Biomedical subjects

H Green

Publications and source records attributed to H Green.

At least 19 recordsLinked to original sources

PubMed2005

A randomized controlled trial of the value of phenotypic testing in addition to genotypic testing for HIV drug resistance: evaluation of resistance assays (ERA) trial investigators.

OBJECTIVES: To assess the clinical utility of phenotypic resistance testing in addition to genotypic resistance testing among HIV-1-infected patients experiencing virologic failure and with limited therapeutic options. DESIGN: Multicenter randomized trial. METHODS: Patients were eligible if a decision had been made to switch antiretroviral therapy, the most recent HIV-1 RNA plasma viral load (VL) exceeded 2000 copies/mL, and the clinician was unable to select a potent regimen of 3 or more drugs without access to a resistance test. Subjects were randomized to genotypic resistance testing alone (G arm) or to genotypic plus phenotypic testing (G + P arm). Patients had access to resistance testing at any time during follow-up (minimum of 1 year) according to the original allocation. The primary end point was change in plasma VL from baseline at 12 months. RESULTS: Three hundred eleven patients were recruited between February 2000 and July 2001. At baseline, mean VL and CD4 count were 4.23 log10 copies/mL and 275 cells/mm, respectively, and subjects had previous exposure to a mean of 7.7 antiretroviral drugs. There was no appreciable difference between the study arms in the drug regimens prescribed after randomization. Mean reduction in VL load at 12 months was similar in the 2 arms (G: 1.37 log10 reduction, G + P: 1.28 log10 reduction; P = 0.77), as was the proportion of subjects with VL <50 copies/mL (G: 35%, G + P: 27%). CONCLUSION: The study did not demonstrate added value of phenotypic resistance testing in conjunction with genotypic resistance testing in patients with limited therapeutic options.

Acquired Immunodeficiency Syndrome↗

PubMed2005

A distinct subset of chemokines dominates the mucosal chemokine response in inflammatory bowel disease.

BACKGROUND: Inflammatory bowel disease (IBD) is characterised by intense mucosal recruitment of activated leukocytes. Chemokines determine inflammatory leukocyte recruitment and retention. AIM: To compare expression of the entire chemokine family within colonic mucosa from IBD patients and uninflamed controls. METHODS: A microarray of cDNAs, representing every member of this superfamily and their cognate receptors, was hybridised with probes derived from colonoscopic biopsies. RESULTS: A distinct subset of chemokines, consisting of CXCLs 1-3 and 8 and CCL20, was upregulated in active colonic IBD, compared with uninflamed areas or tissue from controls. Increased expression of their cognate receptors, CXCR1, CXCR2 and CCR6, was confirmed by quantitative PCR and immunohistochemistry. An identical chemokine response was induced in Caco-2 cells by stimulation with interleukin (IL)-1beta, but not tumour necrosis factor-alpha (TNF-alpha). By contrast, IL-1beta and TNF-alpha were synergistic in an HT29 cell line and primary keratinocytes. CONCLUSIONS: IL-1beta and TNF-alpha appear to be the pivotal mediators of a previously unidentified coordinated epithelial chemokine response that dominates the mucosal chemokine environment in inflamed IBD tissue.

Caco-2 Cells↗

PubMed2004

Effect of free plant sterols in low-fat milk on serum lipid profile in hypercholesterolemic subjects.

OBJECTIVE: To determine the effect of nonesterified, nonhydrogenated plant sterols solubilized in a partly vegetable oil-filled low-fat milk on serum low-density lipoprotein cholesterol (LDL) in mildly hypercholesterolemic patients. DESIGN: Double-blind, randomized, placebo-controlled three-arm crossover study. SETTING: Outpatient clinical trial. SUBJECTS: A total of 138 patients were screened, providing 81 patients for randomization; 71 patients completed the study. INTERVENTIONS: The study product was a 500 ml milk blend with or without nonesterified, nonhydrogenated sterols. The daily consumption of sterols in the three groups was 0 g/day, control group (C); 1.2 g/day, (Lo); or 1.6 g/day, (Hi), respectively. The patients were randomly assigned to one of three different treatment sequences. Each intervention period was 4 weeks. The total study duration was 12 weeks. RESULTS: The milk product was well tolerated. The placebo-adjusted mean reduction in LDL was 7.13+/-12.31 and 9.59+/-12.44% (mean+/-s.d.) for Lo and Hi groups, respectively (P<0.0001); there was no statistically significant difference in LDL lowering for the Lo and Hi groups. There were no significant changes in serum vitamin E or carotenoid concentrations after standardization with LDL cholesterol during the study period. CONCLUSION: The present study shows for the first time a substantial reduction in LDL cholesterol with a new, partly vegetable oil-filled 1.2% low-fat milk product, containing nonesterified plant sterols from soybean oil, in a randomized, placebo-controlled trial. This result encourages further development of novel low-fat dairy products containing free plant sterols for future use in cholesterol-lowering initiatives.

Aged↗

PubMed2004

A prospective multicentre study of discontinuing prophylaxis for opportunistic infections after effective antiretroviral therapy.

OBJECTIVE: To assess the medium-term safety of discontinuing prophylaxis (primary or secondary) for opportunistic infections following an effective response to antiretroviral therapy. METHODS: Participating clinical sites prospectively identified patients in whom the discontinuation of prophylaxis for any opportunistic infection was considered to be clinically indicated, although CD4 levels were not predefined. A follow-up report was subsequently sent every 6 months requesting information on changes in prophylaxis, antiretroviral drugs, new AIDS-defining events, and CD4 cell count results. RESULTS: Prophylaxis for Pneumocystis carinii pneumonia (PCP) was withdrawn in 524 patients (426 primary and 98 secondary prophylaxis), prophylaxis for Mycobacterium avium complex (MAC) was withdrawn in 28 patients (13 primary and 15 secondary), and prophylaxis for cytomegalovirus (CMV) retinitis was withdrawn in 10 patients. CD4 counts were generally maintained above accepted prophylaxis threshold levels during the period of follow up (95-98% of the time). Total follow up to last report or re-continuation of prophylaxis was 680 and 144 person-years for patients discontinuing primary and secondary PCP prophylaxis, respectively. No cases of PCP were reported, giving incidence rates of 0.0 (upper 95% confidence limit 0.4) and 0.0 (2.1) per 100 person-years. No cases of MAC were reported, but one patient had a recurrence of CMV retinitis. PCP prophylaxis was restarted in 30 patients; no patients restarted MAC or CMV prophylaxis. CONCLUSIONS: Previous studies have demonstrated a low risk of PCP in the short term following the withdrawal of prophylaxis in patients who have responded well to antiretroviral therapy. The present study suggests a continuing low level of risk with extended follow up, provided adequate CD4 count levels are maintained. The case of recurrent CMV retinitis in a patient with impressive immunological and virological response indicates the need for close monitoring of patients previously diagnosed with this condition.

AIDS-Related Opportunistic Infections↗

PubMed2003

Role of MC1R variants in uveal melanoma.

Variants of the melanocortin-1 receptor (MC1R) gene have been linked to sun-sensitive skin types and hair colour, and may independently play a role in susceptibility to cutaneous melanoma. To assess the role of MC1R variants in uveal melanoma, we have analysed a cohort of 350 patients for the changes within the major region of the gene displaying sequence variation. Eight variants were detected - V60L, D84E, V92M, R151C, I155T, R160W, R163Q and D294H - 63% of these patients being hetero- or homozygous for at least one variant. Standard melanoma risk factor data were available on 119 of the patients. MC1R variants were significantly associated with hair colour (P=0.03) but not skin or eye colour. The frequency of the variants detected in the 350 patients was comparable with those in the general population, and comparison of the cumulative tumour distribution by age at diagnosis in carriers and noncarriers provided no evidence that MC1R variants confer an increased risk of uveal melanoma. We interpret the data as indicating that MC1R variants do not appear to be major determinants of susceptibility to uveal melanoma.

Adult↗

PubMed2003

Oligomeric and polymeric aggregates formed by proteins containing expanded polyglutamine.

Neurological diseases resulting from proteins containing expanded polyglutamine (polyQ) are characteristically associated with insoluble neuronal inclusions, usually intranuclear, and neuronal death. We describe here oligomeric and polymeric aggregates formed in cells by expanded polyQ. These aggregates are not dissociated by concentrated formic acid, an extremely effective solvent for otherwise insoluble proteins. Perinuclear inclusions formed in cultured cells by expanded polyQ can be completely dissolved in concentrated formic acid, but a soluble protein oligomer containing the expanded polyQ and released by the formic acid is not dissociated to monomer. In Huntington's disease, a formic acid-resistant oligomer is present in cerebral cortex, but not in cerebellum. Cortical nuclei contain a polymeric aggregate of expanded polyQ that is insoluble in formic acid, does not enter polyacrylamide gels, but is retained on filters. This finding shows that the process of polymerization is more advanced in the cerebral cortex than in cultured cells. The resistance of oligomer and polymer to formic acid suggests the participation of covalent bonds in their stabilization.

Animals↗

PubMed2003

Quasi-phase-matched generation of coherent extreme-ultraviolet light.

High-harmonic generation is a well-known method of producing coherent extreme-ultraviolet (EUV) light, with photon energies up to about 0.5 keV (refs 1, 2). This is achieved by focusing a femtosecond laser into a gas, and high harmonics of the fundamental laser frequency are radiated in the forward direction. However, although this process can generate high-energy photons, efficient high-harmonic generation has been demonstrated only for photon energies of the order 50-100 eV (ref. 5). Ionization of the gas prevents the laser and the EUV light from propagating at the same speed, which severely limits the conversion efficiency. Here we report a technique to overcome this problem, and demonstrate quasi-phase-matched frequency conversion of laser light into EUV. Using a modulated hollow-core waveguide to periodically vary the intensity of the laser light driving the conversion, we efficiently generate EUV light even in the presence of substantial ionization. The use of a modulated fibre shifts the energy spectrum of the high-harmonic light to significantly higher photon energies than would otherwise be possible. We expect that this technique could form the basis of coherent EUV sources for advanced lithography and high-resolution imaging applications. In future work, it might also be possible to generate isolated attosecond pulses.

Journal Article↗

PubMed2002

Increases in muscle MCT are associated with reductions in muscle lactate after a single exercise session in humans.

To investigate the effects of a single session of prolonged cycle exercise [60% peak O2 uptake (VO2 peak) for 5-6 h] on metabolic adaptations in working vastus lateralis muscle, nine untrained males (peak O2 uptake = 47.2 +/- 1.1 ml x kg(-1) x min(-1), means +/- SE) were examined before (Pre) and at 2 (Post-2), 4 (Post-4), and 6 (Post-6) days after the training session. On the basis of 15 min of cycle exercise at 59% VO2 peak, it was found that training reduced (P < 0.05) exercise muscle lactate (mM) at Post-2 (6.65 +/- 0.69), Post-4 (7.74 +/- 0.63), and Post-6 (7.78 +/- 1.2) compared with Pre (10.9 +/- 1.3). No effect of training was observed on exercise ATP, phosphocreatine, and glycogen levels. After the single session of training, plasma volumes were elevated (P < 0.05) at Post-2 (6.7 +/- 1.7%), Post-4 (5.86 +/- 1.9), and Post-6 (5.13 +/- 2.5). The single exercise session also resulted in elevations (P < 0.05) in the monocarboxylate transporters MCT1 and MCT4 throughout the 6 days after exercise. Although epinephrine and norepinephrine both increased with exercise, only norepinephrine was reduced (P < 0.05) with training and only at Post-4. These results indicate that regulation of cellular lactate levels occurs rapidly and independently of other metabolic adaptations. It is proposed that increases in MCT and plasma volume are at least partly involved in the lower muscle lactate content observed after the training session by increasing lactate membrane transport and removal, respectively.

Adult↗

PubMed2002

Silver ions induce Ca2+ release from the SR in vitro by acting on the Ca2+ release channel and the Ca2+ pump.

Silver nitrate (AgNO3) is a sulfhydryl oxidizing agent that induces a biphasic Ca2+ release from isolated sarcoplasmic reticulum (SR) vesicles by presumably oxidizing critical sulfhydryl groups in the Ca2+ release channel (CRC), causing the channel to open. To further examine the effects of AgNO3 on the CRC and the Ca2+-ATPase, Ca2+ release was measured in muscle homogenates prepared from rat hindlimb muscle using indo 1. Cyclopiazonic acid (CPA) and ruthenium red (RR) were used to inhibit the Ca2+-ATPase and block the CRC, respectively, before inducing Ca2+ release with both AgNO3 and 4-chloro-m-cresol (4-CMC), a releasing agent specific for the CRC. With AgNO3 and CPA, the early rapid rate of release (phase 1) was increased (P < 0.05) by 42% (314 +/- 5 vs. 446 +/- 39 micromol x g protein(-1) x min(-1)), whereas the slower, more prolonged rate of release (phase 2) was decreased (P < 0.05) by 72% (267 +/- 39 vs. 74 +/- 7.7 micromol x g protein(-1) x min(-1)). RR, in combination with AgNO3, had no effect on phase 1 (P > 0.05) (314 +/- 51 vs. 334 +/- 43 micromol x g protein(-1) x min(-1)) and decreased phase 2 (P < 0.05) by 65% (245 +/- 34 vs. 105 +/- 8.2 micromol x g protein(-1) x min(-1)). With 4-CMC, CPA had no effect (P > 0.05) on either phase 1 or 2. With addition of RR, phase 1 was reduced (P < 0.05) by 59% (2,468 +/- 279 vs. 1,004 +/- 87 micromol x g protein(-1) x min(-1)), and RR completely blocked phase 2. Both AgNO3 and 4-CMC fully inhibited Ca2+-ATPase activity measured in homogenates. These findings indicate that AgNO3, but not 4-CMC, induces Ca2+ release by acting on both the CRC and the Ca2+-ATPase.

Animals↗

PubMed2002

Diet, breakfast, and academic performance in children.

OBJECTIVE: To determine whether nutrient intake and academic and psychosocial functioning improve after the start of a universal-free school breakfast program (USBP). METHODS: Information was gathered from 97 inner city students prior to the start of a USBP and again after the program had been in place for 6 months. Students who had total energy intakes of <50% of the recommended daily allowance (RDA) and/or 2 or more micronutrients of <50% of RDA were considered to be at nutritional risk. RESULTS: Prior to the USBP, 33% of all study children were classified as being at nutritional risk. Children who were at nutritional risk had significantly poorer attendance, punctuality, and grades at school, more behavior problems, and were less likely to eat breakfast at school than children who were not at nutritional risk. Six months after the start of the free school breakfast programs, students who decreased their nutritional risk showed significantly greater: improvements in attendance and school breakfast participation, decreases in hunger, and improvements in math grades and behavior than children who did not decrease their nutritional risk. CONCLUSION: Participation in a school breakfast program enhanced daily nutrient intake and improvements in nutrient intake were associated with significant improvements in student academic performance and psychosocial functioning and decreases in hunger.

Absenteeism↗

PubMed2001

Partial ischemia reduces the efficiency of sarcoplasmic reticulum Ca2+ transport in rat EDL.

To investigate the hypothesis that prolonged partial ischemia would result in a depression in homogenate sarcoplasmic reticulum (SR) Ca2+-sequestering and mechanical properties in muscle, a cuff was placed around the hindlimb of 8 adult Sprague-Dawley rats (267+/-5.8 g; x +/- S.E.) and partially inflated (315 mm Hg) for 2 h. Following occlusion, the EDL was sampled both from the ischemic (I) and contralateral control (C) leg and SR properties compared with the EDL muscles extracted from rats (n = 8) immediately following anaesthetization (CC). Ischemia was indicated by a lower (p < 0.05) concentration (mmol.kg dry wt(-1)) of ATP (19.0+/-0.7 vs. 16.7+/-0.7) and phosphocreatine (58.1+/-5.7 vs. 35.0+/-4.6) in I compared to C. Although Ca2+-ATPase activity (micromol x g protein(-1) x sec(-1)), both maximal and submaximal, was not different between C and I (19.7+/-0.4 vs. 18.5+/-1.3), reductions (p < 0.05) in Ca2+-uptake (mmol x g protein(-1) x sec(-1)) of between 18.2 and 24.7% across a range of submaximal free Ca2+-levels were observed in I compared to C. Lower submaximal Ca2+-ATPase activity and Ca2+-uptake were also observed in the EDL in C compared to CC animals. Time dependent reductions (p < 0.05) were found in peak twitch and maximal tetanic tension in EDL from I but not C. It is concluded that partial ischemia, resulting in modest reductions in energy state in EDL, induces a reduction in Ca2+-uptake independent of changes in Ca2+-ATPase activity. These changes reduce the coupling ratio and the efficiency of Ca2+-transport by SR.

Animals↗

PubMed2001

Minimization in crossover trials with non-prognostic strata: theory and practical application.

To obtain meaningful results in any clinical trial, patients need to be allocated to treatments in such a way that valid analysis can be carried out. Balancing treatment groups before analysis is carried out is more desirable than trying to compensate for incomparability at a later date. Therefore, the development of allocation procedures to produce comparable groups in which prognostic factors are equally represented is important. Minimization, a deterministic allocation method, aims to ensure balance on such factors, particularly in small trials when traditional randomization methods are likely to fail. However, views on the use of conventional analysis following minimization are divided. The use of minimization in two randomised crossover trials is described where, in addition to the comparisons between randomised treatments, it was desired to have balance between groups based on differential trial procedures. Theoretical concerns about the use of minimization are not applicable in this setting, and therefore minimization is shown to be a useful technique for obtaining balance.

Cross-Over Studies↗

PubMed2001

An analysis of factors contributing to a series of deaths caused by exposure to high environmental temperatures.

Autopsy reports at the Forensic Science Centre, Adelaide, South Australia, were reviewed for the 8 years from January 1991 to December 1998 for cases with unusual features in which deaths had been attributed to exposure to high environmental temperatures. Amphetamine-related hyperpyrexial deaths, anesthetic deaths caused by malignant hyperpyrexia, deaths of elderly incapacitated individuals during heat waves, and deaths of children trapped in the back of cars were excluded from the study. In 9 cases, where heat-related deaths had occurred (age range 21 to 77 years; M:F = 8:1). Predisposing factors included lack of familiarity with Australian environmental conditions, excessive clothing, prolonged sun exposure, acute alcohol intoxication, obesity, benztropine and trifluoperazine medication, and underlying dementia, alcoholic liver disease, and possibly epilepsy.

Adult↗

PubMed2001

Germline mutations in TGM2 do not contribute to coeliac disease susceptibility in the Swedish population.

OBJECTIVE: Coeliac disease (CD) shows a strong genetic predisposition involving HLA-DQ2 and non-HLA components. Tissue transglutaminase, encoded by TGM2, occupies a central role in the CD pathogenesis, necessary for the deamidation of specific glutamine residues of alpha-gliadin creating a T-cell epitope that binds with increased affinity to DQ2. To investigate whether germline mutations in TGM2 contribute to disease susceptibility we have carried out a comprehensive analysis of the gene in 52 patients with CD. DESIGN: Blood samples were collected from 52 children with biopsy proven CD attending one Swedish centre. DNA was extracted from lymphocytes and all exons and intron-exon boundaries of the TGM2 gene and the alternatively spliced form of the gene were screened for mutations. METHODS: Mutational analysis was undertaken by a combination of conformational specific gel electrophoresis and direct sequencing. RESULTS: Three novel polymorphisms were identified but no pathogenic mutations were detected. CONCLUSIONS: There is no evidence from this study that mutations in TGM2, which lead to an altered protein, contribute to CD susceptibility.

Adolescent↗

PubMed2001

Cyclooxygenase-2 expression in the Barrett's metaplasia-dysplasia-adenocarcinoma sequence.

OBJECTIVE: Increased expression of the inducible cyclooxygenase 2 (COX-2) enzyme has been detected in esophageal and colonic adenocarcinoma, and intake of aspirin and non-steroidal anti-inflammatory drugs, known COX-2 inhibitors, have been associated with reduced tumor formation. Elevated COX-2 mRNA but variable protein expression has been demonstrated in Barrett's epithelium, and we have, therefore, sought to evaluate the expression of COX-2 protein throughout the Barrett's metaplasia-dysplasia-adenocarcinoma sequence. METHODS: Paraffin-embedded esophageal biopsies from 56 different patients with Barrett's esophagus were analyzed for COX-2 expression by immunohistochemistry. Twenty contained nondysplastic intestinal and gastric metaplasia, 12 demonstrated low-grade dysplasia (LGD), 12 high-grade dysplasia (HGD), and 12 contained invasive adenocarcinoma. RESULTS: Epithelial expression of COX-2 protein was detected in 75% (15/20) of benign cases, 83% (10/12) of cases with LGD, and 100% of cases with HGD or adenocarcinoma. Using a semiquantitative analysis, median staining scores for the groups were 2, 3, 14, and 13, respectively (scale 0-16), with the expression being significantly higher in the HGD and cancer groups compared to benign and LGD groups (p < 0.001). CONCLUSIONS: This study demonstrates clear COX-2 expression in the epithelial cells in Barrett's metaplasia, confirms elevated expression in adenocarcinoma, and shows that the elevation in expression occurs in the progression from LGD to HGD.

Adenocarcinoma↗

PubMed2001

Effects of ischemia on sarcoplasmic reticulum Ca(2+) uptake and Ca(2+) release in rat skeletal muscle.

In this study, we investigated the hypothesis that prolonged ischemia would impair both sarcoplasmic reticulum (SR) Ca(2+) uptake and Ca(2+) release in skeletal muscle. To induce total ischemia (I), a tourniquet was placed around the upper hindlimb in 30 female Sprague-Dawley rats [wt = 256 +/- 6.7 (SE) g] and inflated to 350 mmHg for 4 h. The contralateral limb served as control (C). Immediately after the 4 h of ischemia, mixed gastrocnemius and tibialis anterior muscle was sampled from both limbs, and both crude muscle homogenates and SR vesicles were prepared. In another 10 control animals (CC), muscles were sampled and prepared exactly the same way, but immediately after anesthetization. Ca(2+) uptake and Ca(2+) release were measured in vitro with Indo-I on both homogenates and SR vesicles. As hypothesized, submaximal Ca(2+) uptake was lower (P < 0.05) in I compared with CC and C, by 25 and 45% in homogenates and SR vesicles, respectively. Silver nitrate (AgNO(3))-induced Ca(2+) release, which occurred in two phases (phase 1 and phase 2), was also altered in I compared with CC and C, in both muscle homogenates and SR vesicles. With ischemia, phase 1 peak Ca(2+) release was 26% lower (P < 0.05) in SR vesicles only. For phase 2, peak Ca(2+) release was 54 and 24% lower (P < 0.05) in SR vesicles and homogenates, respectively. These results demonstrate that prolonged skeletal muscle ischemia leads to a reduced SR Ca(2+) uptake in both homogenates and SR vesicles. The effects of ischemia on SR Ca(2+) release, however, depend on both the phase examined and the type of tissue preparation.

Adenosine Triphosphate↗

PubMed2001

Effects of 4-h ischemia and 1-h reperfusion on rat muscle sarcoplasmic reticulum function.

To investigate the hypothesis that ischemia and reperfusion would impair sarcoplasmic reticulum (SR) Ca(2+) regulation in skeletal muscle, Sprague-Dawley rats (n = 20) weighing 290 +/- 3.5 g were randomly assigned to either a control control (CC) group, in which only the effects of anesthetization were studied, or to a group in which the muscles in one hindlimb were made ischemic for 4 h and allowed to recover for 1 h (I). The nonischemic, contralateral muscles served as control (C). Measurements of Ca(2+)-ATPase properties in homogenates and SR vesicles, in mixed gastrocnemius and tibialis anterior muscles, indicated no differences between groups on maximal activity, the Hill coefficient, and Ca(50), defined as the Ca(2+) concentration needed to elicit 50% of maximal activity. In homogenates, Ca(2+) uptake was lower (P < 0.05) by 20-25%, measured at 0.5 and 1.0 microM of free Ca(2+) ([Ca(2+)](f)) in C compared with CC. In SR vesicles, Ca(2+) uptake was lower (P < 0.05) by 30-38% in I compared with CC at [Ca(2+)](f) between 0.5 and 1.5 microM. Silver nitrate induced Ca(2+) release, assessed during both the initial, early rapid (phase 1), and slower, prolonged late (phase 2) phases, in homogenates and SR vesicles, indicated a higher (P < 0.05) release only in phase 1 in SR vesicles in I compared with CC. These results indicate that the alterations in SR Ca(2+) regulation, previously observed after prolonged ischemia by our group, are reversed within 1 h of reperfusion. However, the lower Ca(2+) uptake observed in long-term, nonischemic homogenates suggests that altered regulation may occur in the absence of ischemia.

Animals↗

PubMed2001

Ischemia-induced structural change in SR Ca2+-ATPase is associated with reduced enzyme activity in rat muscle.

In this study, we employed an in vivo model of prolonged ischemia in rat skeletal muscle to investigate the hypothesis that structural modifications to the sarcoplasmic reticulum (SR) Ca2+-ATPase can explain the alterations in Ca2+-ATPase activity that occur with ischemia. To induce total ischemia, a tourniquet was placed around the upper hindlimb in 27 female Sprague-Dawley rats weighing 256 +/- 6.7 g (mean +/- SE) and was inflated to 350 mmHg for 4 h. The contralateral limb served as control (C) to the ischemic limb (I), and the limbs of animals killed immediately after anesthetization served as a double control (CC). Mixed gastrocnemius and tibialis anterior muscles were sampled and used for SR vesicle preparation. Maximal Ca2+-ATPase activity (micromol x g protein(-1) x min(-1)) of C (15,802 +/- 1,246) and I (11,609 +/- 1,029) was 90 and 73% (P < 0.05) of CC (17,562 +/- 1,682), respectively. No differences were found between groups in either the Hill coefficient or the free Ca2+ at half-maximal activity. The fluorescent probes, FITC and N-cyclohexyl-N'-(dimethylamino-alpha-naphthyl) carbodiimide, used to assess structural alterations in the regions of the ATP binding site and the Ca2+ binding sites of the Ca2+-ATPase, respectively, indicated a 26% reduction (P < 0.05) in FITC binding capacity (absolute units) in I (0.22 +/- 0.01) compared with CC (0.29 +/- 0.02) and C (0.29 +/- 0.03). Our results suggest that the reduction in maximal SR Ca2+-ATPase activity in SR vesicles with ischemia is related to structural modification in the region of the nucleotide binding domain by mechanisms that are as yet unclear.

Animals↗