Biomedical subjects

T M Butler

Publications and source records attributed to T M Butler.

At least 19 recordsLinked to original sources

PubMed2004

White monkey syndrome in infant baboons (Papio species).

Over 23 months, zinc toxicosis was diagnosed in 35 baboons aged 5-12 months in one galvanized metal and concrete cage complex with conditions that led to excessive exposure to environmental zinc. Clinical signs included reduced pigmentation of hair, skin, and mucous membranes (whiteness), alopecia, dehydration, emaciation, cachexia, dermatitis, diarrhea and, in six cases, severe gangrenous dermatitis of extremities. The syndrome was characterized by pancytopenia, elevated zinc and low copper serum concentrations, low vitamin D and bone-specific alkaline phosphatase levels, and atypical myelomonocytic proliferation of bone marrow. This syndrome emphasizes the importance of proper husbandry and cage design and indicates the potential of infant baboons as a model to study the effects of excessive zinc on development. This is the first report describing the epidemiologic and clinical presentation of zinc toxicosis in infant baboons in captivity.

Alopecia↗

PubMed2003

Relaxant effects of parathyroid hormone and parathyroid hormone-related peptides on oviduct motility in birds and mammals: possible role of nitric oxide.

Parathyroid hormone (PTH) and PTH-related peptides (PTHrP) have previously been shown to modulate the contractile state of numerous types of smooth muscle. The effects of N-terminal PTH and PTHrP on spontaneous in vitro contractility of oviducal smooth muscle using tissues from egg-laying Japanese quail (10-15 h post ovulation), 4 and 9 days pregnant mouse uterus were investigated. Myometrial tissues from both species contracted vigorously for several hours, when incubated in organ baths in De Jalon's solution gassed with 5%CO2/95%O2. Contractions were enhanced in high (1.2-2.5 mM) compared with low (0.1-0.5 mM) calcium (Ca) containing media. Bovine PTH(1-34) (bPTH(1-34)), human PTH(1-34 amide) (hPTHrP(1-34) amide), and hPTHrP(1-40) caused similar concentration-related inhibition of contractions in media containing 1.2mM Ca over a range of 10(-9) to 10(-7)M, whereas C-terminal hPTHrP(107-139) was devoid of such activity. Responses to bPTH(1-34) in 4 and 9-day pregnant mouse tissues were similar but hPTHrP(1-40) showed substantial loss of activity in 9-day, compared with 4-day pregnant mouse tissues. Repeated exposure of mouse uterine tissue to the peptides resulted in desensitisation of responses. The EC50 responses of mouse tissues were inhibited by the PTH/PTHrP receptor antagonist, hPTHrP(7-34) amide. Responses to bPTH(1-34) were also inhibited by both non-selective and selective neuronal nitric oxide synthase (NOS) inhibitors N(omega)-nitro-L-arginine methyl ester (0.01-1mM) and 7-nitroindazole (0.01-10 microM), respectively. Both NOS inhibitors were more effective in inhibiting bPTH(1-34)-induced relaxation in the absence of L-arginine compared with in the presence of 1mM L-arginine (a NOS substrate) in the incubation media. It is concluded that relaxant responses to N-terminal PTH and PTHrP peptides are well conserved in oviducal and uterine tissues from avian and mammalian species. The results also suggest that NO may be responsible for mediating relaxant activities of these peptides in pregnant mouse uterine tissue.

Animals↗

PubMed2001

Phosphorylation of molluscan twitchin by the cAMP-dependent protein kinase.

Catch in certain molluscan muscles is released by an increase in cAMP, and it was suggested that the target of cAMP-dependent protein kinase (PKA) is the high molecular weight protein twitchin [Siegman, M. J., Funabara, J., Kinoshita, S., Watabe, S., Hartshorne, D. J., and Butler, T. M. (1998) Proc. Natl. Acad. Sci. U.S.A. 95, 5384-5388]. This study was carried out to investigate the phosphorylation of twitchin by PKA. Twitchin was isolated from Mytilus catch muscles and was phosphorylated by PKA to a stoichiometry of about 3 mol of P/mol of twitchin. There was no evidence of twitchin autophosphorylation. Two phosphorylated peptides were isolated and sequenced, termed D1 and D2. Additional cDNA sequence for twitchin was obtained, and the D2 site was located at the C-terminal side of the putative kinase domain in a linker region between two immunoglobulin C2 repeats. Excess PKA substrates, e.g., D1 and D2, blocked the reduction in force on addition of cAMP, confirming the role for PKA in regulating catch. Papain proteolysis of (32)P-labeled twitchin from permeabilized muscles showed that the D1 site represented about 50% of the (32)P labeling. Proteolysis of in-situ twitchin with thermolysin suggested that the D1 and D2 sites were at the N- and C-terminal ends of the molecule, respectively. Thermolysin proteolysis also indicated that D1 and D2 were major sites of phosphorylation by PKA. The direct phosphorylation of twitchin by PKA is consistent with a regulatory role for twitchin in the catch mechanism and probably involves phosphorylation at the D1 and D2 sites.

Amino Acid Sequence↗

PubMed2001

The myosin cross-bridge cycle and its control by twitchin phosphorylation in catch muscle.

The anterior byssus retractor muscle of Mytilus edulis was used to characterize the myosin cross-bridge during catch, a state of tonic force maintenance with a very low rate of energy utilization. Addition of MgATP to permeabilized muscles in high force rigor at pCa > 8 results in a rapid loss of some force followed by a very slow rate of relaxation that is characteristic of catch. The fast component is slowed 3-4-fold in the presence of 1 mM MgADP, but the distribution between the fast and slow (catch) components is not dependent on [MgADP]. Phosphorylation of twitchin results in loss of the catch component. Fewer than 4% of the myosin heads have ADP bound in rigor, and the time course (0.2-10 s) of ADP formation following release of ATP from caged ATP is similar whether or not twitchin is phosphorylated. This suggests that MgATP binding to the cross-bridge and subsequent splitting are independent of twitchin phosphorylation, but detachment occurs only if twitchin is phosphorylated. A similar dependence of detachment on twitchin phosphorylation is seen with AMP-PNP and ATPgammaS. Single turnover experiments on bound ADP suggest an increase in the rate of release of ADP from the cross-bridge when catch is released by phosphorylation of twitchin. Low [Ca(2+)] and unphosphorylated twitchin appear to cause catch by 1) markedly slowing ADP release from attached cross-bridges and 2) preventing detachment following ATP binding to the rigor cross-bridge.

Adenosine Diphosphate↗

PubMed2001

Age-related autoantibody production in a nonhuman primate model.

Autoantibody production increases with ageing. However, the pathological significance of this increase as well as the corresponding underlying mechanisms are poorly understood. To further our understanding of the role that ageing plays in the development of autoantibody responses, we used a novel nonhuman primate model consisting of healthy baboons of ages representing the entire lifespan of this animal species. Results from this study indicate that production of antinuclear antibodies, anticell extract antibodies and natural autoantibodies gradually and significantly increases from young age to old age without a corresponding increase in neither serum immunoglobulin concentration nor in levels of selected markers of immune dysregulation (sTNF-RI, sTNF-RII, IL-2 sR alpha and IFN-gamma). Therefore, in the baboon model, autoantibodies may be produced in absence of recognizable pathological conditions of the ageing immune system.

Aging↗

PubMed2000

Mycobacterium tuberculosis-secreted protein antigens: immunogenicity in baboons.

The effective control of tuberculosis (TB) requires the development of improved vaccines. It is now well established that Mycobacterium tuberculosis-secreted antigens represent promising candidates to be included in subunit vaccine preparations. It also is accepted that studies in nonhuman primate models will be required to further develop these vaccine preparations. As a necessary step in this direction, we have assessed the immunogenicity of M. tuberculosis-secreted antigens in baboons. Animals received a total of three intramuscular injections consisting of M. tuberculosis culture filtrate proteins resuspended in an adjuvant formulation (MPL-SE) and were tested for development of specific antibody and cellular responses. All animals produced antibody and cellular proliferative responses in the absence of detectable delayed-type hypersensitivity reactions. Production of gamma-interferon following stimulation of peripheral blood lymphocytes with culture filtrate proteins was either absent or present at low levels. Results from this study show that, although M. tuberculosis-secreted protein antigens are relatively safe and immunogenic in baboons, alternative immunization approaches must be identified for the induction of T-helper type 1 responses.

Adjuvants, Immunologic↗

PubMed1998

Phosphorylation of a twitchin-related protein controls catch and calcium sensitivity of force production in invertebrate smooth muscle.

"Catch" is a condition of prolonged, high-force maintenance at resting intracellular Ca2+ concentration ([Ca2+]) and very low energy usage, occurring in invertebrate smooth muscles, including the anterior byssus retractor muscle (ABRM) of Mytilus edulis. Relaxation from catch is rapid on serotonergic nerve stimulation in intact muscles and application of cAMP in permeabilized muscles. This release of catch occurs by protein kinase A-mediated phosphorylation of a high (approximately 600 kDa) molecular mass protein, the regulator of catch. Here, we identify the catch-regulating protein as a homologue of the mini-titin, twitchin, based on (i) a partial cDNA of the purified isolated protein showing 77% amino acid sequence identity to the kinase domain of Aplysia californica twitchin; (ii) a polyclonal antibody to a synthetic peptide in this sequence reacting with the phosphorylated catch-regulating protein band from permeabilized ABRM; and (iii) the similarity of the amino acid composition and molecular weight of the protein to twitchin. In permeabilized ABRM, at all but maximum [Ca2+], phosphorylation of twitchin results in a decreased calcium sensitivity of force production (half-maximum at 2.5 vs. 1.3 microM calcium). At a given submaximal force, with equal numbers of force generators, twitchin phosphorylation increased unloaded shortening velocity approximately 2-fold. These data suggest that aspects of the catch state exist not only at resting [Ca2+], but also at higher submaximal [Ca2+]. The mechanism that gives rise to force maintenance in catch probably operates together, to some extent, with that of cycling myosin crossbridges.

Amino Acid Sequence↗

PubMed1998

Regulation of catch muscle by twitchin phosphorylation: effects on force, ATPase, and shortening.

Recent experiments on permeabilized anterior byssus retractor muscle (ABRM) of Mytilus edulis have shown that phosphorylation of twitchin releases catch force at pCa > 8 and decreases force at suprabasal but submaximum [Ca2+]. Twitchin phosphorylation decreases force with no detectable change in ATPase activity, and thus increases the energy cost of force maintenance at subsaturating [Ca2+]. Similarly, twitchin phosphorylation causes no change in unloaded shortening velocity (Vo) at any [Ca2+], but when compared at equal submaximum forces, there is a higher Vo when twitchin is phosphorylated. During calcium activation, the force-maintaining structure controlled by twitchin phosphorylation adjusts to a 30% Lo release to maintain force at the shorter length. The data suggest that during both catch and calcium-mediated submaximum contractions, twitchin phosphorylation removes a structure that maintains force with a very low ATPase, but which can slowly cycle during submaximum calcium activation. A quantitative cross-bridge model of catch is presented that is based on modifications of the Hai and Murphy (1988. Am. J. Physiol. 254:C99-C106) latch bridge model for regulation of mammalian smooth muscle.

Adenosine Triphosphatases↗

PubMed1998

Control of cross-bridge cycling by myosin light chain phosphorylation in mammalian smooth muscle.

This review focuses on experiments in which the single turnover of myosin-bound ADP is used to characterize the regulation of the cross-bridge cycle by myosin light chain phosphorylation in mammalian smooth muscle. Under isometric conditions, at rest, when the myosin light chain is not phosphorylated, myosin cycles very slowly (about 0.004 s-1), while phosphorylation of the light chain results in a 50-fold increase in cycling rate of 0.2 s-1. Experiments consistently show that some myosin does not increase its cycling rate although its light chain is phosphorylated. Studies at low levels of myosin light chain phosphorylation show that phosphorylation also induces an increase in the cycling rate of unphosphorylated myosin. The fast cycling phosphorylated myosin is the main determinant of suprabasal myosin ATPase activity, while the cycling rate of cooperatively activated unphosphorylated myosin is slow and appears to depend on the extent of phosphorylation of the entire thick filament. Single turnover experiments measuring the rate of phosphorylation and dephosphorylation of myosin light chain show that the turnover of light chain phosphate can be very rapid (0.3-0.4 s-1) at suprabasal calcium concentrations. The expected effect of such a rapid turnover of light chain phosphorylation on the turnover of myosin-bound ADP is not observed. The effects of low levels of myosin light chain phosphorylation on the single turnover of myosin suggest that the same small pool of myosin remains phosphorylated for relatively long periods of time rather than the entire pool of myosin spending a small fraction of its cycle time in the phosphorylated state.

Adenosine Diphosphate↗

PubMed1998

Clinical disease associated with simian agent 8 infection in the baboon.

Simian agent 8 (SA8) is an alphaherpesvirus that was first reported as a spontaneous natural infection in a captive baboon colony in 1988. It was first isolated from an African vervet monkey in 1958 and was classified as a simian agent. Simian agent 8 was later isolated from a baboon rectal swab specimen in 1969 and from an oral lesion in a vervet monkey in 1972. Restriction endonuclease analysis was used to identify the virus as SA8. In a 1-year period, 70 baboons housed in two outside 6-acre breeding corrals developed lesions principally on the genitalia and oral cavity. The incidence was the same for males and females, with recurrence rate, severity of the lesions, and duration for the lesions to resolve being greater in the female baboons. Lesions involving the mouth, tongue, and lips were most commonly observed in the juvenile population. The lesions tended to start as small multiple papules or vesicles, which advanced to large pustular or ulcerative areas. Using an every-other-day treatment regimen consisting of Nolvasan cleaning and procaine penicillin G injections, it took an average of 14 to 21 days for the lesions to resolve totally. Thirty-seven percent of the baboons with herpetic lesions experienced another episode of SA8 infection, usually within 1 year of development of the primary lesion. Several complications have been documented to be associated with SA8 infections. Partial or total vaginal obstruction is most common, leading to impaired breeding performance and pyelonephritis. A vaginal corrective surgical procedure has been developed to allow these females to return to productive breeding status within the colony. Penile urethral obstruction, also causing pyelonephritis, was observed in the male baboons. A case of sciatic neuritis was reported in a baboon that presented with self mutilation of the foot; viral isolation revealed the etiologic agent to be SA8. Four female baboons with chronic SA8 infections went on to develop perineal neoplasms. This is an economically important disease entity in captive baboons because it causes severe morbidity, decreased reproductive performance, and ultimately death in 1% of the baboon colony each year. The baboon is a promising animal model in which to study genital herpes as it relates to disease in human beings.

Alphaherpesvirinae↗

PubMed1997

Phosphorylation of a high molecular weight (approximately 600 kDa) protein regulates catch in invertebrate smooth muscle.

A unique property of smooth muscle is its ability to maintain force with a very low expenditure of energy. This characteristic is highly expressed in molluscan smooth muscles, such as the anterior byssus retractor muscle (ABRM) of Mytilus edulis, during a contractile state called 'catch'. Catch occurs following the initial activation of the muscle, and is characterized by prolonged force maintenance in the face of a low [Ca2+]i, high instantaneous stiffness, a very slow cross-bridge cycling rate, and low ATP usage. In the intact muscle, rapid relaxation (release of catch) is initiated by serotonin, and mediated by an increase in cAMP and activation of protein kinase A. We sought to determine which proteins undergo a change in phosphorylation on a time-course that corresponds to the release of catch in permeabilized ABRM. Only one protein consistently satisfied this criterion. This protein, having a molecular weight of approximately 600 kDa and a molar concentration about 30 times lower than the myosin heavy chain, showed an increase in phosphorylation during the release of catch. Under the mechanical conditions studied (rest, activation, catch, and release of catch), changes in phosphorylation of all other proteins, including myosin light chains, myosin heavy chain and paramyosin, are minimal compared with the cAMP-induced phosphorylation of the approximately 600 kDa protein. Under these conditions, somewhat less than one mole of phosphate is incorporated per mole of approximately 600 kDa protein. Inhibition of A kinase blocked both the cAMP-induced increase in phosphorylation of the protein and the release of catch. In addition, irreversible thiophosphorylation of the protein prevented the development of catch. In intact muscle, the degree of phosphorylation of the protein increases significantly when catch is released with serotonin. In muscles pre-treated with serotonin, a net dephosphorylation of the protein occurs when the muscle is subsequently put into catch. We conclude that the phosphorylation state of the approximately 600 kDa protein regulates catch.

Animals↗

PubMed1997

Hypertrophy of colonic smooth muscle: structural remodeling, chemical composition, and force output.

The cellular basis of adaptations occurring during the development of megacolon was studied with the lethal spotted mouse model. Age-dependent changes in the length-force characteristics of the colon reach a steady state by 3-4 mo and include an increased relative force development at very short muscle lengths. In megacolon the following occur: 1) structural remodeling expressed as a greater increase in the fraction of maximum force production at short lengths, a shift of optimum length (Lo) to longer lengths, and no change in force per square centimeter; 2) hypertrophy and hyperplasia of both circular and longitudinal muscle; 3) high resting compliance consistent with no disproportionate change in collagen or elastin composition; 4) marked distension so that in situ circumference approximately 1.8 Lo, where active force production is low, and 5) slack length approximately 0.65 Lo, as in normal colon. Biochemical remodeling in megacolon includes disproportionate increases in ATP and phosphocreatine concentration, with 3.5-fold more preformed phosphagen than in normal colon. The myosin concentration is the same in both muscles, but the actin concentration is 1.5-fold greater in megacolon. Most of the cellular changes in megacolon would facilitate high active force output from the muscle at much larger intestinal diameters.

Actins↗

PubMed1997

Hypertrophy of colonic smooth muscle: contractile proteins, shortening velocity, and regulation.

Smooth muscle in megacolon was studied in the lethal spotted mouse and its normal sibling. In megacolon, structural remodeling and a very large increase in total protein content are associated with some changes in the contractile protein isoform composition. 1) There is a higher actin concentration in megacolon, primarily caused by a larger proportion of gamma-isoforms. 2) There was no difference in myosin concentration or in SM1/SM2 heavy chains in megacolon and normal muscle contractile proteins. 3) Only LC17a essential light chain is present in both normal and megacolon. 4) The 25- to 50-kDa 5'-insert occurs in 15-20% of the myosin in normal colon, compared with 5- to 10-fold lower amounts in megacolon. In permeabilized muscles there was no significant difference in unloaded shortening velocity (Vo) with maximal thiophosphorylation of the 20-kDa light chains, nor was there significant difference in the force vs. Ca2+ and force vs. myosin light chain phosphorylation relationships. At approximately 60% myosin light chain phosphorylation after Ca2+ activation, Vo of megacolon was approximately two times faster than Vo of normal muscle. These cellular changes largely account for the higher propulsive velocity of the colon in situ. The distribution of myosin and actin isoforms and the lack of a simple relationship between myosin light chain phosphorylation and Vo point to the operation of additional regulatory processes.

Actins↗

PubMed1995

Thiophosphorylation of the 130-kDa subunit is associated with a decreased activity of myosin light chain phosphatase in alpha-toxin-permeabilized smooth muscle.

Pretreatment of alpha-toxin-permeabilized smooth muscle with ATP gamma S (adenosine 5'-O-(thiotriphosphate)) under conditions resulting in minimal (< 1%) thiophosphorylation of the myosin light chain increases the subsequent calcium sensitivity of force output and myosin light chain phosphorylation. The change in calcium sensitivity results at least in part from a 5-fold decrease in myosin light chain phosphatase activity. One of the few proteins thiophosphorylated under these conditions is the 130-kDa subunit of myosin light chain phosphatase. These results suggest that thiophosphorylation of this subunit leads to a decrease in the activity of the phosphatase, and that phosphorylation and dephosphorylation of the subunit may play a role in regulating myosin light chain phosphatase activity.

Adenosine Triphosphate↗

PubMed1994

Cross-bridge cycling at rest and during activation. Turnover of myosin-bound ADP in permeabilized smooth muscle.

Single turnover experiments were performed on myosin-bound ADP by measuring the time course of incorporation of [3H]ADP following rapid formation of [3H]ATP by photolysis of caged [3H]ATP. Permeabilized rabbit portal veins were incubated in a solution at 20 degrees C with 1 mM MgATP, 20 mM phosphocreatine, 1 mg/ml creatine phosphokinase, and containing [14C]ATP and high specific activity caged [3H]ATP. At variable times following a UV flash, the muscle was frozen, nucleotides were extracted, and the ratio 3H:14C in ADP was compared to that in ATP. At rest, the exchange of bound ADP occurred with a rate constant of 0.004 s-1. When the myosin light chain was about 80% thiophosphorylated, and the muscle was generating maximum isometric force, there appeared a fast phase of ADP exchange (44% of the total) which had a rate constant of 0.2 s-1. The change in rate of ADP exchange on myosin is sufficient to explain the measured increase in ATPase activity upon thiophosphorylation of the myosin light chain. A simple analysis of the data suggests that there is a 50-fold increase in the cycling rate of cross-bridges in the muscle upon phosphorylation under isometric conditions. The fraction of ADP exchanged at 10 s following photolytic release of [3H]ATP was found to be approximately linearly related to the degree of thiophosphorylation of the myosin light chain. This supports the idea that phosphorylation of the light chain causes the transition of myosin from the resting (slow ATPase) cycle into the activated (fast ATPase) cycle, and that the fraction of myosin in the fast cycle is directly determined by the degree of light chain phosphorylation. The data are also consistent with the cooperativity model described previously by Vyas et al.

Adenosine Diphosphate↗

PubMed1994

Comparison of the effects of 2,3-butanedione monoxime on force production, myosin light chain phosphorylation and chemical energy usage in intact and permeabilized smooth and skeletal muscles.

The primary goal of this study was to determine the utility of 2,3-butanedione monoxime as a tool for determining and separating the chemical energy usage associated with force production from that of force-independent, or 'activation' processes in smooth and skeletal muscles. We determined the effects of 2,3-butanedione monoxime on force production, myosin light chain phosphorylation and high energy phosphate usage in intact and permeabilized smooth (rabbit taenia coli) and skeletal (mouse extensor digitorum longus) muscles. In the intact taenia coli, 2,3-butanedione monoxime depressed the tonic phase of the tetanus, contractures evoked by high potassium (90 mM) and by carbachol (10(-5) M) and the small force response evoked by these agonists after treatment with D-600 (10(-5) M). In the electrically stimulated intact taenia coli 2,3-butanedione monoxime (0-20 mM) caused a proportional inhibition of tetanic force output, myosin light chain phosphorylation and high energy phosphate usage (ED50 approximately 7 mM for all these parameters). At 20 mM 2,3-butanedione monoxime, force and energy usage fell to near zero and the degree of myosin light chain phosphorylation decreased to resting values, indicating a shut-down of both force-dependent and force-independent energy usage at high concentrations of 2,3-butanedione monoxime. In permeabilized taenia coli, 2,3-butanedione monoxime had little or no depressant effects on force production, ATPase activity or calcium sensitivity. 2,3-butanedione monoxime had a very modest inhibitory effect on the in vitro motility of unregulated actin filaments interacting with thiophosphorylated myosin. In solution, 2,3-butanedione monoxime inhibited myosin light chain kinase, but not the phosphatase (SMP-IV). These results suggest that the major effect of 2,3-butanedione monoxime is not on the contractile proteins themselves, but rather on calcium delivery during excitation, thereby reducing the degree of activation of myosin light chain kinase and subsequent activation of myosin by light chain phosphorylation. Thus, 2,3-butanedione monoxime is not useful for the determination of the energetics of activation processes in smooth muscle because of its inhibition of both force-dependent and force-independent processes. In contrast, in the intact mouse extensor digitorum longus, 2,3-butanedione monoxime inhibits tetanic force production (ED50 approximately 2 mM) to a much greater extent than myosin light chain phosphorylation. When 2,3-butanedione monoxime was used to manipulate force production in muscles at L(o), it was found that approximately 60% of the total energy usage was force-independent and the remainder was force-dependent.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenosine Triphosphatases↗

PubMed1994

Xenotransplantation.

Transplantation of solid organs (heart, lung, liver, and kidney) from swine to humans would solve the current critical shortage of cadaver organs needed by patients with end-stage disease of these organs. In addition, transplantation between distant species (discordant xenografting) will require an understanding of a number of unique immunologic features. Discordant xenografts are rejected within minutes to hours after transplantation. This rejection is due to natural immunity by recipients never before exposed to the xenografts. In some species combinations, this fulminant rejection is due to naturally occurring pre-existing antibodies against the xenograft endothelium. In other species combinations, the xenograft activates the alternative pathway of complement. The swine to human species combination is the most clinically relevant. In this combination, natural human and private antibodies recognize alpha-galactosyl residues of glycoproteins and glycolipids. Potential future therapeutic measures to prevent natural immunity include the genetic engineering of human complement inhibitors into swine cell membranes or genetic "knock out" of the enzymes responsible for placing alpha-galactosyl residues on swine cell surfaces. There are also special considerations in acquired immunity against xenografts. Cytokines and adhesion molecules may not work across species lines. Xenograft antigens may have to be processed by host antigen-presenting cells in order to effectively stimulate the immune system.

Animals↗

PubMed1994

Metabolic characteristics of alpha-toxin-permeabilized smooth muscle.

Rabbit portal veins were permeabilized using Staphylococcus aureus alpha-toxin, and adenosinetriphosphatase (ATPase) was measured as the formation of [3H]ADP, [3H]AMP, and [3H]adenosine from [3H]ATP in the solution bathing the muscle. The resting ATPase (1.96 +/- 0.15 mM/min, n = 13) is approximately 5-10 times higher than that measured in Triton X-100-permeabilized muscles (0.28 +/- 0.01 mM/min, n = 4), with nucleotide accumulating as ADP, AMP, and adenosine. The ATPase activity is also seen when the intact muscle is incubated in a Krebs solution containing 1 mM MgATP (2.76 +/- 0.10 mM/min, n = 73). This suggests that it is due primarily to an ecto-ATPase. The ectoenzyme is capable of hydrolyzing both ATP and ADP, and in both cases there is a higher rate at 3 than at 1 mM nucleotide. The high resting ATPase compromises the control of nucleotide concentrations within the permeabilized tissue even in the presence of an ATP-regenerating system consisting of phosphocreatine (PCr, 35mM) and creatine kinase (1 mg/ml). Treatment of the intact muscle with the ectonucleotidase inhibitor 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) followed by alpha-toxin permeabilization and inclusion of sodium azide in subsequent solutions reduces the ecto-ATPase by approximately 70%. Addition of PCr and creatine kinase then results in the maintenance of high [ATP] and low [ADP] in the muscle, and importantly, there are no significant changes in [ATP], [ADP], [adenosine/AMP], or the ADP-to-ATP ratio upon activation of the muscle in pCa 4.5. In general, the force output in high Ca2+ increased as the metabolic profile of the muscle improved. When ATPase was measured as the appearance of [32P]Pi from [32P]PCr and [gamma-32P]ATP, the alpha-toxin-permeabilized muscle subjected to the above treatment showed only approximately 30% higher total ATPase under activated conditions compared with the freeze-glycerinated Triton-treated portal vein. The suprabasal ATPase is similar in both preparations. We conclude that the reduction of the basal ATPase by the DIDS-azide treatment permits both rigorous control of nucleotide contents and accurate measurement of ATPase activity in alpha-toxin-permeabilized smooth muscle.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗