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Biomedical subjects

R D Mitchell

Publications and source records attributed to R D Mitchell.

At least 37 records · Page 2Linked to original sources
PubMed1993

Solution structure of the cAMP-dependent protein kinase catalytic subunit and its contraction upon binding the protein kinase inhibitor peptide.

Small-angle X-ray scattering and Fourier transform infrared (FTIR) spectroscopy experiments have been completed on the catalytic subunit of the cAMP-dependent protein kinase. Measurements were made both with and without the protein kinase inhibitor peptide, PKI alpha(5-22)amide. Binding of the peptide results in an overall contraction of the structure that is characterized by a decrease of 9% in radius of gyration and about 16% in the maximum linear dimension. Both the secondary structure content of the protein/peptide complex, as determined by FTIR, and the solution structure of this binary complex, as determined by X-ray scattering, agree well with the structural characteristics of this complex as elucidated by the crystal structure [Knighton, D.R., Zheng, J., Ten Eyck, L. F., Ashford, V.A., Xuong, N.H., Taylor, S.S., & Sowadsi, J. M. (1991a) Science 253, 407-414]. Further, the contraction of the structure observed by X-ray scattering upon inhibitor peptide binding is not accompanied by any detectable change in secondary structure content of the kinase. We have modeled the contraction of the kinase upon inhibitor peptide binding as a simple rotation of the large and small lobes seen in the crystal structure such that the cleft between them is closed. For a substrate these changes would then allow catalysis to ensue. The hinge for this movement occurs around a glycine that is one of the protein kinase family consensus amino acids.

Amino Acid Sequence↗
PubMed1993

Molar and incisor changes with Wilson rapid molar distalization.

The purpose of this study was to determine the magnitude and direction of maxillary and mandibular first molar and incisor changes that occur during Class II molar correction with Wilson "rapid molar distalization." Nineteen patients (13 females, 6 males) were selected from the clinic at Loma Linda University Department of Orthodontics. All patients received maxillary bimetric distalizing arches (BDA), and either a mandibular three-dimensional lingual arch, with or without a passive 0.016 x 0.016-inch utility arch, or a traditional edgewise full banded and bonded arch. The arches were activated with open coil spring and Class II intermaxillary elastics. A total of five activations were completed or until visual inspection revealed a Class I molar relation. Prestudy and poststudy lateral cephalometric radiographs were taken with molar bands cemented. Dental changes were determined, with Ricketts superimpositions. The mean change in molar relation measured at the occlusal plane was 4.1 mm. The mean maxillary molar distalization was 2.16 mm, with 7.8 degrees of tip. The rate of maxillary molar movement was 0.56 mm per month. The mean mandibular molar mesial movement was 1.38 mm. The maxillary incisors protruded a mean of 0.3 mm, and extruded a mean of 1.6 mm. Except for the edgewise sample, all mandibular molars moved mesially further than the incisors, reducing the mandibular arch length. The presence of erupted maxillary second molars did not correlate with the rate of maxillary first molar movement, magnitude of movement, or amount of tipping that occurred.

Adolescent↗
PubMed1993

The effect of orally administered thyrotropin-releasing hormone on growth and carcass characteristics of seven- to ten-week-old broilers.

In contrast to some earlier studies with young broiler chickens, several recent studies have shown growth responses to exogenous growth hormone (GH) administration in broiler chickens of advanced age (Vasilatos-Younken et al., 1988; Scanes et al., 1990). It was therefore of interest to determine whether intermittent feeding of thyrotropin-releasing hormone (TRH), a known GH secretagogue, would elevate plasma GH of 7- to 10-wk-old broilers and alter their growth characteristics. Four replicate pens of 15 males and four pens of 15 females 7 to 10 wk of age were given access to feed containing 3.5 mg/kg of TRH for four 2-h periods each 24 h with 4-h periods of feed removal between. Control groups either were fed the basal ration on the same intermittent schedule or had continuous access to it. Growth hormone concentrations, measured 45 to 75 min after the start of TRH feeding periods, were significantly elevated in males on the 1st day of the experiment and after 7, 14, and 21 days when they were 7, 8, 9, and 10 wk of age, respectively. Concentrations in females were elevated on the 1st day of treatment, but not thereafter. Treatment with TRH had no effect on BW or relative gain of males at any time. It significantly increased relative gain of females during the 1st wk, but not thereafter. There were no treatment effects on 10-wk shank length, Pectoralis major weight, Gastrocnemius weight, or liver weight in either sex, but the percentage fat pad of males fed TRH was lower than that of controls fed intermittently.

Administration, Oral↗
PubMed1992

Substrate diversity of the cAMP-dependent protein kinase: regulation based upon multiple binding interactions.

The proposition is forwarded that the cAMP-dependent protein kinase is one of quite a small class of enzymes wherein differential modes of binding of its multiple substrates make an important contribution to the end physiological response. It is postulated that a variety of different substrate affinities may have evolved in order to regulate the order of substrate phosphorylation. The recent elucidation of the protein's three-dimensional structure provides the opening to test this as a new concept of cellular regulation.

Amino Acid Sequence↗
PubMed1991

Expression in Escherichia coli and characterization of the heat-stable inhibitor of the cAMP-dependent protein kinase.

Pure heat-stable inhibitor of the cAMP-dependent protein kinase (PKI) has been isolated in high yield by using a bacterial expression vector constructed to synthesize the complete sequence of the rabbit muscle protein kinase inhibitor, plus an amino-terminal initiator methionine and glycine. Bacterially expressed PKI has an inhibitory activity identical to that of the protein isolated from rabbit skeletal muscle and, by gel filtration and gel electrophoresis, has the same physicochemical characteristics as the native physiological form of PKI. Fourier transformed infrared spectroscopy and CD establish that PKI has unusually large amounts of random coil and turn structures, with significantly smaller amounts of alpha-helix and beta structures.

Animals↗
PubMed1990

Digestion of cardiac and skeletal muscle junctional sarcoplasmic reticulum vesicles with calpain II. Effects on the Ca2+ release channel.

The Ca2+ release channel and ryanodine receptor are activities copurifying with the 400,000-450,000 Da high molecular weight protein of cardiac and skeletal junctional sarcoplasmic reticulum. Calpain II, an endogenous cytosolic protease, was used to selectively degrade the high molecular weight protein in cardiac and skeletal muscle sarcoplasmic reticulum vesicles, and its effects on the activity of the Ca2+ release channel and [3H]ryanodine binding sites were analyzed. Degradation of the high molecular weight protein was associated with appearance of 315,000 and 150,000 Da proteolytic fragments and with a change in the ultrastructure of the "feet," extravesicular projections that protrude from the junctional sarcoplasmic reticulum membrane. The maximal number of [3H]ryanodine binding sites and the affinities of the sites for ryanodine were not remarkably affected by calpain II. Ca2+ release channels recorded from nondegraded cardiac and skeletal membrane vesicle preparations had slope conductances of 85 and 110 pS, respectively, measured with 1 microM cis-Ca2+ and 50 mM trans-Ba2+. Proteolysis did not alter the unitary channel conductances but did increase the percentage of channel open times from 36% to more than 90%. After proteolysis, channel opening remained dependent on micromolar cis-Ca2+, and high concentrations of ryanodine (300 microM) still blocked the channel. Our results suggest that proteolysis of the Ca2+ release channel with calpain II selectively impairs its inactivation, leaving its unitary conductance and the requirement for micromolar Ca2+ intact.

Animals↗
PubMed1989

High molecular weight proteins purified from cardiac junctional sarcoplasmic reticulum vesicles are ryanodine-sensitive calcium channels.

The cardiac high molecular weight proteins/ryanodine receptors were purified to homogeneity from junctional sarcoplasmic reticulum membranes and shown to exhibit large conductance calcium channel activity. High molecular weight proteins were solubilized from junctional sarcoplasmic reticulum in zwitterionic detergent and purified by size-exclusion chromatography followed by sucrose density gradient centrifugation. The purified proteins exhibited an apparent Mr = 400,000-350,000, and bound [3H]ryanodine with a Kd of 4.6 nM and a Bmax of 140-280 pmol/mg protein. High molecular weight proteins demonstrated divalent cation channel activity after incorporation into planar lipid bilayers. Two channel types were identified. Large conductance channels had a slope conductance of 96 +/- 13 pS and a Erev of 42 +/- 9 mV (n = 5); small conductance channels had a slope conductance of 5.5 +/- 1 pS [1.0 microM cis CaCl2; 50 mM trans Ba(OH)2]. Reducing cis calcium from 1 microM to 1 nM reduced the large conductance channel open time from 7 +/- 1% to 0.1% (holding potential, -100 mV). Adding ATP (1 mM) to the cis chamber increased channel open time from 6 +/- 1% to 52 +/- 4% (holding potential, -100 mV); 10 nM ryanodine increased and 100 microM ryanodine decreased percent of open time of the 96 pS channel, without altering unitary channel conductance. The large conductance channel was similar to the calcium release channel detected in native canine cardiac junctional sarcoplasmic reticulum vesicles. Our data suggest that the ryanodine receptor, the calcium-release channel, and the high molecular weight proteins are all identical proteins containing allosteric regulatory sites for calcium, ATP, and ryanodine.

Adenosine Triphosphate↗
PubMed1988

Ca2+ binding effects on protein conformation and protein interactions of canine cardiac calsequestrin.

Calsequestrin is a Ca2+-binding protein located intraluminally in the junctional sarcoplasmic reticulum (SR) of striated muscle. In this study, Ca2+ binding to cardiac calsequestrin was assessed directly by equilibrium dialysis and correlated with effects on protein conformation and calsequestrin's ability to interact with other SR proteins. Cardiac calsequestrin bound 800-900 nmol of Ca2+/mg of protein (35-40 mol of Ca2+/mol of calsequestrin). Associated with Ca2+ binding to cardiac calsequestrin was a loss in protein hydrophobicity, as revealed with use of absorbance difference spectroscopy, fluorescence emission spectroscopy, and photoaffinity labeling with the hydrophobic probe 3-(trifluoromethyl)-3-(m-[125]iodophenyl)diazirine. Ca2+ binding to cardiac calsequestrin also caused a large change in its hydrodynamic character, almost doubling the sedimentation coefficient. We observed that cardiac calsequestrin was very resistant to several proteases after binding Ca2+, consistent with a global effect of Ca2+ on protein conformation. Moreover, Ca2+ binding to cardiac calsequestrin completely prevented its interaction with several calsequestrin-binding proteins, which we identified in cardiac junctional SR vesicles for the first time. The principal calsequestrin-binding protein identified in junctional SR vesicles exhibited an apparent Mr of 26,000 in sodium dodecyl sulfate-polyacrylamide gels. This 26-kDa calsequestrin-binding protein was greatly reduced in free SR vesicles and absent from sarcolemmal vesicles and was different from phospholamban, an SR regulatory protein exhibiting a similar molecular weight. Our results suggest that the specific interaction of calsequestrin with this 26-kDa protein may be regulated by Ca2+ concentration in intact cardiac muscle, when the Ca2+ concentration inside the junctional SR falls to submillimolar levels during coupling of excitation to contraction.

Animals↗
PubMed1985

A prospective comparison of 'conventional' and high carbohydrate/high fibre/low fat diets in adults with established type 1 (insulin-dependent) diabetes.

Diabetic associations throughout the world are recommending high carbohydrate/high fibre/low fat diets for diabetic patients as a means of improving general health and metabolic control. We have previously shown that the efficacy of a 'conventional' low carbohydrate diet can be greatly increased by improving the method of dietary education. To test whether further improvement in metabolic control could be achieved by changing to a high carbohydrate/high fibre/low fat diet we have followed a group of 40 Type 1 (insulin dependent) diabetic adults for up to 19 months. They had all been prescribed a conventional low carbohydrate diet taught by one of three methods (dietician only, practical lunchtime demonstration or videotape). Those taught by dietician only were left as a control group for follow-up (group A). The others were randomized either to continue on their present diet (group B) or to change to a high carbohydrate/high fibre/low fat diet (group C). At final assessment those in groups B and C were more knowledgeable, compliant and better controlled than group A. Those in group C achieved a change from 38% carbohydrate/43% fat/20 g fibre daily to 45% carbohydrate/34% fat/32 g fibre daily. However, metabolic control in group C deteriorated while taking the high carbohydrate/high fibre/low fat diet (haemoglobin A1 went from 9.4 +/- 0.5% to 11.2 +/- 0.5%, p less than 0.01, over 4 months). None of the patients in group C changed their eating habits or those of their families nearly as much as was recommended.(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗
PubMed1984

Spontaneous calcium release from sarcoplasmic reticulum. Assessment of other ionic influences.

A form of spontaneous Ca2+ release from purified light sarcoplasmic reticulum has been recently described (Palade, P., Mitchell, R. D., and Fleischer, S. (1983) J. Biol. Chem. 258, 8098-8107). It is characterized by rapid Ca2+ efflux (1-10 mumol/min X mg protein) which begins only after a delay following preloading and depletion of extravesicular Ca2+. In the present study, the influences of a number of ionic factors modulating the spontaneous calcium release phenomenon are described. The divalent metal ions Mn2+ and Sr2+ and higher external [Mg2+] inhibit release. Mn2+ appears to inhibit from outside, whereas Sr2+ must be taken up to inhibit. Decreasing the phosphate concentration during preloading eliminates spontaneous release, but it can be partially restored with salts of other nonprecipitating anions. No such release was obtained with other Ca2+-precipitating anions, i.e. pyrophosphate, oxalate, and fluoride. The release characteristics are strongly dependent on the monovalent cation present. Spontaneous Ca2+ release is optimal at pH 6.8-7.0 and decreases sharply at higher and lower pH. Ca2+ release can be enhanced by addition, during the lag period, of concentrated salt solutions consisting of more permeant cations than anions, which may indicate that an inside positive membrane potential enhances the rate of calcium release. Spontaneous Ca2+ release can take place in the presence of sucrose, and Ca2+ efflux is not accompanied by a corresponding efflux of preloaded sucrose, demonstrating that the vesicles remain sealed during spontaneous release. The spontaneous Ca2+ release process is distinct from several other forms of Ca2+ release from sarcoplasmic reticulum.

Adenosine Triphosphate↗
PubMed1983

Influence of imaginative teaching of diet on compliance and metabolic control in insulin dependent diabetes.

Dietary non-compliance is an important cause of poor metabolic control in insulin dependent diabetes. Patients are often blamed, but teaching methods may be at fault, so a prospective study was set up to compare the effect of three different teaching methods. After a three month run in, 40 adults with longstanding poorly controlled insulin dependent diabetes (mean haemoglobin A1 13.0%) were allocated at random to three teaching methods: conventional diet sheet instruction (group 1); practical lunchtime demonstrations (group 2); videotape education (group 3). Knowledge was assessed by questionnaires, compliance by seven day food records, and glycaemic control by serial glycosylated haemoglobin measurements. During six months of follow up there was no improvement in knowledge, compliance, or HbA1 in group 1, but in groups 2 and 3 both knowledge and compliance improved. In group 2 HbA1 fell to 10.6 (SD 2.1)% and in group 3 to 9.6 (2.3)%. The change in HbA1 showed an appreciable correlation with dietary compliance as judged by day to day consistency in carbohydrate intake. These findings show that new and interesting educational methods can have a major influence on knowledge, compliance, and metabolic control in insulin dependent diabetes.

Adolescent↗
PubMed1983

Spontaneous calcium release from sarcoplasmic reticulum. Effect of local anesthetics.

Spontaneous calcium release from purified light sarcoplasmic reticulum has been previously described (Palade, P., Mitchell, R. D., and Fleischer, S. (1983) J. Biol. Chem. 258, 8098-8107) and found to be distinct from several other forms of Ca2+ release. Ca2+ release occurs after a lag period following active Ca2+ preloading and depletion of extravesicular Ca2+. In the present study, we find that local anesthetics inhibit spontaneous Ca2+ release, in a time-dependent manner, varying considerably in the preincubation time required to exert maximal effect. At pH 7.0, hydrophilic and mostly charged local anesthetics, such as procaine, procainamide, and N-(2,6-dimethylphenyl carbamoyl methyl)triethyl ammonium bromide, inhibit Ca2+ release only after long preincubations (hours), whereas more hydrophobic local anesthetics are effective after only a short incubation (minutes) with sarcoplasmic reticulum. The more hydrophobic anesthetics take somewhat longer to reach equilibrium, as studied by inhibition of unidirectional Ca2+ efflux, and there is a direct relationship between hydrophobic partition coefficient and half-time to reach equilibrium. Agents known to inhibit permeability pathways for monovalent cations i.e. K+ channel blockers (decamethonium and n-dodecane-1, 12-N,N,N,N',N',N'-hexamethyl-bis-ammonium) or the anion blocker (4,4'-diisothiocyanostilbene-2,2'-disulfonic acid), do not inhibit spontaneous Ca2+ release. Carbonyl cyanide m-fluorophenylhydrazone, a protonophore, and gramicidin D, a monovalent cation ionophore, have no effect on Ca2+ release whether local anesthetics are present or not, while the Ca2+ ionophore A23187 relieves inhibition of Ca2+ release by local anesthetics. Ruthenium red does not inhibit spontaneous Ca2+ release. These findings suggest that the binding site(s) for local anesthetics is located on the inner face of the sarcoplasmic reticulum membrane and that local anesthetics interact directly with a Ca2+ channel rather than with other permeability pathways which might indirectly influence Ca2+ channel gating.

Amines↗
PubMed1983

Biochemical characterization, integrity, and sidedness of purified skeletal muscle triads.

The release of Ca2+ from the terminal cisternae of sarcoplasmic reticulum in muscle fiber triggers muscle contraction. The signal for Ca2+ release is mediated via the triad junction, i.e. the junctional association of terminal cisternae and transverse tubule. Recently, highly purified morphologically intact triads were isolated from rabbit skeletal muscle (Mitchell, R. D., Palade, P., and Fleischer, S. (1983) J. Cell Biol. 96, 1008-1016). In this study, biochemical characterization of two variants of purified triad preparations (Pyrophosphate and Standard) is provided. Terminal cisternae of triads sequester Ca2+ at rates comparable to those of purified heavy sarcoplasmic reticulum which is referable to terminal cisternae (Meissner, G. (1975) Biochim. Biophys. Acta 389, 51-68). The permeability for calcium ions, as reflected by a 2-3-fold stimulation of (Ca2+, Mg2+)-ATPase activity in the presence of the Ca2+ ionophore A23187, and by the Ca2+ leak rate, is comparable in triads and heavy sarcoplasmic reticulum. Several transverse tubule characteristics are present in triads. Four of them, i.e. cholesterol content, ouabain binding, dihydroalprenolol binding (beta-adrenergic receptor), and ouabain-sensitive (Na+, K+)-ATPase activity, are comparably enriched in the Pyrophosphate triads and therefore appear to be quantitative indices of the amount of transverse tubule. Adenylate cyclase and basal ATPase are unreliable in this regard. Methodology for analyzing membrane integrity and sidedness was applied (adenylate cyclase activity) and modified (ouabain-sensitive (Na+, K+)-ATPase activity) to characterize the transverse tubule of the triad. In addition, a new method was developed making use of ouabain binding to study sidedness. These studies show that the transverse tubule is largely sealed and inside out in orientation, i.e. with the cytoplasmic face exposed. This report indicates that the t-tubule and sarcoplasmic reticulum components of the triads possess transport capability and retain permeability barriers for ions. Therefore, the isolated triads appear to be suitable for studying the physiological Ca2+ release process in vitro.

Adenylyl Cyclases↗
PubMed1983

Spontaneous calcium release from sarcoplasmic reticulum. General description and effects of calcium.

A form of spontaneous calcium release from purified sarcoplasmic reticulum isolated from rabbit skeletal muscle is described. The conditions utilized for eliciting spontaneous release rely on preloading the vesicles with calcium in the presence of phosphate. Under the conditions of assay, spontaneous release begins only after a time delay following depletion of calcium ions from the extravesicular space. Release rates as high as 10-20 mumol/mg . min have been observed, but only a portion of the calcium accumulated is released. Released calcium is reaccumulated, and successive spontaneous releases of smaller amounts of calcium are observed under some conditions. Release occurs as a consequence primarily of an increase in unidirectional Ca2+ efflux and, secondarily, a decrease in unidirectional Ca2+ influx. Unidirectional efflux is enhanced by calcium preloading, enhanced by low (0.01-0.1 microM) and reduced by moderate (1-10 microM) extravesicular free calcium levels. Spontaneous Ca2+ release is favored by much lower free calcium concentrations than Ca2+-induced Ca2+ release. The inhibition of unidirectional efflux by calcium appears to involve active calcium uptake. Release is not mediated by a reversal of the calcium pump. The temperature dependence of the release process is steep, comparable with that of energized Ca2+ uptake. This may reflect a process involved in the gating of a hypothetical calcium channel in the sarcoplasmic reticulum membrane.

Animals↗