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Biomedical subjects

M Duarte

Publications and source records attributed to M Duarte.

At least 37 records · Page 2Linked to original sources
PubMed2000

Respiratory chain complex I is essential for sexual development in neurospora and binding of iron sulfur clusters are required for enzyme assembly.

We have cloned and disrupted in vivo, by repeat-induced point mutations, the nuclear gene coding for an iron sulfur subunit of complex I from Neurospora crassa, homologue of the mammalian TYKY protein. Analysis of the obtained mutant nuo21.3c revealed that complex I fails to assemble. The peripheral arm of the enzyme is disrupted while its membrane arm accumulates. Furthermore, mutated 21.3c-kD proteins, in which selected cysteine residues were substituted with alanines or serines, were expressed in mutant nuo21. 3c. The phenotypes of these strains regarding the formation of complex I are similar to that of the original mutant, indicating that binding of iron sulfur centers to protein subunits is a prerequisite for complex I assembly. Homozygous crosses of nuo21.3c strain, and of other complex I mutants, are unable to complete sexual development. The crosses are blocked at an early developmental stage, before fusion of the nuclei of opposite mating types. This phenotype can be rescued only by transformation with the intact gene. Our results suggest that this might be due to the compromised capacity of complex I-defective strains in energy production.

Animals↗
PubMed2000

Influence of compression hosiery on physiological responses to standing fatigue in women.

PURPOSE: The purpose of this investigation was to examine the influence of various designs of commercial hosiery, which use graduated compression, on the physiological and performance responses to standing fatigue. METHODS: Twelve healthy women (age = 23.0+/-2.1 yr, height = 165.7+/-5.0 cm, percent body fat = 22.6+/-4.2%, body mass = 60.0+/-8.9 kg) volunteered to participate in this investigation. All subjects completed four identical standing fatigue protocols with different garment conditions each separated by 7 d. The standing fatigue protocol involved a total of 8 h of standing on hard floors during which subjects participated in various tasks and experimental testing procedures. In addition, all activity and dietary profiles of the subjects were carefully controlled 48 h before each experimental session. Before the standing fatigue protocol, subjects completed a battery of tests to establish morning baseline values. Experimental tests included determination of lower leg venous cross-sectional area, blood pressure, heart rate, perceived discomfort ratings, circumferences measurements, total body water, variation in center of pressure during "quiet" standing, vertical jump performance, and specific regional patterns of foot pressures. RESULTS: This investigation demonstrated that commercial hosiery with various forms of graduated compression and construction were effective in mediating a reduction in edema in the ankles and legs while reducing the amount of venous pooling and discomfort in the lower body. Different constructions of garments may mediate these overall effects via different physiological mechanisms related to fluid shifts and muscle tissue damage. CONCLUSION: Wearing various types of graduated compression hose during the day as it relates to women in standing professions may minimize edema and muscle tissue disruption, thereby increasing comfort in the legs.

Adult↗
PubMed2000

The C-terminal domain of rotavirus NSP5 is essential for its multimerization, hyperphosphorylation and interaction with NSP6.

Rotavirus NSP5 is a non-structural phosphoprotein with putative autocatalytic kinase activity, and is present in infected cells as various isoforms having molecular masses of 26, 28 and 30-34 kDa. We have previously shown that NSP5 forms oligomers and interacts with NSP6 in yeast cells. Here we have mapped the domains of NSP5 responsible for these associations. Deletion mutants of the rotavirus YM NSP5 were constructed and assayed for their ability to interact with full-length NSP5 and NSP6 using the yeast two-hybrid assay. The homomultimerization domain was mapped to the 20 C-terminal aa of the protein, which have a predicted alpha-helical structure. A deletion mutant lacking the 10 C-terminal aa (DeltaC10) failed to multimerize both in yeast cells and in an in vitro affinity assay. When transiently expressed in MA104 cells, NSP5 became hyperphosphorylated (30-34 kDa isoforms). In contrast, the DeltaC10 mutant produced forms equivalent to the 26 and 28 kDa species, but was poorly hyperphosphorylated, suggesting that multimerization is important for this proposed activity of the protein. The interaction domain with NSP6 was found to be present in the 35 C-terminal aa of NSP5, overlapping the multimerization domain of the protein, and suggesting that NSP6 might have a regulatory role in the self-association of NSP5. NSP6 was also found to interact with wild-type NSP5, but not with its mutant DeltaC10, in cells transiently transfected with plasmids encoding these proteins, confirming the relevance of the 10 C-terminal aa for the formation of the heterocomplex.

Animals↗
PubMed2000

Rambling and trembling in quiet standing.

The goal of this study was to explore the rambling-trembling decomposition in quiet standing. The center of pressure (COP) and the horizontal ground reaction force (F(hor)) were registered in healthy subjects standing in an upright bipedal posture on a force platform. The COP positions at the instants when F(hor) = 0 were identified (instant equilibrium points, IEP) for the anterior-posterior direction, then the COP time series, were partitioned into its components using 2 different techniques, rambling-trembling decomposition and gravity line decomposition. The two decomposition techniques provided very similar results. An unexpectedly large correlation between the trembling trajectory and the difference between COP and gravity line was found, r = 0.91 (range, 0.83 < r < 0.98). The correlation implies that the GL moves from an IEP to the subsequent IEP along a smooth trajectory that can be predicted by the spline approximation. A substantial negative cross-correlation at a zero time lag was observed between the trembling and the F(hor), -0.90 < r < -0.75. For the rambling trajectory, the coefficients of correlation with F(hor) were low, -0.33 < r < -0.05. The data support the hypothesis that during quiet standing the body sways for two reasons: the migration of the reference point (rambling) and the deviation away from that point (trembling).

Adult↗
PubMed2000

On the inter- and intra-subject variability of the electromyographic signal in isometric contractions.

The objective of the present study was to evaluate the variability of the surface EMG signal of the same muscle in healthy subjects, because of lack of reproducibility of the EMG signal for the same subject and muscle in different trials of maximal isometric voluntary contraction. The results showed an EMG coefficient of variability of 21.61%, indicating that this variability must be considered in experiments with an inappropriate condition for normalization procedures, such as EMG biofeedback in rehabilitation sessions, or normalization procedures by the maximal isometric voluntary contraction.

Adult↗
PubMed1999

Fitts' law in human standing: the effect of scaling.

Fitts' law states that the movement time (MT) of an aiming movement is a linear function of the index of difficulty (ID), where ID = log2(2A/W, A is the movement amplitude, and W is the target width. This law implies that MT should remain unchanged as long as A/W remains constant (i.e. the absence of a scaling effect). The goal of this study was to investigate whether, during upright posture, reciprocal-pointing movements with the center of pressure location follow Fitts' law. Six subjects performed the task with six IDs factorially combined with four As. The results showed that for each A, MT was a linear function of ID. However, the slopes of the linear-regression lines increased with decreases in A. These findings indicate the presence of a scaling effect which violates Fitts' law.

Adult↗
PubMed1999

The 24-kDa iron-sulphur subunit of complex I is required for enzyme activity.

We have cloned the nuclear gene encoding the 24-kDa iron-sulphur subunit of complex I from Neurospora crassa. The gene was inactivated in vivo by repeat-induced point-mutations, and mutant strains lacking the 24-kDa protein were isolated. Mutant nuo24 appears to assemble an almost intact complex I only lacking the 24-kDa subunit. However, we also found reduced levels of the NADH-binding, 51-kDa subunit of the enzyme. Surprisingly, the complex I from the nuo24 strain lacks NADH:ferricyanide reductase activity. In agreement with this, the respiration of intact mitochondria or mitochondrial membranes from the mutant strain is insensitive to rotenone inhibition. These results suggest that the nuo24 complex is not functioning in electron transfer and the 24-kDa protein is absolutely required for complex I activity. This phenotype may explain the findings that the 24-kDa iron-sulphur protein is reduced or absent in human mitochondrial diseases. In addition, selected substitutions of cysteine to alanine residues in the 24-kDa protein suggest that binding of the iron-sulphur centre is a requisite for protein assembly.

Cell Nucleus↗
PubMed1999

Effects of disrupting the 21 kDa subunit of complex I from Neurospora crassa.

We have cloned and inactivated in vivo, by repeat-induced point mutations, the nuclear gene encoding a 21 kDa subunit of complex I from Neurospora crassa. Mitochondria from the nuo21 mutant lack this specific protein but retain other subunits of complex I in approximately normal amounts. In addition, this mutant is able to assemble an almost intact enzyme. The electron transfer activities from NADH to artificial acceptors of mitochondrial membranes from nuo21 differ from those of the wild-type strain, suggesting that the absence of the 21 kDa polypeptide results in conformational changes in complex I. Nevertheless, complex I of nuo21 is able to perform NADH:ubiquinone reductase activity, as judged by the observation that the respiration of mutant mitochondria is sensitive to inhibition by rotenone. We discuss these findings in relation to the involvement of complex I in mitochondrial diseases.

Centrifugation, Density Gradient↗
PubMed1999

Primary structure and characterisation of a 64 kDa NADH dehydrogenase from the inner membrane of Neurospora crassa mitochondria.

A cDNA clone encoding a mitochondrial NADH dehydrogenase from Neurospora crassa was sequenced. The total DNA sequence encompasses 2570 base pairs and contains an open reading frame of 2019 base pairs coding for a precursor polypeptide of 673 amino acid residues. The protein is encoded by a single-copy gene located to the right side of the centromere in linkage group IV of the fungal genome. The N-terminus of the precursor protein has characteristics of a mitochondrial targeting pre-sequence. The protein displays homology with mitochondrial NADH dehydrogenases from yeast. In contrast to these polypeptides, however, analysis of its primary structure revealed that it contains a well-conserved calcium-binding domain. Rabbit antiserum against the protein expressed in an heterologous system recognises a mitochondrial protein of N. crassa with an apparent molecular mass of 64 kDa. Analysis of the fungal mitochondria by swelling, digitonin fractionation and alkaline treatment indicate that the protein is located in the inner membrane of the organelles, possibly facing the matrix side.

Amino Acid Sequence↗
PubMed1999

Characterisation of the last Fe-S cluster-binding subunit of Neurospora crassa complex I.

We have cloned cDNAs encoding the last iron-sulphur protein of complex I from Neurospora crassa. The cDNA sequence contains an open reading frame that codes for a precursor polypeptide of 226 amino acid residues with a molecular mass of 24972 Da. Our results indicate that the mature protein belongs probably to the peripheral arm of complex I and is rather unstable when not assembled into the enzyme. The protein is highly homologous to the PSST subunit of bovine complex I, the most likely candidate to bind iron-sulphur cluster N-2. All the amino acid residues proposed to bind such a cluster are conserved in the fungal protein.

Amino Acid Sequence↗
PubMed1999

Prenatal flutamide treatment eliminates the adult male rat's dependency upon vasopressin when forming social-olfactory memories.

The sexually dimorphic number of cells expressing arginine vasopressin (AVP) in the bed nucleus of the stria terminalis and the density of AVP fibers within the lateral septum appear to be organized by pre- and postnatal androgens. Social recognition behaviors are also sexually dimorphic and AVP-dependent. Whereas AVP antagonists prevent males from recognizing familiar intruders by olfactory investigation of the anal-genital area, they have no effect in females. To test the hypothesis that the male's dependency upon AVP to form social recognition memories begins prior to birth, we compared the effectiveness of an AVP antagonist to block social recognition in control males and females with that seen in male offspring whose mothers were treated prenatally with an androgen antagonist (flutamide). In an initial study we showed that while sexual experience may enhance social recognition in males, virgin males exhibit the ability to recognize conspecifics and are sensitive to the memory blocking actions of AVP antagonists. In a second experiment, pregnant rats were treated daily for the last 10 days of gestation with either flutamide (10 mg) or control vehicle. Within 12 h of birth, male offspring from flutamide litters were injected with either testosterone proprionate (50 microg TP) or vehicle control. AVP-antagonist treatment in adults eliminated the ability of control males to recognize familiar juvenile intruders, but had no effect on males exposed prenatally to flutamide, regardless of whether these males were treated with TP or vehicle on day 1 of life. These data support the hypothesis that the development of the male's dependency upon AVP to express social recognition memories begins with the organizational actions of prenatal androgens.

Androgen Antagonists↗
PubMed1999

Intratumoral injection of bone-marrow derived dendritic cells engineered to produce interleukin-12 induces complete regression of established murine transplantable colon adenocarcinomas.

Stimulation of the antitumor immune response by dendritic cells (DC) is critically dependent on their tightly regulated ability to produce interleukin-12 (IL-12). To enhance this effect artificially, bone marrow (BM)-derived DC were genetically engineered to produce high levels of functional IL-12 by ex vivo infection with a recombinant defective adenovirus (AdCMVIL-12). DC-expressing IL-12 injected into the malignant tissue eradicated 50-100% well established malignant nodules derived from the injection of two murine colon adenocarcinoma cell lines. Successful therapy was dependent on IL-12 transfection and was mediated only by syngeneic, but not allogeneic BM-derived DC, indicating that compatible antigen-presenting molecules were required. The antitumor effect was inhibited by in vivo depletion of CD8+ T cells and completely abrogated by simultaneous depletion with anti-CD4 and anti-CD8 mAbs. Mice which had undergone tumor regression remained immune to a rechallenge with tumor cells, showing the achievement of long-lasting systemic immunity that also was able to reject simultaneously induced concomitant untreated tumors. Tumor regression was associated with a detectable CTL response directed against tumor-specific antigens probably captured by DC artificially released inside tumor nodules. Our results open the possibility of similarly treating the corresponding human malignancies.

Adenocarcinoma↗
PubMed1999

Patterns of center of presure migration during prolonged unconstrained standing.

Prolonged (>30 min) unconstrained standing (PUS) was studied in 10 young healthy subjects. The usual methods of stabilographic analysis assume a random center of pressure (COP) migration. This study was based on the opposite idea and showed that during PUS, specific and consistent patterns of the COP migration can be recognized by a computer algorithm. Three COP migration patterns were found: (a) shifting, a fast displacement of the average position of COP from one region to another; (b) fidgeting, a fast and large displacement and returning of COP to approximately the same position; and (c) drifting, a slow continuous displacement of the average position of COP. A software code was written and default parameter values were chosen for recognizing COP migration patterns. For 30-min PUS the following patterns were identified: Shifting was generally observed every 316 +/- 292 sec in the anterior-posterior (a-p) direction with an average shift amplitude of 17 +/- 15 mm, and every 199 +/- 148 sec in the medial-lateral (m-l) direction with an average shift amplitude of 22 +/- 38 mm. Corresponding time intervals for fidgeting were 59 +/- 15 sec in the a-p direction and 49 +/- 16 sec in the m-l direction. The average drift-to-drift interval was 319 +/- 173 sec in the a-p direction and 529 +/- 333 sec in the m-l direction.

Adult↗
PubMed1999

Instant equilibrium point and its migration in standing tasks: rambling and trembling components of the stabilogram.

A method of decomposing stabilograms into two components, termed rambling, was developed. The rambling component reveals the motion of a moving reference point with respect to which the body's equilibrium is instantly maintained. The trembling component reflects body oscillation around the reference point trajectory. The concepts of instant equilibrium point (IEP) and discrete IEP trajectory are introduced. The rambling trajectory was computed by interpolating the discrete IEP trajectory with cubic spline functions. The trembling trajectory is found as a difference between the approximated rambling trajectory and the COP trajectory. Instant values of the horizontal ground reaction force at a zero time lag. It suggests that trembling is strongly influenced by a restoring force proportional to the magnitude of COP deviation from the rambling trajectory and acts without a time delay. An increment in relative COP position per unit of the restoring force in mm/N, was on average 1.4 +/- 0.4. The contribution of rambling and trembling components in the stabilogram was ascertained. The rambling variability is approximately three times larger than the trembling variability.

Adult↗
PubMed1998

Effect of ranitidine on gastric intramucosal pH in critically ill patients.

OBJECTIVE: To determine whether ranitidine a) increases the values of gastric intramucosal pH (pHi) in critically ill patients, as determined by tonometry; b) reduces the variability of these measurements. DESIGN: Prospective, double blind, randomized, placebo-controlled study. SETTING: General Intensive Care Unit of a teaching hospital. PATIENTS: Twenty-five critically ill, mechanically ventilated patients requiring arterial catheter and nasogastric tube. INTERVENTIONS: Tonometer placement; blind, random administration of intravenous ranitidine (50 mg) or placebo. MEASUREMENTS AND MAIN RESULTS: Tonometer saline PCO2 (PCO2i), arterial blood gases, gastric juice pH and pHi were determined immediately before, and 2, 4, 6 and 8 h after, ranitidine (12 patients) or placebo (13 patients). Ranitidine significantly increased gastric juice pH, but did not affect PCO2i or pHi; pHi was 7.34 +/- 0.14 before ranitidine, and 7.30 +/- 0.12, 7.31 +/- 0.11, 7.31 +/- 0.14 and 7.31 +/- 0.12-2, 4, 6 and 8 h, respectively, after ranitidine administration (p = 0.55). Ranitidine did not modify the coefficients of variation of PCO2i or pHi, either. No significant changes in gastric juice pH, PCO2i or pHi were observed in the placebo group. CONCLUSIONS: In critically ill patients, ranitidine has no effect on pHi values, and does not increase the reproducibility of pHi measurements.

APACHE↗