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Biomedical subjects

M S Moore

Publications and source records attributed to M S Moore.

At least 19 recordsLinked to original sources
PubMed2003

Leptin does not respond to 48 h fat deposition or mobilization in women.

OBJECTIVE: To test the hypothesis that acute responses of plasma leptin concentration to energy balance manipulation are mediated by fat flux. DESIGN: Ten healthy women aged 31-63 y, mass 48-113.5 kg, fat mass 8.5-62.5 kg, were studied for 3 days in a whole-body calorimeter on two occasions. After a control day (D1) during which energy balance was maintained, diet was manipulated to induce fat deposition (FD) or mobilization (FM) of 50 g/day for 2 days (D2 & D3). A difference totalling of 194+/-18.6 g fat was achieved between manipulations without significant effects on carbohydrate or protein balance. Fasting plasma leptin was measured on D2 and D4. RESULTS: After the control day plasma leptin concentration averaged 19.01+/-9.8 ng/ml, and was found to be linearly related to body fat mass. After 2 days manipulation of fat balance, leptin concentrations were 21.4+/-10.3 ng/ml (FD) and 21.2+/-11.3 ng/ml (FM). There was no significant difference between treatments in either control day or postmanipulation leptin concentrations, nor did the treatments induce any differences in glucose or insulin concentration responses. CONCLUSION: Although in states of energy balance leptin concentration is linearly related to fat mass, acute modulation of leptin concentration during energy imbalance is not mediated by fat flux.

Adipose Tissue↗
PubMed2001

Role of importin-beta in coupling Ran to downstream targets in microtubule assembly.

The guanosine triphosphatase Ran stimulates assembly of microtubule asters and spindles in mitotic Xenopus egg extracts. A carboxyl-terminal region of the nuclear-mitotic apparatus protein (NuMA), a nuclear protein required for organizing mitotic spindle poles, mimics Ran's ability to induce asters. This NuMA fragment also specifically interacted with the nuclear transport factor, importin-beta. We show that importin-beta is an inhibitor of microtubule aster assembly in Xenopus egg extracts and that Ran regulates the interaction between importin-beta and NuMA. Importin-beta therefore links NuMA to regulation by Ran. This suggests that similar mechanisms regulate nuclear import during interphase and spindle assembly during mitosis.

Animals↗
PubMed2000

Selective disruption of nuclear import by a functional mutant nuclear transport carrier.

p10/NTF2 is a nuclear transport carrier that mediates the uptake of cytoplasmic RanGDP into the nucleus. We constructed a point mutant of p10, D23A, that exhibited unexpected behavior both in digitonin-permeabilized and microinjected mammalian cells. D23A p10 was markedly more efficient than wild-type (wt) p10 at supporting Ran import, but simultaneously acted as a dominant-negative inhibitor of classical nuclear localization sequence (cNLS)-mediated nuclear import supported by karyopherins (Kaps) alpha and beta1. Binding studies indicated that these two nuclear transport carriers of different classes, p10 and Kap-beta1, compete for identical and/or overlapping binding sites at the nuclear pore complex (NPC) and that D23A p10 has an increased affinity relative to wt p10 and Kap-beta1 for these shared binding sites. Because of this increased affinity, D23A p10 is able to import its own cargo (RanGDP) more efficiently than wt p10, but Kap-beta1 can no longer compete efficiently for shared NPC docking sites, thus the import of cNLS cargo is inhibited. The competition of different nuclear carriers for shared NPC docking sites observed here predicts a dynamic equilibrium between multiple nuclear transport pathways inside the cell that could be easily shifted by a transient modification of one of the carriers.

Active Transport, Cell Nucleus↗
PubMed2000

The nuclear import of RCC1 requires a specific nuclear localization sequence receptor, karyopherin alpha3/Qip.

RCC1 is the only known guanine nucleotide exchange factor for the small GTPase Ran and is normally found inside the nucleus bound to chromatin. In order to analyze in more detail the nuclear import of RCC1, we created a fusion construct in which four IgG binding domains of protein A were fused to the amino terminus of human RCC1 (pA-RCC1). Surprisingly, we found that neither Xenopus ovarian cytosol nor a mixture of recombinant import factors (karyopherin alpha2, karyopherin beta1, Ran, and p10/NTF2) were able to support the import of pA-RCC1 into the nuclei of digitonin-permeabilized cells. Both, in contrast, were capable of supporting the import of a construct containing another classical nuclear localization sequence (NLS), glutathione S-transferase-green fluorescent protein-NLS. Subsequently, we found that only one of the NLS receptors, karyopherin alpha3 (Kapalpha3/Qip), would support significant nuclear import of pA-RCC1 in permeabilized cells, while members of the other two main classes, Kapalpha1 and Kapalpha2, would not. Accordingly, in vitro binding studies revealed that only Kapalpha3 showed significant binding to RCC1 (unlike Kapalpha1 and Kapalpha2) and that this binding was dependent on the basic amino acids present in the RCC1 NLS. In addition to Kapalpha3, we found that the nuclear import of pA-RCC1 also required both karyopherin beta1 and Ran.

Amino Acid Sequence↗
PubMed2000

Dopamine modulates acute responses to cocaine, nicotine and ethanol in Drosophila.

BACKGROUND: Drugs of abuse have a common property in mammals, which is their ability to facilitate the release of the neurotransmitter and neuromodulator dopamine in specific brain regions involved in reward and motivation. This increase in synaptic dopamine levels is believed to act as a positive reinforcer and to mediate some of the acute responses to drugs. The mechanisms by which dopamine regulates acute drug responses and addiction remain unknown. RESULTS: We present evidence that dopamine plays a role in the responses of Drosophila to cocaine, nicotine or ethanol. We used a startle-induced negative geotaxis assay and a locomotor tracking system to measure the effect of psychostimulants on fly behavior. Using these assays, we show that acute responses to cocaine and nicotine are blunted by pharmacologically induced reductions in dopamine levels. Cocaine and nicotine showed a high degree of synergy in their effects, which is consistent with an action through convergent pathways. In addition, we found that dopamine is involved in the acute locomotor-activating effect, but not the sedating effect, of ethanol. CONCLUSIONS: We show that in Drosophila, as in mammals, dopaminergic pathways play a role in modulating specific behavioral responses to cocaine, nicotine or ethanol. We therefore suggest that Drosophila can be used as a genetically tractable model system in which to study the mechanisms underlying behavioral responses to multiple drugs of abuse.

Animals↗
PubMed2000

Interactions between physical activity and diet in the regulation of body weight.

The increasing worldwide prevalence of obesity suggests that there is a chronic positive energy balance. This situation implies poor coupling between energy intake and energy expenditure, but the contribution of each of these factors remains unclear. Epidemiological data suggests that physical activity has a role in the prevention of weight gain, although there is little evidence that it has a beneficial role in weight loss. High-fat diets have also been implicated, evidence from a variety of sources suggests that diets high in fat undermine appetite regulatory mechanisms. There has been much research to investigate the coupling between energy expenditure and energy intake in the short term in an attempt to elucidate some of the mechanisms involved. However, mechanisms regulating appetite are very complex, and it is currently unclear at which points physical activity and diet may have an influence.

Appetite Regulation↗
PubMed2000

The control of gene expression by regulated nuclear transport.

Many proteins show distinct nuclear- and cytoplasmic-localization patterns. For proteins above the diffusion limit of the NPC, this localization is governed by the activity of NLSs and/or NESs contained in the protein. Structural modification of proteins can affect NLS and NES activities. Ligand binding, phosphorylation and proteolysis are each capable of modifying the nucleocytoplasmic distribution of proteins. In the case of transcription factors, control of these structural modifications affects access of the transcription factor to the chromatin. This management of cellular distribution, in turn, regulates gene expression.

Active Transport, Cell Nucleus↗
PubMed1999

Engineered mutants in the switch II loop of Ran define the contribution made by key residues to the interaction with nuclear transport factor 2 (NTF2) and the role of this interaction in nuclear protein import.

Nuclear protein import requires a precisely choreographed series of interactions between nuclear pore components and soluble factors such as importin-beta, Ran, and nuclear transport factor 2 (NTF2). We used the crystal structure of the GDPRan-NTF2 complex to design mutants in the switch II loop of Ran to probe the contribution of Lys71, Phe72 and Arg76 to this interaction. X-ray crystallography showed that the F72Y, F72W and R76E mutations did not introduce major structural changes into the mutant Ran. The GDP-bound form of the switch II mutants showed no detectable binding to NTF2, providing direct evidence that salt bridges involving Lys71 and Arg76 and burying Phe72 are all crucial for the interaction between Ran and NTF2. Nuclear protein accumulation in digitonin-permeabilzed cells was impaired with Ran mutants deficient in NTF2 binding, confirming that the NTF2-Ran interaction is required for efficient transport. We used mutants of the yeast Ran homologue Gsp1p to investigate the effect of the F72Y and R76E mutations in vivo. Although neither mutant was viable when integrated into the genome as a single copy, yeast mildly overexpressing the Gsp1p mutant corresponding Ran F72Y on a centromeric plasmid were viable, confirming that this mutant retained the essential properties of wild-type Ran. However, yeast expressing the Gsp1p mutant corresponding to R76E to comparable levels were not viable, although strains overexpressing the mutant to higher levels using an episomal 2micrometers plasmid were viable, indicating that the R76E mutation may also have interfered with other interactions made by Gsp1p.

Animals↗
PubMed1999

Getting across the nuclear pore complex.

The nuclear pore complex (NPC) connects the cytoplasm and nucleus through the nuclear envelope and serves as the pipeline for moving material between the two compartments. Macromolecules that move through the NPC range in size from the very small (for example, ions and ATP) to the very large (for example, ribonucleoprotein particle complexes). Unlike translocation across other organelle membranes, proteins do not have to be unfolded to be transported through the NPC, and the NPC also routinely transports large, multicomponent substrates in both directions. This review focuses on current understanding of the different mechanisms by which macromolecules move across the NPC.

Animals↗
PubMed1999

Contribution of a sedentary lifestyle and inactivity to the etiology of overweight and obesity: current evidence and research issues.

PURPOSE: The etiology of overweight and obesity is clearly multifactorial, but ultimately it is determined by the long-term balance between energy intake and expenditure. This review will consider the effects on body weight and the risk of obesity of sedentary lifestyles, within the context of dietary habits. METHODS: The data from ecological, cross-sectional, and prospective studies that have assessed physical activity and dietary intake and their relationship to body weight were reviewed. RESULTS: Ecological analyses imply that the increase in the prevalence of obesity is more strongly related to lower levels of physical activity than higher energy intakes. However, there is a paucity of pertinent data from cross-sectional or prospective studies. There is some evidence that both a high proportion of dietary fat and low levels of physical activity may increase the likelihood of weight gain. However, even the most comprehensive studies are unable to account for more than a small proportion of the interindividual variance in weight gain, so it is difficult to usefully assess their relative importance. Furthermore, there are insufficient data that pertain to "sedentary lifestyles" to segregate any putative effect from a protective effect of exercise. All the data in this review is NHLBI Evidence category C. CONCLUSIONS: This review provides clear evidence that low levels of physical activity are associated with an increased risk of weight gain and obesity. On balance, the evidence is suggestive of a causal link, but the experimental designs are too weak is provide conclusive evidence. The potential effect of interactions between diet and activity have largely been ignored. To make progress in this area, a number of key issues need to be resolved with regard to the methodology, study design, and statistical analysis of prospective epidemiological studies. In the meantime, data need to be drawn from other sources, particularly those studies designed to elucidate the mechanism of action of diet and physical activity in the etiology of obesity, to establish rational interventions to guide public health policies.

Cross-Sectional Studies↗
PubMed1999

The porous-coated anatomic total hip prosthesis, inserted without cement. A prospective study with a minimum of ten years of follow-up.

One hundred consecutive primary total hip arthroplasties performed with use of a porous-coated anatomic total hip prosthesis, fixed without cement, in ninety-one patients were followed prospectively for a minimum of ten years. At the time of the most recent follow-up, twenty patients (twenty-three hips) had died and seventy-one patients (seventy-seven hips) were living. The average age of the living patients was sixty-six years (range, thirty-two to ninety-two years), and their average Harris hip score was 84 points (range, 33 to 100 points). Twelve percent (nine) of the seventy-seven hips were found to be associated with pain in the thigh when the patients were specifically questioned by the examiner. Eleven hips were revised during the follow-up period. Only the acetabular component was revised in six hips, only the femoral component was revised in one hip, and both the femoral and the acetabular components were revised in four hips. Of the ten acetabular revisions, one was performed because of acute dissociation of the component and eight, because of a combination of polyethylene wear, osteolysis, and loosening; the tenth acetabular revision consisted of exchange of the liner and curettage and bone-grafting of the osteolytic area. Of the five femoral revisions, two were performed because of loosening and three, because of extensive osteolysis of the proximal aspect of the femur. Including the revised components, twelve acetabular components and five femoral components had radiographic evidence of aseptic loosening. Acetabular osteolysis occurred in seventeen hips. Femoral osteolysis occurred in thirty-nine hips: in the proximal aspect of thirty-one hips, in the distal aspect of four, and in both the proximal and the distal aspect of four. The durability of the femoral fixation documented in this study is especially encouraging in view of the fact that this was our initial experience with devices fixed without cement and that a so-called first-generation femoral component was used. However, the study also demonstrated that not all acetabular components fixed without cement function well over the long term and that specific design considerations (adequate initial fixation, congruency between the liner and the shell, an optimum shell-liner capturing mechanism, and a smaller femoral head) are warranted.

Aged↗
PubMed1998

Ran-dependent signal-mediated nuclear import does not require GTP hydrolysis by Ran.

Nuclear import of classical nuclear localization sequence-containing proteins involves the assembly of an import complex at the cytoplasmic face of the nuclear pore complex (NPC) followed by movement of this complex through the NPC and release of the import substrate into the nuclear interior. This process has historically been thought to require nucleotide hydrolysis as a source of energy. We found, using hydrolysis-resistant GTP analogs and a mutant Ran unable to hydrolyze GTP, that transport of classical nuclear localization sequence containing substrate through the NPC and release of the substrate into the nucleus did not require hydrolysis of GTP by Ran. In fact, for movement of this type of import substrate into the nuclear interior we did not observe a requirement for hydrolysis of any nucleotide triphosphate. We did, however, find that a pool of free GTP (or its structural equivalent) must be added, probably because the GDP Ran that is added must be converted to GTP Ran during the import process. We found that a requirement for GTP hydrolysis can be restored to an import mixture consisting of recombinant import factors by the addition of RCC1, the Ran guanine nucleotide exchange factor.

Biological Transport↗
PubMed1998

Ethanol intoxication in Drosophila: Genetic and pharmacological evidence for regulation by the cAMP signaling pathway.

Upon exposure to ethanol, Drosophila display behaviors that are similar to ethanol intoxication in rodents and humans. Using an inebriometer to measure ethanol-induced loss of postural control, we identified cheapdate, a mutant with enhanced sensitivity to ethanol. Genetic and molecular analyses revealed that cheapdate is an allele of the memory mutant amnesiac. amnesiac has been postulated to encode a neuropeptide that activates the cAMP pathway. Consistent with this, we find that enhanced ethanol sensitivity of cheapdate can be reversed by treatment with agents that increase cAMP levels or PKA activity. Conversely, genetic or pharmacological reduction in PKA activity results in increased sensitivity to ethanol. Taken together, our results provide functional evidence for the involvement of the cAMP signal transduction pathway in the behavioral response to intoxicating levels of ethanol.

Adenylyl Cyclases↗
PubMed1998

Viral protein R regulates nuclear import of the HIV-1 pre-integration complex.

Replication of human immunodeficiency virus type 1 (HIV-1) in non-dividing cells critically depends on import of the viral pre-integration complex into the nucleus. Genetic evidence suggests that viral protein R (Vpr) and matrix antigen (MA) are directly involved in the import process. An in vitro assay that reconstitutes nuclear import of HIV-1 pre-integration complexes in digitonin-permeabilized cells was used to demonstrate that Vpr is the key regulator of the viral nuclear import process. Mutant HIV-1 pre-integration complexes that lack Vpr failed to be imported in vitro, whereas mutants that lack a functional MA nuclear localization sequence (NLS) were only partially defective. Strikingly, the import defect of the Vpr- mutant was rescued when recombinant Vpr was re-added. In addition, import of Vpr- virus was rescued by adding the cytosol of HeLa cells, where HIV-1 replication had been shown to be Vpr-independent. In a solution binding assay, Vpr associated with karyopherin alpha, a cellular receptor for NLSs. This association increased the affinity of karyopherin alpha for basic-type NLSs, including that of MA, thus explaining the positive effect of Vpr on nuclear import of the HIV-1 pre-integration complex and BSA-NLS conjugates. These results identify the biochemical mechanism of Vpr function in transport of the viral pre-integration complex to, and across, the nuclear membrane.

Biological Transport↗
PubMed1998

The nuclear import factor p10 regulates the functional size of the nuclear pore complex during oogenesis.

Previtellogenic, stage-1 Xenopus oocytes produce mainly 5S and tRNA, whereas vitellogenic oocytes, stages 2-6, synthesize predominantly 18S and 28S rRNA. Using nucleoplasmin-coated gold as a transport substrate, it was determined that the shift in synthesis from small to large RNAs during oogenesis is accompanied by an increase in both the rates of signal-mediated nuclear import and the functional size of nuclear pores. It was observed that, despite the reduction in transport capacity, gold still accumulated at the cytoplasmic surface of the pores in stage-1 oocytes. This suggested that transport in these cells is limited by translocation factors, rather than by cytoplasmic binding factors. Analysis of extracts prepared from stage-1 and vitellogenic oocytes revealed that the transport factor p10 is more abundant in stage-1 cells. Microinjection of purified p10 into stage-2 oocytes reduced the nuclear import of large gold particles to the level observed in stage-1 cells. It is concluded that p10 can modulate transport through the pores by regulating the functional size of the central transporter element.

Animals↗
PubMed1998

The role of dual energy x-ray absorptiometry in aiding the diagnosis of pediatric osteogenesis imperfecta.

The role of dual energy x-ray absorptiometry (DEXA) in the evaluation of the pediatric patient with multiple fractures has not been well established. We retrospectively examined the medical records of 45 patients who had presented to our institution with multiple fractures of unknown cause, who were not known to have osteogenesis imperfecta, and who had obtained DEXA as part of their evaluation. Of these, 26 patients had sufficient clinical data for inclusion in this study. Patients underwent DEXA of the anteroposterior spine and whole body. A z score was calculated to normalize the DEXA values for age. The diagnosis of osteogenesis imperfecta was correlated with the outcome of each DEXA scan to assess the validity of DEXA as a diagnostic tool. The DEXA of the anteroposterior spine had the highest sensitivity at 91.7%, while DEXA of the whole body had the highest specificity at 100.0%. Decreased bone mineral density may be associated with osteogenesis imperfecta, and DEXA is helpful in detecting low bone mineral density that may be missed on plain radiographs of children with milder forms of osteogenesis imperfecta.

Absorptiometry, Photon↗
PubMed1997

A T42A Ran mutation: differential interactions with effectors and regulators, and defect in nuclear protein import.

Ran, the small, predominantly nuclear GTPase, has been implicated in the regulation of a variety of cellular processes including cell cycle progression, nuclear-cytoplasmic trafficking of RNA and protein, nuclear structure, and DNA synthesis. It is not known whether Ran functions directly in each process or whether many of its roles may be secondary to a direct role in only one, for example, nuclear protein import. To identify biochemical links between Ran and its functional target(s), we have generated and examined the properties of a putative Ran effector mutation, T42A-Ran. T42A-Ran binds guanine nucleotides as well as wild-type Ran and responds as well as wild-type Ran to GTP or GDP exchange stimulated by the Ran-specific guanine nucleotide exchange factor, RCC1. T42A-Ran.GDP also retains the ability to bind p10/NTF2, a component of the nuclear import pathway. In contrast to wild-type Ran, T42A-Ran.GTP binds very weakly or not detectably to three proposed Ran effectors, Ran-binding protein 1 (RanBP1), Ran-binding protein 2 (RanBP2, a nucleoporin), and karyopherin beta (a component of the nuclear protein import pathway), and is not stimulated to hydrolyze bound GTP by Ran GTPase-activating protein, RanGAP1. Also in contrast to wild-type Ran, T42A-Ran does not stimulate nuclear protein import in a digitonin permeabilized cell assay and also inhibits wild-type Ran function in this system. However, the T42A mutation does not block the docking of karyophilic substrates at the nuclear pore. These properties of T42A-Ran are consistent with its classification as an effector mutant and define the exposed region of Ran containing the mutation as a probable effector loop.

Amino Acid Substitution↗