Transgenic cardiac-targeted overexpression of human thymidylate kinase.

Thymidylate kinase (TMPK) is a nucleoside monophosphate kinase that catalyzes phosphorylation of thymidine monophosphate to thymidine diphosphate. TMPK also mediates phosphorylation of monophosphates of thymidine nucleoside analog (NA) prodrugs on the pathway to their active triphosphate antiviral or antitumor moieties. Novel transgenic mice (TG) expressing human (h) TMPK were genetically engineered using the alpha-myosin heavy chain promoter to drive its cardiac-targeted overexpression. In '2 by 2' protocols, TMPK TGs and wild-type (WT) littermates were treated with the NA zidovudine (a deoxythymidine analog, 3'-azido-3'deoxythymidine (AZT)) or vehicle for 35 days. Alternatively, TGs and WTs were treated with a deoxycytidine NA (racivir, RCV) or vehicle. Changes in mitochondrial DNA (mtDNA) abundance and mitochondrial ultrastructure were defined quantitatively by real-time PCR and transmission electron microscopy, respectively. Cardiac performance was determined echocardiographically. Results showed TMPK TGs treated with either AZT or RCV exhibited decreased cardiac mtDNA abundance. Cardiac ultrastructural changes were seen only with AZT. AZT-treated TGs exhibited increased left ventricle (LV) mass. In contrast, LV mass in RCV-treated TGs and WTs remained unchanged. In all cohorts, LV end-diastolic dimension remained unchanged. This novel cardiac-targeted overexpression of hTMPK helps define the role of TMPK in mitochondrial toxicity of antiretrovirals.

Murine cardiac mtDNA: effects of transgenic manipulation of nucleoside phosphorylation.

Mitochondrial toxicity results from pyrimidine nucleoside reverse transcriptase inhibitors (NRTIs) for HIV/AIDS. In the heart, this can deplete mitochondrial (mt) DNA and cause cardiac dysfunction (eg, left ventricle hypertrophy, LVH). Four unique transgenic, cardiac-targeted overexpressors (TGs) were generated to determine their individual impact on native mitochondrial biogenesis and effects of NRTI administration on development of mitochondrial toxicity. TGs included cardiac-specific overexpression of native thymidine kinase 2 (TK2), two pathogenic TK2 mutants (H121N and I212N), and a mutant of mtDNA polymerase, pol-gamma (Y955C). Each was treated with antiretrovirals (AZT-HAART, 3 or 10 weeks, zidovudine (AZT) + lamivudine (3TC) + indinavir, or vehicle control). Parameters included left ventricle (LV) performance (echocardiography), LV mtDNA abundance (real-time PCR), and mitochondrial fine structure (electron microscopy, EM) as a function of duration of treatment and presence of TG. mtDNA abundance significantly decreased in Y955C TG, increased in TK2 native and I212N TGs, and was unchanged in H121N TGs at 10 weeks regardless of treatment. Y955C and I212N TGs exhibited LVH during growth irrespective of treatment. Y955C TGs exhibited cardiomyopathy (CM) at 3 and 10 weeks irrespective of treatment, whereas H121N and I212N TGs exhibited CM only after 10 weeks AZT-HAART. EM features were consistent with cardiac dysfunction. mtDNA abundance and cardiac functional changes were related to TG expression of mitochondrially related genes, mutations thereof, and NRTIs.

Tenofovir renal toxicity targets mitochondria of renal proximal tubules.

Tenofovir disoproxil fumarate (TDF) is an analog of adenosine monophosphate that inhibits HIV reverse transcriptase in HIV/AIDS. Despite its therapeutic success, renal tubular side effects are reported. The mechanisms and targets of tenofovir toxicity were determined using '2 x 2' factorial protocols, and HIV transgenic (TG) and wild-type (WT) littermate mice with or without TDF (5 weeks). A parallel study used didanosine (ddI) instead of TDF. At termination, heart, kidney, and liver samples were retrieved. Mitochondrial DNA (mtDNA) abundance, and histo- and ultrastructural pathology were analyzed. Laser-capture microdissection (LCM) was used to isolate renal proximal tubules for molecular analyses. Tenofovir increased mtDNA abundance in TG whole kidneys, but not in their hearts or livers. In contrast, ddI decreased mtDNA abundance in the livers of WTs and TGs, but had no effect on their hearts or kidneys. Histological analyses of kidneys showed no disruption of glomeruli or proximal tubules with TDF or ddI treatments. Ultrastructural changes in renal proximal tubules from TDF-treated TGs included an increased number and irregular shape of mitochondria with sparse fragmented cristae. LCM-captured renal proximal tubules from TGs showed decreased mtDNA abundance with tenofovir. The results indicate that tenofovir targets mitochondrial toxicity on the renal proximal tubule in an AIDS model.

Transgenic mitochondrial superoxide dismutase and mitochondrially targeted catalase prevent antiretroviral-induced oxidative stress and cardiomyopathy.

Transgenic mice (TG) were used to define mitochondrial oxidative stress and cardiomyopathy (CM) induced by zidovudine (AZT), an antiretroviral used to treat HIV/AIDS. Genetically engineered mice either depleted or overexpressed mitochondrial superoxide dismutase (SOD2(+/-) KOs and SOD2-OX, respectively) or expressed mitochondrially targeted catalase (mCAT). TGs and wild-type (WT) littermates were treated (oral AZT, 35 days). Cardiac mitochondrial H(2)O(2), aconitase activity, histology and ultrastructure were analyzed. Left ventricle (LV) mass and LV end-diastolic dimension were determined echocardiographically. AZT induced cardiac oxidative stress and LV dysfunction in WTs. Cardiac mitochondrial H(2)O(2) increased and aconitase was inactivated in SOD2(+/-) KOs, and cardiac dysfunction was worsened by AZT. Conversely, the cardiac function in SOD2-OX and mCAT hearts was protected. In SOD2-OX and mCAT TG hearts, mitochondrial H(2)O(2), LV mass and LV cavity volume resembled corresponding values from vehicle-treated WTs. AZT worsens cardiac dysfunction and increases mitochondrial H(2)O(2) in SOD2(+/-) KO. Conversely, both SOD2-OX and mCAT TGs prevent or attenuate AZT-induced cardiac oxidative stress and LV dysfunction. As dysfunctional changes are ameliorated by decreasing and worsened by increasing H(2)O(2) abundance, oxidative stress from H(2)O(2) is crucial pathogenetically in AZT-induced mitochondrial CM.

Multiple sex pheromones and receptors of a mushroom-producing fungus elicit mating in yeast.

The mushroom-producing fungus Schizophyllum commune has thousands of mating types defined, in part, by numerous lipopeptide pheromones and their G protein-linked receptors. Compatible combinations of pheromones and receptors encoded by different mating types regulate a pathway of sexual development leading to mushroom formation and meiosis. A complex set of pheromone-receptor interactions maximizes the likelihood of outbreeding; for example, a single pheromone can activate more than one receptor and a single receptor can be activated by more than one pheromone. The current study demonstrates that the sex pheromones and receptors of Schizophyllum, when expressed in Saccharomyces cerevisiae, can substitute for endogenous pheromone and receptor and induce the yeast pheromone response pathway through the yeast G protein. Secretion of active Schizophyllum pheromone requires some, but not all, of the biosynthetic machinery used by the yeast lipopeptide pheromone a-factor. The specificity of interaction among pheromone-receptor pairs in Schizophyllum was reproduced in yeast, thus providing a powerful system for exploring molecular aspects of pheromone-receptor interactions for a class of seven-transmembrane-domain receptors common to a wide range of organisms.

A mushroom-inducing DNA sequence isolated from the Basidiomycete, Schizophyllum commune.

A DNA sequence capable of inducing the de novo development of fruiting bodies (mushrooms) when integrated into the genome of unmated, nonfruiting strains of the Basidiomycete Schizophyllum commune has been isolated and partially characterized. This sequence, designated FRT1, overrides the normal requirement of a mating interaction for fruiting in this organism. It has been shown to integrate stably in different chromosome locations and appears to be trans-acting. It also enhances the normal process of fruiting that occurs after mating. Additional DNA sequences with similarity to FRT1 were detected within the genome of the strain of origin by hybridization of labeled FRT1 DNA to blots of digested genomic DNAs. FRT1 and the genomic sequences similar to it were shown to be genetically linked. Southern hybridization experiments suggested sequence divergence at the FRT1 locus between different strains of S. commune. A testable model for how FRT1 may act as a key element in the pathway for the differentiation of fruiting bodies is presented as a working hypothesis for further investigation.

Multiple genes encoding pheromones and a pheromone receptor define the B beta 1 mating-type specificity in Schizophyllum commune.

The genes defining multiple B mating types in the wood-rotting mushroom Schizophyllum commune are predicted to encode multiple pheromones and pheromone receptors. These genes are clustered in each of two recombinable and independently functioning loci, B alpha and B beta. A difference in specificity at either locus between a mated pair of individuals initiates an identical series of events in sexual morphogenesis. The B alpha 1 locus was recently found to contain genes predicted to encode three lipopeptide pheromones and a pheromone receptor with a seven-transmembrane domain. These gene products interact in hetero-specific pairs, the pheromone of one B alpha specificity with the receptor of any one of the other eight B alpha specificities, and are likely to activate a signaling cascade similar to that known for mating in Saccharomyces cerevisiae. We report here that the B beta 1 locus also contains at least three pheromone genes and one pheromone receptor gene, which function similarly to the genes in the B alpha 1 locus, but only within the series of B beta specificities. A comparison of the DNA sequences of the B alpha 1 and B beta 1 loci suggests that each arose from a common ancestral sequence, allowing us to speculate about the evolution of this unique series of regulatory genes.

Changes in mate recognition through alterations of pheromones and receptors in the multisexual mushroom fungus Schizophyllum commune.

Schizophyllum commune has thousands of mating types defined in part by numerous lipopeptide pheromones and their G-protein-coupled receptors. These molecules are encoded within multiple versions of two redundantly functioning B mating-type loci, B alpha and B beta. Compatible combinations of pheromones and receptors, produced by individuals of different B mating types, trigger a pathway of fertilization required for sexual development. Analysis of the B beta 2 mating-type locus revealed a large cluster of genes encoding a single pheromone receptor and eight different pheromones. Phenotypic effects of mutations within these genes indicated that small changes in both types of molecules could significantly alter their specificity of interaction. For example, a conservative amino acid substitution in a pheromone resulted in a gain of function toward one receptor and a loss of function with another. A two-amino-acid deletion from a receptor precluded the mutant pheromone from activating the mutant receptor, yet this receptor was activated by other pheromones. Sequence comparisons provided clues toward understanding how so many variants of these multigenic loci could have evolved through duplication and mutational divergence. A three-step model for the origin of new variants comparable to those found in nature is presented.

Soft drugs. 3. A new class of anticholinergic agents.

A new class of antimuscarinic drugs was designed and synthesized. The compounds are "soft" quaternary ammonium esters in which there is only one carbon atom separating the ester oxygen and the quaternary head. The compounds are potent anticholinergics when derived from hindered "umbrella" acids and cholinergics when derivatives of simple aliphatic acids. The more potent anticholinergics have up to 10 times higher acetylcholine antagonist activity than atropine, but they have a much shorter duration of action. The compounds cleave hydrolytically with simultaneous destruction of the quaternary head. The compounds are promising as selective, local agents, particularly as inhibitors of eccrine sweating.

Heparin-neutralizing activity, total progressive antithrombin and ethanol gel test in acute chest pain.

92 patients admitted to a cardiac monitoring unit with chest pain were investigated. The mean heparin-neutralizing activity of the 34 patients with severe myocardial infarction was found to be significantly different from the mean of 70 normal controls. However, there was considerable overlap between individual observations and the normal range. Only 5 of the 34 patients with severe myocardial infarction had antithrombin levels outside the normal range. In patients with a severe myocardial infarct tested on the 2nd and 3rd day after infarction there was s significant increase in the number of positive ethanol gel tests compared with the other patients studied.

Some studies on peripheral actions of mephenesin, methocarbamol and diazepam.

1. Mephenesin, methocarbamol and diazepam abolished polysynaptic reflex contractions of the cat tibialis anterior muscle elicited by stimulation of the homolateral femoral nerve.2. Mephenesin and methocarbamol caused a prolongation of the mean refractory period of directly or indirectly stimulated skeletal muscle. These effects were due to a direct action on the muscle fibres. There was no effect on responses to single stimuli.3. The increase in refractory period produced by mephenesin was greater in indirectly than in directly stimulated rat diaphragms. Experiments using the isolated phrenic nerve suggest that this difference is due to the local anaesthetic action of mephenesin.4. In the indirectly stimulated cat tibialis anterior muscle high frequency stimulation resulted in a non-maintained tetanus in the presence of mephenesin and methocarbamol.5. Diazepam was without peripheral effects on the responses of skeletal muscle.6. The results with mephenesin and methocarbamol are discussed in relation to their mode of action in reducing muscle spasm.

Prejunctional actions of some beta-adrenoreceptor antagonists in the vas deferens preparation of the guinea-pig.

1. The beta-adrenoceptor antagonists propranolol, pronethalol, MJ 1999 and Ciba 39089-Ba reduced responses to field stimulation of the guinea-pig isolated vas deferens preparation without significantly affecting responses to exogenously added noradrenaline.2. This prejunctional blocking action of the drugs cannot be correlated with their action as beta-adrenoceptor antagonists or non-specific depressants.3. The blockade produced was more pronounced at low (5-20 Hz) than at high (50 Hz) frequencies of stimulation.4. The blockade was slow in onset, and once established was poorly reversed by washing the preparation over a period of 1 to 2 h.5. The blockade produced could be reversed by dexamphetamine and cocaine.6. These experiments suggest that the beta-adrenoceptor antagonists may have some actions which closely resemble those of the adrenergic neurone blocking agent guanethidine.

Mephenesin, methocarbamol, chlordiazepoxide and diazepam: actions on spinal reflexes and ventral root potentials.

1. Dose levels of mephenesin, methocarbamol, chlordiazepoxide and diazepam which abolished polysynaptic reflex contractions had no effect on monosynaptic knee-jerk reflexes in chloralose anaesthetized cats.2. Ventral root potentials were recorded following stimulation of the corresponding dorsal root (L7 or S1), and the areas of the mono- and polysynaptic components were measured by planimetry.3. Dose levels of the drugs which abolished polysynaptic reflex contractions reduced the areas of the polysynaptic component of the ventral root potentials by about 50%. Mephenesin and methocarbamol reduced the area of the monosynaptic component to a similar extent. Chlordiazepoxide was less potent in this respect while diazepam was without effect at this dose level.4. Linear regression lines were calculated for the reduction in the mono- and polysynaptic components of ventral root potentials with increasing doses of each of the four drugs. With methocarbamol and mephenesin the lines were parallel and coincident. With chlordiazepoxide and diazepam they were parallel but not coincident. Large doses of diazepam were required to reduce the area of the monosynaptic component, this drug being the only one of the four tested to have a differential action on the two components which was statistically significant.5. The results are discussed in terms of depressant actions of the drugs on alpha-motorneurones, effects of the drugs at higher centres concerned with motor function, and the lack of evidence that spinal interneurones represent a specific site of action for centrally acting skeletal muscle relaxants.

Pyrrolizidine alkaloids: actions on muscarinic receptors in the guinea-pig ileum.

1. Eleven pyrrolizidine alkaloids have been tested on the isolated guinea-pig ileum preparation.2. Platyphylline, supinine, heleurine and cynaustraline were more potent in antagonizing responses to acetylcholine and carbachol than responses to histamine. Their anticholinergic activity appeared to involve a competitive mechanism.3. Lasiocarpine, monocrotaline, spectabiline, sarracine, 7-angelylheliotridine, heliotrine and senecionine had similar antagonistic potencies against responses to both acetylcholine and histamine.4. The alkaloids had no appreciable activity as antagonists of acetylcholine in the isolated toad rectus abdominis preparation.5. These results are discussed with respect to interactions of the alkaloids at receptor sites involved in anticholinergic activity at the muscarinic receptor.

Use of cumulative dose-response curves in potency comparisons of sympathomimetic amines on the cat soleus muscle.

The ability of beta-adrenoceptor agonists to reduce the fusion of incomplete tetanic contractions of the soleus muscle of the cat has been used previously as a model to assess the potential tremor producing effect of sympathomimetic bronchodilators. The ability of (-)-isoprenaline, (-)-adrenaline, (+/-)-soterenol and (+/-)-quinterenol to depress incomplete tetanic contractions of the soleus has now been assessed using cumulative administration of the amines. The method quickly produced accurate and reproducible dose-response curves. It is particularly useful for evaluating the potency of long-acting compounds.

Comparison of pre-junctional alpha-adrenoceptors at the neuromuscular junction with vascular post-junctional alpha-receptors in cat skeletal muscle.

1 Activation of pre-junctional alpha-adrenoceptors at the skeletal neuromuscular junction enhances acetylcholine release whereas activation of such receptors at autonomic nerve endings inhibits transmitter output. In the present study the characteristics of pre-junctional alpha-adrenoceptors at motor nerve terminals have been compared with post-junctional (vascular) alpha-adrenoceptors in the cat hind limb.2 Reversal of partial (+)-tubocurarine blockade of contractions of the tibialis anterior muscle was used to monitor pre-junctional activity and increases in hindlimb vascular resistance to assess post-junctional actions at alpha-adrenoceptors.3 Responses to intra-arterial injections of noradrenaline, adrenaline, phenylephrine, oxymetazoline, methoxamine and clonidine were monitored. Dose-response lines for all the compounds except clonidine were parallel. The latter agent produced only weak and inconsistent effects.4 Ratios of the doses of the agents required to produce pre- and post-junctional effects indicated that oxymetazoline and adrenaline possessed some preferential activity at post-junctional sites, whereas the remaining agents were non-selective in their actions. If dose-ratios with respect to noradrenaline were compared at the two sites none of the compounds possessed a marked degree of selectivity.5 In the presence of phentolamine or tolazoline, dose-response curves to the pre- and post-junctional effects of phenylephrine were shifted to a similar extent. Thymoxamine showed preferential activity as a pre-junctional alpha-receptor antagonist.6 In comparing the results of this study with those of other authors, it is apparent that there are marked differences in the characteristics of pre-junctional alpha-receptors at the skeletal neuromuscular junction and at autonomic nerve endings. The pre- and post-junctional alpha-receptors in skeletal muscle show less divergence.

Post-tetanic and drug-induced repetitive firing in the soleus muscle of the cat.

1. The effects of a high frequency indirect tetanus on the responses to subsequent infrequently applied nerve shocks have been compared in the tibialis anterior and soleus muscles of cats and rabbits.2. Post-tetanic augmentation of twitches in the cat soleus muscle was shown to be partly due to repetitive firing and partly due to increased synchronization of the muscle fibre response. Post-tetanic repetitive firing was not evident in the responses of the other three muscles studied.3. Post-tetanic repetitive responses in the cat soleus muscle and nerve did not originate in the nerve trunk and were not produced by direct muscle stimulation; they were abolished by doses of tubocurarine smaller than those necessary to reduce the twitch tension below the pre-tetanic level. These findings support the conclusion of others that the repetitive firing originates at the neuromuscular junction.4. The repetitive firing could not be explained by an increase in the sensitivity of the motor endplates to acetylcholine, suggesting that an increase in and/or a prolongation of the output of transmitter from the motor nerve contributes to it.5. The cat soleus muscle was shown to be more sensitive to neostigmine than were the other three muscles studied, and acetylcholinesterase determinations showed that this muscle possesses less enzyme activity.6. It is concluded that an increase in transmitter output, coupled with a weaker cholinesterase activity, probably accounts for the post-tetanic repetitive activity in the cat soleus muscle.7. Post-tetanic repetitive firing was absent in cat soleus muscles which had been cross-innervated with the nerve formerly innervating a fast-contracting muscle.

Catechol-substituted phenoxypropanolamines: adrenoceptor activity in the anaesthetized cat.

1 The pharmacological actions of racemic noradrenaline, adrenaline, isoprenaline and N-t-butylnoradrenaline have been compared with those of their corresponding derivatives containing an oxymethylene (OXY) link between the ring and ethanolamine side chain. 2 The compounds were tested in the anaesthetized cat for their ability to produce positive chronotropic effects, bronchodilator actions, changes in perfusion pressure in the perfused hind limb and decreases in soleus muscle contractions. 3 All the OXY-derivatives were potent beta-adrenoceptor agonists. The inclusion of the oxymethylene link promotes selectivity for beta1-as opposed to beta2-adrenoceptor activity. 4 In comparison with the parent compounds, the OXY-derivatives of adrenaline and noradrenaline had very weak alpha-adrenoceptor stimulant effects.

The in vitro pharmacology of xamoterol (ICI 118,587).

The effect of xamoterol and (-)-isoprenaline have been compared for their activity at beta-adrenoceptor sites in a number of in vitro cardiac and smooth muscle preparations. Xamoterol produced weak positive chronotropic effects in guinea-pig, rat and cat atria (intrinsic activity less than 0.55, (-)-isoprenaline = 1). Positive inotropic effects were obtained in driven left atria of the cat but were absent in guinea-pig left atrial and right ventricular strip preparations. Agonistic effects were due to beta 1-adrenoceptor stimulation. Xamoterol was without beta-adrenoceptor-mediated inhibitory effects in guinea-pig ileal, tracheal and uterine preparations and in the rat vas deferens and oestrogen-primed uterus. Weak beta 2-adrenoceptor-mediated relaxation was obtained in progesterone-primed rat uteri. Xamoterol produced non-specific inhibitory effects in guinea-pig ileal and tracheal preparations. Xamoterol acted as a competitive antagonist at beta 1-(pA2 range = 7.4 to 7.8) and beta 2-adrenoceptors (pA2 range 5.2 to 6.2) and displaced [125I]-iodocyanopindolol from guinea-pig left atrial (pKD = 7.25) and uterine (pKD 5.24) membrane preparations. It is concluded that xamoterol displays a selective affinity for beta 1-adrenoceptors. Although its partial agonistic actions are more evident at beta 1-adrenoceptor sites, like prenalterol, xamoterol displays a degree of tissue rather than receptor-dependent selectivity.

Pre- and postjunctional effects of clonidine- and oxymetazoline-like compounds in guinea-pig ileal preparations.

1 Noradrenaline and 28 imidazolidine (clonidine-like) and imidazoline (oxymetazoline-like) compounds with various phenyl ring substituents have been examined for their ability to inhibit responses to transmural stimulation and exogenous acetylcholine in ileal preparations from reserpine-treated guinea-pigs.2 The bathing solution contained propranolol, mepyramine, cimetidine and desipramine to preclude interference with the responses by other than the alpha-receptor-mediated actions of the compounds.3 In transmurally stimulated preparations the inhibitory response to noradrenaline is due to a combination of prejunctional alpha-adrenoceptor stimulation and a postjunctional depressant effect that does not involve adrenoceptor activation.4 Of the 28 imidazolidines and imidazolines studied, 21 inhibited transmurally elicited responses. In the various compounds studied this effect involved actions at pre- or postjunctional sites as indicated by (a) the frequency-dependence of the inhibitory response, (b) its susceptibility to blockade by alpha-receptor antagonists and (c) the relative concentrations required to inhibit responses to transmural stimulation and exogenous acetylcholine.