes of Acinetobacter baumannii from Chilean hospitals.

Class 1 and class 2 integrons were investigated by hybridisation in 100 isolates of multiresistant biotypes of Acinetobacter baumannii from Chilean hospitals. Most isolates of A. baumannii biotype 9, the prevalent biotype, harboured integrons of class 2 (Tn7-like) whereas no integrons were detected in infrequent biotypes. Integron-carrying isolates exhibited broader antibiotic resistance patterns as well as higher resistance levels to various antimicrobials.

Acinetobacter↗
PubMed1998

Inhibition of esterases in the marine gastropod Littorina littorea exposed to cadmium.

Electrophoretic analysis of Littorina littorea esterases indicated the presence of a cathodal migration isoenzyme (EST-C), which was identified in extracts obtained from the gonad-digestive gland complex. A loss of EST-C enzyme activity was observed in individuals exposed to Cd2+. This loss of activity was complete in 86% of individuals dead by the action of Cd2+ and in 68% of those individuals that survived Cd2+ exposure. This difference was statistically significant. In vitro inhibition of esterases by different concentrations of Cd2+ and Cu2+ was studied in individuals not experimentally exposed to heavy metals, to determine whether EST-C inhibition was caused by direct binding of Cd2+ to the esterase molecule or by Cd2+ displacement of a metal ion from essential Cu2+- or Zn2+-containing proteins, which may then be responsible for the inhibition of esterase. Contrary to what happened when individuals were exposed to Cd2+, in the in vitro experiment with Cd2+, inhibition of anodal but not cathodal systems was observed. At the same time, when Cu2+ was used in the in vitro experiment, both anodal and cathodal systems were inhibited. These results suggest that the inhibition of EST-C activity by Cd2+ takes place in vivo and seems not to be due to the direct action of Cd2+ on the molecule, but rather is a process in which transcriptional, posttranscriptional, and other inhibitory processes may be involved.

Animals↗