Succinate-tetrazolium Reductase Research Articles

Gefitinib Enhances Mitochondrial Biological Functions in NSCLCs with EGFR Mutations at a High Cell Density.

Gefitinib is a tyrosine kinase inhibitor of epidermal growth factor receptor (EGFR) and has been approved for the treatment of non-small cell lung cancers (NSCLCs) with EGFR mutations. Here we demonstrated that gefitinib induced a significantly enhanced biological activity of succinate-tetrazolium reductase (STR) in mitochondria and mitochondrial membrane potential in HCC827 cells (EGFR mutation NSCLCs, sensitive to gefitinib) at a high cell density. We assessed the biological activity (STR, mitochondrial membrane potential, expression level of Bcl-2 family proteins) of gefitinib on NSCLCs at different cell densities. The 3D cell culture experiments showed the enhanced mitochondrial biological activity in clustered cell culture treated with gefitinib. Interestingly, the expression levels of Bcl-xL and Bax, were affected by the cellular number and gefitinib treatment. We also found that gefitinib prevented additive anticancer activity in the combinational treatment with doxorubicin, which induces mitochondria-dependent apoptotic cell death. Our results indicate that gefitinib may work as a mitochondrial protector against combinational treatment with mitochondria-dependent anticancer agents in high-cell-density.

Protomitohondria from liver cells: Similarities with and differences from mitochondria

In this study, we continue the investigation of a number of the properties of protomitochondria, which are young mitochondria of a smaller size that are present in animal cells. The protomitochondria were obtained from the rat liver by filtration of a total mitochondrial suspension through Millipore filters. The organelles contain the active respiratory chain as shown by the high rate of oxygen consumption during succinate and NADH oxidation. The shunt activity of succinate-tetrazolium reductase was lower and the activity of NADH-tetrazolium reductase was higher as compared with those in mitochondria. According to gel electrophoresis and gel filtration no qualitative differences between protomitochondria (0.25–0.45 µm) and mitochondria in their major protein composition were found. Nevertheless, there were some quantitative differences in several bands. Perhaps these differences reflect the process of intracellular maturation of protomitochondria to mitochondria. The data we obtained are important for understanding mitochondriogenesis in the animal cell.

Hepatocyte growth factor increases mitochondrial mass in glioblastoma cells

Hepatocyte growth factor/scatter factor (HGF) is a multifunctional growth factor that is linked to the initiation and/or progression of numerous malignancies. HGF also alters cancer cell responses to DNA damaging cytotoxic agents. Many cell responses to Met activation require alterations in metabolic activity but how the metabolic machinery responds to Met activation remains poorly defined. Treating human glioblastoma cells with HGF followed by the topoisomerase inhibitor camptothecin was found to increase the activity per cell of the mitochondrial respiratory chain enzyme succinate-tetrazolium reductase (>80% increase, p < 0.05) and the tricarboxylic acid cycle enzyme succinate dehydrogenase (>25% increase, p < 0.05). Treatment with either HGF or camptothecin alone had no effect on enzyme activity. The mitochondrial enzymatic response to HGF was dose- and time-dependent with the maximum increase occurring in cells pre-treated with 30 ng/ml HGF for 48 h prior to camptothecin exposure. This enzymatic response was associated with a concurrent increase in mitochondrial mass of comparable magnitude (∼56%, p < 0.05) as measured by fluorescent mitochondrial staining and flow cytometry. The mitochondrial mass response to HGF was prevented by the MAP-kinase pathway inhibitor PD98059 and was unaffected by the phosphatidylinositol 3-kinase inhibitors LY294002 and wortmannin. These findings suggest that HGF influences cell responses to chemotherapeutic stress, in part, by altering mitochondrial functions through a MAP-kinase dependent increase in mitochondrial mass.

Mitochondrial Transcription Factor A Induction by Redox Activation of Nuclear Respiratory Factor 1

The nuclear expression of mitochondrial transcription factor A (Tfam), which is required for mitochondrial DNA (mtDNA) transcription and replication, must be linked to cellular energy needs. Because respiration generates reactive oxygen species as a side-product, we tested the idea that reactive oxygen species regulate Tfam expression through phosphorylation of nuclear respiratory factor (NRF-1) and binding to the Tfam promoter. In mitochondria-rich rat hepatoma cells that overexpress NRF-1, basal and oxidant-induced increases were found in Tfam expression and mtDNA content. Specific binding of NRF-1 to Tfam promoter was demonstrated by electrophoretic mobility shift assay and chromatin immunoprecipitation. NRF-1-Tfam binding was augmented under pro-oxidant conditions. NRF-1 gene silencing produced 1:1 knockdown of Tfam expression and decreased mtDNA content. To evaluate oxidation-reduction (redox) regulation of NRF-1 in Tfam expression, blockade of upstream phosphatidylinositol 3-kinase was used to demonstrate loss of oxidant stimulation of NRF-1 phosphorylation and Tfam expression. The oxidant response was also abrogated by specific inhibition of Akt/protein kinase B. Examination of the NRF-1 amino acid sequence revealed an Akt phosphorylation consensus at which site-directed mutagenesis abolished NRF-1 phosphorylation by Akt. Finally, Akt phosphorylation and NRF-1 translocation predictably lacked oxidant regulation in a cancer line having no PTEN tumor suppressor (HCC1937 cells). This study discloses novel redox regulation of NRF-1 phosphorylation and nuclear translocation by phosphatidylinositol 3,4,5-triphosphate kinase/Akt signaling in controlling Tfam induction by an anti-oxidant pro-survival network.

Metabolic Correction of Cerebral Radiation Syndrome

DNA strand breaks that occur after irradiation activate the repair enzyme adenosine diphosphoribosyl transferase, which consumes NAD as a substrate and causes depletion first of neuronal NAD and then of the ATP pool. This is considered to be the crucial link in the mechanism underlying the cerebral radiation syndrome (CRS). In this study, two ways to correct the CRS metabolically were examined: (a) prevention of depletion of NAD after irradiation by administration of the enzyme inhibitor nicotinamide and (b) shunting the NAD-dependent oxidative phosphorylation pathway of ATP resynthesis by administration of a substrate of NAD-independent oxidation, succinate. Cerebral lesions induced by radiation were modeled by irradiation of rats or rat brain homogenates with 150 Gy of X rays. The manifestations of CRS in rats (excitement, convulsions, etc.) closely resembled those seen after acute hypoxia. In brain homogenates, pyruvate tetrazolium-reductase activity decreased after irradiation and could be corrected by addition of NAD after irradiation. Succinate tetrazolium-reductase activity was not affected by irradiation. Oxygen consumption by brain homogenates after irradiation in vitro and in situ decreased, as did oxygen consumption by rats in vivo after cranio-caudal irradiation. Administration of nicotinamide or succinate prevented both the postirradiation decrease in respiration (in both rats in vivo and brain homogenates in vitro) and the development of cerebral radiation syndrome. These results help to clarify the mechanisms underlying CRS and its metabolic correction.

Effects of 4-tertiary butylphenol on the tyrosinase activity in human melanocytes.

Vitiligo is a common dermatological disorder characterized by the development of complete pigment loss from focal lesions that tends to increase in size over time. The etiology of vitiligo, resulting in the disappearance of functional melanocytes from involved skin, is not clearly understood. As a consequence, no satisfactory therapy has been developed. A subtype of vitiligo, termed 'occupational' or 'contact' vitiligo, is increased in individuals who are exposed to materials containing phenolic derivatives, such as 4-tertiary butylphenol (4-TBP). Phenolic derivatives are structurally similar to tyrosine, the initial substrate of tyrosinase in the biochemical synthesis of melanin. Therefore, it has been proposed that phenolic derivatives compete with tyrosine for hydroxylation by tyrosinase and interfere with the completion of melanin synthesis and/or generate cytotoxic intermediates. Our results demonstrated that 4-TBP competitively inhibited both tyrosine hydroxylase and dihydroxyphenylalanine (DOPA) oxidase activities of tyrosinase, i.e., the first two catalytic steps in the biochemical conversion of tyrosine to melanin in cultured human melanocytes. This inhibition occurred at concentrations that did not influence the viability of melanocytes. The tyrosinase activity inhibited by 4-TBP was recovered after removing the treatment. 4-TBP did not affect the function of other enzymes, such as succinate-tetrazolium reductase, acid phosphatase and sulfatase. Since depigmentation occurred without a cytotoxic response after exposure of melanocytes to low concentration of 4-TBP, it is unclear whether the interaction between 4-TBP and tyrosinase leads to the destruction of the melanocytes in 'contact/occupational' vitiligo.

Isolation and characterization of membranes from bovine liver which are highly enriched in mannose 6-phosphate receptors.

We have developed a method for the isolation of the subcellular organelles from bovine liver which are enriched in the cation-independent mannose 6-phosphate receptor (CI-MPR) and the cation-dependent mannose 6-phosphate receptor (CD-MPR). The purification scheme consists of sedimentation of a postnuclear supernatant fraction on a sucrose gradient followed by immunoisolation using specific anti-peptide antibodies conjugated to magnetic polystyrene beads. Antibodies that recognize the cytoplasmic domain of either the CI-MPR or the CD-MPR routinely give membrane preparations that are approximately 50-fold enriched in each of the respective receptors, as determined by quantitative Western blotting. The immunoisolated membranes are also enriched in the other MPR, as well as in the asialoglycoprotein receptor. They contain significantly lower levels of enzyme activities representative of the plasma membrane (5' nucleotidase) or the Golgi complex (galactosyltransferase and sialyltransferase). There is little or no enrichment for either the lysosomal enzymes beta-hexosaminidase and tartrate-resistant acid phosphatase, or the mitochondrial enzyme succinate-tetrazolium reductase. These data, together with electron microscopy of the immunoisolated material, suggest that the bulk of MPR-containing membranes we have isolated from bovine liver correspond to endosomes. Analysis by SDS-PAGE indicates that several proteins, including two with apparent molecular weights of 170 K and 400 K, are significantly enriched in the purified fractions and may represent potential markers for MPR-containing endosomes.

Toxicity of diminazene aceturate (Berenil) to camels

Ten mg per kg of diminazene aceturate (Berenil) was highly toxic to camels. The main signs of berenil poisoning were hyperaesthesia, salivation, intermittent convulsions, frequent urination and defaecation, itching and sweating. The damage to the liver and kidney was accompanied by increases in the activity of ASAT and in the concentration of ammonia and by decreases in the concentration of calcium and magnesium in serum. The activity of adenosine triphosphatase, 5-nucleotidase, succinic tetrazolium reductase and glucose-6-phosphatase was reduced in the hepatocytes and the cells of the renal tubules.

Adaptation of the small intestine during pregnancy and lactation in the rat.

During pregnancy and lactation in the rat the small intestine in general and the mucosal epithelium in particular gain weight. The specific activities of sucrase, lactate dehydrogenase and succinate-tetrazolium reductase remain constant and those of glucose 6-phosphate dehydrogenase and isocitrate dehydrogenase increase. There is no evidence that the reported decrease in absorption per unit area or weight of mucosal epithelium during pregnancy and lactation is due to decreases in enzyme activities within the epithelium. The pattern of enzyme change shows that the response of the gut to the stimuli of pregnancy and lactation must be a complex one, possibly involving increases in the specific activities of some enzymes.

Susceptibility of desert sheep to infection with Schistosoma mansoni of Northern Sudan.

Each of two Desert Sheep was infected with 1500 cercariae of Schistosoma mansoni of Northern Sudan. Signs of infection were anorexia, soft faces, progressive weakness and loss of wool. The sheep were killed 254 and 269 days after infection. The findings were heavy infiltration of the lamina propria with inflammatory cells, numerous ova in the submucosa, hyperplasia of lymphoid tissue, oedema of the mesenteric lymph nodes, and focal pulmonary oedema and congestion. There were egg granulomas, focal necrosis, schistosomal pigment, fatty change, depletion of glycogen and reduction in the activity of adenosine triphosphatase, succinic tetrazolium reductase and glucose-6-phosphatase in the liver. In one sheep 1330 cercariae penetrated and 700 matured to produce males and females in a 5:2 ratio. In the other sheep, about one third of the cercariae penetrated and matured. The ratio of males to females was 3:1.

Studies in succinate dehydrogenase histochemistry.

The activity of succinate tetrazolium reductase was investigated in liver and kidney from the rat and mouse. The results obtained were related to the cellular level of succinate dehydrogenase (SDH) as well as to the level of CoQ.

The intramitochondrial localization of succinate-yellow tetrazolium reductase with the electron microscope.

A new tetrazolium salt, yellow tetrazolium, has been used to localise succinatetetrazolium reductase with the electron microscope. As expected, the formazan did not give high contrast in the optical microscope, but localization with the EM was good. The size of the formazan granules was 60–100A; lead staining was essential to secure good contrast.

The effect of cortisone on brown adipose tissue of young rats.

Rats aged 7 to 9 days were injected for 1 to 3 days with 5 mg/100 g body weight cortisone acetate per day. This led to an increase in the weight of brown adipose tissue, an increase in its fat content, and a decrease in its protein content. The content of norepinephrine in brown adipose tissue was decreased by cortisone treatment. The in vitro increase in O2 consumption following the addition of 5 μg nore-pinephrine/ml in vitro to brown adipose tissue from control animals was abolished by cortisone administration. Lipolysis due to norepinephrine was, however, not affected and was even slightly increased in cortisone-treated rats. Mitochondrial succinate–tetrazolium reductase activity was decreased after treatment with cortisone. It is concluded that cortisone alters the mitochondria of brown adipose tissue but has little effect on lipolysis as such. The content of norepinephrine in brown adipose tissue was found to decrease immediately after birth and then to return rapidly to higher levels.

The effects of autolysis on the histochemical demonstration of succinic tetrazolium reductase and adenosine triphosphatase in rat liver.

The Journal of Pathology and BacteriologyVolume 96, Issue 2 p. 504-508 Short Article The effects of autolysis on the histochemical demonstration of succinic tetrazolium reductase and adenosine triphosphatase in rat liver E. Thorpe, E. Thorpe Department of Veterinary Pathology, University of LiverpoolSearch for more papers by this authorC. Gopinath, C. Gopinath Department of Veterinary Pathology, University of LiverpoolSearch for more papers by this author E. Thorpe, E. Thorpe Department of Veterinary Pathology, University of LiverpoolSearch for more papers by this authorC. Gopinath, C. Gopinath Department of Veterinary Pathology, University of LiverpoolSearch for more papers by this author First published: October 1968 https://doi.org/10.1002/path.1700960233Citations: 2AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Citing Literature Volume96, Issue2October 1968Pages 504-508 RelatedInformation

The Comparative Histochemistry of some Hydrolytic and Oxidative Enzymes in the Livers of Several Domestic and Laboratory Animals

The distribution of various hydrolytic and oxidative enzymes was studied histochemically in the livers of rats, mice, dogs, cats, and cattle. The enzymes investigated comprised non-specific alkaline phosphatase, adenosine triphosphatase, glucose-6-phosphatase, succinic tetrazolium reductase, DPN diaphorase, glutamic, lactic and sorbitol dehydrogenases. The comparative merits of 2 techniques for the demonstration of succinic tetrazolium reductase were evaluated. It was found that the optimal incubation period for the demonstration of adenosine triphosphatase in the canaliculi of dog liver was considerably in excess of that required for the other species investigated.

Succinyl lecithin as a substrate for succinic dehydrogenase.

Succinyl lecithin has been used as substrate in the histochemical succinate-tetrazolium reductase system; it is as acceptable to the tissue system as is succinate. Cholesterol succinate, however, is not utilized by this system. It is inferred that the amphophilic nature of succinyl lecithin allows this substrate to penetrate lipid-rich mitochondrial membranes, whereas cholesterol succinate's more hydrophobic nature impedes its movement at such lipid-water interfaces. It must be emphasized that this study does not reveal whether or not succinyl lecithin is a physiologic substrate.

The effect of coenzyme Q on the histochemical succinic tetrazolium reductase reaction.

The role played by coenzyme Q (CoQ) in the succinic tetrazolium reductase reaction was investigated. Fresh cryostat sections of normal rat tissues were extracted with acetone to remove CoQ from the tissue sections and the acetone-extracted sections were reconstituted with CoQ-lecithin complexes. The incubation film method for tetrazolium reductases was used as a test system, using succinate as a substrate. Sections from which CoQ had been selectively extracted did not reduce the tetrazolium to its formazan, whereas those acetone-extracted sections that were treated with CoQ-lecithin complexes or with the electron carrier phenazine methosulfate reacted positively. It was concluded that the succinic tetrazolium reductase reaction requires intermediate electron carriers, mainly CoQ. It follows that nitro blue tetrazolium cannot accept electrons directly from the flavins. Therefore, the tetrazolium reductase reaction depends not only on the amount of succinic dehydrogenase and flavins but also on the amount of CoQ in the tissue.

The isolation of whole cells from the ciliary epithelium together with some observations on the metabolism of the two cell types

The cells of bovine ciliary epithelium have been isolated from the underlying stroma and separated into pigmented and non-pigmented fractions. Enzymic digestion of the tissue by papain or pronase, followed by collagenase, was required to free the cells from the tissue and, after filtration, the cell suspension was fractionated by centrifugation on a concentration gradient of colloidal silica. The cells were recovered from the gradient in zones which had only a 10% contamination by the minority cell type. In addition to the enzymic method small sheets of non-pigmented cells were obtained from calf eyes by manual dissection. Respiratory and glycolytic activities were determined and both were found to be higher in the non-pigmented cells than in the pigmented cells. Succinate-tetrazolium reductase activity was also found to be higher in the non-pigmented cells, but lactic dehydrogenase was equally distributed in the two types of cell. The rates of respiration and glycolysis of the pigmented cells were, respectively, little affected by succinate and by anaerobic conditions, whereas the non-pigmented cells showed much greater changes in metabolic activity in response to variations in substrate and oxygen supply. These findings lead to the conclusion that it is the non-pigmented cells that play the major role in the provision of energy for sodium transport by the ciliary epithelium.

Respiratory Enzyme Activities in Neurons and Glial Cells of the Hypoglossal Nucleus during Nerve Regeneration

AbstractRespiratory enzyme activities were measured in the hypoglossal nucleus of the rabbit, 6, 14, 28, and 48 days after unilateral hypoglossal nerve crush in order to evaluate the changes in nerve and glial cells during axon regeneration. Succinoxidase activity was measured by the Warburg technique on homogenates of the hypoglossal nuclei and by Zeuthen's micro‐diver technique in single nerve and glial cells. Activity of succinate tetrazolium reductase, cytochrome oxidase and NADH2‐tetrazolium reductase were analyzed histochemically. Succinoxidase activity increased in single neurons up to the 14th postoperative day, both on the unoperated and operated side and was on a high level until the 28th day. A striking increase in NADH2‐tetrazolium reductase was demonstrated in the neurons on the operated side from the beginning of the second postoperative week. In the micro‐diver experiments, the glial cells of the operated side revealed a significant increase in Succinoxidase activity from the sixth to the 48th postoperative day. The histochemical study of respiratory enzymes did not reveal any change in glial enzyme activity in the hypoglossal nucleus, although morphological studies have shown hypertrophic astrocytes and reactive microglial cells in this region.The importance of analyzing nerve and glial cells separately and to consider volume and mass changes in single cells is stressed.

Studies on succinate-tetrazolium reductase systems IV. Histochemical aspects

Summary A study has been made concerning the composition of three different succinate-tetrazolium reductase systems present in 6-μ sections of rat liver. The tetrazolium salts studied were C,N -diphenyl- N' -4,5-dimethylthiazol-2-yl tetrazolium bromide (MTT, with and without the addition of CoCl 2 ) and nitro-blue tetrazolium. In the case of MTT, both in the presence and in the absence of added Co 2+ , the succinate-MTT reductase system showed close similarities to the system previously studied in tissue specimens. Co 2+ produced a marked inhibition of the overall reaction in sections though not to such an extent as found in suspensions. It is concluded that the routine histochemical procedure using MTT demonstrates a similar sequence of reactions to that occurring in liver homogenates where MTT is reduced via two main interactions with the respiratory chain. In the case of nitro-blue tetrazolium, a similar close resemblance was found between the results obtained in this study with frozen sections and those previously obtained with suspensions.