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J R Patel

Publications and source records attributed to J R Patel.

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

Polyamines decrease Ca(2+) sensitivity of tension and increase rates of activation in skinned cardiac myocytes.

Owing in part to their interactions with membrane proteins, polyamines (e.g., spermine, spermidine, and putrescine) have been identified as potential modulators of membrane excitability and Ca(2+) homeostasis in cardiac myocytes. To investigate whether polyamines also affect cardiac myofilament proteins, we assessed the effects of polyamines on contractility using rat myocytes and trabeculae that had been permeabilized with Triton X-100. Spermine, spermidine, and putrescine reversibly increased the [Ca(2+)] required for half-maximal tension (i.e., right-shifted tension pCa curves), with the following order of efficacy: spermine (+4) > spermidine (+3) > putrescine (+2). However, synthetic analogs that differed from spermine in charge distribution were not as effective as spermine in altering isometric tension. None of the polyamines had a significant effect on maximal tension, except at high concentrations. After flash photolysis of DM-Nitrophen (a caged Ca(2+) chelator), spermine accelerated the rate of tension development at low and intermediate but not high [Ca(2+)]. These results indicate that polyamines, especially spermine, interact with myofilament proteins to reduce apparent Ca(2+) binding affinity and speed cross-bridge cycling kinetics at submaximal [Ca(2+)].

Adenosine Triphosphate↗
PubMed1999

Impaired cardiomyocyte relaxation and diastolic function in transgenic mice expressing slow skeletal troponin I in the heart.

1. To assess the specific functions of the cardiac isoform of troponin I (cTnI), we produced transgenic mice that expressed slow skeletal troponin I (ssTnI) specifically in cardiomyocytes. Cardiomyocytes from these mice displayed quantitative replacement of cTnI with transgene-encoded ssTnI. 2. The ssTnI transgenic mice were viable and fertile and did not display increased mortality or detectable cardiovascular histopathology. They exhibited normal ventricular weights and heart rates. 3. Permeabilized transgenic cardiomyocytes demonstrated an increased Ca2+ sensitivity of tension and a lack of contractile responsiveness to cAMP-dependent protein kinase (PKA). Isolated cardiomyocytes from transgenic mice had normal velocities of unloaded shortening but unlike wild-type controls exhibited no enhancement of the velocity of shortening in response to treatment with isoprenaline. Transgenic cardiomyocytes exhibited greater extents of shortening than non-transgenic cardiomyocytes at baseline and after treatment with isoprenaline. 4. The rates of rise of intracellular [Ca2+] and the peak amplitudes of the intracellular [Ca2+] transients were similar in transgenic and wild-type myocytes. However, the half-time of intracellular [Ca2+] decay was significantly greater in the transgenic myocytes. This change in decay of intracellular [Ca2+] was correlated with an increase in the re-lengthening time of the transgenic cells. 5. These changes in cardiomyocyte function in vitro were manifested in vivo as impaired diastolic function both at baseline and after stimulation with isoprenaline. 6. Thus, cTnI has important roles in regulating the Ca2+ sensitivity of cardiac myofibrils and controlling cardiomyocyte relaxation and cardiac diastolic function. cTnI is also required for the normal responsiveness of cardiomyocytes to beta-adrenergic receptor stimulation.

Animals↗
PubMed1999

Aging-dependent depression in the kinetics of force development in rat skinned myocardium.

Normal aging of the rodent heart results in prominent prolongation of the twitch. We tested the hypothesis that increased expression of beta-myosin heavy chain (MHC), as occurs in the normal aging process in the rodent heart, contributes to the prolongation of the twitch by depressing the kinetics of cross-bridge interaction. Using 3-, 9-, 21-, and 33-mo-old male Fischer 344 x Brown Norway F1 hybrid rats, we examined both the rate of tension development (kCa) and unloaded shortening velocity in chemically skinned myocardium. Although kCa in all four age groups was dependent on the level of Ca2+ activation, both submaximal and maximal kCa were significantly slower in 9-, 21-, and 33-mo-old rats relative to 3-mo-old rats. Furthermore, unloaded shortening velocity was significantly reduced in 9-, 21-, and 33-mo-old rats compared with 3-mo-old rats. Collectively, these data strongly suggest that the aging-related increase in beta-MHC expression results in a progressive slowing of cross-bridge interaction kinetics in skinned myocardium, which most likely contributes to the overall aging-dependent reduction in myocardial functional capacity.

Acetates↗
PubMed1999

Cardiac myosin heavy chains lacking the light chain binding domain cause hypertrophic cardiomyopathy in mice.

Myosin is a chemomechanical motor that converts chemical energy into the mechanical work of muscle contraction. More than 40 missense mutations in the cardiac myosin heavy chain (MHC) gene and several mutations in the two myosin light chains cause a dominantly inherited heart disease called familial hypertrophic cardiomyopathy. Very little is known about the biochemical defects in these alleles and how the mutations lead to disease. Because removal of the light chain binding domain in the lever arm of MHC should alter myosin's force transmission but not its catalytic function, we tested the hypothesis that such a mutant MHC would act as a dominant mutation in cardiac muscle. Hearts from transgenic mice expressing this mutant myosin are asymmetrically hypertrophied, with increases in mass primarily restricted to the cardiac anterior wall. Histological examination demonstrates marked cellular hypertrophy, myocyte disorganization, small vessel coronary disease, and severe valvular pathology that included thickening and plaque formation. Skinned myocytes and multicellular preparations from transgenic hearts exhibited decreased Ca2+ sensitivity of tension and decreased relaxation rates after flash photolysis of diazo 2. These experiments demonstrate that alterations in myosin force transmission are sufficient to trigger the development of hypertrophic cardiomyopathy.

Animals↗
PubMed1998

Role of myosin heavy chain composition in kinetics of force development and relaxation in rat myocardium.

1. The effects of ventricular myosin heavy chain (MHC) composition on the kinetics of activation and relaxation were examined in both chemically skinned and intact myocardial preparations from adult rats. Thyroid deficiency was induced to alter ventricular MHC isoform expression from approximately 80% alpha-MHC/20% beta-MHC in euthyroid rats to 100% beta-MHC, without altering the expression of thin-filament-associated regulatory proteins. 2. In single skinned myocytes, increased expression of beta-MHC did not significantly affect either maximal Ca2+-activated tension (P0) or the Ca2+ sensitivity of tension (pCa50). However, unloaded shortening velocity (V0) decreased by 80% due to increased beta-MHC expression. 3. The kinetics of activation and relaxation were examined in skinned multicellular preparations using the caged Ca2+ compound DM-nitrophen and caged Ca2+ chelator diazo-2, respectively. Myocardium expressing 100% beta-MHC exhibited apparent rates of submaximal and maximal tension development (kCa) that were 60% lower than in control myocardium, and a 2-fold increase in the half-time for relaxation from steady-state submaximal force. 4. The time courses of cell shortening and intracellular Ca2+ transients were assessed in living, electrically paced myocytes, both with and without beta-adrenergic stimulation (70 nM isoproterenol (isoprenaline)). Thyroid deficiency had no affect on either the extent of myocyte shortening or the resting or peak fura-2 fluorescence ratios. However, induction of beta-MHC expression by thyroid deficiency was associated with increased half-times for myocyte shortening and relengthening and increased half-time for the decay of the fura-2 fluorescence ratio. Qualitatively similar results were obtained in both the absence and the presence of beta-adrenergic stimulation although the beta-agonist accelerated the kinetics of the twitch and the Ca2+ transient. 5. Collectively, these data provide evidence that increased beta-MHC expression contributes significantly to the observed depression of contractile function in thyroid deficient myocardium by slowing the rates of both force development and force relaxation.

Adrenergic beta-Agonists↗
PubMed1998

Phosphorylation of myosin regulatory light chain eliminates force-dependent changes in relaxation rates in skeletal muscle.

The rate of relaxation from steady-state force in rabbit psoas fiber bundles was examined before and after phosphorylation of myosin regulatory light chain (RLC). Relaxation was initiated using diazo-2, a photolabile Ca2+ chelator that has low Ca2+ binding affinity (K(Ca) = 4.5 x 10(5) M(-1)) before photolysis and high affinity (K(Ca) = 1.3 x 10(7) M(-1)) after photolysis. Before phosphorylating RLC, the half-times for relaxation initiated from 0.27 +/- 0.02, 0.51 +/- 0.03, and 0.61 +/- 0.03 Po were 90 +/- 6, 140 +/- 6, and 182 +/- 9 ms, respectively. After phosphorylation of RLC, the half-times for relaxation from 0.36 +/- 0.03 Po, 0.59 +/- 0.03 Po, and 0.65 +/- 0.02 Po were 197 +/- 35 ms, 184 +/- 35 ms, and 179 +/- 22 ms. This slowing of relaxation rates from steady-state forces less than 0.50 Po was also observed when bundles of fibers were bathed with N-ethylmaleimide-modified myosin S-1, a strongly binding cross-bridge derivative of S1. These results suggest that phosphorylation of RLC slows relaxation, most likely by slowing the apparent rate of transition of cross-bridges from strongly bound (force-generating) to weakly bound (non-force-generating) states, and reduces or eliminates Ca2+ and cross-bridge activation-dependent changes in relaxation rates.

Animals↗
PubMed1997

Ca2+ binding to troponin C in skinned skeletal muscle fibers assessed with caged Ca2+ and a Ca2+ fluorophore. Invariance of Ca2+ binding as a function of sarcomere length.

Ca2+ sensitivity of tension varies with sarcomere length in both skeletal and cardiac muscles. One possible explanation for this effect is that the Ca2+ affinity of the regulatory protein troponin C decreases when sarcomere length is reduced. To examine length dependence of Ca2+ binding to troponin C in skeletal muscle, we developed a protocol to simultaneously monitor changes in sarcomere length, tension, and Ca2+ concentration following flash photolysis of caged Ca2+. In this protocol, [Ca2+] was rapidly increased by flash photolysis of caged Ca2+, and changes in [Ca2+] due to photolysis and the subsequent binding to troponin C were assessed using a Ca2+ fluorophore. Small bundles of fibers from rabbit skinned psoas muscles were loaded with Ca2+ fluorophore (Fluo-3) and caged Ca2+ (dimethoxynitrophenamine or o-nitrophenyl-EGTA). The bundles were then transferred to silicone oil, where [Ca2+]free, tension, and sarcomere length were monitored before and after photolysis of caged Ca2+. Upon photolysis of caged Ca2+, fluorescence increased and then decayed to a new steady-state level within approximately 1 s, while tension increased to a new steady-state level within approximately 1.5 s. After extracting troponin C, fibers did not generate tension following the flash, but steady-state post-flash fluorescence was significantly greater than when troponin C was present. The difference in [Ca2+]free represents the amount of Ca2+ bound to troponin C. In fibers that were troponin C-replete, Ca2+ binding to troponin C did not differ at short (approximately 1.97 microm) and long (approximately 2.51 microm) sarcomere length, yet tension was approximately 50% greater at the long sarcomere length. These results show that the affinity of troponin C for Ca2+ is not altered by changes in sarcomere length, indicating that length-dependent changes in Ca2+ sensitivity of tension in skeletal muscle are not related to length-dependent changes in Ca2+ binding affinity of troponin C.

Aniline Compounds↗
PubMed1996

Chloride-induced Ca2+ release from the sarcoplasmic reticulum of chemically skinned rabbit psoas fibers and isolated vesicles of terminal cisternae.

There is increasing evidence that Ca2+ release from sarcoplasmic reticulum (SR) of mammalian skeletal muscle is regulated or modified by several factors including ionic composition of the myoplasm. We have studied the effect of Cl- on the release of Ca2+ from the SR of rabbit skeletal muscle in both skinned psoas fibers and in isolated terminal cisternae vesicles. Ca2+ release from the SR in skinned fibers was inferred from increases in isometric tension and the amount of release was assessed by integrating the area under each tension transient. Ca2+ release from isolated SR was measured by rapid filtration of vesicles passively loaded with 45Ca2+. Ca2+ release from SR was stimulated in both preparations by exposure to a solution containing 191 mm choline-Cl, following pre-equilibration in Ca2+-loading solution that had propionate as the major anion. Controls using saponin (50 microg/ml), indicated that the release of Ca2+ was due to direct action of Cl- on the SR rather than via depolarization of T-tubules. Procaine (10 mM) totally blocked Cl-- and caffeine-elicited tension transients recorded using loading and release solutions having ([Na+] + [K+]) x [Cl-] product of 6487.69 mm2 and 12361.52 mm2, respectively, and blocked 60% of Ca2+ release in isolated SR vesicles. Surprisingly, procaine had only a minor effect on tension transients elicited by Cl- and caffeine together. The data from both preparations suggests that Cl- induces a relatively small amount of Ca2+ release from the SR by activating receptors other than RYR-1. In addition, Cl- may increase the Ca2+ sensitivity of RYR-1, which would then allow the small initial release of Ca2+ to facilitate further release of Ca2+ from the SR by Ca2+-induced Ca2+ release.

Animals↗
PubMed1996

Altered kinetics of contraction in skeletal muscle fibers containing a mutant myosin regulatory light chain with reduced divalent cation binding.

We examined the kinetic properties of rabbit skinned skeletal muscle fibers in which the endogenous myosin regulatory light chain (RLC) was partially replaced with a mutant RLC (D47A) containing a point mutation within the Ca2+/Mg2+ binding site that severely reduced its affinity for divalent cations. We found that when approximately 50% of the endogenous RLC was replaced by the mutant, maximum tension declined to approximately 60% of control and the rate constant of active tension redevelopment (ktr) after mechanical disruption of cross-bridges was reduced to approximately 70% of control. This reduction in ktr was not an indirect effect on kinetics due to a reduced number of strongly bound myosin heads, because when the strongly binding cross-bridge analog N-ethylmaleimide-modified myosin subfragment1 (NEM-S1) was added to the fibers, there was no effect upon maximum ktr. Fiber stiffness declined after D47A exchange in a manner indicative of a decrease in the number of strongly bound cross-bridges, suggesting that the force per cross-bridge was not significantly affected by the presence of D47A RLC. In contrast to the effects on ktr, the rate of tension relaxation in steadily activated fibers after flash photolysis of the Ca2+ chelator diazo-2 increased by nearly twofold after D47A exchange. We conclude that the incorporation of the nondivalent cation-binding mutant of myosin RLC decreases the proportion of cycling cross-bridges in a force-generating state by decreasing the rate of formation of force-generating bridges and increasing the rate of detachment. These results suggest that divalent cation binding to myosin RLC plays an important role in modulating the kinetics of cross-bridge attachment and detachment.

Animals↗
PubMed1996

Myosin regulatory light chain modulates the Ca2+ dependence of the kinetics of tension development in skeletal muscle fibers.

To determine the role of myosin regulatory light chain (RLC) in modulating contraction in skeletal muscle, we examined the rate of tension development in bundles of skinned skeletal muscle fibers as a function of the level of Ca(2+) activation after UV flash-induced release of Ca(2+) from the photosensitive Ca(2+) chelator DM-nitrophen. In control fiber bundles, the rate of tension development was highly dependent on the concentration of activator Ca(2+) after the flash. There was a greater than twofold increase in the rate of tension development when the post-flash [Ca(2+)] was increased from the lowest level tested (which produced a steady tension that was 42% of maximum tension) to the highest level (producing 97% of maximum tension). However, when 40-70% of endogenous myosin RLC was extracted from the fiber bundles, tension developed at the maximum rate, regardless of the post-flash concentration of Ca(2+). Thus, the Ca(2+) dependence of the rate of tension development was eliminated by partial extraction of myosin RLC, an effect that was partially reversed by recombination of RLC back into the fiber bundles. The elimination of the Ca(2+) dependence of the kinetics of tension development was specific to the extraction of RLC rather than an artifact of the co-extraction of both RLC and Troponin C, because the rate of tension development was still Ca(2+) dependent, even when nearly 50% of endogenous Troponin C was extracted from fiber bundles fully replete with RLC. Thus, myosin RLC appears to be a key component in modulating Ca(2+) sensitive cross-bridge transitions that limit the rate of force development after photorelease of Ca(2+) in skeletal muscle fibers.

Animals↗
PubMed1996

Response of cardiac myocytes to a ramp increase of diacylglycerol generated by photolysis of a novel caged diacylglycerol.

To test the responsiveness of living cells to the intracellular messenger diacylglycerol, we developed a prototype caged diacylglycerol compound, 3-O-(alpha-carboxyl-2,4-dinitrobenzyl)-1 ,2-dioctanoyl-rac-glycerol (designated alpha-carboxyl caged diC(8)), that produces dioctanoylglycerol (diC(8)) on photolysis. Alpha-Carboxyl caged diC(8) is biologically inert toward diacylglycerol kinase and protein kinase C in vitro and is readily incorporated into cardiac myocyte membranes, where it has no effect before irradiation. Exposure to near-UV light releases biologically active diC8 in good yield (quantum efficiency = 0.2). Here we examine a cellular response to controlled elevation of diC8 within single cardiac myocytes. Twitch amplitude was monitored in electrically stimulated myocytes, and a ramp increase in the concentration of diC(8) was generated by continuous irradiation of cells loaded with the caged compound. The myocyte response was biphasic with a positive inotropic phase (39% increase in twitch amplitude), followed by a large negative inotropic phase (>80% decrease). The time to peak inotropy for both phases depended on the light intensity, decreasing from 376 +/- 51 S to 44 +/- 5 s (positive phase) and 422 +/- 118 S to 51 +/- 9 S (negative phase) as the light intensity was increased eightfold. Both phases were inhibited by the protein kinase C inhibitor chelethyrine chloride. An increase in extracellular K+ from 5 mM to 20 mM to partially depolarize the cell membrane eliminated the positive inotropic phase, but the negative inotropic response was largely unaltered. The results reveal new features in the response of cardiac muscle to diacylglycerol, including a positive inotropic phase and a complex responsiveness to a simple linear increase in diacylglycerol. The effects of photoreleased diC(8) were similar to the effects of opiate agonists selective for kappa receptors, consistent with a major role for diacylglycerol in these responses.

3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cycloh↗
PubMed1995

Enrichment in Fraser broth supplemented with catalase or Oxyrase, combined with the microcolony immunoblot technique, for detecting heat-injured Listeria monocytogenes in foods.

The microcolony immunoblot technique using monoclonal antibodies to Listeria monocytogenes was evaluated for its suitability to detect heat-injured cells. Pasteurized milk and filtrates of homogenized raw ground beef slurry and cabbage were inoculated with L. monocytogenes Scott A, heated, diluted, inoculated into Fraser broth (FB) supplemented with 400 micrograms of catalase ml-1 or 0.01 unit of Oxyrase ml-1, and incubated at 30 degrees C for 6 h. Three inoculum populations (high, medium, and low) were used. The extent of injury was dependent on the heating menstruum. Forty percent of the cells were injured in beef slurry filtrate, whereas 79 and 94% were injured in milk and cabbage filtrate, respectively, when foods were heated at 52 degrees C for 20 min. Populations of viable cells were determined using the immunoblot technique and by surface plating on modified Oxford (mMOX) agar. Recovery of cells from heated foods was enhanced in FB supplemented with catalase or Oxyrase compared to recovery in control broth. Essentially all unheated (control) cells could be detected within about 30 h using enrichment and the immunoblot technique; 54 h were required to easily detect colonies on mMOX. In most cases, the number of cells detected in heated milk or filtrates of homogenized beef after enrichment in FB supplemented with catalase or Oxyrase was significantly higher than populations detected using unsupplemented FB; however, enrichment in FB supplemented with catalase or Oxyrase did not significantly increase cell populations in heated cabbage filtrate. Within each heat treatment and level of inoculum, cell populations detected on mMOX agar after incubating plates for 48 h or on immunoblots after 24 h were not significantly different. Results indicate that the immunoblot technique in conjunction with enrichment in FB containing either catalase or Oxyrase can be successfully used to detect healthy and heat-injured cells of L. monocytogenes in diverse types of foods within 34 h.

Animals↗
PubMed1995

Primary structure and properties of helothermine, a peptide toxin that blocks ryanodine receptors.

Helothermine, a protein from the venom of the Mexican beaded lizard (Heloderma horridum horridum), was found to inhibit [3H]ryanodine binding to cardiac and skeletal sarcoplasmic reticulum, to block cardiac and skeletal ryanodine receptor channels incorporated into planar bilayers, and to block Ca(2+)-induced Ca2+ release triggered by photolysis of nitr-5 in saponin-permeabilized trabeculae from rat ventricle. Cloning of the helothermine cDNA revealed that the protein is composed of 223 amino acids with a molecular mass of 25,376 daltons, and apparently is stabilized by eight disulfide bridges. The peptide sequence showed significant homology with a family of cysteine-rich secretory proteins found in the male genital tract and in salivary glands. The interaction of helothermine and ryanodine receptors should serve to define functional domains within the channel structure involved in the control of Ca2+ release from sarcoplasmic reticulum.

Amino Acid Sequence↗
PubMed1995

Evaluation of enrichment broths for their ability to recover heat-injured Listeria monocytogenes.

Listeria selective enrichment broth (LEB), University of Vermont (UVM) broth, modified UVM (MUVM) broth and Fraser broth (FB) were compared for their ability to recover cells of L. monocytogenes from heated tryptose phosphate broth. Three strains of L. monocytogenes were heated at 54 degrees C for 30 min, inoculated into enrichment broths supplemented with 400 micrograms catalase ml-1, and incubated for 8 h at 30 degrees C. After incubation for 4 h, the total viable cell populations either decreased or did not change, whereas the number of healthy (non-injured) cells of all strains increased significantly in all broths except FB inoculated with the LCDC strain. With an increase in incubation time to 8 h, the number of healthy cells of all strains increased in all broths. At 8 h, the difference between populations of total (injured plus healthy cells) and healthy cells detected in LEB inoculated with two strains was not significant. Overall, recovery of heat-treated cells was significantly higher in LEB, followed by MUVM broth, UVM broth and FB. The addition of catalase to enrichment broths significantly enhanced recovery of heat-injured cells. A slight reduction of catalase activity of heated cells of all test strains in all enrichment broths except FB was observed by extending the incubation period from 4 to 8 h. A test strain that produces relatively higher catalase activity compared to the other strains exhibited the greatest resistance to exogenous hydrogen peroxide. Enumeration of viable L. monocytogenes cells in heated foods should be done using LEB supplemented with 400 micrograms catalase ml-1 to maximize the recovery of injured cells.

Catalase↗
PubMed1995

Cardiac sarcoplasmic reticulum phosphorylation increases Ca2+ release induced by flash photolysis of nitr-5.

Effects on Ca(2+)-induced Ca2+ release due to phosphorylation of sarcoplasmic reticulum (SR) proteins were investigated in isoproterenol-treated saponin-permeabilized trabeculae from rat ventricles. In these experiments, Ca2+ release from the SR was induced by a rapid change in concentration of free Ca2+ (ie, trigger Ca2+) achieved by flash photolysis of nitr-5, and the amount of Ca2+ released was assessed by measuring isometric tension. Ca2+ uptake by the SR was more rapid, and the amount of Ca2+ released by a given concentration of trigger Ca2+ was greater in isoproterenol-treated trabeculae compared with control trabeculae. However, under the same conditions of Ca2+ loading, the amplitudes of caffeine-elicited tension transients in control trabeculae were similar to those in isoproterenol-treated trabeculae, suggesting that the Ca2+ available for release was similar in the two cases. Control experiments showed that there were no significant differences in Ca2+ sensitivity of tension between isoproterenol-treated and control trabeculae. Also, application of alkaline phosphatase to trabeculae that had previously been treated with isoproterenol returned SR Ca2+ release to control levels. We conclude that the greater release of Ca2+ in isoproterenol-treated trabeculae in response to a given concentration of trigger Ca2+ is due to phosphorylation of SR proteins, most likely the Ca2+ release channel.

Alkaline Phosphatase↗
PubMed1995

Using computers to diagnose and plan treatment of approximal caries. Detected in radiographs.

Two of the authors developed a computer-based software system that is capable of diagnosing approximal caries and making decisions about restorative care. The system was compared with decisions made by a group of experienced clinicians. The results indicate that the clinician viewers overall correctly identified a similar number of surfaces as the software, but from viewer to viewer, there was more variability in the number of surfaces identified correctly than in those identified correctly by the software users. Software packages such as this have the potential to raise overall accuracy by increasing the consistency of treatment decisions over time.

Bicuspid↗
PubMed1993

Cleft mandible.

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Child↗