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Biomedical subjects

D G Allen

Publications and source records attributed to D G Allen.

At least 55 records · Page 3Linked to original sources
PubMed1999

The use of caged adenine nucleotides and caged phosphate in intact skeletal muscle fibres of the mouse.

The effects of 1-(2-nitrophenyl)ethyl ester of ATP (NPE-caged ATP), NPE-caged ADP, NPE-caged phosphate (Pi) and desoxybenzoinyl phosphate (desyl-caged Pi) on mouse skeletal muscle function were studied. All these caged compounds, when microinjected into intact single mouse muscle fibres, reduced the myoplasmic calcium during a tetanus (tetanic [Ca2+]i) and reduced force. Flash photolysis partially reversed this reduction of tetanic [Ca2+]i and force. In fibres fatigued by repeated tetani, flash photolysis of NPE-caged ATP, ADP and Pi, also caused a transient recovery of tetanic [Ca2+]i, and force. Because photolytic release of ATP, ADP and Pi produced comparable effects it seems that the mechanism of action is the reduction in concentration of the caged compound rather than the release of the biologically active molecule. Experiments on mechanically skinned rat skeletal muscle fibres with intact T-tubular/sarcoplasmic reticulum coupling showed that 1 mM NPE-caged ATP had no effect on depolarization-induced force. This result suggests that the depressant effects of the NPE-caged compounds are neither on voltage-activated Ca2+ release from the sarcoplasmic reticulum nor on myofibrillar function. Thus all the caged compounds tested inhibit excitation-contraction coupling in muscle by an unknown mechanism and this limits their value as sources of biologically important molecules. This inhibitory effect was smallest for desyl-caged Pi and under conditions of maximal activation photolytic release of Pi caused a direct inhibition of the contractile proteins. This inhibition amounted to a 1% reduction of maximum force with an increase of [Pi] of about 0.3 mM. The mean rate of force decline under these conditions was 55 s-1, which reflects the rate of cross-bridge cycling during a maximal tetanus.

Adenosine Diphosphate↗
PubMed1999

Survival of patients with epithelial ovarian cancer and the effect of lymphadenectomy in those with stage 3 disease.

This study looks at the 10-year survival data in patients with epithelial ovarian carcinoma at the Mercy Hospital for Women, Melbourne. An ovarian cancer database was established at the hospital in 1980, since then 253 patients have been diagnosed with epithelial ovarian cancer. The 5-year survival rates for Stages 1 to 4 are 75%, 55%, 24% and 21% respectively. The 10-year survival rates are 65%, 55%, 16% and 15% respectively. One hundred and thirty patients have been diagnosed with Stage 3 disease. The Gynaecologic Oncology Department at the Mercy Hospital for Women was formalized in 1987. Patients treated for ovarian cancer by the unit since 1987 had a significantly better survival (median 47 months) than patients treated prior to 1987 (median 17 months), p = 0.03. There is much debate as to whether lymphadenectomy in patients with ovarian cancer is of therapeutic value. In this study the patients with Stage 3 disease and who had a lymphadenectomy performed had a better 5-year survival rate of 38% compared to 22% in the group who did not have a lymphadenectomy (p < 0.05). The Stage 3 patients who had negative retroperitoneal nodes had a 10-year survival rate of 51%. There was no difference in survival rate between patients with endometrioid and serous papillary carcinomas.

Australia↗
PubMed1999

Changes in intracellular Na+ and pH in rat heart during ischemia: role of Na+/H+ exchanger.

The role of the Na+/H+ exchanger in rat hearts during ischemia and reperfusion was investigated by measurements of intracellular Na+ concentration ([Na+]i) and intracellular and extracellular pH. Under our standard conditions (2-Hz stimulation), 10 min of ischemia caused no significant rise in [Na+]i but an acidosis of 1.0 pH unit, suggesting that the Na+/H+ exchanger was inactive during ischemia. This was confirmed by showing that the Na+/H+ exchange inhibitor methylisobutyl amiloride (MIA) had no effect on [Na+]i or on intracellular pH during ischemia. However, there was a short-lived increase in [Na+]i of 8.2 +/- 0.6 mM on reperfusion, which was reduced by MIA, showing that the Na+/H+ exchanger became active on reperfusion. To investigate the role of metabolic changes, we measured [Na+]i during anoxia. The [Na+]i did not change during 10 min of anoxia, but there was a small, transient rise of [Na+]i on reoxygenation, which was inhibited by MIA. In addition, we show that the Na+/H+ exchanger, tested by sodium lactate exposure, was inhibited during anoxia. These results show that the Na+/H+ exchanger is inhibited during ischemia and anoxia, probably by an intracellular metabolic mechanism. The exchanger activates rapidly on reperfusion and can cause a rapid rise in [Na+]i.

Acidosis↗
PubMed1999

Early events in stretch-induced muscle damage.

Unaccustomed exercise involving stretch of active muscle at long length causes an immediate loss of tension-generating capacity, a shift of optimum length, and changes in excitation-contraction coupling. Eventually, fiber damage may be observed, resulting in pain and tenderness. The subject of this review is the early stage in this process, particularly the cause of the immediate drop in tension. There is strong evidence pointing to sarcomere length instabilities and nonuniformities as important contributors to these changes. The evidence includes the influence of initial length, electron microscopy of rapidly fixed active fibers, the shift in optimum length in single fibers, and the effects of training on sacomere numbers. Experiments using Ca(2+)-sensitive dyes clearly show changes in excitiation-contraction coupling, but cross-species comparisons indicate that these are not always able to explain the consequences seen. We conclude that sarcomere length instabilities provide the most comprehensive explanation of the early consequences of eccentric exercise.

Animals↗
PubMed1998

The contribution of pH-dependent mechanisms to fatigue at different intensities in mammalian single muscle fibres.

1. The contribution of intracellular pH (pHi) to the failure of Ca2+ release and inhibition of contractile proteins observed during fatigue was assessed in single intact mouse muscle fibres at 22 C. Fatigue was induced by repeated tetani at intensities designed to induce different levels of intracellular acidosis. Force and either intracellular free Ca2+ concentration ([Ca2+]i; measured using indo-1) or pHi (measured using SNARF-1) were recorded in fibres fatigued at two different intensities. 2. Intensity was varied by the repetition rate of tetani and quantified by the duty cycle (the fraction of time when the muscle was tetanized). Stimulation at the low intensity (duty cycle approximately 0.1) reduced force to 30 % of initial values in 206 +/- 21 s (60 +/- 7 tetani); at the high intensity (duty cycle approximately 0.3) force was reduced to 30% in 42 +/- 7 s (43 +/- 7 tetani) (P < 0.05; n = 14). 3. When force was reduced to 30 % of initial values, tetanic [Ca2+]i had fallen from 648 +/- 87 to 336 +/- 64 nM (48% decrease) at the low intensity but had only fallen from 722 +/- 84 to 468 +/- 60 nM (35% decrease) at the higher intensity (P < 0.05 low vs. high intensity; n = 7). 4. Fatigue resulted in reductions in Ca2+ sensitivity of the contractile proteins which were greater at the high intensity (pre-fatigue [Ca2+]i required for 50 % of maximum force (Ca50) = 354 +/- 23 nM; post-fatigue Ca50 = 421 +/- 48 nM and 524 +/- 43 nM for low and high intensities, respectively). Reductions in maximum Ca2+-activated force (Fmax) were similar at the two intensities (pre-fatigue Fmax = 328 +/- 22 microN; post-fatigue Fmax = 271 +/- 20 and 265 +/- 19 microN for low and high intensities, respectively). 5. Resting pHi was 7.15 +/- 0.05. During fatigue at the low intensity, pHi was reduced by 0.12 +/- 0.02 pH units and at the high intensity pHi was reduced by 0.34 +/- 0.07 pH units (P < 0.05; n = 5). 6. Our results indicate that the more rapid fall in force at a high intensity is due to a reduction in Ca2+ sensitivity of the contractile proteins, probably related to the greater acidosis. Our data also indicate that the failure of Ca2+ release and reduced maximum Ca2+-activated force observed during fatigue are not due to reductions in intracellular pH.

Acidosis↗
PubMed1998

Effect of hydrogen peroxide and dithiothreitol on contractile function of single skeletal muscle fibres from the mouse.

1. We used intact single fibres from a mouse foot muscle to study the role of oxidation-reduction in the modulation of contractile function. 2. The oxidant hydrogen peroxide (H2O2, 100-300 microM) for brief periods did not change myoplasmic Ca2+ concentrations ([Ca2+]i) during submaximal tetani. However, force increased by 27 % during the same contractions. 3. The effects of H2O2 were time dependent. Prolonged exposures resulted in increased resting and tetanic [Ca2+]i, while force was significantly diminished. The force decline was mainly due to reduced myofibrillar Ca2+ sensitivity. There was also evidence of altered sarcoplasmic reticulum (SR) function: passive Ca2+ leak was increased and Ca2+ uptake was decreased. 4. The reductant dithiothreitol (DTT, 0.5-1 mM) did not change tetanic [Ca2+]i, but decreased force by over 40 %. This was completely reversed by subsequent incubations with H2O2. The force decline induced by prolonged exposure to H2O2 was reversed by subsequent exposure to DTT. 5. These results show that the elements of the contractile machinery are differentially responsive to changes in the oxidation-reduction balance of the muscle fibres. Myofibrillar Ca2+ sensitivity appears to be especially susceptible, while the SR functions (Ca2+ leak and uptake) are less so.

Animals↗
PubMed1998

Effect of nitric oxide on single skeletal muscle fibres from the mouse.

1. Single skeletal muscle fibres from a mouse foot muscle were used to investigate the effects of nitric oxide on contractile function. 2. We measured force production and myoplasmic free Ca2+ concentration ([Ca2+]i) in single fibres exposed to the nitric oxide donors S-nitroso-N-acetylcysteine (SNAC) and nitroprusside. 3. The nitric oxide donors reduced myofibrillar Ca2+ sensitivity, whereas [Ca2+]i transients were increased during submaximal tetani. Force was largely unchanged. SNAC did not change maximum shortening velocity, the rate of force redevelopment, or force production at saturating [Ca2+]i. 4. The guanylyl cyclase inhibitor LY83583 increased tetanic [Ca2+]i but had no effect on Ca2+ sensitivity. LY83583 did not prevent the decrease in myofibrillar Ca2+ sensitivity in response to SNAC. The oxidizer sodium nitrite increased tetanic [Ca2+]i and decreased myofibrillar Ca2+ sensitivity. 5. We conclude that under our experimental conditions nitric oxide impairs Ca2+ activation of the actin filaments which results in decreased myofibrillar Ca2+ sensitivity.

Acetylcysteine↗
PubMed1998

Intracellular calcium and Na+-Ca2+ exchange current in isolated toad pacemaker cells.

1. Single pacemaker cells were isolated from the sinus venosus of cane toad (Bufo marinus) in order to study the mechanisms involved in the spontaneous firing rate of action potentials. Intracellular calcium concentration ([Ca2+]i) was measured with indo-1 to determine whether [Ca2+]i influenced firing rate. A rapid transient rise of [Ca2+]i was recorded together with each spontaneous action potential. [Ca2+]i at the peak of systole was 655 +/- 64 nM and the minimum at the end of diastole was 195 +/- 15 nM. 2. Reduction of extracellular Ca2+ concentration from 2 to 0.5 mM caused a reduction in both systolic and diastolic [Ca2+]i and the spontaneous firing rate also gradually declined. 3. Application of the acetoxymethyl (AM) ester of BAPTA (10 microM), in order to increase intracellular calcium buffering, caused a decline in systolic and diastolic [Ca2+]i. The firing rate declined progressively until the cells stopped firing after 10-15 min. At the time that firing ceased, the diastolic [Ca2+]i had declined by 141 +/- 38 nM. 4. In the presence of ryanodine (2 microM), which interferes with Ca2+ release from the sarcoplasmic reticulum, the systolic and diastolic [Ca2+]i both declined and the firing rate decreased until the cells stopped firing. At quiescence diastolic [Ca2+]i had declined by 93 +/- 20 nM. 5. Exposure of the cells to Na+-free solution caused a rise in [Ca2+]i which exceeded the systolic level after 4.8 +/- 0.3 s. This rise is consistent with Ca2+ entry on a Na+-Ca2+ exchanger. 6. Rapid application of caffeine (10-20 mM) to cells clamped at -60 mV caused a rapid increase in [Ca2+]i which then spontaneously declined. An inward current with a similar time course to that of [Ca2+]i was also generated. Application of Ni2+ (5 mM) or 2,4-dichlorobenzamil (25 microM) reduced the amplitude of the inward current produced by caffeine by 96 +/- 1 % and 74 +/- 10 %, respectively. In a Na+-free solution the caffeine-induced current was reduced by 93 +/- 7 %. 7. Under a variety of circumstances the diastolic [Ca2+]i showed a close association with pacemaker firing rate. The existence of a Na+-Ca2+ exchanger and its estimated contribution to inward current during the pacemaker potential suggest that the Na+-Ca2+ exchange current makes a contribution to pacemaker activity.

Action Potentials↗
PubMed1998

Panniculectomy: a useful technique for the obese patient undergoing gynecological surgery.

OBJECTIVES: To establish the safety and efficacy of panniculectomy at the time of surgery for benign and malignant gynecological disease. METHODS: Retrospective review of the course of 57 patients undergoing radical gynecological surgery and panniculectomy between January 1992 and January 1997 at the Mercy Hospital for Women, Melbourne. Data were collected regarding indication for treatment, operative details, and complications of surgery. RESULTS: Of 57 patients in the study, 32 had a primary gynecological malignancy, 11 had benign gynecological disease, 3 had cervical dysplasia, 5 had endometrial hyperplasia, and the remaining 6 had incisional hernia repair. The mean age of patients was 55 years with a mean weight of 101 kg (range 70-145 kg). The mean operative time was 2 h 24 min, and blood transfusion was undertaken in 23 (41%) patients. Four (7.1%) individuals had a minor wound infection and 3 (5.4%) a moderate wound infection. One patient experienced a nonfatal pulmonary embolus and 2 patients experienced a deep vein thrombosis. There were no postoperative deaths. Long term, 6 patients developed an incisional hernia. CONCLUSIONS: Panniculectomy is a useful technique in obese patients. It improves surgical access facilitating radical surgery and is cosmetically pleasing to the patient. It has acceptable morbidity when compared to conventional midline vertical or transverse incisions in comparable populations.

Adipose Tissue↗
PubMed1998

Mechanisms underlying the reduction of isometric force in skeletal muscle fatigue.

A decline of isometric force production is one characteristic of skeletal muscle fatigue. In fatigue produced by repeated short tetani, this force decline can be divided into two components: a reduction of the cross-bridges' ability to generate force, which comes early; and a reduction of the sarcoplasmic reticulum Ca2+ release, which develops late in fatigue. Acidification due to lactic acid accumulation has been considered as an important cause of the reduced cross-bridge force production. However, in mammalian muscle it has been shown that acidification has little effect on isometric force production at physiological temperatures. By exclusion, in mammalian muscle fatigue, the reduction of force due to impaired cross-bridge function would be caused by accumulation of inorganic phosphate ions, which results from phosphocreatine breakdown. The reduction of sarcoplasmic reticulum Ca2+ release in late fatigue correlates with a decline of ATP and we speculate that the reduced Ca2+ release is caused by a local increase of the ADP/ATP ratio in the triads.

Animals↗
PubMed1998

Evidence for Na+/Ca2+ exchange in intact single skeletal muscle fibers from the mouse.

The myoplasmic free Ca2+ concentration ([Ca2+]i) was measured in intact single fibers from mouse skeletal muscle with the fluorescent Ca2+ indicator indo 1. Some fibers were perfused in a solution in which the concentration of Na+ was reduced from 145.4 to 0.4 mM (low-Na+ solution) in an attempt to activate reverse-mode Na+/Ca2+ exchange (Ca2+ entry in exchange for Na+ leaving the cell). Under normal resting conditions, application of low-Na+ solution only increased [Ca2+]i by 5.8 +/- 1.8 nM from a mean resting [Ca2+]i of 42 nM. In other fibers, [Ca2+]i was elevated by stimulating sarcoplasmic reticulum (SR) Ca2+ release with caffeine (10 mM) and by inhibiting SR Ca2+ uptake with 2,5-di(tert-butyl)-1,4-benzohydroquinone (TBQ; 0.5 microM) in an attempt to activate forward-mode Na+/Ca2+ exchange (Ca2+ removal from the cell in exchange for Na+ influx). These two agents caused a large increase in [Ca2+]i, which then declined to a plateau level approximately twice the baseline [Ca2+]i over 20 min. If the cell was allowed to recover between exposures to caffeine and TBQ in a solution in which Ca2+ had been removed, the increase in [Ca2+]i during the second exposure was very low, suggesting that Ca2+ had left the cell during the initial exposure. Application of caffeine and TBQ to a preparation in low-Na+ solution produced a large, sustained increase in [Ca2+]i of approximately 1 microM. However, when cells were exposed to caffeine and TBQ in a low-Na+ solution in which Ca2+ had been removed, a sustained increase in [Ca2+]i was not observed, although [Ca2+]i remained higher and declined slower than in normal Na+ solution. This suggests that forward-mode Na+/Ca2+ exchange contributed to the fall of [Ca2+]i in normal Na+ solution, but when extracellular Na+ was low, a prolonged elevation of [Ca2+]i could activate reverse-mode Na+/Ca2+ exchange. The results provide evidence that skeletal muscle fibers possess a Na+/Ca2+ exchange mechanism that becomes active in its forward mode when [Ca2+]i is increased to levels similar to that obtained during contraction.

Animals↗
PubMed1998

Molecular analysis of bacterial isolates and total community DNA from kraft pulp mill effluent treatment systems.

Chloroaliphatics are major components of bleached kraft mill effluents. Gene probes and oligonucleotide primers were developed to monitor kraft pulp mill effluent treatment systems for the presence of key genes (dehalogenases) responsible for the dehalogenation of chloroaliphatic organics. The primers were used for polymerase chain reaction (PCR) analysis of genomic DNA extracted from dehalogenating bacterial isolates and from total community DNA extracted from water and sediments of mill effluent treatment system. PCR amplification with oligonucleotide primers designed from dhlB, encoding the haloacid dehalogenase from Xanthobacter autotrophicus, revealed the presence of dehalogenase genes in both aerated lagoons and stabilization basins. Similarly, positive results were obtained with mmoX primers designed from the soluble methane monooxygenase gene of Methylococcus capsulatus Bath. The haloacetate dehalogenase encoding gene (dehH2) from Moraxella sp. was typically not detected in mill effluent treatment systems unless the biomass was selectively enriched. DNA sequence analysis of several PCR fragaments revealed significant similarity to known dehalogenase amd methane monooxygenase genes. The results indicated a broad distribution of known dehalogenation genes and bacteria with chloroorganic-degrading potential in the mill effluent treatment systems.

Amino Acid Sequence↗
PubMed1997

Distribution of sarcomere length and intracellular calcium in mouse skeletal muscle following stretch-induced injury.

1. The effect on sarcomere organization of stretching intact single skeletal muscle fibres by 50% of their optimum length (Lo) during ten consecutive short tetani was investigated. Stretch reduced tetanic force to 36 +/- 4% of the pre-stretch condition. Sarcomere organization was analysed using both electron and confocal microscopy. For confocal microscopy the striation pattern was examined by fluorescently staining F-actin with rhodamine-phalloidin. 2. Electron microscopy revealed that fibres which had been stretched during contraction contained areas of severe sarcomere disorganization, as well as adjacent sarcomeres of normal appearance. 3. Confocal images of stretched fibres, which had been fixed and stained with rhodamine-phalloidin, showed focal regions of overstretched sarcomeres and regions where sarcomeres of adjacent myofibrils were out of alignment with each other. Analysis of all sarcomeres along the length of fibres showed regions of sarcomere inhomogeneity were distributed throughout the fibre length and cross-section. 4. Fibres were microinjected with the fluorescent [Ca2+]i indicator fura-2 before being stretched. Conventional wide-field fluorescence imaging microscopy showed that the tetanic [Ca2+]i was reduced after stretching but remained uniformly distributed. 5. This study confirms the finding that stretch-induced muscle injury has components caused by disorganization of the myofibrillar array and by failure of tetanic Ca2+ release. The structural damage is spatially heterogeneous whereas the changes in Ca2+ release appear to be spatially homogeneous.

Animals↗