Mechanisms to prevent caspase activation in rotenone-induced dopaminergic neurodegeneration: role of ATP depletion and procaspase-9 degradation

The evidence implicating a mode of cell death that either favors or argues against caspase-dependent apoptosis is available in studies that used experimental models of Parkinson’s disease. We sought to investigate the mechanisms by which release of cytochrome c is not linked to caspase activation during rotenone-induced dopaminergic (DA) neurodegeneration. Unlike caspase activation in 6-hydroxydopamine-treated cells, both MN9D DA neuronal cells and primary cultures of mesencephalic neurons showed no obvious signs of caspase activation upon exposure to rotenone. We found that intracellular levels of ATP significantly decreased at the early phase of neurodegeneration (<~24 h) and therefore external addition of ATP to the lysates obtained at this stage reconstituted caspase-3 activity. At a later phase of cell death (>~24 h), both decreased levels of ATP and procaspase-9 contributed to the lack of caspase-3 activation. Under this condition, calpain and the proteasome system were responsible for the degradation of procaspase-9. Consequently, external addition of ATP and procaspase-9 to the lysates harvested at the later phase was required for activation of caspase-3. Similarly, caspase-3 activity was also reconstituted in the lysates harvested from cells co-treated with inhibitors of these proteases and incubated in the presence of external ATP. Taken together, our findings provided a sequential mechanism underlying how DA neurons may undergo caspase-independent cell death, even in the presence of cytoplasmic cytochrome c following inhibition of mitochondrial complex I.     

Equol induces apoptosis through cytochrome c-mediated caspases cascade in human breast cancer MDA-MB-453 cells

This study investigated the role of the caspase activation cascade in extrinsic and intrinsic apoptosis induced by equol in human breast cancer MDA-MB cells. First, the antiproliferative effect of equol was determined in cells treated with 1-100 μM equol for 24, 48, and 72 h. Equol significantly inhibited cell proliferation in a dose-dependent manner (p < 0.05). Exposure to 50 or 100 μM equol for 72 h strongly promoted apoptosis. Under the same conditions, remarkable cytochrome c release was observed. Subsequently, caspase-9, which acts in mitochondria-mediated apoptosis, was cleaved by equol at high concentrations, but caspase-8 activation of receptor-mediated apoptosis was not observed. At both equol concentrations, the caspase-8 and -9 activity assays showed similar patterns. In addition, equol treatment activated caspase-3, which is downstream from caspase-9, and this was accompanied by the cleavage of capase-6 and -7. Activation of these caspases leads to increased activation of PARP, lamin, and ICAD. This study suggests that equol induces the intrinsic pathway of apoptosis via caspase-9 and cytochrome c, independent of caspase-8, in human breast cancer MDA-MB-453 cells.     

Intramuscular adaptations to eccentric exercise and antioxidant supplementation

Prophylactic supplementation of N-acetyl-cysteine (NAC) and epigallocatechin gallate (EGCG) was studied for physiological and cellular changes in skeletal muscle after eccentric muscle contractions. Thirty healthy, active males (20.0 ± 1.8 years, 160 ± 7.1 cm, 76.1 ± 17.0 kg) ingested for 14 days either 1,800 mg of NAC, 1,800 mg of EGCG, or 1,000 mg of fiber (glucomannan) placebo (PLC) in a double blind, prophylactic fashion. Subjects completed one eccentric exercise bout (100 repetitions at 30°/s) using the dominant knee extensors. Strength and soreness were assessed, and blood and muscle samples obtained before and 6, 24, 48, and 72 h with no muscle sample being collected at 72 h. Separate mixed factorial repeated measures ANOVA (P < 0.05) were used for all statistical analysis. All groups experienced significantly reduced peak torque production after 6 and 24 h, increased soreness at all time points from baseline [with even greater soreness levels 24 h after exercise in PLC when compared to EGCG and NAC (P < 0.05)], increased lactate dehydrogenase at 6 h, and increased creatine kinase 6, 24 and 48 h after exercise. No significant group × time interaction effects were found for serum cortisol, neutrophil counts, and the neutrophil:lymphocyte ratio; although, all values experienced significant changes 6 h after exercise (P < 0.05), but at no other time points. At 48 h after the exercise bout the Neu:Lym ratio in EGCG was significantly less than NAC (P < 0.05), whereas there was a trend (P = 0.08) for the EGCG values to be less when compared to PLC at this time point. Markers of intramuscular mitochondrial and cytosolic apoptosis were assessed (e.g., bax, bcl-2, cytochrome C, caspase-3 content/enzyme activity, and total DNA content). Significant increases (P < 0.05) in muscle levels of bax and bcl-2 were observed in all groups with no significant differences between groups, whereas no changes (P > 0.05) were reported for cytochrome C, caspase-3 content, caspase-3 enzyme activity, and total DNA. Caspase-3 enzyme activity was significantly greater in all groups 48 h after exercise when compared to baseline (P < 0.05) and 6 h (P < 0.05) after exercise. An eccentric bout of muscle contractions appears to significantly increase muscle damage, markers of mitochondrial apoptosis, apoptotic enzyme activity, and whole-blood cell markers of inflammation with no changes in oxidative stress. While soreness ratings were blunted in the two supplementation groups 24 h after exercise when compared to PLC values, more research is needed to determine the potential impact of EGCG and NAC supplementation on changes related to oxidative stress, apoptosis, and eccentric exercise.     

Effect of progesterone on the expression of bax and bcl-2 and on caspase activity in bovine luteal cells

Bovine luteal cells from days 6-10 and 11-15 of the estrous cycle were exposed (6 h) to factors that support or disrupt steroidogenesis. The expression of bcl-2 and bax and level of active caspase-3 in cells was measured. Progesterone (P4) increased (P<0.01) while staurosporine decreased (P<0.01-P<0.001) bcl-2 expression at both stages of the estrous cycle studied. In cells from 11-15 days of the estrous cycle expression of bcl-2 was stimulated (P<0.05) by prostaglandin (PG)E2 and inhibited (P<0.01) by 3,3',4,4'-tertrachlorobiphenyl (PCB)-77. Treatment with aminoglutethimide (blocker of cytochrome P450scc; 1.5 x 10(-4)M), nitric oxide donor (spermine NONOate), and staurosporine increased bax expression in cells collected from both experimental periods. The influence of these factors was greater in cells from days 11-15 (P<0.001) than by cells on days 6-10 (P<0.05) of the estrous cycle. PCB-77 stimulated expression of bax in cells from 11-15 days of cycle (P<0.01) only. Treatment of luteal cells with P4 and PGE2 for 24 h decreased (P<0.05) level of active caspase-3 while aminoglutethimide (P<0.05), spermine NONOate (P<0.05), and staurosporine (P<0.001) increased caspase-3 activity in the cells. Moreover, P4 decreased (P<0.05) while staurosporine increased (P<0.01) the ratio of bax/bcl-2 at both stages of the cycle. Aminoglutethimide, spermine NONOate and PCB increased (0<0.05) this ratio in cells on days 11-15 of the cycle. These results suggest that P4 concentrations in luteal cells protects against apoptosis, while disruption of steroidogenesis and reduced ability of luteal cells to produce P4 can induce cell death.     

Interleukin-10 modulates pro-apoptotic effects of TNF-α in human articular chondrocytes in vitro

The aim of this study is to determine if there is an antagonistic effect between tumour necrosis factor (TNF)-α and the immunoregulatory interleukin (IL)-10 on chondrocytes survival. Serum-starved primary human articular chondrocytes were stimulated with either 10 ng/ml recombinant TNF-α, IL-10 or a combination of both (at 10 ng/ml each). Chondrocyte apoptosis was determined by measuring caspase-3/7, -8 and -9 activities using caspase assays. Mitochondrial apoptotic inducer bax, and the suppressor bcl-2 were evaluated using western blotting at 48 h. Results indicated that TNF-α increased caspase activities and resulted in a significant (p = 0.001) increase in bax/bcl-2 ratio. Stimulation with IL-10 did not alter caspase activities, while co-treatment with IL-10 and TNF-α inhibited TNF-α induced caspase activities and significantly (p > 0.004) impaired bax/bcl-2 ratio. At 24 h, mRNA levels for collagen type II, TNF-α and IL-10 were determined using real-time RT-PCR. Stimulation with TNF-α or TNF-α and IL-10 significantly inhibited collagen type II and increased IL-10 and TNF-α mRNA expression. IL-10 modulated the pro-apoptotic capacity of TNF-α in chondrocytes as shown by the decrease in caspase activities and bax/bcl-2 ratio compared to TNF-α stimulated chondrocytes, suggesting a mostly antagonistic interplay of IL-10 and TNF-α on mitochondrial apoptotic pathways.     

The prognostic significance of apoptosis-related biological markers in Chinese gastric cancer patients

Background and Objective

The prognosis varied among the patients with the same stage, therefore there was a need for new prognostic and predictive factors. The aim of this study was to evaluate the relationship of apoptosis-related biological markers such as p53, bcl-2, bax, and c-myc, and clinicopathological features and their prognostic value.

Methods

From 1996 to 2007, 4426 patients had undergone curative D2 gastrectomy for gastric cancer at Fudan University Shanghai Cancer Center. Among 501 patients, the expression levels of p53, bcl-2, bax, and c-myc were examined by immunohistochemistry. The prognostic value of biological markers and the correlation between biological markers and other clinicopathological factors were investigated.

Results

There were 339 males and 162 females with a mean age of 57. The percentages of positive expression of p53, bcl-2, bax, and c-myc were 65%, 22%, 43%, and 58%, respectively. There was a strong correlation between p53, bax, and c-myc expression (P = 0.00). There was significant association between bcl-2, and bax expression (P<0.05). p53 expression correlated with histological grade (P = 0.01); bcl-2 expression with pathological stage (P = 0.00); bax expression with male (P = 0.02), histological grade (P = 0.01), Borrmann type (P = 0.01), tumor location (P = 0.00), lymph node metastasis (P = 0.03), and pathological stage (P = 0.03); c-myc expression with Borrmann type (P = 0.00). bcl-2 expression was related with good survival in univariate analysis (P = 0.01). Multivariate analysis showed that bcl-2 expression and pathological stage were defined as independent prognostic factors. There were significant differences of overall 5-year survival rates according to bcl-2 expression or not in stage IIB (P = 0.03).

Conclusion

The expression of bcl-2 was an independent prognostic factor for patients with gastric cancer; it might be a candidate for the gastric cancer staging system.     

Characterization of cell clones stably transfected with short form caspase-9: Apoptotic resistance and Bcl-XL expression

Caspases play important roles in the initiation and progression of apoptosis. In experimental models of ATP depletion, we have demonstrated the activation of caspase-9, -8, and -3, which is followed by the development of apoptotic morphology. To determine the specific contribution of caspase-9 to ATP depletion-induced apoptosis, we transfected renal epithelial cells with its endogenous dominant-negative inhibitor caspase-9S. Two cell clones with stable transfection were obtained. These clones expressed caspase-9S, and the cytosol isolated from these cells was resistant to cytochrome c-induced caspase activation in vitro. The clones were then examined for ATP depletion-induced apoptosis. Compared with the wild-type cells, the caspase-9S clones were markedly resistant to apoptosis in this model. Caspase activation was also inhibited. Surprisingly, these clones also showed significantly less cytochrome c release during ATP-depletion. Moreover, Bax translocation to mitochondria was inhibited, suggesting that these clones were resistant to apoptosis not only at the cytosolic caspase activation level but also at the upstream mitochondrial level. To gain insights into the mitochondrial resistance, we analyzed the expression of Bcl-2 family proteins. While the expression of Bax, Bak, and Bcl-2 was comparable to the wild-type cells, the selected clones showed specific up-regulation of Bcl-XL, an anti-apoptotic protein. We conclude that the selected clones were resistant to apoptosis at two levels. In the cytosol, they expressed dominant negative caspase-9, and at the mitochondria they up-regulated Bcl-XL.     

Hand-held lactate analyzer as a tool for the real-time measurement of physical fatigue before slaughter and pork quality prediction

The objectives of this study were to assess the relationship between blood lactate variation measured at the plant, and pork quality variation on a large sample size and under commercial preslaughter handling conditions. A total of 600 pigs were randomly chosen on arrival at a commercial slaughter plant and blood samples taken from the ear vein at unloading (UN), after lairage (LA), in the restrainer (RE; before stunning) and at exsanguination (EX) were analysed for lactate content using a Lactate Scout Analyzer (LSA). In order to have a large range of measures, pigs were distributed into two groups; one kept in lairage overnight (G1) and the other for 2 to 3 h (G2) before slaughter. Meat quality was assessed in the Longissimus thoracis (LT), Semimembranosus (SM) and Adductor (AD) muscles by measuring the pH 30 min postmortem (pH1) and at 24 h postmortem (pHu), the colour and the drip loss. Blood lactate levels did not differ between G1 and G2 (P>0.05). A reduced muscle lactate and glucose contents (P=0.02 and P=0.004, respectively) resulting in a lower (P<0.001) glycolytic potential (GP) was observed in the LT muscle of G1 pigs when compared with G2 loins. In the LT muscle of G1 pigs, the lower GP resulted in an increased pHu (r=−0.67; P<0.001), decreased drip loss (r=0.57; P<0.001) and darker colour (r=0.50; P<0.001) compared with G2. In both G1 and G2 pigs, the lower GP was correlated to higher pHu value in the SM and AD muscles (r=−0.73; P<0.001). The greatest correlation was observed in G2 between blood lactate levels at LA and pHu value of the SM and AD muscles (r=0.46 and r=0.44, respectively; P<0.001 for both muscles). The second greatest correlation was found between blood lactate levels at EX and pH1 value in the SM muscle in both groups (r=−0.37 and r=−0.41, respectively; P<0.001 for both groups). Based on the results of this study, it appears that blood lactate levels, as measured by the LSA, reliably reflect the physiological response of pigs to perimortem stress and may help explain the variation in pork quality.     

Down-regulation of platelet-derived growth factor-D expression blockades NF-κB pathway to inhibit cell proliferation and invasion as well as induce apoptosis in esophageal squamous cell carcinoma

Substantial evidence has demonstrated that platelet-derived growth factor-D (PDGF-D) is tightly associated with the development and progression of tumors. However, its biological functions in esophageal squamous cell carcinoma (ESCC) remain to be delineated. In this study, we found that expressions of PDGF-D mRNA and protein in ESCC tissues and cells were significantly higher than that in normal esophageal epithelial tissues (P < 0.05), further investigation showed that PDGF-D protein level in EC1 cells was obviously higher than those in EC9706 and Eca109 cells (P < 0.05). Elevated PDGF-D level was closely associated with TNM staging, tumor differentiation and lymph node metastasis (P < 0.05), but not related to the patients’ age and gender (P > 0.05). In addition, down-regulation of PDGF-D expression markedly inhibited proliferation, reduced invasion and induced apoptosis in EC1 cells. More importantly, reduced PDGF-D level evoked the down-regulation of p65 and p-IκBα proteins and elevation of IκBα protein of NF-κB pathway, accompanied with the decreases of bcl-2 and MMP-9 protein expressions and increases of bax protein level and caspase-3 activities. Correctively, our data suggest that PDGF-D plays pivotal roles in the development and progression of ESCC, and combinations with PDGF-D and NF-κB pathway may be effective and feasible molecular targets for therapy of ESCC.     

Neuroprotective effect of methanol extract of Phellodendri Cortex against 1-methyl-4-phenylpyridinium (MPP)-induced apoptosis in PC-12 cells

Phellodendri Cortex (PC) is a traditional herbal medicine, widely used in Korea and China. The effects of the methanol extract of Phellodendri Cortex (PC extract) on 1-methyl-4-phenylpyridinium (MPP⁺)-induced neuronal apoptosis in PC-12 cells have been investigated. MPP⁺-induced apoptosis in PC-12 cells was accompanied by an increased bax/bcl-2 ratio, release of cytochrome c to the cytosol and activation of caspase-3. PC extract inhibited the downregulation of bcl-2 and the upregulation of bax, as well as the release of mitochondrial cytochrome c into the cytosol. In addition, PC extract attenuated caspase-3 activation and cleavage of poly (ADP-ribose) polymerase (PARP). These results suggest that the PC extract has protective effects against MPP⁺-induced neuronal apoptosis in PC-12 cells.     

Expression of basic fibroblast growth factor,protein kinase C and members of the apoptotic pathway in skeletal muscle of streptozotocin-induced diabetic rats

This study investigated the potential mechanisms that may underlie diabetes induced amyoatrophy. Sprague-Dawley rats were either injected intraperiotneally with STZ (test group; N = 8) to induce diabetic-like symptoms (blood glucose level ≥16.65 mmol/L) or with buffer (control group; N = 8). Differences in muscle structure between the STZ-induced diabetic and control groups were evaluated by histochemistry. Protein and mRNA levels of basic FGF (bFGF), bax, bcl-2, and caspase 3 in skeletal muscle were compared between the 2 groups using immunohistochemistry and quantitative PCR, respectively. Serum level of insulin and protein kinase C (PKC) were measured by competitive RIA and ELISA, respectively. Unlike control animals, the skeletal muscle fibers from STZ-induced diabetic animals were broken and pyknotic, the sarcomeric structure disrupted, and mild hyperplasia of interstitial adipose tissues was detected. The serum level of PKC was higher (P = 0.003) and the protein and mRNA levels of bFGF in skeletal muscle were lower (P = 0.001) in STZ-induced diabetic versus control animals. Protein and mRNA levels of the apoptosis promoting genes caspase-3 and bax were higher in skeletal muscle from STZ-induced diabetic rats as compared to control animals (P < 0.001 and P = 0.037, respectively), while mRNA and protein levels of bcl-2, an inhibitor of apoptosis, was lower in STZ-induced diabetic rats versus control animals (P = 0.026). Increasing apoptosis in skeletal muscle from STZ-induced diabetic rats was further demonstrated by TNNEL assay. Our findings suggest that enhanced PKC levels, reduction of bFGF expression, and increased in apoptosis might be associated with the development of diabetes-induced myoatrophy.     

Delay in Apoptosome Formation Attenuates Apoptosis in Mouse Embryonic Stem Cell Differentiation

Differentiation is an inseparable process of development in multicellular organisms. Mouse embryonic stem cells (mESCs) represent a valuable research tool to conduct in vitro studies of cell differentiation. Apoptosis as a well known cell death mechanism shows some common features with cell differentiation, which has caused a number of ambiguities in the field. The research question here is how cells could differentiate these two processes from each other. We have investigated the role of the mitochondrial apoptotic pathway and cell energy level during differentiation of mESCs into the cardiomyocytes and their apoptosis. p53 expression, cytochrome c release, apoptosome formation, and caspase-3/7 activation are observed upon induction of both apoptosis and differentiation. However, remarkable differences are detected in time of cytochrome c appearance, apoptosome formation, and caspase activity upon induction of both processes. In apoptosis, apoptosome formation and caspase activity were observed rapidly following the cytochrome c release. Unlike apoptosis, the release of cytochrome c upon differentiation took more time, and the maximum caspase activity was also postponed for 24 h. This delay suggests that there is a regulatory mechanism during differentiation of mESCs into cardiomyocytes. The highest ATP content of cells was observed immediately after cytochrome c release 6 h after apoptosis induction and then decreased, but it was gradually increased up to 48 h after differentiation. These observations suggest that a delay in the release of cytochrome c or delay in ATP increase attenuate apoptosome formation, and caspase activation thereby discriminates apoptosis from differentiation in mESCs.     

BCL-2 antisense and cisplatin combination treatment of MCF-7 breast cancer cells with or without functional p53

Summary Chemotherapy has been used for treatment of breast cancer but with limited success. We characterized the effects of bcl-2 antisense and cisplatin combination therapy in two human isogenic breast carcinoma cells p53(+)MCF-7 and p53(−)MCF-7/E6. The transferrin-facilitated lipofection strategy we have developed yielded same transfection efficiency in both cells. Bcl-2 antisense delivered with this strategy significantly induced more cell death, apoptosis, and cytochrome c release in MCF-7/E6 than in MCF-7, but did not affect Fas level in both cells and activated caspase-8 equally. Cisplatin exerted same effects on cell viability and apoptosis in both cells, but released smaller amounts of cytochrome c while activated more caspase-8 in MCF-7/E6. The combination treatment yielded greater effects on cell viability, apoptosis, cytochrome c release, and caspase-8 activation than individual treatments in both cells although p53(−) cells were more sensitive. The potentiated activation of caspase-8 in the combination treatment suggested that caspase-8-mediated (but cytochrome c-independent) apoptotic pathway is the major contributor of the enhanced cell killing. Thus, bcl-2 antisense delivered with transferrin-facilitated lipofection can achieve the efficacy of killing breast cancer cells and sensitizing them to chemotherapy. Bcl-2 antisense and cisplatin combination treatment is a potentially useful therapeutic strategy for breast cancer irrespective of p53 status. Hesham Basma and Hesham El-Refaey contributed equally     

Cytochrome c associated apoptosis during ATP recovery after hypoxia in neonatal rat cerebrocortical slices

Cellular injury was evaluated in superfused cerebrocortical slices (350 micro m) from 7-day-old Sprague-Dawley rats exposed to 30 min hypoxia followed by 4 h of reoxygenation. At the end of hypoxia homogenous cytosolic immunoreactivity of cytochrome c increased approximately fourfold, cytochrome c intensity in western blot analyses increased more than fivefold, and whole cell and cytosolic cleaved caspase-9 underwent 50% and 100% increases, respectively. Immunostaining of sections taken 1.5 h after hypoxia showed: (i) more than a threefold increase in cleaved caspase-9; (ii) localization of cleaved caspase-9 to the interior and peripheral exterior of nuclei; and (iii) homogeneously distributed cytochrome c in the cytosol. Western blot analysis for 1.5 h after hypoxia showed that cytosolic caspase-9 returned to control values, while whole cell caspase-9 stayed approximately the same, suggesting translocation of caspase-9 to nuclei. By 4 h after hypoxia there was significant nuclear fragmentation and an increase in TUNEL positive staining. 31P/1H nuclear magnetic resonance (NMR) confirmed substantial decreases of ATP and phosphocreatine during hypoxia, with rapid but incomplete recovery being close to steady state 1 h after reoxygenation. At all time points after hypoxia the primary injury was cytochrome c associated apoptosis.     

Inhibition of Nitric-Oxide-Mediated Apoptosis in Jurkat Leukemia Cells despite Cytochrome c Release

We have recently shown that nitric-oxide (NO)-induced apoptosis in Jurkat human leukemia cells requires degradation of mitochondria phospholipid cardiolipin, cytochrome c release, and activation of caspase-9 and caspase-3. Moreover, an inhibitor of lipid peroxidation, Trolox, suppressed apoptosis in Jurkat cells induced by NO donor glycerol trinitrate. Here we demonstrate that this antiapoptotic effect of Trolox occurred despite massive release of the mitochondrial protein cytochrome c into the cytosol and mitochondrial damage. Incubation with Trolox caused a profound reduction of intracellular ATP concentration in Jurkat cells treated by NO. Trolox prevented cardiolipin degradation and caused its accumulation in Jurkat cells. Furthermore, Trolox markedly downregulated the NO-mediated activation of caspase-9 and caspase-3. Caspase-9 is known to be activated by released cytochrome c and together with caspase-3 is considered the most proximal to mitochondria. Our results suggest that the targets of the antiapoptotic effect of Trolox are located downstream of the mitochondria and that caspase activation and subsequent apoptosis could be blocked even in the presence of cytochrome c released from the mitochondria.     

Bcl-2 and tBid proteins counter-regulate mitochondrial potassium transport

The mechanism of cytochrome c release from mitochondria in apoptosis remains obscure, although it is known to be regulated by bcl-2 family proteins. Here we describe a set of novel apoptotic phenomena—stimulation of the mitochondrial potassium uptake preceding cytochrome c release and regulation of such potassium uptake by bcl-2 family proteins. As a result of increased potassium uptake, mitochondria undergo moderate swelling sufficient to release cytochrome c. Overexpression of bcl-2 protein prevented the mitochondrial potassium uptake as well as cytochrome c release in apoptosis. Bcl-2 was found to upregulate the mitochondrial potassium efflux mechanism—the K/H exchanger. Specific activation of the mitochondrial K-uniporter led to cytochrome c release, which was inhibited by bcl-2. tBid had an opposite effect—it stimulated mitochondrial potassium uptake resulting in cytochrome c release. The described counter-regulation of mitochondrial potassium transport by bcl-2 and Bid suggests a novel view of a mechanism of cytochrome c release from mitochondria in apoptosis.     

Influence of oilseed supplement ranging in n-6/n-3 ratio on fatty acid composition and Δ5-, Δ6-desaturase protein expression in steer muscles

This study investigated effects of roasted or extruded oilseed supplementation ranging in n-6/n-3 ratios from 0.3 to 5.0 on the fatty acid composition and expression of delta-5 desaturase (Δ5d) and Δ6-desaturase (Δ6d) protein in commercial steer cheek (m. masseter) and diaphragm (pars costalis diaphragmatis) muscles. In general, the n-6/n-3 ratio of the diet had a subsequent effect on the muscle n-6/n-3 ratio (P < 0.05), with muscle 18:2n-6 and 18:3n-3 content relating to proportion of dietary soya bean and linseed (P < 0.01). Compared with canola, pure linseed and soya bean diets reduced 14:1c-9 and 16:1c-9 (P < 0.05) but increased 18:1t-11 and c-9,t-11 conjugated linoleic acid (CLA) content (P < 0.01). Oilseed processing had a minor influence but extruded oilseeds increase 18:1t-11 and c-9,t-11 CLA compared with roasted (P < 0.05). Polar lipid 18:3n-3 and n-3 long-chain polyunsaturated fatty acid (LC, ⩾20 carbons PUFA) derivative content increased in relation to dietary linseed supplementation in the diaphragm (P < 0.01), whereas only 18:3n-3 was increased in the cheek (P < 0.01). Protein expression did not differ between diets; however, in each muscle the Δ5d protein expression had a stronger association with the desaturase products rather than the precursors. The relationship between Δ5d protein expression and the muscle LC n-6/n-3 ratio was negative in both muscles (P < 0.05). The relationship between Δ6d protein expression and the LC n-6/n-3 ratio was positive in the cheek (P < 0.001) and negative in the diaphragm (P < 0.05). In conclusion, diet n-6/n-3 ratio affected muscle 18:2n-6 and 18:3n-3 deposition, whereas the Δ5d and Δ6d protein expression had some influence on the polar lipid LC-PUFA profile. Results reaffirm that processed oilseeds can be used to increase the proportion of fatty acids potentially beneficial for human health, by influencing the formation of LC-PUFA and reducing the n-6/n-3 ratio.     

Spectrophotometric Studies on Intact Muscle : II. Recovery from contractile activity

The kinetics of the mitochondrial respiratory chain of intact muscle and the concomitant changes of the intercellular pH were investigated. Addition of lactate and pyruvate under resting conditions produces reductions of DPN and cytochrome b, and, occasionally, of cytochrome c and flavoprotein. Succinate gives similar but smaller changes. In recently excised muscles moderate contractile activity produces a reduction of cytochrome c and oxidations of DPNH, cytochrome b, and sometimes of the flavoproteins. Tetanic contractions and larger numbers of twitches produce reductions of DPN and of cytochromes b and c. In sartorii of the tropical toad, stored for approximately 2 days at 0–3°C, contractile activity always gives rise to long lasting oxidations of DPNH and cytochrome b. Addition of pyruvate or lactate shortens these oxidation cycles with a concomitant reduction of cytochrome c. These responses to contractions agree with those of mitochondria isolated from leg muscles of the toad upon the addition of ADP. Apparently the mitochondria in resting, excised muscles are not supplied with an excess of substrate. Measurements on the intercellular pH showed that even limited activity ( < 5 twitches) initiates glycolysis. The primary control of respiration resides, nevertheless, in the ADP concentration, rather than in the levels of substrate or inorganic phosphate. The results are quantitatively consistent with the view that ATP is the primary energy donor for muscular contraction.     

Mechanism of Activation of Caspase-9 and Caspase-3 during Hypoxia in the Cerebral Cortex of Newborn Piglets: The Role of Nuclear Ca<Superscript>2+</Superscript>-Influx

In previous studies, we have shown that cerebral hypoxia results in increased activity of caspase-9, the initiator caspase, and caspase-3, the executioner of programmed cell death. We have also shown that cerebral hypoxia results in high affinity Ca²⁺–ATPase-dependent increase in nuclear Ca²⁺-influx in the cerebral cortex of newborn piglets. The present study tests the hypothesis that inhibiting nuclear Ca²⁺-influx by pretreatment with clonidine, an inhibitor of high affinity Ca²⁺–ATPase, will prevent the hypoxia-induced increase in caspase-9 and caspase-3 activity in the cerebral cortex of newborn piglets. Thirteen newborn piglets were divided into three groups, normoxic (Nx, n = 4), hypoxic (Hx, n = 4), and hypoxic treated with clonidine (100 mg/kg) (Hx–Cl, n = 5). Anesthetized, ventilated animals were exposed to an FiO₂ of 0.21 (Nx) or 0.07 (Hx) for 60 min. Cerebral tissue hypoxia was documented biochemically by determining levels of ATP and phosphocreatine (PCr). Caspase-9 and -3 activity were determined spectrofluoro-metrically using specific fluorogenic synthetic substrates. ATP (μmoles/g brain) was 4.6 ± 0.3 in Nx, 1.7±0.4 in Hx (P < 0.05 vs. Nx), and 1.5 ± 0.2 in Hx–Cl (P < 0.05 vs. Nx). PCr (μmoles/g brain) was 3.6 ± 0.4 in Nx, 1.1 ± 0.3 in Hx (P < 0.05 vs. Nx), and 1.0 ± 0.2 in Hx–Cl (P < 0.05 vs. Nx). Caspase-9 activity (nmoles/mg protein/h) was 0.548 ± 0.0642 in Nx and increased to 0.808 ± 0.080 (P < 0.05 vs. Nx and Hx–Cl) in the Hx and 0.562 ± 0.050 in the Hx–Cl group (p = NS vs. Nx). Caspase-3 activity (nmoles/mg protein/h) was 22.0 ± 1.3 in Nx and 32 ± 6.3 in Hx (P < 0.05 vs. Nx) and 18.8 ± 3.2 in the Hx–Cl group (P < 0.05 vs. Hx). The data demonstrate that clonidine administration prior to hypoxia prevents the hypoxia-induced increase in the activity of caspase-9 and caspase-3. We conclude that the high afinity Ca²⁺–ATPase-dependent increased nuclear Ca²⁺ during hypoxia results in increased caspase-9 and caspase-3 activity.     

Ataxia telangiectasia mutated influences cytochrome c oxidase activity

Cells lacking ataxia telangiectasia mutated (ATM) have impaired mitochondrial function. Furthermore, mammalian cells lacking ATM have increased levels of reactive oxygen species (ROS) as well as mitochondrial DNA (mtDNA) deletions in the region encoding for cytochrome c oxidase (COX). We hypothesized that ATM specifically influences COX activity in skeletal muscle. COX activity was ∼40% lower in tibialis anterior from ATM-deficient mice than for wild-type mice (P < 0.01, n = 9/group). However, there were no ATM-related differences in activity of succinate dehydrogenase, isocitrate dehydrogenase, alpha-ketoglutarate dehydrogenase, mitochondrial glycerol 3-phosphate dehydrogenase, or complex III. Incubation of wild-type extensor digitorum longus muscles for 1 h with the ATM inhibitor KU55933 caused a ∼50% reduction (P < 0.05, n = 5/group) in COX activity compared to muscles incubated with vehicle alone. Among the control muscles and muscles treated with the ATM inhibitor, COX activity was correlated (r = 0.61, P < 0.05) with activity of glucose 6-phosphate dehydrogenase, a key determinant of antioxidant defense through production of NADPH. Overall, the findings suggest that ATM has a protective role for COX activity.