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Biomedical subjects

R Wade

Publications and source records attributed to R Wade.

At least 19 recordsLinked to original sources
PubMed2000

Isolation of U(VI) reduction-deficient mutants of Shewanella putrefaciens.

A U(VI) reduction-deficient mutant (Urr) screening technique was developed and combined with chemical mutagenesis procedures to identify a Urr mutant of Shewanella putrefaciens strain 200. The Urr mutant lacked the ability to grow anaerobically on U(VI) and NO(2)(-), yet retained the ability to grow anaerobically on eight other compounds as terminal electron acceptor. All 11 members of previously isolated sets of Fe(III) and Mn(IV) reduction-deficient mutants of S. putrefaciens 200 displayed Urr-positive phenotypes with the Urr screen and were capable of anaerobic growth on U(VI). This is the first reported isolation of a respiratory mutant that is unable to grow anaerobically on U(VI) as terminal electron acceptor.

Anaerobiosis↗
PubMed2000

Patients' and providers' perceptions of outpatient treatment termination in a managed behavioral health organization.

OBJECTIVE: A common complaint about managed care is that treatment decisions of patients and providers are frequently altered by concurrent review of ongoing outpatient treatment. The objective of this study was to examine this perception from the perspectives of patients and providers. METHODS: A total of 190 patients and their providers were surveyed about the reason that outpatient treatment was terminated. The sample was randomly drawn from completed outpatient treatment episodes of a large national managed behavioral health organization. RESULTS: In more than three-quarters of the cases, outpatient treatment ended because patients and providers agreed that treatment goals were partially or completely met. Only 5 percent of patients and 3 percent of providers said that treatment ended because the managed care organization denied ongoing treatment. Agreement between patient-provider pairs was generally poor regarding the perceived reason for termination, except when termination was attributed to concurrent review by the managed behavioral health organization. CONCLUSIONS: In this study of a single large managed behavioral health organization, outpatient treatment was most likely to end based on the decisions of patients and providers rather than utilization review decisions.

Adult↗
PubMed1999

Involvement of conserved, acidic residues in the N-terminal domain of troponin C in calcium-dependent regulation.

We have mutated eight conserved, charged amino acid residues in the N-terminal, regulatory domain of troponin C (TnC) so we could investigate their role in troponin-linked Ca2+ regulation of muscle contraction. These residues surround a hydrophobic pocket in the N-terminal domain of TnC which, when Ca2+ binds to regulatory sites in this domain, is exposed and interacts with the inhibitory region of troponin I (TnI). We constructed three double mutants (E53A/E54A, E60A/E61A, and E85A/D86A) and two single mutants (R44A and R81A) of rabbit fast skeletal muscle troponin C (TnC) in which the charged residues were replaced with neutral alanines. All five of these mutants retained TnC's ability to bind TnI in a Ca2+-dependent manner, to neutralize TnI's inhibition of actomyosin S1 ATPase activity, and to form a ternary complex with TnI and troponin T (TnT). Ternary complexes formed with TnC(R44A) or TnC(R81A) regulated actomyosin S1 ATPase activity normally, with TnI-based inhibition in the absence of Ca2+ and TnT-based activation in the presence of Ca2+. TnC(E53A/E54A) and TnC(E85A/D86A) interacted weakly with TnT, as judged by native gel electrophoresis. Ternary complexes formed with these mutants inhibited actomyosin S1 ATPase activity in both the presence and absence of Ca2+, and did not undergo Ca2+-dependent structural changes in TnI which can be detected by limited chymotryptic digestion. TnC(E60A/E61A) interacted normally with TnT. Its ternary complex showed Ca2+-dependent structural changes in TnI, inhibited actomyosin S1 ATPase in the absence of Ca2+, but did not activate ATPase in the presence of Ca2+. This is the first demonstration that selective mutation of TnC can abolish the activating effect of troponin while its inhibitory function is retained. Our results suggest the existence of an elaborate network of protein-protein interactions formed by TnI, TnT, and the N-terminal domain of TnC, all of which are important in the Ca2+-dependent regulation of muscle contraction.

Actomyosin↗
PubMed1999

Ca2+-dependent interaction of the inhibitory region of troponin I with acidic residues in the N-terminal domain of troponin C.

Ca2+ regulation of vertebrate striated muscle contraction is initiated by conformational changes in the N-terminal, regulatory domain of the Ca2+-binding protein troponin C (TnC), altering the interaction of TnC with the other subunits of troponin complex, TnI and TnT. We have investigated the role of acidic amino acid residues in the N-terminal, regulatory domain of TnC in binding to the inhibitory region (residues 96-116) of TnI. We constructed three double mutants of TnC (E53A/E54A, E60A/E61A and E85A/D86A), in which pairs of acidic amino acid residues were replaced by neutral alanines, and measured their affinities for synthetic inhibitory peptides. These peptides had the same amino acid sequence as TnI segments 95-116, 95-119 or 95-124, except that the natural Phe-100 of TnI was replaced by a tryptophan residue. Significant Ca2+-dependent increases in the affinities of the two longer peptides, but not the shortest one, to TnC could be detected by changes in Trp fluorescence. In the presence of Ca2+, all the mutant TnCs showed about the same affinity as wild-type TnC for the inhibitory peptides. In the presence of Mg2+ and EGTA, the N-terminal, regulatory Ca2+-binding sites of TnC are unoccupied. Under these conditions, the affinity of TnC(E85A/D86A) for inhibitory peptides was about half that of wild-type TnC, while the other two mutants had about the same affinity. These results imply a Ca2+-dependent change in the interaction of TnC Glu-85 and/or Asp-86 with residues (117-124) on the C-terminal side of the inhibitory region of TnI. Since Glu-85 and/or Asp-86 of TnC have also been demonstrated to be involved in Ca2+-dependent regulation through interaction with TnT, this region of TnC must be critical for troponin function.

Amino Acid Sequence↗
PubMed1999

RNA oxidation is a prominent feature of vulnerable neurons in Alzheimer's disease.

In this study we used an in situ approach to identify the oxidized nucleosides 8-hydroxydeoxyguanosine (8OHdG) and 8-hydroxyguanosine (8OHG), markers of oxidative damage to DNA and RNA, respectively, in cases of Alzheimer's disease (AD). The goal was to determine whether nuclear and mitochondrial DNA as well as RNA is damaged in AD. Immunoreactivity with monoclonal antibodies 1F7 or 15A3 recognizing both 8OHdG and 8OHG was prominent in the cytoplasm and to a lesser extent in the nucleolus and nuclear envelope in neurons within the hippocampus, subiculum, and entorhinal cortex as well as frontal, temporal, and occipital neocortex in cases of AD, whereas similar structures were immunolabeled only faintly in controls. Relative density measurement showed that there was a significant increase (p < 0.0001) in 8OHdG and 8OHG immunoreactivity with 1F7 in cases of AD (n = 22) as compared with senile (n = 13), presenile (n = 10), or young controls (n = 4). Surprisingly, the oxidized nucleoside was associated predominantly with RNA because immunoreaction was diminished greatly by preincubation in RNase but only slightly by DNase. This is the first evidence of increased RNA oxidation restricted to vulnerable neurons in AD. The subcellular localization of damaged RNA showing cytoplasmic predominance is consistent with the hypothesis that mitochondria may be a major source of reactive oxygen species that cause oxidative damage in AD.

8-Hydroxy-2'-Deoxyguanosine↗
PubMed1998

Comparative kinetic analysis of FLP and cre recombinases: mathematical models for DNA binding and recombination.

The integrase class site specific recombinases FLP from Saccharomyces cerevisiae, and Cre from bacteriophage P1, have been extensively used to direct DNA rearrangements in heterologous organisms. Although their reaction mechanisms have been relatively well characterised, little comparative analysis of the two enzymes has been published. We present a comparative kinetic analysis of FLP and Cre, which identifies important differences. Gel mobility shift assays show that Cre has a higher affinity for its target, loxP (7. 4x10(10) M-1), than FLP for its target, FRT (8.92x10(8) M-1). We show that both recombinases bind the two halves of their target sites cooperatively, and that Cre shows approximately threefold higher cooperativity than FLP. Using a mathematical model describing the sequential binding of recombinase monomers to DNA, we have determined values for the association and dissociation rate constants for FLP and Cre.FLP and Cre also showed different characteristics in in vitro recombination assays. In particular, approximately tenfold more active FLP was required than Cre to optimally recombine a given quantity of excision substrate. FLP was able to reach maximum excision levels approaching 100%, whilst Cre-mediated excision did not exceed 75%. To investigate possible reasons for these differences a mathematical model describing the excision recombination reaction was established. Using measured DNA binding parameters for FLP and Cre in the model, and comparing simulated and experimental recombination data, the values of the remaining unknown parameters were determined. This analysis indicates that the synaptic complex is more stable for Cre than for FLP.

Allosteric Regulation↗
PubMed1998

The sarcoplasmic reticulum Ca2+ pump: inhibition by thapsigargin and enhancement by adenovirus-mediated gene transfer.

The role of the sarcoplasmic reticulum Ca2+ pump in the excitation-contraction coupling of cardiac muscle fibers was evaluated in experiments on SR ATPase inhibition with thapsigargin or, alternatively, on Ca2+ pump enhancement by SR ATPase transgenic expression. We found that thapsigargin, a highly specific and potent inhibitor of the SR ATPase, produces a strong reduction of cytosolic Ca2+ transient and contractile activation in neonatal rat myocytes, in the absence of any other functional effect. On the other hand, Ca2+ pump enhancement by ATPase transgenic expression affects dramatically Ca2+ transient and twitches, resulting in shorter duration and more rapid decay rates. Of particular interest is gene transfer mediated by recombinant adenovirus vectors under control of a cell-specific promoter, resulting in transgenic expression of all myocytes in culture, and no expression in fibroblasts.

Adenoviridae↗
PubMed1998

The motivation to learn: efficacy and relevance of the Oswestry postgraduate orthopaedic training programme.

The efficacy and relevance of medical education has come under increased scrutiny in recent years. The shortening of basic surgical training and the introduction of 'seamless' higher surgical training has placed greater emphasis on the quality of education provided/facilitated by trainers. Additionally, study leave budgets are under increasing pressure from trainees wishing to attend courses or conferences, and regional postgraduate deans who wish to see a proportionally greater amount of training on an 'in-house' basis. Against this background we have reviewed the learning opportunities available on the Oswestry postgraduate programme to see if these opportunities provide adequate motivation to learn for the trainee.

Attitude of Health Personnel↗
PubMed1997

Sublytic terminal complement attack on myotubes decreases the expression of mRNAs encoding muscle-specific proteins.

Activation of inflammatory and cytotoxic complement effectors that include the C5b-9 complex plays an important pathogenic role in myasthenia gravis, an inflammatory autoimmune disease of the muscle. Altered muscle-specific gene expression has been observed in experimental myasthenic rats. In this study, we have examined the effect of sublytic C5b-9 on myotubes differentiated from C2C12 myoblasts, by generating C5b-9 with C7-deficient serum with or without C7. Within 2 h, C7-deficient serum plus C7, compared with C7-deficient serum alone, induced markedly decreased levels of mRNAs encoding alpha-actin, troponin I slow twitch isoform, acetylcholine receptor alpha, and muscle aldolase A, whereas the heat shock protein 83 mRNA level remained constant, by northern analysis. Because the half-life of the acetylcholine receptor alpha was estimated to be > 8 h, the C5b-9 effect was, in part, due to enhanced mRNA decay. Because C5b-9 also induced c-jun mRNA and reduced the myoD mRNA level, a possible inhibition of muscle gene transcription by C5b-9 was examined in myotubes transfected with troponin promoter-luciferase gene constructs. Luciferase activity was reduced to 50% in response to C5b-9 at 2 h. Thus, C5b-9 appears to inhibit the muscle-specific gene expression by stimulating mRNA decay and by decreasing the transcription process. The data also indicate a possible pathogenic role of C5b-9 in immune-mediated inflammatory muscle disorders in which complement activation has been implicated.

Animals↗
PubMed1996

Isolation and characterization of an avian slow myosin heavy chain gene expressed during embryonic skeletal muscle fiber formation.

We have isolated and begun characterization of the quail slow myosin heavy chain (MyHC) 3 gene, the first reported avian slow MyHC gene. Expression of slow MyHC 3 in skeletal muscle is restricted to the embryonic period of development, when the fiber pattern of future fast and slow muscle is established. In embryonic hindlimb development, slow MyHC 3 gene expression coincides with slow muscle fiber formation as distinguished by slow MyHC-specific antibody staining. In addition to expression in embryonic appendicular muscle, slow MyHC 3 is expressed continuously in the atria. Transfection of slow MyHC 3 promoter-reporter constructs into embryonic myoblasts that form slow MyHC-expressing fibers identified two regions regulating expression of this gene in skeletal muscle. The proximal promoter, containing potential muscle-specific regulatory motifs, permits expression of a reporter gene in embryonic slow muscle fibers, while a distal element, located greater than 2600 base pairs upstream, further enhances expression 3-fold. The slow muscle fiber-restricted expression of slow MyHC 3 during embryonic development, and expression of slow MyHC 3 promoter-reporter constructs in embryonic muscle fibers in vitro, makes this gene a useful marker to study the mechanism establishing the slow fiber lineage in the embryo.

Amino Acid Sequence↗
PubMed1995

Voltage-dependent calcium channel beta-subunits in combination with alpha 1 subunits, have a GTPase activating effect to promote the hydrolysis of GTP by G alpha o in rat frontal cortex.

The dihydropyridine-sensitive calcium channel agonist (-)-BayK 8644 was found to produce an enhancement of the intrinsic hydrolysis of GTP by Go in rat frontal cortex membranes. An anti-calcium channel beta-subunit antiserum abolished the (-)-BayK 8644-stimulated hydrolysis of GTP by Go and reduced the dihydropyridine binding capacity of the cortical membranes. A peptide which mimics the beta-subunit binding domain of the calcium channel complex, also attenuated (-)-BayK 8644 activation of GTPase. This study suggests that the calcium channel beta-subunit is the principal component of the channel complex involved in linking dihydropyridine agonist binding to enhanced hydrolysis of GTP by Go. This may be a mechanism by which calcium channels can normally act to limit the duration of a G-protein modulatory signal.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1995

Calcium-induced troponin flexibility revealed by distance distribution measurements between engineered sites.

The contraction of vertebrate striated muscle is regulated by Ca2+ binding to troponin C (TnC). This causes conformational changes which alter the interaction of TnC with the inhibitory protein TnI and the tropomyosin-binding protein TnT. We have used the frequency domain method of fluorescence resonance energy transfer to measure TnT-TnC and TnT-TnI distances and distance distributions, in the presence of Ca2+, Mg2+, or EGTA, in TnC.TnI.TnT complexes. We reconstituted functional, ternary troponin complexes using the following recombinant subunits whose sequences were based on those of rabbit skeletal muscle: wild-type TnC; TnT25, a mutant C-terminal 25-kDa fragment of TnT containing a single Trp212 which was used as the sole donor for fluorescence energy transfer measurements; Trp-less TnI mutants which contained either no Cys or a single Cys at position 9, 96, or 117. Energy acceptor groups were introduced into TnC or TnI by labeling with dansyl aziridine or N-(iodoacetyl)-N'-(1-sulfo-5-naphthyl)ethylenediamine. Our results indicate that the troponin complex is relatively rigid in relaxed muscle, but becomes much more flexible when Ca2+ binds to regulatory sites in TnC. This increased flexibility may be propagated to the whole thin filament, releasing the inhibition of actomyosin ATPase activity and allowing the muscle to contract. This is the first report of distance distribution measurements between troponin subunits.

Adenosine Triphosphatases↗
PubMed1995

Delineation of a slow-twitch-myofiber-specific transcriptional element by using in vivo somatic gene transfer.

Contractile proteins are encoded by multigene families, most of whose members are differentially expressed in fast- versus slow-twitch myofibers. This fiber-type-specific gene regulation occurs by unknown mechanisms and does not occur within cultured myocytes. We have developed a transient, whole-animal assay using somatic gene transfer to study this phenomenon and have identified a fiber-type-specific regulatory element within the promoter region of a slow myofiber-specific gene. A plasmid-borne luciferase reporter gene fused to various muscle-specific contractile gene promoters was differentially expressed when injected into slow- versus fast-twitch rat muscle: the luciferase gene was preferentially expressed in slow muscle when fused to a slow troponin I promoter, and conversely, was preferentially expressed in fast muscle when fused to a fast troponin C promoter. In contrast, the luciferase gene was equally well expressed by both muscle types when fused to a nonfiber-type-specific skeletal actin promoter. Deletion analysis of the troponin I promoter region revealed that a 157-bp enhancer conferred slow-muscle-preferential activity upon a minimal thymidine kinase promoter. Transgenic analysis confirmed the role of this enhancer in restricting gene expression to slow-twitch myofibers. Hence, somatic gene transfer may be used to rapidly define elements that direct myofiber-type-specific gene expression prior to the generation of transgenic mice.

Animals↗
PubMed1995

Developmental regulation of troponin I isoform genes in striated muscles of transgenic mice.

The differentiation and diversification of striated muscle is a complex process involving numerous temporal and spatial alterations in the pattern of contractile protein isoform gene expression. In order to gain insight into the regulation of contractile protein isoform changes during skeletal and cardiac muscle formation, the expression of a transgene comprising a chloramphenicol acetyltransferase (CAT) reporter gene linked with sequences from -4200 to +12 of the human slow skeletal troponin I (TnIs) gene, and all three endogenous mouse troponin I (TnI) isoform genes, was investigated in embryonic, neonatal, and postnatal mice. The -4200 TnIsCAT transgene was properly activated in the limb and trunk skeletal muscle primordia and the early embryonic atrium and ventricle of the heart. Along with the endogenous mouse TnIs gene, expression of the CAT transgene began to segregate into the presumptive slow-twitch myofibers at late fetal stages and expression declined in the neonatal and postnatal heart except for the conductive tissues, in which expression persisted into adulthood. However, expression of the CAT transgene during development did not completely follow the endogenous mouse TnIs gene. The expression of the CAT transgene was aberrantly low in the embryonic cardiac outflow tract and the ventricles of the fetal heart. In addition to its expression in striated muscles, the transgene was expressed aberrantly in the primordial axial skeleton. We conclude that the upstream sequences from the human TnIs gene contain sufficient regulatory information to confer appropriate transgene expression during the early differentiation of skeletal muscles and during the establishment of fiber type upon the maturation of myofibers. However, additional regulatory elements are likely to be required for correct temporal and spatial regulation in the heart and somitic mesoderm during development. In vitro DNA transfection of cultured skeletal and cardiac muscle cells identified a cell type-specific enhancer element within the first intron of the TnIs gene whose absence in the transgene may account for the aberrant expression observed in vivo. In addition, we provide the first evidence that the fast-twitch skeletal muscle isoform of troponin I, TnIf, is transiently expressed during early cardiac muscle development.

Animals↗
PubMed1995

Use of thapsigargin to study Ca2+ homeostasis in cardiac cells.

Several reports have documented that thapsigargin is a potent inhibitor of the SR Ca2+ ATPase isolated from cardiac or skeletal muscle. We have characterized the specificity of this agent in intact rat cardiac myocytes using cells maintained in the whole cell voltage clamp configuration. We have shown that thapsigargin decreases the magnitude of the Ca2+ transient and the twitch by about 80% while it slows the decay rate for these responses. These changes were not accompanied by any alterations in sarcolemmal currents or in the trigger Ca2+ generated by the inward calcium current. Taken together these results reveal that the action of thapsigargin is restricted to the SR Ca2+ ATPase in intact cardiac myocytes. Furthermore, it is demonstrated unambiguously that SR intracellular Ca2+ stores are an absolute requirement for the development of contractile tension in rat heart myocytes. It is shown that thapsigargin is a valuable probe to examine the importance of SR pools of Ca2+ and the role of the Ca2+ ATPase in intact myocytes as well as in genetically altered heart cells.

Animals↗