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A Gutierrez

Publications and source records attributed to A Gutierrez.

At least 37 records · Page 2Linked to original sources
PubMed2001

Kinetics of CO binding and CO photodissociation in Pseudomonas stutzeri cd(1) nitrite reductase: probing the role of extended N-termini in fast structural relaxation upon CO photodissociation.

cd(1) nitrite reductase from Pseudomonas stutzeri is a di-haem- containing enzyme, comprising a c-type haem and a d-type haem. Studies with the highly related cd(1) nitrite reductase of Pseudomonas aeruginosa have established that this enzyme undergoes fast (microsecond) and global structural relaxation upon CO photodissociation from the reduced enzyme. A key difference between the Ps. aeruginosa and Ps. stutzeri enzyme is the absence of a flexible N-terminal extension in the Ps. stutzeri enzyme. In Ps. aeruginosa cd(1) nitrite reductase the N-terminal extension wraps around the second subunit of the homodimer and with Tyr(10) stabilizing a water molecule co-ordinated to the d(1)-haem. Given the intimate association of the N-terminal extension with the d(1)-haem, we hypothesized that the presence of the N-terminal extension likely contributes to the fast structural reorganization seen during photodissociation of CO from the reduced enzyme. In the present study we have investigated the kinetics of CO association and CO photodissociation of Ps. stutzeri cd(1) nitrite reductase (which lacks the N-terminal arm seen in the Ps. aeruginosa enzyme) to probe the role and influence of the N-terminal arm in the fast global structural reorganization seen with Ps. aeruginosa. Surprisingly, we find that Ps. stutzeri cd(1) nitrite reductase also undergoes fast structural reorganization during CO photodissociation. We also show, in stopped-flow experiments, that the kinetics of CO binding and dissociation with reduced Ps. stutzeri cd(1) nitrite reductase are similar to those observed with Ps. aeruginosa enzyme, thus ruling out a major role for the N-terminal flexible arm found in Ps. aeruginosa in the kinetics of these processes. Our data indicate that global structural reorganization following CO photodissociation is an intrinsic property of the haem domains in cd(1) nitrite reductases. The absence of an N-terminal extension, as in the Ps. stutzeri cd(1) nitrite reductase, does not lead to loss of global structural reorganization following CO photodissociation.

Carbon Monoxide↗
PubMed2001

Stopped-flow kinetic studies of flavin reduction in human cytochrome P450 reductase and its component domains.

The reduction by NADPH of the FAD and FMN redox centers in human cytochrome P450 reductase and its component domains has been studied by rapid-mixing, stopped-flow spectroscopy. Reduction of the isolated FAD-domain occurs in three kinetically resolvable steps. The first represents the rapid formation (>500 s(-)(1)) of a charge-transfer species between oxidized FAD and NADPH. This is followed by an isomerization ( approximately 200 s(-)(1)) to a second charge-transfer species, characterized by a more intense absorption in the long-wavelength region. The third step represents hydride transfer from NADPH to FAD and is accompanied by a change in the tryptophan fluorescence of the FAD-domain. Flavin reduction is reversible, and the observed rate of hydride transfer displays a complex dependence on NADPH concentration. Two-electron-reduced FAD-domain is active in electron transfer reactions with the isolated FMN domain through the formation of a weakly associating electron transfer complex. Reduction of the CPR by NADPH occurs without direct spectral evidence for the formation of charge-transfer species, although the presence of such species is inferred indirectly. Transfer of the first hydride ion leads to the accumulation of a blue di-semiquinoid species of the reductase, indicating rapid transfer of one electron to the FMN domain. The di-semiquinoid species decays on transfer of the second hydride ion. A third phase is seen following prolonged incubation with NADPH and is assigned to a series of equilibration reactions between different redox species of the enzyme as the system relaxes to its thermodynamically most stable state. As with the isolated FAD-domain, the first hydride transfer in the reductase shows a complex dependence on NADPH concentration. At high NADPH concentration, the observed rate of hydride transfer is slow (approximately 20 s(-1)), and this attenuated rate is attributed to the reversible formation of an less active complex resulting from the binding of a second molecule of NADPH. The kinetic data are discussed with reference to the potentiometric studies on the enzyme and its component domains presented in the preceding paper in this issue [Munro, A., Noble, M., Robledo, L., Daff, S., and Chapman, S. (2001) Biochemistry 40, 1956-1963].

Animals↗
PubMed2001

The combined effect of isolation and Fasciola hepatica infection on the life history traits of Fossaria cubensis.

Life history traits of Fossaria cubensis were compared between isolated and paired snails after infection with three miracidia of Fasciola hepatica. Four experimental groups were tested: isolated-unexposed, paired-unexposed, isolated-infected, and paired-infected. A repeated-measures ANOVA showed statistically significant interactions among isolation, infection, and age effects for shell size, number of egg masses per snail, number of eggs per snail, and number of viable eggs per snail. Isolated-unexposed snails exhibited the higher values of these variables and those of survival and finite and intrinsic rates of natural increase. Infection stimulated shell growth during the prepatent period, but differences were present only in paired snails since isolation causes a similar effect. Reproduction, in terms of the number of egg masses per snail and the number of eggs per mass per snail, decreases in the presence of parasitic infection, whereas isolation stimulates it. These effects were observed from early stages of infection.

Animals↗
PubMed2001

Evolutionary relationships and biogeography of Biomphalaria (Gastropoda: Planorbidae) with implications regarding its role as host of the human bloodfluke, Schistosoma mansoni.

The wide geographic distribution of Schistosoma mansoni, a digenetic trematode and parasite of humans, is determined by the occurrence of its intermediate hosts, freshwater snails of the genus Biomphalaria (Preston 1910). We present phylogenetic analyses of 23 species of Biomphalaria, 16 Neotropical and seven African, including the most important schistosome hosts, using partial mitochondrial ribosomal 16S and complete nuclear ribosomal ITS1 and ITS2 nucleotide sequences. A dramatically better resolution was obtained by combining the data sets as opposed to analyzing each separately, indicating that there is additive congruent signal in each data set. Neotropical species are basal, and all African species are derived, suggesting an American origin for the genus. We confirm that a proto-Biomphalaria glabrata gave rise to all African species through a trans-Atlantic colonization of Africa. In addition, genetic distances among African species are smaller compared with those among Neotropical species, indicating a more recent origin. There are two species-rich clades, one African with B. glabrata as its base, and the other Neotropical. Within the African clade, a wide-ranging tropical savannah species, B. pfeifferi, and a Nilotic species complex, have both colonized Rift Valley lakes and produced endemic lacustrine forms. Within the Neotropical clade, two newly acquired natural hosts for S. mansoni (B. straminea and B. tenagophila) are not the closest relatives of each other, suggesting two separate acquisition events. Basal to these two species-rich clades are several Neotropical lineages with large genetic distances between them, indicating multiple lineages within the genus. Interesting patterns occur regarding schistosome susceptibility: (1) the most susceptible hosts belong to a single clade, comprising B. glabrata and the African species, (2) several susceptible Neotropical species are sister groups to apparently refractory species, and (3) some basal lineages are susceptible. These patterns suggest the existence of both inherent susceptibility and resistance, but also underscore the ability of S. mansoni to adapt to and acquire previously unsusceptible species as hosts. Biomphalaria schrammi appears to be distantly related to other Biomphalaria as well as to Helisoma, and may represent a separate or intermediate lineage.

Animals↗
PubMed2001

Toluene alters mu-opioid receptor expression in the rat brainstem.

Toluene is an ototoxic organic solvent widely used in industry and could be a cause of sleep apnea. Acute toluene administration in rats induces an increase in the number of neural cells immunostained for mu-opioid receptors in several brainstem nuclei, such as the inferior colliculus, dorsal and lateral periaqueductal gray and dorsal raphe, without changes in the superior colliculus and the interpeduncular and lateral reticular nuclei. These data suggest that mu-opioid receptors could be involved in toluene-induced neurotoxic effects on the physiological regulation of breathing during sleep, and auditive function.

Animals↗
PubMed2000

Trp-676 facilitates nicotinamide coenzyme exchange in the reductive half-reaction of human cytochrome P450 reductase: properties of the soluble W676H and W676A mutant reductases.

The kinetics of flavin reduction in two mutant forms of human cytochrome P450 reductase have been studied by stopped-flow spectroscopy with absorption and fluorescence detection. The mutant enzymes were altered at the position of Trp-676, which, by analogy with the structure of rat CPR, is close to the isoalloxazine ring of the enzyme-bound FAD. We show that mutant CPRs in which Trp-676 has been changed to histidine (W676H) and alanine (W676A) can be reduced by NADPH only to the two-electron level in single mixing stopped-flow experiments. The concentration dependence of the rate of hydride transfer indicates that the second, noncatalytic NADPH-binding site present in wild-type CPR is retained in the mutant enzymes. Detailed studies of W676H CPR indicate that further reduction of the enzyme beyond the two electron level is prevented due to the slow release of NADP(+) from the active site following the first hydride transfer from NADPH, owing to the stability of a reduced enzyme-NADP(+) charge-transfer complex. Reduction to the four-electron level is achieved in a sequential mixing stopped-flow experiment. In this procedure, W676H CPR is reacted first with a stoichiometric amount of NADPH, and then, following a delay of 100 ms, with excess NADPH. The data indicate that occupancy of the noncatalytic coenzyme site also hinders NADP(+) release from reduced enzyme. Fluorescence stopped-flow studies of the W676H and wild-type CPR enzymes reveal that the complex signals associated with reduction of wild-type CPR by NADPH are attributable to changes in the environment of residue W676. From these studies, a model is proposed for nicotinamide binding in wild-type CPR. In this model W676 serves as a trigger to release NADP(+) from the active site following hydride transfer. In the W676H enzyme, the slow release of NADP(+) is a consequence of the combined effects of (i) removing W676 by mutagenesis (thus removing the trigger for displacement) and (ii) the binding of NADPH in the noncatalytic site, thus trapping NADP(+) in the catalytic site.

Alanine↗
PubMed2000

Liquid chromatography/electron capture atmospheric pressure chemical ionization/mass spectrometry: analysis of pentafluorobenzyl derivatives of biomolecules and drugs in the attomole range.

The corona discharge used to generate positive and negative ions under conventional atmospheric pressure chemical ionization conditions also provides a source of gas-phase electrons. This is thought to occur by displacement of electrons from the nitrogen sheath gas. Therefore, suitable analytes can undergo electron capture in the gas phase in a manner similar to that observed for gas chromatography/electron capture negative chemical ionization/mass spectrometry. This technique, which has been named electron capture atmospheric pressure chemical ionization/mass spectrometry, provided an increase in sensitivity of 2 orders of magnitude when compared with conventional atmospheric pressure chemical ionization methodology. It is a simple procedure to tag many biomolecules and drugs with an electron-capturing group such as the pentafluorobenzyl moiety before analysis. Pentafluorobenzyl derivatives have previously been used as electron capturing derivatives because they undergo dissociative electron capture in the gas phase to generate negative ions through the loss of a pentafluorobenzyl radical. A similar process was found to occur under electron capture atmospheric pressure chemical ionization conditions. By monitoring the negative ions that were formed, it was possible to obtain attomole sensitivity for pentafluorobenzyl derivatives of a representative steroid, steroid metabolite, prostaglandin, thromboxane, amino acid, and DNA-adduct.

Chromatography, Liquid↗
PubMed2000

Unusual electrophysiological findings in X-linked dominant Charcot-Marie-Tooth disease.

X-linked Charcot-Marie-Tooth disease (CMTX) is the second most common form of Charcot-Marie-Tooth disease. Variable histopathological and nerve conduction velocity (NCV) results have suggested either a primary demyelinating or axonal polyneuropathy. We identified five individuals across three generations in a family with CMTX associated with a mutation in the gene coding for connexin 32. All individuals were studied by clinical neurological examination, DNA analysis, and nerve conduction studies. The proband (1174/KD) also underwent a sural nerve biopsy. As expected, all the affected males were more clinically affected than the females. All affected males and obligate female carriers exhibited some electrophysiological characteristics of demyelination. However, striking heterogeneity of nerve conduction velocities was seen. This family shows that CMTX is a heterogeneous and distinctly nonuniform demyelinating polyneuropathy, the severity of which varies with sex and age. Such electrophysiological variability is unique among hereditary neuropathies.

Charcot-Marie-Tooth Disease↗
PubMed2000

Plasmodium vivax: polymorphism in the merozoite surface protein 1 gene from wild Colombian isolates.

The Plasmodium vivax merozoite surface protein-1 (PvMSP-1) has been considered a candidate for a malaria vaccine against erythrocytic stages. PvMSP-1 is immunogenic during natural infections and exhibits antigenic polymorphism. The extent of genetic polymorphism in a region between the so-called interspecies conserved blocks (ICBs) 2 and 4 of the PvMSP-1 was analyzed in 20 isolates taken from patients from two different areas in Colombia. Variation is unevenly distributed along this gene segment among the isolates. Comparative analysis of these sequences led to the definition of five sequence types (ST1 to 5). ST1 to ST4 exhibit a variation pattern associated with sequences present in the Salvador or Belem sequences. However, ST5 has clusters of sequence that have not been previously described. The changes found along the five variants confirm the important role of recombinational and/or gene conversion events in generating allelic diversity.

Amino Acid Sequence↗
PubMed2000

The influence of beta-adrenergic activation on noradrenergic alpha1 activation of rabbit afferent arterioles.

The aim of the present investigation was to examine the effect of beta-adrenergic stimulation on smooth muscle calcium concentration ([Ca2+]i) in resting conditions and after administration of norepinephrine (NE) to stimulate alpha-adrenoceptors in isolated rabbit afferent arterioles loaded with the calcium-sensitive fluorescent probe fura-2. [Ca2+]i was evaluated in the proximal and distal parts of the arterioles. NE (4x10(-7) M) increased [Ca2+]i in both these regions. The alpha1-adrenoceptor antagonist prazosin (10(-7) M) totally abolished the NE-induced increase in [Ca2+]i, while the alpha2-adrenoceptor antagonist yohimbine (5x10(-7) M) had no influence on the response to NE. When beta-adrenoceptors were stimulated, using isoproterenol (10(-7) M), the NE-induced increase in [Ca2+]i was significantly lower in both regions. Activation of beta-adrenoceptors with isoproterenol did not affect the [Ca2+]i increase in response to depolarization with K+. Since beta-adrenoceptor stimulation raises the smooth muscle cell levels of cAMP, an adenylate cyclase stimulator, forskolin (10(-5) M) was administered prior to NE application. This maneuver also blunted the increase in [Ca2+]i in both regions. We conclude that the calcium response to NE in the isolated rabbit afferent arteriole is mediated by an alpha1-adrenoceptor. beta-Adrenoceptor stimulation and forskolin blunt the increase in [Ca2+]i induced by NE stimulation, indicating that cAMP counteracts the NE-induced activation of alpha1-adrenoceptors.

Adenylyl Cyclases↗
PubMed2000

Renal NO production and the development of hypertension.

The juxtaglomerular apparatus (JGA) has the very important functions of detecting the fluid flow rate to the distal tubule and thus controlling the glomerular filtration rate (GFR) (tubuloglomerular feedback mechanism [TGF]) and renin release from the afferent arteriole. In studies of the TGF it has been evident that the sensitivity of this mechanism can be reset. Volume expansion will reset it to a low sensitivity leading to a high GFR and urine excretion rate, while dehydration will sensitize the TGF mechanism, giving rise to a low GFR and low urine excretion rate. Furthermore, we have found that in animals that spontaneously develop hypertension there is initially a sensitization of the TGF, leading to a reduced GFR and urine excretion rate, with fluid volume retention in the body and a consequent rise in blood pressure. When the pressure is raised, the TGF characteristics are normalized. In the macula densa (MD) cells in the JGA, there is a large production of NO from neuronal NOS. This production continuously reduces TGF sensitivity and is apparently impaired in animals that spontaneously develop hypertension. When we added an nNOS inhibitor to the drinking water for several weeks while measuring blood pressure, we found an increase in blood pressure after 3-4 weeks of treatment. This effect was abolished by a high salt diet. From these investigations, it also appeared as if nNOS-derived NO inhibited renin release. Experiments have also indicated that NO may resensitize inhibited G-protein coupled purinergic receptors.

Animals↗
PubMed2000

Modulation of activity and substrate specificity by modifying the backbone length of the distant interdomain loop of D-amino acid aminotransferase.

The activity and substrate specificity of D-amino acid aminotransferase (D-AAT) (EC 2.6.1.21) can be rationally modulated by replacing the loop core (P119-R120-P121) with glycine chains of different lengths: 1, 3, or 5 glycines. The mutant enzymes were much more active than the wild-type enzyme in the overall reactions between various amino acids and pyruvate. The presteady-state kinetic analyses of half-reactions revealed that the 5-glycine mutant has the highest affinity (Kd) among all mutant enzymes and the wild-type enzyme towards various amino acids except D-aspartate. The 5-glycine mutant was much more efficient as a catalyst than the wild-type enzyme because the mutant enzyme showed the highest value of specificity constant (kmax/Kd) for all amino acids except D-aspartate and D-glutamate. The kmax/Kd values of the three mutants decreased with decrease in glycine chain length for each amino acid examined. Our findings may provide a new approach to rational modulation of enzymes.

Alanine Transaminase↗
PubMed2000

A bacteriophage lambda-based genetic screen for characterization of the activity and phenotype of the human immunodeficiency virus type 1 protease.

Human immunodeficiency virus type 1 (HIV-1) resistance to antiretroviral drugs is the main cause of patient treatment failure. Despite the problems associated with interpretation of HIV-1 resistance testing, resistance monitoring should help in the rational design of initial or rescue antiretroviral therapies. It has previously been shown that the activity of the HIV-1 protease can be monitored by using a bacteriophage lambda-based genetic assay. This genetic screening system is based on the bacteriophage lambda regulatory circuit in which the viral repressor cI is specifically cleaved to initiate the lysogenic to lytic switch. We have adapted this simple lambda-based genetic assay for the analysis of the activities and phenotypes of different HIV-1 proteases. Lambda phages that encode HIV-1 proteases either from laboratory strains (strain HXB2) or from clinical samples are inhibited in a dose-dependent manner by the HIV-1 protease inhibitors indinavir, ritonavir, saquinavir, and nelfinavir. Distinct susceptibilities to different drugs were also detected among phages that encode HIV-1 proteases carrying different resistance mutations, further demonstrating the specificity of this assay. Differences in proteolytic processing activity can also be directly monitored with this genetic screen system since two phage populations compete in culture with each other until one phage outgrows the other. In summary, we present here a simple, safe, and rapid genetic screening system that may be used to predict the activities and phenotypes of HIV-1 proteases in the course of viral infection and antiretroviral therapy. This assay responds appropriately to well-known HIV-1 protease inhibitors and can be used to search for new protease inhibitors.

Amino Acid Sequence↗
PubMed1999

Amino acid concentration in the interstitium of human skeletal muscle: a microdialysis study.

BACKGROUND: The microdialysis technique has been widely used for in vivo monitoring of the interstitial composition of several tissues. Remarkably high concentrations of taurine and glycerol were reported in a recent human study. As taurine and glycerol are predominantly present in the intracellular space, cellular trauma after probe insertion may have resulted in elevated interstitial concentrations. With the present study we wanted to investigate the impact of the initial trauma on the interstitial concentrations of amino acids and glycerol. METHODS: Microdialysis probes were inserted into the vastus lateralis muscle in eight subjects. Using a slow perfusion rate of 0.3 muL min-1, dialysate samples were collected in five 75-min periods. Simultaneously, plasma samples were taken from a peripheral vein for amino acid determination. RESULTS: During the first collection period, the dialysate concentration for 21 measured amino acids was on average 180% +/- 51% higher than the concentration in plasma water. This difference decreased to 52% +/- 15%, 32% +/- 8%, 37% +/- 8% and 31% +/- 7% during periods 2, 3, 4 and 5 respectively. Carnosine, which is not present in plasma, was detected in high concentrations in the interstitium during the first collection period and decreased subsequently. CONCLUSION: In the post-absorptive phase, the concentrations of most amino acids in muscle interstitium are slightly higher than in venous plasma water. The leakage of intracellular amino acids, because of probe insertion, will initially lead to an overestimation of the actual interstitial concentration of amino acids. Therefore, reliable baseline values of amino acids cannot be obtained until 120-150 min after probe insertion. The dialysate concentration of carnosine may be used as a marker of cellular leakage.

Amino Acids↗
PubMed1999

Live transmission of neonatal echocardiograms from underserved areas: accuracy, patient care, and cost.

OBJECTIVE: Echocardiography is an important tool in the diagnosis and management of critically ill neonates. The authors hypothesized that live telemedicine guidance and interpretation of neonatal echocardiograms from underserved areas would improve management, prevent unnecessary transports, enhance sonographer proficiency, and result in monetary savings. MATERIALS AND METHODS: Using personal computers capable of real-time transmission of echocardiograms over three integrated services digital network (ISDN) telephone lines, pediatric cardiologists interpreted echocardiograms, suggested views to sonographers, and made recommendations to neonatologists 200 miles away. Analyses of accuracy, management, echocardiogram quality, time, and costs were carried out prospectively. RESULTS: Sixty studies were transmitted over 7 months. Indications for echocardiography were suspected congenital heart disease (n = 29), suspected patent ductus arteriosus (PDA) (n = 27), and hemodynamic instability (n = 4). Diagnoses were critical congenital heart disease (n = 4), noncritical heart disease (n = 8), PDA (n = 21), ventricular dysfunction (n = 5), persistent pulmonary hypertension (n = 3), and normal (n = 19). Videotape review confirmed all telemedicine interpretations. The echocardiogram led to immediate change in management in 25 cases (42%), and echocardiogram quality was improved in 53 studies (88%). Time from request to completion of echocardiography was 43+/-30 min. Monetary savings from five avoided transports exceeded all expenses. CONCLUSION: Live transmission of neonatal echocardiograms over three ISDN lines is diagnostic, improves patient care and echocardiography quality, and is cost effective.

Ductus Arteriosus, Patent↗