Changes in Activity of Mitochondrial Enzymes during the Development of Xenopus laevis

Changes in activity of mitochondrial enzymes were studied during the embryonic development of Xenopus laevis.
The following enzymes were determined: malate dehydrogenase (MDH), isocitrate dehydrogenase (NAD⁺-dependent) (IDH), aspartate aminotransferase (GOT), cytochrome oxidase (COX), succinate dehydrogenase (SDH), rotenone-insensitive NADH cytochrome c reductase (NADH-red) and monoamine oxidase (MAO). IDH is constant throughout the period studied. COX and SDH, two enzymes of the inner membrane, are constant in pregastrula stages, and subsequently decrease significantly. MDH and NADH-red are highly active in the pregastrula stages and decline thereafter, while MAO is undetectable during early development and increases significantly only in the larvae. GOT increases during the cleavage stages, being most active in the gastrula stages, and decreases subsequently.
The results are discussed in the sense of mitochondrial differentiation during the early development of the amphibian embryo.     

Methodological aspects of the histochemical localization and activity of some cerebellar dehydrogenases

Summary In a detailed study focused on the methodological problems in dehydrogenase histochemistry [e.g., fixation, diffusion of enzymes and of reduced inermediates, conversion of NADPH and NADP to NADH and NAD, respectively, penetration of tetrazolium salt and formazan substantivity, nothing dehydrogenase reaction, use of exogenous CoQ₁₀ and of flavoprotein substitute (PMS)], the distribution and activity of succinate dehydrogenase, NAD(P)H-tetrazolium reductase, glucose-6-phosphate dehydrogenase, lactate dehydrogenase (H and M types), and of l-glutamate dehydrogenase (E.C. 1.4.1.2 and E.C. 1.4.1.3) have been investigated in the rat cerebellum.It was evident from the study that reliable results could only be obtained if all the aforementioned factors had been considered. The image of actual concentration of SDH in the neuropil of the molecular layer could only be recorded by adding CoQ₁₀, while other structures exhibited greater balance between SDH and endogenous mitochondrial CoQ. Contrary to previous studies, a reversed localization of the activity of G-6-PDH and LDH was noticed. The elements of molecular and Purkinje layers were rich in G-6-PDH, while the granular layer was nearly depleted. The actual level of LDH could only be recorded if NADH-tetrazolium reductase was bypassed with PMS. The H and M types of LDH coexisted in the three cortical layers, the H type being prevalent and the M type attaining its highest level in synaptic glomeruli followed by the structures of the molecular layer and the Purkinje cells. High activity of GDH was noticed in Bergmann glia followed by synaptic glomeruli, while most other structures showed weak to moderate activity. The two GDH types coexisted in all structures showing activity, except for Bergmann cells, which only showed presence of the E.C. 1.4.1.3 type.Furthermore, Bergmann glia was exceptional by showing no activity of SDH and LDH, but strong activity of G-6-PDH and NADPH-tetrazolium reductase. The granular cells were exceptional by showing weak or no activity of all enzymes in question.     

Histochemical studies of enzymes of the energy metabolism in postimplantation rat embryos

Using histochemical procedures for the detection of lactate dehydrogenase (LDH), succinate dehydrogenase (SDH), and cytochrome c oxidase (cytox), we investigated the levels of these enzymes of the energy metabolism in postimplantation rat embryos (9.5-12.5 days of gestation). On day 10.5 of gestation, the neural tube, somites, myocardium, and mesenchyme displayed moderate levels of LDH activity; this activity gradually increased in strength, so that, on day 12.5 of gestation, intense LDH activity was uniformly distributed in these intraembryonic tissues. In contrast to LDH, distinct regional differences in the distribution of SDH and cytox were detected. On day 10.5 of gestation, the myocardium exhibited weak to moderate SDH and cytox activity, and on day 11.5, the myocardial activity of these enzymes had become moderate to intense. However, in all other embryonic tissues, e.g., the neural tube and somites, only weak SDH and cytox activity was present. On day 12.5 of gestation, the myocardium displayed very intense SDH and cytox activity, whereas the mantle layer of the neural tube, the spinal ganglia, and the myotomes exhibited only moderate levels of SDH and cytox activity. In the matrix of the neural tube and mesenchyme, these enzyme activities remained at low levels. At electron microscopy, cytox activity was detectable in the spaces between the inner and outer membranes as well as in the intracristal spaces of mitochondria. In general, cytox activity increased in parallel with the differentiation of mitochondria (i.e., increased mitochondrial numbers and size, and the development of mitochondrial cristae), but when the distribution of the cytox activity was considered in detail, it was found to differ among mitochondria.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cytochemical study of cells of primary and disseminated ascite Yoshida tumor cells

The different distribution of cytochemically demonstrable enzymes: lactate dehydrogenase (LDH, 1.1.1.27), succinate dehydrogenase (SDH, 1.3.99.1), dihydrofolate reductase (DHFR, 1.5.1.3), acid phosphatase (AcP, 3.1.3.2) and alkaline phosphatase (ALP, 3.1.3.1), has been documented in Yoshida ascites hepatoma cells in vivo or stored at 80 degrees C. The dehydrogenase activities (LDH, SDH, DHFR) show a strong reaction in all samples. An increased level of these enzyme activities has been observed in the malignant cells spreading through the organs of tumor bearing rats. On the contrary, in the same samples, acid and alkaline phosphatase activities are very low. The strong dehydrogenase activities observed in Yoshida ascite cells stress the rapid turnover of tumor cells. Our results indicate that the histochemical method may be a useful tool to detect the scattered tumor cells. Furthermore, the cytochemical methods allow the characterization of the metabolic pathways employed by the primary and disseminated tumor cells.     

Histochemical profiles of motoneurons innervating muscle fibres with different activity patterns in the zebrafish, Brachydanio rerio.

Summary Enzyme histochemical profiles of spinal motoneurons in the zebrafish were determined. Five enzymes of glucose metabolism were chosen: glucose-6-phosphate dehydrogenase (G6PDH), hexokinase (HK), phosphofructokinase (PFK), succinate dehydrogenase (SDH) and NADH tetrazolium reductase (NADH-TR). Motoneurons were traced with Fluorogold and classified as those that innervate white muscle fibres (W-MNs) and those that innervate red and intermediate muscle fibres (R/ I-MNs). The average enzyme activities per volume of tissue in the somata of both populations differed at most by 25%. Both the average soma volume and the average number of muscle fibres innervated are three times larger for the W-MNs than for the a/I-MNs. This suggests that the total amount of enzyme activity within a neuron soma matches target size.In the R/I-MNs, the activities of SDH and NADH-TR were closely correlated (correlation coefficient, r=0.99;p<0.05) and HK activity correlated well with G6PDH activity (r=0.94;p<0.05), butnot with PFK (r=0.64;p>0.05). In the W-MNs, there was no correlation between SDH and NADH-TR (r=–0.59;p>0.05) or between HK and G6PDH (r=0.50;p>0.05) and the correlation coefficient between HK and PFK activity was close to zero (r=0.04;p>0.05).It was concluded that in the R/I-MNs gwhich are continuously ctive, firing activity is fuelled by oxidative metabolsm. We suggest that in the W-MNs glucose is stored in the form of glycogen and that, despite high levels of NADH-TR present, the energy for intermittent firing activity is provided by glycolysis.     

Structural and histochemical aspects of perirenal adipose tissue in fetal pigs: relationships between stromal-vascular characteristics and fat cell concentration and enzyme activity

Samples of perirenal adipose tissue were obtained from four fetuses from each of seven crossbred gilts at each of three stages of gestation: 70, 90, and 110 days. Samples were routinely prepared for histochemistry and histology. At each age, the largest fat cell clusters were consistently located near points where large blood vessels entered the loose connective tissue. Cell-cluster size decreased with distance from the entry points of large blood vessels. Fat cells proximal to entry points of large arterioles and fat cells distal to entry points of large arterioles were the same size. Enzyme cytochemistry disclosed that reactions for glucose-6-phosphate dehydrogenase (G6PDH), lipoprotein lipase (LPL) and NADH-TR enzymes were reduced in distal (relative to entry points of large arterioles) adipocytes compared with proximal adipocytes. Reactions for succinate dehydrogenase (SDH) and lactate dehydrogenase (LDH) in adipocytes were not influenced by location within the tissue. Small fat cell clusters with sparse capillary beds surround arterioles in distal areas of sections from fetuses at 70, 90, and 110 days of gestation. In the proximal areas of sections from 110-day-old fetuses, arterioles were surrounded by large fat cell clusters with dense capillary beds. These characteristics serve to distinguish perirenal depots from subcutaneous depots in the fetus.     

Histochemical studies of enzymes of the energy metabolism in postimplantation rat embryos

Summary Using histochemical procedures for the detection of lactate dehydrogenase (LDH), succinate dehydrogenase (SDH), and cytochrome c oxidase (cytox), we investigated the levels of these enzymes of the energy metabolism in postimplantation rat embryos (9.5–12.5 days of gestation). On day 10.5 of gestation, the neural tube, somites, myocardium, and mesenchyme displayed moderate levels of LDH activity; this activity gradually increased in strength, so that, on day 12.5 of gestation, intense LDH activity was uniformly distributed in these intraembryonic tissues. In contrast to LDH, distinet regional differences in the distribution of SDH and cytox were detected. On day 10.5 of gestation, the myocardium exhibited weak to moderate SDH and cytox activity, and on day 11.5, the myocardial activity of these enzymes had become moderate to intense. However, in all other embryonic tissues, e.g., the neural tube and somites, only weak SDH and cytox activity was present. On day 12.5 of gestation, the myocardium displayed very intense SDH and cytox activity, whereas the mantle layer of the neural tube, the spinal ganglia, and the myotomes exhibited only moderate levels of SDH and cytox activity. In the matrix of the neural tube and mesenchyme, these enzyme activities remained at low levels. At electron microscopy, cytox activity was detectable in the spaces between the inner and outer membranes as well as in the intracristal spaces of mitochondria. In general, cytox activity increased in paralled with the differentiation of mitochondria (i.e., increased mitochondrial numbers and size, and the development of mitochondrial cristae), but when the distribution of the cytox activity was considered in detail, it was found to differ among mitochondria. The relationship between, on the one hand, changes in the enzymatic patterns with a bearing on the energy-yielding metabolism and, on the other hand, cellular differentiation during major organogenesis in rat embryos is discussed.Dedicated to Professor Dr. T.H. Schiebler on the occasion of his 65th birthday     

Qualitative and quantitative histochemical evaluation of the dehydrogenase activity in human glioblastoma cells treated with beta-interferon

In an attempt to clarify the role of beta interferon in vitro cell systems, the metabolic expression of dihydrofolate reductase (DHFR), succinate dehydrogenase (SDH) and lactate dehydrogenase (LDH), by histochemical methods was studied. DHFR was also quantified by flow cytometry. Glioblastoma cell line with or without beta interferon was used. Three days after the treatment the DHFR reaction was much less intense than in the control. Quantitative data confirmed these results. Immediately afterwards, most cells exhibited much more intense reaction. These facts underlined that, in this biological model in vitro, beta interferon could reduce the synthesis of these enzymes only for a short period.     

Effect of thyroidectomy on fast and slow muscle fibres of rat gastrocnemius muscle

A combined histochemical, biochemical and electrophoretic study with respect to the enzymes succnic dehydrogenase(SDH), myofibrillar adenosine triphosphatase (m-ATPase), lactate dehydrogenase (LDH) isozymes and myosin light chains was carried out to investigate the response of rat gastrocnemius muscle (medial head). Twelve weeks after thyroidectomy, the results indicated a shift from fast to slow type pattern of LDH isozymes, fibre type transformation from Type II to Type I and a decrease in SDH and m-ATPase activity. The results suggest, possible thyroidal involvement in determining the phenotypic properties of skeletal muscle.     

Hormonal secretion and enzyme activity of cultured roe-deer (Capreolus capreolus L.) Leydig cells, as measured by radio-immunological and histochemical assays

Leydig cells from roe-deer collected according to Steinberger's (1975) technique were cultured as monolayers in Leighton tubes for 10 days. Cultures were grown in medium 199 supplemented with 10% calf serum. Androgen and oestrogen secretion by Leydig cells into the culture medium was measured using appropriate radio-immunoassays. Using histochemical tests the activity of the following oxydoreductive enzymes in cultured Leydig cells was shown: delta 5, 3 beta-hydroxysteroid dehydrogenase (delta 5, 3 beta-HSD), 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD), succinate and lactate dehydrogenases (SDH and LDH). Strong activity of the enzymes investigated during the first 4 days of culture was observed. The androgen level was high throughout the second and fourth day of culture. A decrease in hormone secretion after day 4 occurred, and this was closely correlated with enzyme activity. The oestrogen level was very low during culture. The direct effect of the luteinizing hormone (LH) added into the culture medium caused an increase in not only enzyme activity but also androgen and oestrogen levels.     

Effect of ochratoxin A (OTA) on the activity of some enzymes in developing eggs of<Emphasis Type="Italic">Ascaris suum</Emphasis>

The studies focused on the effect of 1-ppm ochratoxin A solution on the activity of some enzymes during successive embryogenesis stages ofAscaris suum. As a result of OTA-affected incubation ofA suum eggs, inhibition of AcP reactions was observed throughout the embryonic development, reduction in of SDH and LDH activity during cleavage and gastrulation, and enhancement of the SDH and LDH activity was found in larvae. Moreover, inhibition of the processes of cleavage, gastrulation and organogenesis was recorded. The observed morphological and metabolic embryo disorders demonstrated teratogenic properties of the studied mycotoxin, and the presence of granules of various sizes in the developing eggs may indicate that OTA penetrated into the eggs.     

Histochemical demonstration of enzymatic heterogeneity within the mesocoxal and metacoxal muscles of Periplaneta americana

Enzymatic activity in the mesocoxal and metacoxal muscles of the cockroach, Periplaneta americana, has been characterized using histochemical techniques. Our interpretation of the histochemical stains for myosin-ATPase (pre-incubated at pH 9.4, 10.4 and 4.3). NADH reductase, succinic dehydrogenase (SDH), and lactic dehydrogenase (LDH) has enabled us to classify fibres within these muscles according to established fibre typing strategies. The results reaffirm the biochemical heterogeneity reported for the mesocoxal muscles and allow us to confirm the expected heterogenity in the homologous metacoxal muscles. These techniques have proven to be an effective tool for assessing the biochemical properties of muscle fibres.     

Dehydrogenase activity of the pineal gland during the estrous cycle

An investigation of the relationship of succinic dehydrogenase (SDH) and lactate dehydrogenase (LDH) in the pineal gland of the adult female albino rats was carried out during seven definitive stages of the estrous cycle: Early Estrus (E1), Late Estrus (E2), Metestrus (ME), Early Diestrus (D1), Late Diestrus (D2), Preproestrus (PPE) and Proestrus (PE). Controlled housing conditions were maintained on 7:00 A.M. to 7:00 P.M. light-dark cycle. The animals were necropsied at precise times after the onset of estrus and subsequent stages of the cycle. Comparative histochemical and biochemical analysis revealed the following for SDH activity: E2 greater than E1 greater than D2 greater than D1 greater than PPE greater than PE greater than ME; and for LDH activity: PE greater than E2 greater than ME greater than D1 greater than PPE greater than E1 greater than D2. Interrelationships are postulated between pineal SDH, GOT, GPT, and estrogen concentrations in the rat.     

Differentiation of blood vessels in the adipose tissue of lean and obese fetal pigs, studied by differential enzyme histochemistry

Subcutaneous adipose tissue was obtained from fetuses removed from pregnant obese (Ossabaw) and lean (crossbred) sows at three stages of gestation (70, 90, and 110 days). Histochemical analysis for nucleo-side phosphatase (NPase), alkaline phosphatase (APase), and NADH tetrazoleum reductase (NADH-TR) was conducted on fresh-frozen cryostat sections. Age- associated changes in NPase and NADH-TR reactions in the arteriolar system were correlated with the morphological development of the medial layer of arterioles and arteries. For instance, a strong NPase reaction in small arterioles was associated temporally with the assumption of a normal smooth muscle cell morphology and arrangement in the medial layer. In the youngest fetuses, strong NADH-TR reactions were only evident in small and presumptive arterioles and venules (associated with fat cells). Little NADH-TR reactivity was evident in larger arterioles and venules in 70-day tissue. Arteries and large arterioles were distinguished from veins and venules (strong reactions vs. weak reactions) with NADH-TR and NPase reactions in the oldest fetuses. In the younger fetuses, the NPase distinction (arterioles vs. veinules) was obvious before NADH-TR distinction. Small adipocyte-associated vessels were APase positive in the youngest fetuses, but APase reactivity was limited to short segments of vessel between arterioles and capillaries in the oldest fetuses. With the following exceptions, all the above observations were independent of fetal strain. In obese fetuses (110 day) small venules and small arterioles were equally reactive for NPase activity. Capillaries in obese fetuses (110 day) were NADH-TR reactive, whereas no activity was evident in capillaries from lean fetuses (110 day).(ABSTRACT TRUNCATED AT 250 WORDS)     

Lysine catabolism in Haemonchus contortus and Teladorsagia circumcincta

Catabolism of lysine through the pipecolate, saccharopine and cadaverine pathways has been investigated in L3 and adult Haemonchus contortus and Teladorsagia circumcincta. Both enzymes of the saccharopine pathway (lysine ketoglutarate reductase (LKR) and saccharopine dehydrogenase (SDH)) were active in L3 and adult worms of both species. All three enzymes which catabolise lysine to α-amino adipic semialdehyde via pipecolate (lysine oxidase (LO), Δ(1)-piperideine-2-carboxylate reductase (Pip2CR) and pipecolate oxidase (PipO)) were present in adult worms, whereas the pathway was incomplete in L3 of both species; Pip2CR activity was not detected in the L3 of either parasite species. In adult worms, the saccharopine pathway would probably be favoured over the pipecolate pathway as the K(m) for lysine was lower for LKR than for LO. Neither lysine dehydrogenase nor lysine decarboxylase activity was detected in the two parasite species. Enzyme activities and substrate affinities were higher for all five enzymes in adult worms than in L3. An unexpected finding was that both LKR and SDH were dual co-factor enzymes and not specific for either NAD(+) or NADP(+), as is the case in other organisms. This novel property of LKR/SDH suggests it could be a good candidate for anthelmintic targeting.     

Enzyme histochemistry of the mesocoxal muscles of Periplaneta americana

Histochemical techniques have been employed to characterize enzymatic activity in the mesocoxal muscles of the cockroach, Periplaneta americana. Through our studies of the enzymes myosin-ATPase, NADH reductase, succinic dehydrogenase (SDH), and lactic dehydrogenase (LDH), we were able to classify fibers within these muscles according to criteria established for muscle fibers of vertebrates. Many of the mesocoxal muscles possess two different and distinct populations of fibers, whereas the remaining muscles are homogeneous with respect to their constituent fibers. The data presented here indicate biochemical heterogeneity for muscles of differing structural and functional features and possible neurotrophic influences upon oxidative enzymes and myosin-ATPase isozymes.     

Identification of cells secreting a thymostimulin-like substance and examination of some histoenzymatic pathways in aging avian primary lymphatic organs: II. Bursa of Fabricius

The Bursa of Fabricius of 15 day, 1-, 3-, and 6 month-old adult chickens (White Leghorn strain) were studied by histological and histochemical staining, histoenzymatic reactions (LDH, SDH, a-GPDH, NAD, NADPH, Ca++-dependent ATP-ase, pH 8.5) and by anti-thymostimulin immunoreaction. Positive reactions for mucopolysaccharides and enzymatic activities were located in the epithelia of the follicles, i.e. in follicle-associated-epithelium (FAE), inter-follicle-epithelium (IFE) and in different epithelial compartments of cortical and medullary zones. Positive reaction for thymostimulin-like (TS-like) substance was restricted to FAE cells and weakly to the basal lamina of IFE. In 6-month-old chickens, the FAE cells disappeared; the phenomenon of bursal regression was evident, although not all the follicles were involved. In the few still normal follicles, the good reactivity to the enzymes tested suggests that residual physiological activity is still present, even if reduced.     

An enzyme histochemical and biochemical study of the activity of three oxidative enzymes in the developing rat parotid gland

Information on ductal differentiation in the developing rat parotid gland is sparse. One of the main functions of the striated and excretory ducts in this gland is the selective exchange of electrolytes from the primary fluid secreted by the acini. These ducts are rich in a number of enzymes involved in this task, suggesting that they might be useful as markers of ductal differentiation. The objective of this investigation was to delineate the developmental changes in activity of three of these, cytochrome C oxidase (CCO), succinate dehydrogenase (SDH), nicotinamide adenine phosphate dinucleotide (reduced form)-dehydrogenase (NADPH-DH). Histochemical localization of all three enzymes in fresh frozen sections was complemented by biochemical assays of CCO and SDH and cytochemical localization of CCO. Biochemically, CCO- and SDH-specific activity in gland homogenates increased progressively after birth, reaching adult levels at 21-28 days. Histochemically, deposits of reaction products of all three enzymes increased more in the striated and excretory ducts, especially in their basal cytoplasm, than in other glandular structures between 19 days in utero and 28 days after birth. During the same age span, the mitochondria in the striated and excretory ducts increased markedly in both number and size, migrated to a mostly basal location, and increased from many to virtually all showing strong cytochemical CCO reactions. These histochemical and cytochemical patterns of changes in enzyme activity at the cellular level accounted for the overall increases in CCO and SDH seen in the biochemical assays. Only the SDH histochemical reaction was consistently weak in the acini and intercalated ducts, and thus provided the most contrast with the progressively stronger reactions in the larger ducts. We conclude that of the three enzymes evaluated in these experiments, SDH is the best marker of the functional differentiation of the striated and excretory ducts in the developing rat parotid gland.     

An enzyme histochemical and biochemical study of the activity of three oxidative enzymes in the developing rat parotid gland

Information on ductal differentiation in the developing rat parotid gland is sparse. One of the main functions of the striated and excretory ducts in this gland is the selective exchange of electrolytes from the primary fluid secreted by the acini. These ducts are rich in a number of enzymes involved in this task, suggesting that they might be useful as markers of ductal differentiation. The objective of this investigation was to delineate the developmental changes in activity of three of these, cytochrome C oxidase (CCO), succinate dehydrogenase (SDH), nicotinamide adenine phosphate dinucleotide (reduced form)-dehydrogenase (NADPH-DH). Histochemical localization of all three enzymes in fresh frozen sections was complemented by biochemical assays of CCO and SDH and cytochemical localization of CCO. Biochemically, CCO- and SDH-specific activity in gland homogenates increased progressively after birth, reaching adult levels at 21-28 days. Histochemically, deposits of reaction products of all three enzymes increased more in the striated and excretory ducts, especially in their basal cytoplasm, than in other glandular structures between 19 days in utero and 28 days after birth. During the same age span, the mitochondria in the striated and excretory ducts increased markedly in both number and size, migrated to a mostly basal location, and increased from many to virtually all showing strong cytochemical CCO reactions. These histochemical and cytochemical patterns of changes in enzyme activity at the cellular level accounted for the overall increases in CCO and SDH seen in the biochemical assays. Only the SDH histochemical reaction was consistently weak in the acini and intercalated ducts, and thus provided the most contrast with the progressively stronger reactions in the larger ducts. We conclude that of the three enzymes evaluated in these experiments, SDH is the best marker of the functional differentiation of the striated and excretory ducts in the developing rat parotid gland.     

黑斑蛙早期胚胎发育过程中乳酸脱氨酶同工酶的初步研究

本文采用聚丙烯酰胺凝胶电泳方法,对河南新乡地区黑班蛙（Rananigromaculata）早期胚胎发育过程中（受精后0－324h）乳酸脱氢酶（LDH）同工酶进行了研究。结果表明：LDH1自始至终存在,且活性一直占绝对优势;LDH5比于心跳期出现,该期以后其活性仅次于LDH1;LDH2于襄胚早期开始出现,其活性一直较弱;LDH3和LDH4均于开口期少量出现,以后前者活性一直极弱,后者则呈现一定的活性。与哺乳类及鱼类资料比较,提示两栖类黑斑蛙LDH同工酶有其独特的表达谱式。