Lower Succinate Dehydrogenase Activity Research Articles

Seasonal cold induces divergent structural/biochemical adaptations in different skeletal muscles of Columba livia: evidence for nonshivering thermogenesis in adult birds.

Birds are endothermic homeotherms even though they lack the well-studied heat producing brown adipose tissue (BAT), found in several clades of eutherian mammals. Earlier studies in ducklings have demonstrated that skeletal muscle is the primary organ of nonshivering thermogenesis (NST) plausibly via futile calcium (Ca2+)-handling through ryanodine receptor (RyR) and sarco-endoplasmic reticulum Ca2+-ATPase (SERCA). However, recruitment of futile Ca2+-cycling in adult avian skeletal muscle has not been documented. Studies in mammals show remarkable mitochondrial remodeling concurrently with muscle NST during cold. Here, we wanted to define the mitochondrial and biochemical changes in the muscles in free-ranging adult birds and whether different skeletal muscle groups undergo similar seasonal changes. We analyzed four different muscles (pectoralis, biceps, triceps and iliotibialis) from local pigeon (Columba livia) collected during summer and winter seasons in two consecutive years. Remarkable increase in mitochondrial capacity was observed as evidenced from succinate dehydrogenase (SDH) and cytochrome c oxidase (COX) activity staining in all the muscles. Interestingly, fibers with low SDH activity exhibited greater cross-sectional area during winter in all muscles except iliotibialis and became peripherally arranged in individual fascicles of pectoralis, which might indicate increased shivering. Furthermore, gene expression analysis showed that SERCA, sarcolipin and RyR are up-regulated to different levels in the muscles analyzed indicating muscle NST via futile Ca2+-cycling is recruited to varying degrees in winter. Moreover, proteins of mitochondrial-SR-tethering and biogenesis also showed differential alterations across the muscles. These data suggest that tropical winter (∼15°C) is sufficient to induce distinct remodeling across muscles in adult bird.

Abstract 2327: Dysregulated TCA cycle is associated with endocrine therapy resistance in ER+ breast cancer cells

Abstract Estrogen receptor positive (ER+) breast cancer is the most common breast cancer subtype. Endocrine therapies, such as selective estrogen receptor modulators (SERM) and aromatase inhibitors (AI), along with cyclin-dependent kinase 4/6 (CDK4/6) inhibitors, are the current mainstays of treatment. Despite being initially effective, the majority of advanced ER+ breast cancers become resistant to the endocrine therapies. Therefore, it is important to understand the underlying molecular mechanisms that enable or promote resistance to endocrine therapies. Rapidly proliferating cells have a high energy demand and consequently consume greater quantities of glucose to fulfill their energy and anabolic demands to support cell growth. Using endocrine therapy sensitive and resistant breast cancer cell models we investigated the metabolic flux of glucose by growing the cells in the presence of the stable glucose isotope, UC13-Glucose and measured the C13-labelled metabolites of glycolysis and the TCA cycle. Glucose consumption was higher in endocrine therapy resistant MCF7/LCC9 (LCC9) cells compared with parental MCF7 (MCF7) and estrogen independent, endocrine therapy sensitive MCF7/LCC1 (LCC1) cells. The TCA cycle was noticeably impaired in LCC9 cells as the C-13 labelled citrate (m+4 and m+6) isotopomer was absent in these cells. In addition, C-13 labelled fumarate was very low in LCC9 cells with a concurrent increase in the succinate:fumarate ratio. These observations show that in LCC9 cells the TCA cycle is dysregulated at the succinate to fumarate conversion step, which is catalyzed by the succinate dehydrogenase (SDH) enzyme. SDH is a multimeric protein comprised of four different subunits and SDH assembly factor 2 (SDHAF2), which is a tumor suppressor gene. Further investigation revealed that SDHAF2 was under expressed in LCC9 cells compared with LCC1 cells, likely contributing to lower SDH activity. Intriguingly, SDH inhibition by dimethyl-malonate re-sensitized LCC9 cells to both Fulvestrant and 4-hydroxytamoxifen. Clinically, lower expression levels of SDHAF2 correlates with poor relapse free survival in ER+ breast cancer patients. In summary, our study suggests that accumulation of succinate driven by deficient SDH activity is functionally associated with endocrine therapy resistance in ER+ breast cancer cells. Citation Format: Surojeet Sengupta, Karla Andrade de Oliviera, Lu Jin, Robert Clarke. Dysregulated TCA cycle is associated with endocrine therapy resistance in ER+ breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2327.

PECULIARITIES OF MORPHOFUNCTIONAL CHANGES OF MASTICATORY MUSCLES IN IODINE-DEFICIENT CONDITIONS

The problem of iodine deficiency is becoming increasingly important in Ukraine. According to the United Nations Children’s Fund (UNICEF), each and all regions of Ukraine suffer from “hidden hunger”. The World Health Assembly has stated that the elimination of iodine deficiency will be as much a triumph for health care system as the eradication of smallpox and poliomyelitis. The goal of this study was to establish the peculiarities of structural organization of the masticatory muscles of rats in experimentally induced iodine deficiency. The research material involved masticatory muscle of 36 white outbred male rats, which were divided into groups: group І – intact animals; group ІІ – kept in iodine deficiency condition; group III – exposed to iodine deficiency with the addition of goitriferous food. All manipulations with test animals were performed in agreement with the regulations on the protection of vertebrate animals. The methods used in the study included: biochemical, histological, histochemical, submicroscopic, morphometric and statistical research methods. It has been established that under the conditions of iodine deficiency the lumen of arterial vessels in masticatory muscles becomes narrowed, the vascular vessels become thickened due to membrane oedema. The number of hemocapillaries per unit area of muscle fibre goes down in all the studied muscles. The number of muscle fibres with intermediate succinate dehydrogenase activity decreases with the simultaneous increase in muscle fibres with low succinate dehydrogenase activity. Furthermore, the oedematous changes are the most prominent in muscle fibres with low succinate dehydrogenase activity. In conditions of iodine deficiency with the addition of goitriferous products, we can observe the progression of luminal narrowing of the artery and wall thickening in masticatory muscles where oedematous-dystrophic changes are considerably marked. There is a tendency in the growth in haemocapillaries number per 1 μm2. Histostructurally, the loss of cross striation is quite noticeable. The cross-section diameter increases, especially in muscle fibres with low succinate dehydrogenase activity and muscle fibres with high succinate dehydrogenase activity in the masticatory muscle proper of immature animals. Ultrastructurally, the oedematous-dystrophic changes of myofibrils and mitochondria were revealed. Thus, under the conditions of iodine deficient diet the lumen of arterial vessels narrows in the masticatory muscles, their wall thickens due to oedema of the membranes, which progresses in iodine deficient diet with the addition of goitriferous products. The number of hemocapillaries per unit area of muscle fibre decreases in both study groups. Changes in vascular bed lead to the redistribution of the number of muscle fibres and their significant oedema.

P1603INVOLVEMENT OF SUCCINATE DEHYDROGENASE (SDH) IN DECEASED RENAL TRANSPLANT DONORS' INFAMMATION AND FIBROSIS

Abstract Background and Aims Succinate is a Krebs cycle intermediate that is converted to fumarate by succinate dehydrogenase (SDH). SDH is part of the electron-transport chain of inner mitochondrial membrane (complex II) and its activity depends indirectly on the oxygen availability. Low oxygen states, such as ischemia, can increase circulating succinate levels due to low SDH activity. In renal transplantation, ischemia and hypoxia can increase renal succinate formation and induce the infiltration of immune cells producing TNF-α, increasing inflammation, renal epithelial apoptosis, as well as leukocyte recruitment, binding and migration, which can lead to renal graft damage. Method Samples of pre-implantation renal tissue, 17 from living donors and 40 from deceased donors were analyzed by Real-time PCR. Results In kidneys from deceased donors SDH expression is downregulated before transplantation and it lasts for at least the first year. Low SDH gene expression at 4 months after kidney transplant is positively correlated with graft renal function (CKD-EPI) at 12 and 24 months (p=0.046, r= 0.6511 and p= 0.0351, r= 0.4731; respectively). In kidneys from deceased and living donors, SDHD is negatively associated with monocyte recruitment (MCP-1) and activation (IL-1β) and positively with the expression of SOD1. Moreover, only in deceased donors, SDHD gene expression correlates inversely with pro-fibrotic (TGF-β1 and vimentin) and inflammatory (TNF-α) molecules, as well as with M1 (CD80, CD86) and M2 (CD163) macrophage markers, all of which are found to be higher expressed in deceased donor kidneys than in living donor ones. Conclusion Our results indicate that low gene expression of SDH in kidneys from deceased donors could result in reduced activity of SDH that would reduce the ability to transform succinate to fumarate resulting in succinate accumulation, increasing inflammation and reparation through fibrosis worsening transplant outcome.

17 Generation of myostatin gene knockout boars by somatic cell nuclear transfer

Skeletal muscle is the most economically valuable tissue part in meat-producing animals, and enhancing muscle growth in these species may increase the efficiency of meat production. Skeletal muscle mass is negatively regulated by myostatin (MSTN), and nonfunctional mutations of this MSTN gene in various animal species have led to dramatic hypermuscularity. A porcine fetal fibroblast cell line with biallelic MSTN mutations (MSTN −/−), consisting of a 2-bp deletion in one allele and a 4-bp deletion in the other allele, was used as a donor to generate cloned pigs via somatic cell nuclear transfer. Cloned embryos were transferred into seven surrogates and 38 live piglets were delivered. Using these MSTN null piglets, the detailed morphological, gene and protein expression analyses were performed not only in biceps femoris, semitendinosus, and diaphragm of MSTN null piglets but also in heart, liver, spleen, lung, kidney, and tongue. The results showed that MSTN −/− boars have visually clear hypermuscular characteristics, which was supported by the increased carcass dressing percentage and loin eye size and decreased backfat thickness. The fibre cross-sectional areas of the semitendinosus and semimembranosus muscles were much larger in MSTN −/− piglets than in wild-type (WT) piglets (98.0±8.2 vs. 62.7±4.7, 105.7±11.6 vs. 72.3±8.4mm2, respectively; P<0.05). MSTN −/− pigs showed a higher proportion of fast-type fibres (Type-II fibre%, 95±1.8 vs. 86±2.4; P<0.05) and lower succinate dehydrogenase activity in muscles than WT pigs. The mRNA expression of myosin heavy-chain IIB in both two muscles was higher in MSTN −/− pigs compared with WT pigs (P<0.05). In organs, the heart and liver were lighter in MSTN −/− piglets than in WT piglets (8.1±0.3 vs. 10.3±0.4, 26.9±1.8 vs. 34.2±1.2g kg−1, respectively; P<0.05), whereas the tongue was heavier in MSTN −/− piglets than in WT piglets and myofibres of the tongue were significantly larger in the former piglets than in the latter piglets (P<0.01). Messenger RNA expression of MSTN in all organs was significantly lower in MSTN −/− piglets than in WT piglets (P<0.01) and follistatin in the heart and liver was significantly higher in MSTN −/− piglets than in WT piglets (P<0.05). Protein expression of MSTN in the heart, kidneys, and tongue was significantly lower in MSTN −/− piglets than in WT piglets (P<0.01). In conclusion, MSTN −/− pigs showed a phenotype of supermuscular ratio is associated with increased muscle fibres and smaller internal organs and has a complicated gene expression patterns.

Fiber-type distribution and expression of myosin heavy chain isoforms in newborn heterozygous myostatin-knockout pigs.

To explore the effects of heterozygous myostatin-knockout (MSNT+/-) on muscle characteristics, specifically fiber-type distribution and expression of myosin heavy chain isoforms in pigs. The fiber cross-sectional area of the semitendinosus and semimembranosus muscles were much larger in MSTN+/- pigs at birth than in wild-type (WT) pigs. MSTN+/- pigs had a higher proportion of fast-type fibers and lower succinate dehydrogenase activity in muscles than WT pigs. The myosin heavy chain IIB mRNA level in both two muscles was~threefold higher in MSTN+/- pigs compared with WT pigs. MSTN+/- pigs exhibit a disproportionate increase in muscle mass and can have a higher body weight due to fiber hypertrophy, a change in the fiber-type distribution, and alteration of myosin heavy chain isoforms levels, leading to more fast glycolytic fibers.

The effect of radiation dose on mouse skeletal muscle remodeling.

BackgroundThe purpose of this study was to determine the effect of two clinically relevant radiation doses on the susceptibility of mouse skeletal muscle to remodeling.Materials and methods.Alterations in muscle morphology and regulatory signaling were examined in tibialis anterior and gastrocnemius muscles after radiation doses that differed in total biological effective dose (BED). Female C57BL/6 (8-wk) mice were randomly assigned to non-irradiated control, four fractionated doses of 4 Gy (4x4 Gy; BED 37 Gy), or a single 16 Gy dose (16 Gy; BED 100 Gy). Mice were sacrificed 2 weeks after the initial radiation exposure.ResultsThe 16 Gy, but not 4x4 Gy, decreased total muscle protein and RNA content. Related to muscle regeneration, both 16 Gy and 4x4 Gy increased the incidence of central nuclei containing myofibers, but only 16 Gy increased the extracellular matrix volume. However, only 4x4 Gy increased muscle 4-hydroxynonenal expression. While both 16 Gy and 4x4 Gy decreased IIB myofiber mean cross-sectional area (CSA), only 16 Gy decreased IIA myofiber CSA. 16 Gy increased the incidence of small diameter IIA and IIB myofibers, while 4x4 Gy only increased the incidence of small diameter IIB myofibers. Both treatments decreased the frequency and CSA of low succinate dehydrogenase activity (SDH) fibers. Only 16 Gy increased the incidence of small diameter myofibers having high SDH activity. Neither treatment altered muscle signaling related to protein turnover or oxidative metabolism.ConclusionsCollectively, these results demonstrate that radiation dose differentially affects muscle remodeling, and these effects appear to be related to fiber type and oxidative metabolism.

Abnormalities in the Fiber Composition and Capillary Architecture in the Soleus Muscle of Type 2 Diabetic Goto-Kakizaki Rats

Type 2 diabetes mellitus is linked to impaired skeletal muscle glucose uptake and storage. This study aimed to investigate the fiber type distributions and the three-dimensional (3D) architecture of the capillary network in the skeletal muscles of type 2 diabetic rats. Muscle fiber type transformation, succinate dehydrogenase (SDH) activity, capillary density, and 3D architecture of the capillary network in the soleus muscle were determined in 36-week-old Goto-Kakizaki (GK) rats as an animal model of nonobese type 2 diabetes and age-matched Wistar (Cont) rats. Although the soleus muscle of Cont rats comprised both type I and type IIA fibers, the soleus muscle of GK rats had only type I fibers. In addition, total SDH activity in the soleus muscle of GK rats was significantly lower than that in Cont rats because GK rats had no high-SDH activity type IIA fiber in the soleus muscle. Furthermore, the capillary diameter, capillary tortuosity, and microvessel volume in GK rats were significantly lower than those in Cont rats. These results indicate that non-obese diabetic GK rats have muscle fiber type transformation, low SDH activity, and reduced skeletal muscle capillary content, which may be related to the impaired glucose metabolism characteristic of type 2 diabetes.

Regional skeletal muscle remodeling and mitochondrial dysfunction in right ventricular heart failure

Exercise intolerance is a cardinal symptom of right ventricular heart failure (RV HF) and skeletal muscle adaptations play a role in this limitation. We determined regional remodeling of muscle structure and mitochondrial function in a rat model of RV HF induced by monocrotaline injection (MCT; 60 mg·kg(-1); n = 11). Serial sections of the plantaris were stained for fiber type, succinate dehydrogenase (SDH) activity and capillaries. Mitochondrial function was assessed in permeabilized fibers using respirometry, and isolated complex activity by blue native gel electrophoresis (BN PAGE). All measurements were compared with saline-injected control animals (CON; n = 12). Overall fiber cross-sectional area was smaller in MCT than CON: 1,843 ± 114 vs. 2,322 ± 120 μm(2) (P = 0.009). Capillary-to-fiber ratio was lower in MCT in the oxidative plantaris region (1.65 ± 0.09 vs. 1.93 ± 0.07; P = 0.03), but not in the glycolytic region. SDH activity (P = 0.048) and maximal respiratory rate (P = 0.012) were each ∼15% lower in all fibers in MCT. ADP sensitivity was reduced in both skeletal muscle regions in MCT (P = 0.032), but normalized by rotenone. A 20% lower complex I/IV activity in MCT was confirmed by BN PAGE. MCT-treatment was associated with lower mitochondrial volume density (lower SDH activity), quality (lower complex I activity), and fewer capillaries per fiber area in oxidative skeletal muscle. These features are consistent with structural and functional remodeling of the determinants of oxygen supply potential and utilization that may contribute to exercise intolerance and reduced quality of life in patients with RV HF.

Effect of castration on mouse skeletal muscle oxidative capacity

Low testosterone is often observed in male patients with cancer, COPD and HIV, and associated with impaired oxidative metabolism in skeletal muscle. Although testosterone has well described effects on muscle mass, less is known about the effect of testosterone on muscle's metabolic capacity. The purpose of this study was to determine if oxidative capacity is reduced in wasting skeletal muscle due to castration and if anabolic steroid (ND) administration can rescue this capacity. 8‐week male C57BL/6 mice were placed into three treatment groups: sham castration (CON, n = 6), castration (CAS, n = 6), and castration with ND administration (n = 6). Gastrocnemius (GAS) muscle succinate dehydrogenase (SDH) activity and PGC‐1 expression were examined. Castrated GAS muscle increased the percentage of low SDH activity fibers (78.2±1.5% vs. 84.5±1.1%, p < .05) and decreased the incidence of high SDH activity fibers (5.0±0.6% vs. 1.3±0.2%, p < .05) compared to CON. ND administration significantly attenuated these changes. PGC‐1 mRNA expression decreased 54% with castration (p = .029), but increased 141% with ND administration (p = .036). These data indicate testosterone availability may regulate oxidative capacity in mouse hindlimb muscle. This study was funded by NIH Grant 5R03AR051434‐02.

Cold Acclimation Responses of Three Cool-season Turfgrasses and the Role of Proline-associated Pentose Phosphate Pathway

The role of the antioxidant response system in association with the proline-associated pentose phosphate pathway for cold adaptation was investigated in three cool-season turfgrasses during a cold acclimation period. As phenolic biosynthesis and antioxidant stimulation is proposed to be linked to the proline-associated pentose phosphate pathway, this study was aimed to determine the active role of proline in metabolic regulation and its relationship with the cold stress tolerance mechanism of cool-season turfgrasses. In this study, significant accumulation of total soluble phenolics and higher total antioxidant activity was observed in creeping bentgrass (Agrostis stolonifera L.), kentucky bluegrass (Poa pratensis L.), and perennial ryegrass (Lolium perenne L.) during cold acclimation, confirming the direct and indirect role of phenolics to counter low temperature-induced oxidative stress. A positive correlation between high phenolic content and the proline-associated pentose phosphate pathway was also found in investigated turfgrass species during a cold acclimation period. Low succinate dehydrogenase activity along with the high glucose-6-phosphate dehydrogenase activity in cold-acclimated turfgrass species suggested a probable shift of carbon flux from the energy-consuming tricarboxylic cycle to the alternative energy-efficient proline-associated pentose phosphate pathway to induce a better cold stress tolerance mechanism in these cool-season turfgrasses. Higher proline accumulation in cold-acclimated turfgrass species also supported the above findings and a probable proline oxidation to support mitochondrial oxidative phosphorylation was observed in acclimated kentucky bluegrass based on the activity of proline dehydrogenase, which likely supports the active metabolic role of proline in stress-induced situations. Through this study, a significant variation in cold stress tolerance mechanisms was observed among three investigated cool-season turfgrass species during cold acclimation. Furthermore, a high cold stress tolerance characteristic was observed in kentucky bluegrass by adapting a more efficient pathway for an antioxidant response linked to proline accumulation.

A study on thestunning phenomenon of nerve cells after transient anoxia-serum deprivation/reperfusion

：Objective To identifywhether there is nerve cells stunning after transient anoxia-serum deprivation/reperfusionand to explore the probable underlying mechanism of the phenomenon.Methods The PC12 cellswere randomly divided into a control group and a anoxia group, which were divided into 3subgroups respectively, according to the time of reperfusion after 15 min of anoxia-serumdeprivation. The content of adenosine triphosphate, mitochondria membrane potential andcell activity were determined at different time points after transient anoxia-serumdeprivation/reperfusion.Results The content of adenosine triphosphate(1.05±0.34),mitochondria membrane potential (41.39±1.242) and cell activity after 15 min ofanoxia-serum deprivation plus 1h of reperfusion in the anoxia group were significantlylower than those in the control group(P ＜0.05), and recovered to level of the control group after6h.Conclusion There was lower energy status after transient of anoxia-serumdeprivation/reperfusion, and the lower energy status can recover to the normal level.There might be nerve cell stunning. This phenomenon probably results from lower succinatedehydrogenase activity in the respiratory chain of the cells and the changes ofmitochondria membrane potential. Key words: Anoxia; Serum deprivation; Reperfusion; Nerve cell stunning; Energymetabolism

Relation of systemic and local muscle exercise capacity to skeletal muscle characteristics in men with congestive heart failure

The present study was undertaken to further characterize changes in skeletal muscle morphology and histochemistry in congestive heart failure and to determine the relation of these changes to abnormalities of systemic and local muscle exercise capacity. Abnormalities of skeletal muscle appear to play a role in the limitation of exercise capacity in congestive heart failure, but information on the changes in muscle morphology and biochemistry and their relation to alterations in muscle function is limited. Eighteen men with predominantly mild to moderate congestive heart failure (mean +/- SEM New York Heart Association functional class 2.6 +/- 0.2, ejection fraction 24 +/- 2%) and eight age- and gender-matched sedentary control subjects underwent measurements of peak systemic oxygen consumption (VO2) during cycle ergometry, resistance to fatigue of the quadriceps femoris muscle group and biopsy of the vastus lateralis muscle. Peak VO2 and resistance to fatigue were lower in the patients with heart failure than in control subjects (15.7 +/- 1.2 vs. 25.1 +/- 1.5 ml/min-kg and 63 +/- 2% vs. 85 +/- 3%, respectively, both p < 0.001). Patients had a lower proportion of slow twitch, type I fibers than did control subjects (36 +/- 3% vs. 46 +/- 5%, p = 0.048) and a higher proportion of fast twitch, type IIab fibers (18 +/- 3% vs. 7 +/- 2%, p = 0.004). Fiber cross-sectional area was smaller, and single-fiber succinate dehydrogenase activity, a mitochondrial oxidative marker, was lower in patients (both p < or = 0.034). Likewise, the ratio of average fast twitch to slow twitch fiber cross-sectional area was lower in patients (0.780 +/- 0.06 vs. 1.05 +/- 0.08, p = 0.019). Peak VO2 was strongly related to integrated succinate dehydrogenase activity in patients (r = 0.896, p = 0.001). Peak VO2, resistance to fatigue and strength also correlated significantly with several measures of fiber size, especially of fast twitch fibers, in patients. None of the skeletal muscle characteristics examined correlated with exercise capacity in control subjects. These results indicate that congestive heart failure is associated with changes in the characteristics of skeletal muscle and local as well as systemic exercise performance. There are fewer slow twitch fibers, smaller fast twitch fibers and lower succinate dehydrogenase activity. The latter finding suggests that mitochondrial content of muscle is reduced in heart failure and that impaired aerobic-oxidative capacity may play a role in the limitation of systemic exercise capacity.

Enzymic pattern of preneoplastic and neoplastic lesions induced in the kidney of CBA mice by 1,2-dimethylhydrazine.

Mouse renal cell tumors (RCTs) were induced in male CBA mice by 5 subcutaneous injections of 8 mg 1,2-dimethylhydrazine (DMH)/kg body weight once a week. After a lag period of 2 yr kidneys were removed, and serial cryostat sections of the kidneys were histochemically analyzed for the following parameters: glycogen content, basophilia, and the activities of glycogen synthase (SYN), glycogen phosphorylase (PHO), glucose-6-phosphatase (G6Pase), glucose-6-phosphate dehydrogenase (G6PDH), hexokinase (HK), pyruvate kinase (PK), lactate dehydrogenase (LDH), malic enzyme (ME), succinate dehydrogenase (SDH), alkaline phosphatase (ALPase) and gamma-glutamyltranspeptidase (GGT). RCTs displayed the same histochemical profile irrespective of their size and growth pattern. In comparison with the normal kidney epithelium, the neoplastic cells exhibited elevated activities of enzymes for glycolysis (HK, PK, LDH) and the pentose phosphate pathway (G6PDH), while negative G6Pase and low SDH activity were observed in these cells. The majority of RCTs showed high PHO activity and weak staining for SYN. Activities of ALPase and GGT were negative in most of the RCTs. Markedly enlarged cells with atypical nuclei were detected in some advanced RCTs. Higher activities of glycolytic and mitochondrial enzymes and G6PDH were found in these enlarged cells than in other tumor cells. Tubular preneoplastic lesions were similar to neoplastic lesions in morphological and histochemical characteristics. The present study revealed that a markedly elevated capacity for glycolysis and the pentose phosphate pathway occurred in RCTs in mice. A similar histochemical pattern in the few preneoplastic tubular lesions observed suggests that these metabolic aberrations emerge early during carcinogenesis, but additional studies on early stages of renal carcinogenesis are needed to substantiate this assumption.

Separation of outer and cytoplasmic membranes of Fibrobacter succinogenes and membrane and glycogen granule locations of glycanases and cellobiase

The outer membrane (OM) of Fibrobacter succinogenes was isolated by a combination of salt, sucrose, and water washes from whole cells grown on either glucose or cellulose. The cytoplasmic membrane (CM) was isolated from OM-depleted cells after disruption with a French press. The OM and membrane vesicles isolated from the extracellular culture fluid of cellulose-grown cells had a higher density, much lower succinate dehydrogenase activity, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis protein profiles different from those of the CM. The OM from both glucose- and cellulose-grown cells and the extracellular membrane vesicles from cellulose-grown cultures exhibited higher endoglucanase, xylanase, and acetylesterase activities than the CM and other cell fractions. Endoglucanase 2 was absent from the isolated OM fractions of glucose- and cellulose-grown cells and from the extracellular membrane vesicles of cellulose-grown cells but was present in the CM and intracellular glycogen granule fractions, while endoglucanase 3 was enriched in the OM. Cellobiosidase was located primarily in the periplasm as previously reported, while cellobiase was mainly present in the glycogen granule fraction of glucose-grown cells and in a nongranular glycogen and CM complex in cellulose-grown cells. The cellobiase was not eluted from glycogen granules by cellobiose, maltose, and maltotriose nor from either the granules or the cell membranes by nondenaturing detergents but was eluted from both glycogen granules and cell membranes by high concentrations of salts. The eluted cellobiase rebound almost quantitatively when diluted and mixed with purified glycogen granules but exhibited a low affinity for Avicel cellulose. Thus, we have documented a method for isolation of OM from F. succinogenes, identified the OM origin of the extracellular membrane vesicles, and located glycanases and cellobiase in membrane and glycogen fractions.

Low succinate dehydrogenase (SDH) activity in a patient with a hereditary myopathy with paroxysmal myoglobinuria

An unusual hereditary myopathy with paroxysmal myoglobinuria has been described previously. We have studied muscle biopsy specimens taken before and after exercise to exhaustion (24 min at 20-25 W) in a young woman with this condition. Marked glycogenolysis with lactate production and marked phosphagen breakdown (ATP + CP) were observed after exercise, and almost all type I fibres were found to be depleted of glycogen. Succinate dehydrogenase (SDH) activity was low, while the activities of 3-OH-acyl-CoA-dehydrogenase, phosphofructokinase, phosphorylase and lactate dehydrogenase were normal. On electron microscopy, the mitochondria showed abnormalities typical of mitochondrial myopathy. The findings in our patient suggest a limitation of mitochondrial function, probably related to SDH in the tricarboxylic acid cycle and complex II in the electron transport chain. This may explain the inability of ATP regeneration to keep pace with ATP utilization during exercise. Other metabolic defects may coexist.

Differential distribution of B16F10 melanoma cells in the liver lobule.

The distribution pattern and the number of tumor cells arrested in the liver were studied in mouse livers. Mice were perfused intravascularly with a suspension of B16F10 melanoma cells. The animals were sacrificed at 0, 1, 5, and 20 min after tumor cell perfusion. The pattern of tumor cell distribution was studied by morphological methods, and by a combined method of fluorescent-tumor cell labelling and histochemical succinate dehydrogenase activity on frozen sections, in order to define the localization of tumor cells arrested in the liver lobule. The results show that the tumor cells have an exclusive distribution in the periportal regions of the liver lobule (identified as the high succinate dehydrogenase activity areas), and that the cells are not arrested in the pericentral regions (identified as the low succinate dehydrogenase activity areas). In addition, indomethacin treatment (2 mg/kg/day) induced an increase in the number of melanoma cells arrested in the liver, but a different distribution with respect to controls was not observed. These results show that periportal regions of the liver lobule constitute a particular domain in which the B16F10 melanoma cells present a special retention ability that can be modulated by indomethacin treatment.

Slow type muscle cells in the earthworm gizzard with a distinct, Ca2+-regulated myosin isoform

Histochemical staining of the gizzard from the earthworm,Lumbricus terrestris, reveals low ATPase and high succinic dehydrogenase activity for all muscle cells as compared to the main part of the body wall. In accordance with the presence of slow type muscle cells in the gizzard, isolated actomyosin shows an ATPase activity three times lower than the body wall actomyosin. Gizzard myosin represents an isoform, distinct from those of the body wall muscle, by comparison of the light chain pattern in isoelectric focusing. No difference was observed in the Ca2+-regulatory properties between gizzard and body wall actomyosin. Gizzard actomyosin is dual-regulated, and the myosin contains a regulatory light chain which is reversibly dissociated by EDTA. Isolated gizzard binds two molecules of Ca2+ per molecule, in the same range of free Ca2+ concentrations over which actomyosin is activated, suggesting that the myosin-linked regulatory system is mediated by direct binding of Ca2+. The molar ratios of the major contractile proteins of body wall and gizzard actomyosins differ considerably, indicating a structural diversity of fast and slow type muscle cells.

Succinate dehydrogenase and peroxidase activities in leaf epidermis and stem of some angiosperm species growing near a fertilizer complex

The enzymes succinate dehydrogenase and peroxidase were localized histochemically in the leaf epidermal cells and in the young stems of:Annona squamosa L.,Coccinia indica W. and A.,Bougainvillea spectabilis Willd.,Azadirechta indica L.,Ficus benghalensis L.,Lantana camara L. var.aculeata, Mangifera indica L.,Manilkara hexandra (Roxb) Dub.,Streblus asper Lour,Syzygium cumini L.,Tamarindus indica L., andZizyphus oenoplia L., from the areas around the Gujarat State Fertilizer Company, near Baroda (22°−30′ N latitude, 73°−60′ E longitude), and nearly-normal area of Vallabh Vidyanagar (22°−50′ N latitude, 73°−10′ E longitude). The soil types, temperature, rainfall, humidity and agricultural patterns of the two localities are identical. Intensities of enzyme activity vary in different cell types of the epidermis and in various tissues of the stem. Intensities of succinate dehydrogenase and peroxidase in epidermal cells is low in majority of the pollution-affected plants. Except inAzadirechta indica andManilkara hexandra, stem of all the other pollution-affected species showed lower succinate dehydrogenase activity than in the normal. Vascular parenchyma in the stems of pollution-affected plants ofAzadirechta indica andManilkara hexandra showed high succinate dehydrogenase activity than that in the normal, whereas in extravascular regions it is almost identical with that of the normal stem. Peroxidase activity in the stem of pollution-affected plants ofManilkara hexandra andStreblus asper is higher than that in normal, and in rest of the species it is lower than or almost the same as in the normal.

Low platelet monoamine oxidase activity in headache: no correlation with phenolsulphotransferase, succinate dehydrogenase, platelet preparation method or smoking.

Platelet monoamine oxidase activity in male migrainous and cluster headache patients was significantly lower than in male controls, confirming our previous study. The activity range showed a normal distribution and low mean values could not be attributed to a subgroup with particularly low activity. When Corash 's platelet preparation method was used, with its high platelet yield, specific enzyme activities of a similar order were obtained. Thus, the low values encountered were not due to abnormal recovery within the platelet population. Two other enzyme activities, phenolsulphotransferase M and succinate dehydrogenase, were also measured in the same platelet samples. Although low succinate dehydrogenase activity was identified in the headache groups, it appeared to represent a separate phenomenon and there was no significant correlation between activity of either enzyme and that of monoamine oxidase. This shows that the low activity of platelet monoamine oxidase in headache is not related to a generalised platelet enzyme deficit. It was also shown that the low monoamine oxidase activity in the headache patients could not be attributed to smoking.