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Biomedical subjects

P D Allen

Publications and source records attributed to P D Allen.

At least 19 recordsLinked to original sources
PubMed2006

Automated osteoporosis risk assessment by dentists: a new pathway to diagnosis.

General dental practitioners use a vast amount of panoramic radiography in their routine clinical work, but valuable information about patients' osteoporotic status is not collected. There are many reasons for this, but one of the prime reasons must be the disruption involved in clinical routine with lengthy manual radiographic assessment. We have developed computer software, based on active shape modeling that will automatically detect the mandibular cortex on panoramic radiographs, and then measure its width. Automatic or semi-automatic measurement of the cortical width will indicate the osteoporotic risk of the patient. The aim of our work was to assess the computer search technique's ability to measure the mandibular cortical width and to assess its potential for detection of osteoporosis of the hip, spine and femoral neck. Mandibular cortical width was measured using the manually initialized (semi-automatic) method and, when assessed for diagnosing osteoporosis at one of the three measurement sites, gave an area under the ROC curve (A(z))=0.816 (95% CI=0.784 to 0.845) and for the automatically initialized searches, A(z)=0.759 (95% CI=0.724 to 0.791). The difference between areas=0.057 (95% Confidence interval=0.025 to 0.089), p<0.0001. For diagnosing osteoporosis at the femoral neck, mandibular cortical width derived from the manually initialized fit gave an area under the ROC curve (A(z))=0.835 (95% CI=0.805 to 0.863) and for the automatically initialized searches A(z)=0.805 (95% CI=0.773 to 0.835). The difference in A(z) values between active shape modeling search methods=0.030 (95% CI=-0.010 to 0.070), and this was not significant, p=0.138. We concluded that measurement of mandibular cortical width using active shape modeling is capable of diagnosing skeletal osteoporosis with good diagnostic ability and repeatability.

Absorptiometry, Photon↗
PubMed2006

Structural determinants of 4-chloro-m-cresol required for activation of ryanodine receptor type 1.

4-Chloro-m-cresol (4-CmC) is a clinically relevant activator of the intracellular Ca2+ release channel, the ryanodine receptor isoform 1 (RyR1). In this study, the chemical moieties on the 4-CmC molecule required for its activation of RyR1 were determined using structure-activity relationship analysis with a set of commercially available 4-CmC analogs. Separate compounds each lacking one of the three functional groups of 4-CmC (1-hydroxyl, 3-methyl, or 4-chloro) were poor activators of RyR1. Substitution of different chemical groups for the 1-hydroxyl of 4-CmC resulted in compounds that were poor activators of RyR1, suggesting that the hydroxyl group is preferred at this position. Substitution of hydrophobic groups at the 3-position enhanced bioactivity of the compound relative to 4-CmC, whereas substitution with hydrophilic groups abolished bioactivity. Likewise, 4-CmC analogs with hydrophobic groups substituted into the 4-position enhanced bioactivity, whereas hydrophilic or charged groups diminished bioactivity. 4-CmC analogs containing a single hydrophobic group at either the 3- or 4-position as well as 3,5-disubstituted or 3,4,5-trisubstituted phenols were also effective activators of RyR1. These results indicate that the 1-hydroxyl group of 4-CmC is required for activation of RyR1 and that hydrophobic groups at the 3,4- and 5-positions are preferred. These findings suggest that the 4-CmC binding site on RyR1 most likely consists of a hydrophilic region to interact with the 1-hydroxyl as well as a hydrophobic region(s) to interact with chemical groups at the 3- and/or 4-positions of 4-CmC.

Animals↗
PubMed2006

Functional coupling between TRPC3 and RyR1 regulates the expressions of key triadic proteins.

We have shown that TRPC3 (transient receptor potential channel canonical type 3) is sharply up-regulated during the early part of myotube differentiation and remains elevated in mature myotubes compared with myoblasts. To examine its functional roles in muscle, TRPC3 was "knocked down" in mouse primary skeletal myoblasts using retroviral-delivered small interference RNAs and single cell cloning. TRPC3 knockdown myoblasts (97.6 +/- 1.9% reduction in mRNA) were differentiated into myotubes (TRPC3 KD) and subjected to functional and biochemical assays. By measuring rates of Mn(2+) influx with Fura-2 and Ca(2+) transients with Fluo-4, we found that neither excitation-coupled Ca(2+) entry nor thapsigargin-induced store-operated Ca(2+) entry was significantly altered in TRPC3 KD, indicating that expression of TRPC3 is not required for engaging either Ca(2+) entry mechanism. In Ca(2+) imaging experiments, the gain of excitation-contraction coupling and the amplitude of the Ca(2+) release seen after direct RyR1 activation with caffeine was significantly reduced in TRPC3 KD. The decreased gain appears to be due to a decrease in RyR1 Ca(2+) release channel activity, because sarcoplasmic reticulum (SR) Ca(2+) content was not different between TRPC3 KD and wild-type myotubes. Immunoblot analysis demonstrated that TRPC1, calsequestrin, triadin, and junctophilin 1 were up-regulated (1.46 +/- 1.91-, 1.42 +/- 0.08-, 2.99 +/- 0.32-, and 1.91 +/- 0.26-fold, respectively) in TRPC3 KD. Based on these data, we conclude that expression of TRPC3 is tightly regulated during muscle cell differentiation and propose that functional interaction between TRPC3 and RyR1 may regulate the gain of SR Ca(2+) release independent of SR Ca(2+) load.

Acetylcysteine↗
PubMed2006

The relationship between acetabular retroversion and osteoarthritis of the hip.

Excessive acetabular cover secondary to a retroverted acetabulum causes pincer impingement, which may cause early osteoarthritis of the hip. Our aim was to determine if there was a relationship between acetabular version and osteoarthritis of the hip. Using image processing and analysis software we studied 117 CT images of the hip in patients aged less than 65 years who had undergone a CT virtual colonoscopy. The mean CT joint space of the 18 hips with acetabular retroversion was narrower compared with the 99 hips with normal acetabular alignment (p < 0.0001). A correlation of r = 0.46 (p < 0.01) was found between right hip acetabular version and the mean right hip joint space and of r = 0.31 (p = 0.02) between left hip acetabular version and the mean left hip joint space. Acetabular retroversion is associated with radiological evidence of osteoarthritis of the hip. An understanding of the mechanical basis of osteoarthritis of the hip allows early treatment of the underlying structural abnormality and prevents progression of the degenerative condition.

Acetabulum↗
PubMed2005

Internal structure and visualization of transmembrane domains of the RyR1 calcium release channel by cryo-EM.

RyR1 is an intracellular calcium channel with a central role in muscle contraction. We obtained a three-dimensional reconstruction of the RyR1 in the closed state at a nominal resolution of approximately 10 A using cryo-EM. The cytoplasmic assembly consists of a series of interconnected tubular structures that merge into four columns that extend into the transmembrane assembly. The transmembrane assembly, which has at least six transmembrane alpha-helices per monomer, has four tilted rods that can be fitted with the inner helices of a closed K(+) channel atomic structure. The rods splay out at the lumenal side and converge into a dense ring at the cytoplasmic side. Another set of four rods emerges from this ring and shapes the inner part of the four columns. The resulting constricted axial structure provides direct continuity between cytoplasmic and transmembrane assemblies, and a possible mechanism for control of channel gating through conformational changes in the cytoplasmic assembly.

Animals↗
PubMed2004

Mutational analysis of putative calcium binding motifs within the skeletal ryanodine receptor isoform, RyR1.

The functional relevance of putative Ca(2+) binding motifs previously identified with Ca(2+) overlay binding analysis within the skeletal muscle ryanodine receptor isoform (RyR1) was examined using mutational analysis. EF hands between amino acid positions 4081 and 4092 (EF1) and 4116 and 4127 (EF2) were scrambled singly or in combination within the full-length rabbit RyR1 cDNA. These cDNAs were expressed in 1B5 RyR-deficient myotubes and channel function assessed using Ca(2+)-imaging techniques, [(3)H]ryanodine binding measurements, and single channel experiments. In intact myotubes, these mutations did not affect functional responses to either depolarization or RyR agonists (caffeine, 4-chloro-m-cresol) compared with wtRyR1. However, in [(3)H]ryanodine binding measurements, both Ca(2+) activation and inhibition of the EF1 mutant was significantly altered compared with wtRyR1. No high affinity [(3)H]ryanodine binding was observed in membranes expressing the EF2 mutation, although in single channel measurements, the EF2-disrupted channel could be activated by micromolar Ca(2+) concentrations. In addition, micromolar levels of ryanodine placed these channels into the classical half-conductance state, thus indicating that occupancy of high affinity ryanodine binding sites is not required for ryanodine-induced subconductance states in RyR1. Disruption of three additional putative RyR1 calcium binding motifs located between amino acid positions 4254 and 4265 (EF3), 4407 and 4418 (EF4), or 4490 and 4502 (EF5) either singly or in combination (EF3-5) did not affect functional responses in 1B5 myotubes except that the EC(50) for caffeine activation for the EF3 construct was significantly increased compared with wtRyR1. However, in [(3)H]ryanodine binding experiments, the Ca(2+)-dependent activation and inactivation of mutated RyRs containing EF3, EF4, or EF5 was unaffected when compared with wtRyR1.

Amino Acid Motifs↗
PubMed2003

Conformational coupling of DHPR and RyR1 in skeletal myotubes is influenced by long-range allosterism: evidence for a negative regulatory module.

Four ryanodine receptor type 1 and 2 chimeras (R4, R9, R10, and R16) and their respective wild-type ryanodine receptors (type 1 and 2; wtRyR1 and wtRyR2) were expressed in dyspedic 1B5 to identify possible negative regulatory modules of the Ca2+ release channel that are under the influence of the dihydropyridine receptor (DHPR). Responses of intact 1B5 myotubes expressing each construct to caffeine in the absence or presence of either La3+ and Cd2+ or the organic DHPR blocker nifedipine were determined by imaging single 1B5 myotubes loaded with fluo 4. The presence of La3+ and Cd2+ or nifedipine in the external medium at concentrations known to block Ca2+ entry through the DHPRs significantly decreased the caffeine EC50 of wtRyR1 (2.80 +/- 0.12 to 0.83 +/- 0.09 mM; P < 0.05). On the other hand, DHPR blockade did not significantly alter the caffeine EC50 values of wtRyR2, chimeras R10 and R16, whereas the caffeine EC50 values of chimeras R4 and R9 were significantly increased (1.27 +/- 0.05 to 2.60 +/- 0.16 mM, and 1.15 +/- 0.03 to 2.11 +/- 0.32 mM, respectively; P < 0.05). Despite the fact that all the chimeras form fully functional Ca2+ release channels in situ, sarcoplasmic reticulum (SR) containing R4, R10, and R16 did not possess high-affinity binding of [3H]ryanodine regardless of Ca2+ concentration. These results suggest the presence of an interaction between RyR1 and the DHPR, which is not present in RyR2, that contributes negative control of SR Ca2+ release induced by direct agonists such as caffeine. Although we were unable to define the negative module using RyR1-RyR2 chimeras, they further demonstrated that the RyR is very sensitive to long-range allosterism.

Allosteric Regulation↗
PubMed2003

FKBP12 binding to RyR1 modulates excitation-contraction coupling in mouse skeletal myotubes.

The skeletal muscle sarcoplasmic reticulum (SR) Ca2+ release channel or ryanodine receptor (RyR1) binds four molecules of FKBP12, and the interaction of FKBP12 with RyR1 regulates both unitary and coupled gating of the channel. We have characterized the physiologic effects of previously identified mutations in RyR1 that disrupt FKBP12 binding (V2461G and V2461I) on excitation-contraction (EC) coupling and intracellular Ca2+ homeostasis following their expression in skeletal myotubes derived from RyR1-knockout (dyspedic) mice. Wild-type RyR1-, V246I-, and V2461G-expressing myotubes exhibited similar resting Ca2+ levels and maximal responses to caffeine (10 mm) and cyclopiazonic acid (30 microm). However, maximal voltage-gated Ca2+ release in V2461G-expressing myotubes was reduced by approximately 50% compared with that attributable to wild-type RyR1 (deltaF/Fmax = 1.6 +/- 0.2 and 3.1 +/- 0.4, respectively). Dyspedic myotubes expressing the V2461I mutant protein, that binds FKBP12.6 but not FKBP12, exhibited a comparable reduction in voltage-gated SR Ca2+ release (deltaF/Fmax = 1.0 +/- 0.1). However, voltage-gated Ca2+ release in V2461I-expressing myotubes was restored to a normal level (deltaF/Fmax = 2.9 +/- 0.6) following co-expression of FKBP12.6. None of the mutations that disrupted FKBP binding to RyR1 significantly affected RyR1-mediated enhancement of L-type Ca2+ channel activity (retrograde coupling). These data demonstrate that FKBP12 binding to RyR1 enhances the gain of skeletal muscle EC coupling.

Amino Acid Substitution↗
PubMed2003

Genomics, proteomics and bioinformatics of human heart failure.

Unraveling the molecular complexities of human heart failure, particularly end-stage failure, can be achieved by combining multiple investigative approaches. There are several parts to the problem. Each patient is the product of a complex set of genetic variations, different degrees of influence of diets and lifestyles, and usually heart transplantation patients are treated with multiple drugs. The genomic status of the myocardium of any one transplant patient can be analysed using gene arrays (cDNA- or oligonucleotide-based) each with its own strengths and weaknesses. The proteins expressed by these failing hearts (myocardial proteomics) were first investigated over a decade ago using two-dimensional polyacrylamide gel electrophoresis (2DGE) which promised to resolve several thousand proteins in a single sample of failing heart. However, while 2DGE is very successful for the abundant and moderately expressed proteins, it struggles to identify proteins expressed at low levels. Highly focused first dimension separations combined with recent advances in mass spectrometry now provide new hope for solving this difficulty. Protein arrays are a more recent form of proteomics that hold great promise but, like the above methods, they have their own drawbacks. Our approach to solving the problems inherent in the genomics and proteomics of heart failure is to provide experts in each analytical method with a sample from the same human failing heart. This requires a sufficiently large number of samples from a sufficiently large pool of heart transplant patients as well as a large pool of non-diseased, non-failing human hearts. We have collected more than 200 hearts from patients undergoing heart transplantations and a further 50 non-failing hearts. By combining our expertise we expect to reduce and possibly eliminate the inherent difficulties of each analytical approach. Finally, we recognise the need for bioinformatics to make sense of the large quantities of data that will flow from our laboratories. Thus, we plan to provide meaningful molecular descriptions of a number of different conditions that result in terminal heart failure.

Animals↗
PubMed2003

Targeted transgene integration into transgenic mouse fibroblasts carrying the full-length human AAVS1 locus mediated by HSV/AAV rep(+) hybrid amplicon vector.

Herpes simplex virus type 1/adeno-associated virus (HSV/AAV) rep(+) hybrid amplicon vectors containing AAV inverted terminal repeats (ITRs) and rep gene sequences can mediate site-specific integration into the human genome. In this study, we have generated and characterized the first transgenic mice that bear the full-length (8.2 kb) human AAVS1 locus. Immortalized mouse embryonic fibroblasts from this mouse line were transduced with the rep(+), rep(-) (containing only ITRs flanking the transgene) hybrid amplicon vectors, and the standard amplicon vector to determine stable integration frequency and the site of integration. Transduction of transgenic fibroblasts resulted in a 10-fold higher stable integration frequency with rep(+) hybrid amplicon vector than with rep(-) or standard amplicon vectors. Southern blot analysis of genomic DNA from transgenic cells stably transduced with the rep(+) hybrid amplicon vector revealed site-specific integration of transgenes at the AAVS1 locus in 50% of clones. Some site-specific and random integration events were limited to the ITR-flanked transgene cassette. In contrast, transduction of transgenic mouse cells with the rep(-) or standard amplicon vectors resulted in random integrations of the entire rep(-) hybrid amplicon or amplicon DNA that were incorporated into the host genome as a concatenate of various sizes. These results demonstrate for the first time that the genome of transgenic mice bearing the human AAVS1 locus serves as a platform for site-specific integration of AAV ITR-flanked transgene cassettes within the hybrid amplicon vector in the presence of Rep.

Animals↗
PubMed2002

Herpes simplex virus type 1/adeno-associated virus rep(+) hybrid amplicon vector improves the stability of transgene expression in human cells by site-specific integration.

Herpes simplex virus type 1 (HSV-1) amplicon vectors are promising gene delivery tools, but their utility in gene therapy has been impeded to some extent by their inability to achieve stable transgene expression. In this study, we examined the possibility of improving transduction stability in cultured human cells via site-specific genomic integration mediated by adeno-associated virus (AAV) Rep and inverted terminal repeats (ITRs). A rep(-) HSV/AAV hybrid amplicon vector was made by inserting a transgene cassette flanked with AAV ITRs into an HSV-1 amplicon backbone, and a rep(+) HSV/AAV hybrid amplicon was made by inserting rep68/78 outside the rep(-) vector 3' AAV ITR sequence. Both vectors also had a pair of loxP sites flanking the ITRs. The resulting hybrid amplicon vectors were successfully packaged and compared to a standard amplicon vector for stable transduction frequency (STF) in human 293 and Gli36 cell lines and primary myoblasts. The rep(+), but not the rep(-), hybrid vector improved STF in all three types of cells; 84% of Gli36 and 40% of 293 stable clones transduced by the rep(+) hybrid vector integrated the transgene into the AAVS1 site. Due to the difficulty in expanding primary myoblasts, we did not assess site-specific integration in these cells. A strategy to attempt further improvement of STF by "deconcatenating" the hybrid amplicon DNA via Cre-loxP recombination was tested, but it did not increase STF. These data demonstrate that introducing the integrating elements of AAV into HSV-1 amplicon vectors can significantly improve their ability to achieve stable gene transduction by conferring the AAV-like capability of site-specific genomic integration in dividing cells.

Cell Line↗
PubMed2001

Calcium transients in 1B5 myotubes lacking ryanodine receptors are related to inositol trisphosphate receptors.

Potassium depolarization of skeletal myotubes evokes slow calcium waves that are unrelated to contraction and involve the cell nucleus (Jaimovich, E., Reyes, R., Liberona, J. L., and Powell, J. A. (2000) Am. J. Physiol. 278, C998-C1010). Studies were done in both the 1B5 (Ry53-/-) murine "dyspedic" myoblast cell line, which does not express any ryanodine receptor isoforms (Moore, R. A., Nguyen, H., Galceran, J., Pessah, I. N., and Allen, P. D. (1998) J. Cell Biol. 140, 843-851), and C(2)C(12) cells, a myoblast cell line that expresses all three isoforms. Although 1B5 cells lack ryanodine binding, they bind tritiated inositol (1,4,5)-trisphosphate. Both type 1 and type 3 inositol trisphosphate receptors were immuno-located in the nuclei of both cell types and were visualized by Western blot analysis. After stimulation with 47 mm K(+), inositol trisphosphate mass raised transiently in both cell types. Both fast calcium increase and slow propagated calcium signals were seen in C(2)C(12) myotubes. However, 1B5 myotubes (as well as ryanodine-treated C(2)C(12) myotubes) displayed only a long-lasting, non-propagating calcium increase, particularly evident in the nuclei. Calcium signals in 1B5 myotubes were almost completely blocked by inhibitors of the inositol trisphosphate pathway: U73122, 2-aminoethoxydiphenyl borate, or xestospongin C. Results support the hypothesis that inositol trisphosphate mediates slow calcium signals in muscle cell ryanodine receptors, having a role in their time course and propagation.

Animals↗
PubMed2001

Ryanodine receptor point mutant E4032A reveals an allosteric interaction with ryanodine.

The ryanodine receptor (RyR) family of proteins constitutes a unique type of calcium channel that mediates Ca(2+) release from endoplasmic reticulum/sarcoplasmic reticulum stores. Ryanodine has been widely used to identify contributions made by the RyR to signaling in both muscle and nonmuscle cells. Ryanodine, through binding to high- and low-affinity sites, has been suggested to block the channel pore based on its ability to induce partial conductance states and irreversible inhibition. We examined the effect of ryanodine on an RyR type 1 (RyR1) point mutant (E4032A) that exhibits a severely compromised phenotype. When expressed in 1B5 (RyR null/dyspedic) myotubes, E4032A is relatively unresponsive to stimulation by cell membrane depolarization or RyR agonists, although the full-length protein is correctly targeted to junctions and interacts with dihydropyridine receptors (DHPRs) inducing their arrangement into tetrads. However, treatment of E4032A-expressing cells with 200-500 microM ryanodine, concentrations that rapidly activate and then inhibit wild-type (wt) RyR1, restores the responsiveness of E4032A-expressing myotubes to depolarization and RyR agonists. Moreover, the restored E4032A channels remain resistant to subsequent exposure to ryanodine. In single-channel studies, E4032A exhibits infrequent (channel-open probability, P(o) < 0.005) and brief (<250 micros) gating events and insensitivity to Ca(2+). Addition of ryanodine restores Ca(2+)-dependent channel activity exhibiting full, 3/4, 1/2, and 1/4 substates. This evidence suggests that, whereas ryanodine does not occlude the RyR pore, it does bind to sites that allosterically induce substantial conformational changes in the RyR. In the case of E4032A, these changes overcome unfavorable energy barriers introduced by the E4032A mutation to restore channel function.

Allosteric Regulation↗
PubMed2001

Protein kinase C-alpha and -epsilon modulate connexin-43 phosphorylation in human heart.

We have previously demonstrated that protein kinase C (PKC)- alpha expression is significantly elevated in failing human left ventricle, with immunostaining showing increased PKC- alpha localization at the intercalated disks of cardiomyocytes. In the present study we sought to determine, in the failing heart, if PKC- alpha interacted with connexin-43 (Cx-43) both spatially and functionally, and to compare the association of PKC- alpha/Cx-43 with that of PKC- epsilon, a PKC isozyme that does not significantly increase in failing hearts. The possibility of a PKC- alpha or PKC- epsilon/Cx-43 association in non-failing hearts was also investigated. Co-immunoprecipitation of PKC- alpha or PKC- epsilon and Cx-43 in non-failing and failing left ventricle was achieved using antibodies to PKC- alpha or Cx-43. Confocal microscopy confirmed that PKC- alpha distribution within the cardiomyocyte included co-localization with connexin-43 in both failing and non-failing myocardium. In a similar manner, confocal imaging of PKC- epsilon showed cardiomyocyte distribution in both cytosol and membrane, and colocalization of PKC- epsilon with Cx-43. Recombinant PKC- alpha or - epsilon increased PKC activity significantly above endogenous levels in the co-immunoprecipitated Cx-43 complexes (P<0.05). However, phosphorylation of purified human Cx-43 (isolated from failing human left ventricle) by recombinant PKC- alpha or PKC- epsilon resulted in only PKC- epsilon mediated Cx-43 phosphorylation. Thus, in the human heart PKC- alpha, PKC- epsilon, and Cx-43 appear to form a closely associated complex. Whereas only PKC- epsilon directly phosphorylates Cx-43, both PKC isoforms result in increased phosphorylation within the Cx-43 co-immunoprecipitated complex.

Blotting, Western↗
PubMed2001

Organochlorine contaminants and biomarker response in double-crested cormorants nesting in Green Bay and Lake Michigan, Wisconsin, USA.

Double-crested cormorant (Phalacrocorax auritus) eggs at pipping and sibling 10-day-old chicks were collected from two colonies in Green Bay, WI, one colony in Lake Michigan, WI, and reference colonies in South Dakota and Minnesota. Egg contents and chicks were analyzed for organochlorine contaminants including polychlorinated biphenyl (PCB) congeners. Livers of embryos and chicks were assayed for hepatic microsomal ethoxyresorufin-O-dealkylase (EROD) activity. Eggshell thickness and the physical dimensions of embryo brains were measured. Concentrations of organochlorines, including p,p'-DDE (p,p'-dichlorodiphenyldichloroethylene), PCBs, and PCB congeners were generally an order of magnitude higher in eggs and chicks from Wisconsin than from reference locations. Total PCBs averaged 10-13 microg/g wet weight in eggs from three Wisconsin colonies compared to 0.9 microg/g PCBs from reference locations. Double-crested cormorant chicks accumulated on average 33-66 microg PCBs/day and 7-12 microg p,p'-DDE/day in the Wisconsin colonies compared to 0 microg PCBs/day and 1 microg p,p'-DDE/day in the reference colonies. At pipping, EROD activity in the livers of cormorant embryos was significantly higher in the Wisconsin colonies and significantly correlated with PCBs and the toxic equivalents (TEQs) of aryl hydrocarbon-active PCB congeners relative to 2,3,7,8-tetrachlorodibenzo-p-dioxin. However, in 10-day-old chicks EROD activity was not consistently different among colonies and was not correlated with PCBs or TEQs. A significant negative relationship between embryo brain asymmetry and the size of the egg suggested that physical constraint might be an important factor influencing the response of this bioindicator. Thinner eggshells in two colonies located near Door County, Wisconsin, suggested that historic p,p'-DDE residues associated with orchards are still an important source of p,p'-DDE in the local environment.

Abnormalities, Drug-Induced↗
PubMed2001

Type 1 and type 3 ryanodine receptors generate different Ca(2+) release event activity in both intact and permeabilized myotubes.

In this investigation we use a "dyspedic" myogenic cell line, which does not express any ryanodine receptor (RyR) isoform, to examine the local Ca(2+) release behavior of RyR3 and RyR1 in a homologous cellular system. Expression of RyR3 restored caffeine-sensitive, global Ca(2+) release and causes the appearance of relatively frequent, spontaneous, spatially localized elevations of [Ca(2+)], as well as occasional spontaneous, propagating Ca(2+) release, in both intact and saponin-permeabilized myotubes. Intact myotubes expressing RyR3 did not, however, respond to K(+) depolarization. Expression of RyR1 restored depolarization-induced global Ca(2+) release in intact myotubes and caffeine-induced global release in both intact and permeabilized myotubes. Both intact and permeabilized RyR1-expressing myotubes exhibited relatively infrequent spontaneous Ca(2+) release events. In intact myotubes, the frequency of occurrence and properties of these RyR1-induced events were not altered by partial K(+) depolarization or by application of nifedipine, suggesting that these RyR1 events are independent of the voltage sensor. The events seen in RyR1-expressing myotubes were spatially more extensive than those seen in RyR3-expressing myotubes; however, when analysis was limited to spatially restricted "Ca(2+) spark"-like events, events in RyR3-expressing myotubes were larger in amplitude and duration compared with those in RyR1. Thus, in this skeletal muscle context, differences exist in the spatiotemporal properties and frequency of occurrence of spontaneous release events generated by RyR1 and RyR3. These differences underscore functional differences between the Ca(2+) release behavior of RyR1 and RyR3 in this homologous expression system.

Animals↗