945 resultados para Motor Function Recovery
Resumo:
The Saccharomyces cerevisiae CDC9 gene encodes a DNA ligase protein that is targeted to both the nucleus and the mitochondria. While nuclear Cdc9p is known to play an essential role in nuclear DNA replication and repair, its role in mitochondrial DNA dynamics has not been defined. It is also unclear whether additional DNA ligase proteins are present in yeast mitochondria. To address these issues, mitochondrial DNA ligase function in S.cerevisiae was analyzed. Biochemical analysis of mitochondrial protein extracts supported the conclusion that Cdc9p was the sole DNA ligase protein present in this organelle. Inactivation of mitochondrial Cdc9p function led to a rapid decline in cellular mitochondrial DNA content in both dividing and stationary yeast cultures. In contrast, there was no apparent defect in mitochondrial DNA dynamics in a yeast strain deficient in Dnl4p (Δdnl4). The Escherichia coli EcoRI endonuclease was targeted to yeast mitochondria. Transient expression of this recombinant EcoRI endonuclease led to the formation of mitochondrial DNA double-strand breaks. While wild-type and Δdnl4 yeast were able to rapidly recover from this mitochondrial DNA damage, clones deficient in mitochondrial Cdc9p were not. These results support the conclusion that yeast rely upon a single DNA ligase, Cdc9p, to carry out mitochondrial DNA replication and recovery from both spontaneous and induced mitochondrial DNA damage.
Resumo:
p75/AIRM-1 is a recently identified inhibitory receptor expressed by natural killer and myeloid cells displaying high homology with CD33. Crosslinking of p75/AIRM-1 or CD33 has been shown to sharply inhibit the in vitro proliferation of both normal myeloid cells and chronic myeloid leukemias. In this study, we analyzed acute myeloid leukemic cells for the expression of p75/AIRM-1. p75/AIRM-1 marked the M5 (11/12) and M4 (2/2) but not the M1, M2, and M3 subtypes according to the French–American–British classification. Cell samples from 12 acute myeloid leukemias were cultured in the presence of granulocyte/macrophage colony-stimulating factor. Addition to these cultures of anti-CD33 antibody resulted in ≈70% inhibition of cell proliferation as assessed by [3H]thymidine uptake or by the recovery of viable cells. Anti-p75/AIRM-1 antibody exerted a strong inhibitory effect only in two cases characterized by a high in vitro proliferation rate. After crosslinking of CD33 (but not of p75/AIRM-1), leukemic cells bound Annexin V and displayed changes in their light-scattering properties and nucleosomal DNA fragmentation, thus providing evidence for the occurrence of apoptotic cell death. Remarkably, when anti-CD33 antibody was used in combination with concentrations of etoposide insufficient to induce apoptosis when used alone, a synergistic effect could be detected in the induction of leukemic cell death. These studies provide the rationale for new therapeutic approaches in myeloid leukemias by using both chemotherapy and apoptosis-inducing mAbs.
Resumo:
The F1F0 ATP synthase is the smallest motor enzyme known. Previous studies had established that the central stalk, made of the γ and ɛ subunits in the F1 part and c subunit ring in the F0 part, rotates relative to a stator composed of α3β3δab2 during ATP hydrolysis and synthesis. How this rotation is regulated has been less clear. Here, we show that the ɛ subunit plays a key role by acting as a switch of this motor. Two different arrangements of the ɛ subunit have been visualized recently. The first has been observed in beef heart mitochondrial F1-ATPase where the C-terminal portion is arranged as a two-α-helix hairpin structure that extends away from the α3β3 region, and toward the position of the c subunit ring in the intact F1F0. The second arrangement was observed in a structure determination of a complex of the γ and ɛ subunits of the Escherichia coli F1-ATPase. In this, the two C-terminal helices are apart and extend along the γ to interact with the α and β subunits in the intact complex. We have been able to trap these two arrangements by cross-linking after introducing appropriate Cys residues in E. coli F1F0, confirming that both conformations of the ɛ subunit exist in the enzyme complex. With the C-terminal domain of ɛ toward the F0, ATP hydrolysis is activated, but the enzyme is fully coupled in both ATP hydrolysis and synthesis. With the C-terminal domain toward the F1 part, ATP hydrolysis is inhibited and yet the enzyme is fully functional in ATP synthesis; i.e., it works in one direction only. These results help explain the inhibitory action of the ɛ subunit in the F1F0 complex and argue for a ratchet function of this subunit.
Resumo:
Stable mammalian cell lines harboring a synthetic bovine opsin gene have been derived from the suspension-adapted HEK293 cell line. The opsin gene is under the control of the immediate-early cytomegalovirus promoter/enhancer in an expression vector that also contains a selectable marker (Neo) governed by a relatively weak promoter. The cell lines expressing the opsin gene at high levels are selected by growth in the presence of high concentrations of the antibiotic geneticin. Under the conditions used for cell growth in suspension, opsin is produced at saturated culture levels of more than 2 mg/liter. After reconstitution with 11-cis-retinal, rhodopsin is purified to homogeneity in a single step by immunoaffinity column chromatography. Rhodopsin thus prepared (> 90% recovery at concentrations of up to 15 microM) is indistinguishable from rhodopsin purified from bovine rod outer segments by the following criteria: (i) UV/Vis absorption spectra in the dark and after photobleaching and the rate of metarhodopsin II decay, (ii) initial rates of transducin activation, and (iii) the rate of phosphorylation by rhodopsin kinase. Although mammalian cell opsin migrates slower than rod outer segment opsin on SDS/polyacrylamide gels, presumably due to a different N-glycosylation pattern, their mobilities after deglycosylation are identical. This method has enabled the preparation of several site-specific mutants of bovine opsin in comparable amounts.
Resumo:
To examine the hypothesis that surface P-selectin-positive (degranulated) platelets are rapidly cleared from the circulation, we developed novel methods for tracking of platelets and measurement of platelet function in vivo. Washed platelets prepared from nonhuman primates (baboons) were labeled with PKH2 (a lipophilic fluorescent dye), thrombin-activated, washed, and reinfused into the same baboons. Three-color whole blood flow cytometry was used to simultaneously (i) identify platelets with a mAb directed against glycoprotein (GP)IIb-IIIa (integrin alpha 11b beta 3), (ii) distinguish infused platelets by their PKH2 fluorescence, and (iii) analyze platelet function with mAbs. Two hours after infusion of autologous thrombin-activated platelets (P-selectin-positive, PKH2-labeled), 95 +/- 1% (mean +/- SEM, n = 5) of the circulating PKH2-labeled platelets had become P-selectin-negative. Compared with platelets not activated with thrombin preinfusion, the recovery of these circulating PKH2-labeled, P-selectin-negative platelets was similar 24 h after infusion and only slightly less 48 h after infusion. The loss of platelet surface P-selectin was fully accounted for by a 67.1 +/- 16.7 ng/ml increase in the plasma concentration of soluble P-selectin. The circulating PKH2-labeled, P-selectin-negative platelets were still able to function in vivo, as determined by their (i) participation in platelet aggregates emerging from a bleeding time wound, (ii) binding to Dacron in an arteriovenous shunt, (iii) binding of mAb PAC1 (directed against the fibrinogen binding site on GPIIb-IIIa), and (iv) generation of procoagulant platelet-derived microparticles. In summary, (i) circulating degranulated platelets rapidly lose surface P-selectin to the plasma pool, but continue to circulate and function; and (ii) we have developed novel three-color whole blood flow cytometric methods for tracking of platelets and measurement of platelet function in vivo.
Resumo:
Cytolytic lymphocytes are of cardinal importance in the recovery from primary viral infections. Both natural killer cells and cytolytic T cells mediate at least part of their effector function by target cell lysis and DNA fragmentation. Two proteins, perforin and granzyme B, contained within the cytoplasmic granules of these cytolytic effector cells have been shown to be directly involved in these processes. A third protein contained within these granules, granzyme A, has so far not been attributed with any biological relevance. Using mice deficient for granzyme A, we show here that granzyme A plays a crucial role in recovery from the natural mouse pathogen, ectromelia, by mechanisms other than cytolytic activity.
Resumo:
While there are many instances of single neurons that can drive rhythmic stimulus-elicited motor programs, such neurons have seldom been found to be necessary for motor program function. In the isolated central nervous system of the marine mollusc Tritonia diomedea, brief stimulation (1 sec) of a peripheral nerve activates an interneuronal central pattern generator that produces the long-lasting (approximately 30-60 sec) motor program underlying the animal's rhythmic escape swim. Here, we identify a single interneuron, DRI (for dorsal ramp interneuron), that (i) conveys the sensory information from this stimulus to the swim central pattern generator, (ii) elicits the swim motor program when driven with intracellular stimulation, and (iii) blocks the depolarizing "ramp" input to the central pattern generator, and consequently the motor program itself, when hyperpolarized during the nerve stimulus. Because most of the sensory information appears to be funneled through this one neuron as it enters the pattern generator, DRI presents a striking example of single neuron control over a complex motor circuit.
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Adrogenesis, development from paternal but not maternal chromosomes, can be induced to occur in some organisms, including vertebrates, but has only been reported to occur naturally in interspecific hybrids of the Sicilian stick insect. Androgenesis has not been described previously in Drosophila. We now report the recovery of androgenetic offspring from Drosophila melanogaster females mutant for a gene that affects an oocyte- and embryo-specific alpha-tubulin. The androgenetic exceptions are X,X diploid females that develop from haploid embryos and express paternal markers on all 4 chromosomes. The exceptional females arise by fusion of haploid cleavage nuclei or failure of newly replicated haploid chromosomes to segregate, rather than fusion of two inseminating sperm. The frequency of androgenetic offspring is greatly enhanced by a partial loss-of-function mutant of the NCD (nonclaret disjunctional) microtubule motor protein, suggesting that wild-type NCD functions is pronuclear fusion. Diploidization of haploid paternal chromosome complements results in complete genetic homozygosity, which could facilitate studies of gene variation and mutational load in populations.
Resumo:
Poly(ADP-ribose) polymerase [PARP; NAD+ ADP-ribosyltransferase; NAD+:poly(adenosine-diphosphate-D-ribosyl)-acceptor ADP-D-ribosyltransferase, EC 2.4.2.30] is a zinc-dependent eukaryotic DNA-binding protein that specifically recognizes DNA strand breaks produced by various genotoxic agents. To study the biological function of this enzyme, we have established stable HeLa cell lines that constitutively produce the 46-kDa DNA-binding domain of human PARP (PARP-DBD), leading to the trans-dominant inhibition of resident PARP activity. As a control, a cell line was constructed, producing a point-mutated version of the DBD, which has no affinity for DNA in vitro. Expression of the PARP-DBD had only a slight effect on undamaged cells but had drastic consequences for cells treated with genotoxic agents. Exposure of cell lines expressing the wild-type (wt) or the mutated PARP-DBD, with low doses of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) resulted in an increase in their doubling time, a G2 + M accumulation, and a marked reduction in cell survival. However, UVC irradiation had no preferential effect on the cell growth or viability of cell lines expressing the PARP-DBD. These PARP-DBD-expressing cells treated with MNNG presented the characteristic nucleosomal DNA ladder, one of the hallmarks of cell death by apoptosis. Moreover, these cells exhibited chromosomal instability as demonstrated by higher frequencies of both spontaneous and MNNG-induced sister chromatid exchanges. Surprisingly, the line producing the mutated DBD had the same behavior as those producing the wt DBD, indicating that the mechanism of action of the dominant-negative mutant involves more than its DNA-binding function. Altogether, these results strongly suggest that PARP is an element of the G2 checkpoint in mammalian cells.
Resumo:
Background: Acetylation and deacetylation at specific lysine (K) residues is mediated by histone acetylases (HATs) and deacetylases (HDACs), respectively. HATs and HDACs act on both histone and non-histone proteins, regulating various processes, including cardiac impulse propagation. Aim of the present work was to establish whether the function of the Ca2+ ATPase SERCA2, one of the major players in Ca2+ reuptake during excitation-contraction coupling in cardiac myocytes (CMs), could be modulated by direct K acetylation. Materials and methods: HL-1 atrial mouse cells (donated by Prof. Claycomb), zebrafish and Streptozotocin-induced diabetic rat CMs were treated with the pan-inhibitor of class I and II HDACs suberanilohydroxamic acid (SAHA) for 1.5 hour. Evaluation of SERCA2 acetylation was analyzed by co-immunoprecipitation. SERCA2 activity was measured on microsomes by pyruvate/NADH coupled reaction assay. SERCA2 mutants were obtained after cloning wild-type and mutated sequences into the pCDNA3 vector and transfected into HEK cells. Ca2+ transients in CMs (loading with Fluo3-AM, field stimulation, 0.5 Hz) and in transfected HEK cells (loading with FLUO-4, caffeine pulse) were recorded. Results: Co-Immunoprecipitation experiments performed on HL-1 cells demonstrated a significant increase in the acetylation of SERCA2 after SAHA-treatment (2.5 µM, n=3). This was associated with an increase in SERCA2 activity in microsomes obtained from HL-1 cells, after SAHA exposure (n=5). Accordingly, SAHA-treatment significantly shortened the Ca2+ reuptake time of adult zebrafish CMs. Further, SAHA 2.5 nM restored to control values the recovery time of Ca2+ transients decay in diabetic rat CMs. HDAC inhibition also improved contraction parameters, such as fraction of shortening, and increased pump activity in microsomes isolated from diabetic CMs (n=4). Notably, the K464, identified by bioinformatic tools as the most probable acetylation site on human SERCA2a, was mutated into Glutamine (Q) or Arginine (R) mimicking acetylation and deacetylation respectively. Measurements of Ca2+ transients in HEK cells revealed that the substitution of K464 with R significantly delayed the transient recovery time, thus indicating that deacetylation has a negative impact on SERCA2 function. Conclusions: Our results indicate that SERCA2 function can be improved by pro-acetylation interventions and that this mechanism of regulation is conserved among species. Therefore, the present work provides the basis to open the search for novel pharmacological tools able to specifically improve SERCA2 activity in diseases where its expression and/or function is impaired, such as diabetic cardiomyopathy.
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This paper presents a new mathematical programming model for the retrofit of heat exchanger networks (HENs), wherein the pressure recovery of process streams is conducted to enhance heat integration. Particularly applied to cryogenic processes, HENs retrofit with combined heat and work integration is mainly aimed at reducing the use of expensive cold services. The proposed multi-stage superstructure allows the increment of the existing heat transfer area, as well as the use of new equipment for both heat exchange and pressure manipulation. The pressure recovery of streams is carried out simultaneously with the HEN design, such that the process conditions (streams pressure and temperature) are variables of optimization. The mathematical model is formulated using generalized disjunctive programming (GDP) and is optimized via mixed-integer nonlinear programming (MINLP), through the minimization of the retrofit total annualized cost, considering the turbine and compressor coupling with a helper motor. Three case studies are performed to assess the accuracy of the developed approach, including a real industrial example related to liquefied natural gas (LNG) production. The results show that the pressure recovery of streams is efficient for energy savings and, consequently, for decreasing the HEN retrofit total cost especially in sub-ambient processes.
Resumo:
BACKGROUND Gastrointestinal (GI) complications often delay recovery after radical cystectomy with urinary diversion. The authors investigated if perioperative administration of a potassium-enriched, chloride-depleted 5% glucose solution (G5K) accelerates recovery of GI function. METHODS This randomized, parallel-group, single-center double-blind trial included 44 consecutive patients undergoing radical cystectomy and pelvic lymph node dissection with urinary diversion. Patients were randomized to receive either a G5K (G5K group) solution or a Ringer's maleate solution (control group). Fluid management aimed for a zero fluid balance. Primary endpoint was time to first defecation. Secondary endpoints were time to normal GI function, need for electrolyte substitution, and renal dysfunction. RESULTS Time to first defecation was not significantly different between groups (G5K group, 93 h [19 to 168 h] and control group, 120 h [43 to 241 h]); estimator of the group difference, -16 (95% CI, -38 to 6); P = 0.173. Return of normal GI function occurred faster in the G5K group than in the control group (median, 138 h [range, 54 to 262 h] vs. 169 h [108 to 318 h]); estimator of the group difference, -38 (95% CI, -74 to -12); P = 0.004. Potassium and magnesium were less frequently substituted in the G5K group (13.6 vs. 54.5% [P = 0.010] and 18.2 vs. 77.3% [P < 0.001]), respectively. The incidence of renal dysfunction (Risk, Injury, Failure, Loss and End-stage kidney disease stage "risk") at discharge was 9.1% in the G5K group and 4.5% in the control group; P = 1.000. CONCLUSIONS Perioperative administration of a G5K did not enhance first defecation, but may accelerate recovery of normal GI function, and reduces potassium and magnesium substitution after radical cystectomy and urinary diversion.
Resumo:
Many authors report changes in the control of the trunk muscles in people with low back pain (LBP). Although there is considerable disagreement regarding the nature of these changes, we have consistently found differential effects on the deep intrinsic and superficial muscles of the lumbopelvic region. Two issues require consideration; first, the potential mechanisms for these changes in control, and secondly, the effect or outcome of changes in control for lumbopelvic function. Recent data indicate that experimentally induced pain may replicate some of the changes identified in people with LBP. While this does not exclude the possibility that changes in control of the trunk muscles may lead to pain, it does argue that, at least in some cases, pain may cause the changes in control. There are many possible mechanisms, including changes in excitability in the motor pathway, changes in the sensory system, and factors associated. with the attention demanding, stressful and fearful aspects of pain. A new hypothesis is presented regarding the outcome from differential effects of pain on the elements of the motor system. Taken together these data argue for strategies of prevention and rehabilitation of LBP (C) 2003 Elsevier Science Ltd. All rights reserved.
Resumo:
The speech characteristics, oromotor function and speech intelligibility of a group of children treated for cerebellar tumour (CT) was investigated perceptually. Assessment of these areas was performed on 11 children treated for CT with dysarthric speech as well as 21 non-neurologically impaired controls matched for age and sex to obtain a comprehensive perceptual profile of their speech and oromotor mechanism. Contributing to the perception of dysarthria were a number of deviant speech dimensions including imprecision of consonants, hoarseness and decreased pitch variation, as well as a reduction in overall speech intelligibility for both sentences and connected speech. Oromotor assessment revealed deficits in lip, tongue and laryngeal function, particularly relating to deficits in timing and coordination of movements. The most salient features of the dysarthria seen in children treated for CT were the mild nature of the speech disorder and clustering of speech deficits in the prosodic, phonatory and articulatory aspects of speech production.
Perceptual and instrumental analysis of laryngeal function after traumatic brain injury in childhood
Resumo:
Objective: To investigate laryngeal function and phonatory disturbance in children with traumatic brain injury (TBI), using both perceptual and instrumental techniques. Design and participants: The performance of 16 individuals with moderate to severe TBI acquired in childhood and 16 nonneurologicatly impaired control subjects was compared on a battery of perceptual (Frenchay Dysarthria Assessment, speech sample analysis) and instrumental (Aerophone II, laryngograph) assessments. Results and conclusions: As a group, the children with TBI demonstrated normal, or only minimally impaired laryngeal function, when compared with the control group, which contrasts with the significant laryngeal impairment noted in adults after TBI. Several reasons for the different findings in relation to laryngeal function in adults and children after TBI are postulated: (1) differing types of injury usually incurred by adults and children may result in a relatively decreased degree of neurologic impairment in these children, (2) differences in recovery potential between adults and children, and (3) the pediatric larynx is still developing, hence it may be better able to compensate for any impairment incurred.