Experimentally induced colon cancer metastases in rat liver increase the proliferation rate and capacity for purine catabolism in liver cells

Metastases in rat liver were generated experimentally by intraportal injection of colon cancer cells to investigate the effects of cancerous growth on the metabolism of surrounding liver tissue. Maximum activities (capacity) of glucose-6-phosphate dehydrogenase, phosphogluconate dehydrogenase, lactate dehydrogenase, succinate dehydrogenase, alkaline phosphatase, 5-nucleotidase, xanthine oxidoreductase, purine nucleoside phosphorylase and adenosine triphosphatase have been determined. Two types of metastases were found, a small type surrounded by stroma and a larger type in direct contact with hepatocytes. Both types affected the adjacent tissue in a similar way suggesting that the interactions were not mediated by stroma. High capacity of the degradation pathway of extracellular purines released from dead cells of either tumours or host tissue was found in stroma and sinusoidal cells. Metastases induced both an increase in the number of Kupffer cells and proliferation of hepatocytes. The distribution pattern in the liver lobulus of most enzymes investigated did not change distinctly. However, activity of alkaline phosphatase, succinate dehydrogenase and phosphogluconate dehydrogenase was increased in hepatocytes directly surrounding metastases. These data imply that the overall metabolic zonation in liver lobuli is not dramatically disturbed by the presence of cancer cells despite the fact that various metabolic processes in liver cells are affected.In honour of Prof. Dr. Z. Lojda for his 65th birthday     

Odorant receptor proteins in olfactory axons and in cells of the cribriform mesenchyme may contribute to fasciculation and sorting of nerve fibers

Odorant receptors (ORs) have been shown to be present not only in the chemosensory cilia of the olfactory sensory neurons, but also in their axon terminals. This observation has emphasized the notion that the receptor protein may contribute to the precise receptor-specific targeting of olfactory axons in the olfactory bulb. This concept implies a particularly important role for the axonal receptor protein during the onset and early phase of the wiring process during development. In the present study, we have demonstrated, by means of specific antibodies, that, as early as mouse embryonic day E12, the OR protein can be visualized in outgrowing axonal processes of the olfactory epithelium and in cells located in the cribriform mesenchyme. On their trajectory from the olfactory epithelium through the cribriform mesenchyme toward the forebrain, axons with strong OR immunoreactivity have only been seen in the dorsal part of the mesenchyme where they traverse the region of OR-positive cells. Upon visualization by specific antibodies, these cells have been revealed to have long protrusions extending along the surface of nerve fascicles. They are often located at bifurcations where two small axon fascicles merge to form a stronger bundle. Within this region, fascicles coalesce forming a coherent nerve. Moreover, within the now compact nerve bundle, axons visualized by the OR-specific antibody are no longer distributed evenly but are segregated from other axonal populations within the nerve. These findings suggest that OR proteins in the membrane of axonal processes and of cells in the cribriform mesenchyme are involved in crucial processes such as fasciculation and the sorting of outgrowing axons, both of which are fundamental for the initiation and establishment of the precise wiring of the olfactory system during early development. This work was supported by the Deutsche Forschungsgemeinschaft (SFB 495).     

Calibration of a flow cytometric assay of glucose-6-phosphate dehydrogenase activity.

The reduction of tetrazolium salts to colored formazans is a reaction which has been exploited both in histo- and cytochemistry. Tetrazolium salts forming fluorescent formazans prove suitable for measuring defined cellular dehydrogenase activities in automated processes. This study considers an important aspect of formazan measurement in flow cytometry, namely, calibration. Calibration is performed by correlating the number (and fluorescence intensity) of formazan-bearing cells measured by flow cytometry with simultaneously performed biochemical analyses of the same material. The method is demonstrated by an example of glucose-6-phosphate dehydrogenase. Using the data of a typical experiment, the enzyme activity is expressed in femtomol of hydrogen transferred per cell during incubation time. Furthermore, through spatially resolved double excitation of formazan and nuclear DAPI fluorescence, an independent analysis of cell cycle and cellular enzymatic activity is established.     

Histological and histochemical studies of the cells of the human foetal adrenal and hypophysis in tissue culture

Summary The histological and histochemical changes taking place in expiants and outgrowing cells of the human foetal adrenals and hypophyses have been studied on the 5th, 10th, 20th, and 28th day of the culture period. In the outgrowth from adrenals, both the epithelial-type and the fibroblast-type cells contain 3-ol steroid dehydrogenase activity. The cells growing out from hypophyseal expiants appear to be morphologically undifferentiated. Expiants contain differentiated cells in the first 10 to 13 days of the culture period.     

Mathematical modelling of metabolic pathways affected by an enzyme deficiency. Energy and redox metabolism of glucose-6-phosphate-dehydrogenase-deficient erythrocytes

The effects of various forms of glucose-6-phosphate dehydrogenase deficiency on erythrocyte metabolism have been studied on the basis of a complex mathematical model which comprises the main pathways of this cell: glycolysis, pentose pathway, reactions of the glutathione and adenine nucleotide metabolism. The calculated flux rates through the oxidative pentose pathway with and without methylene blue are in good accord with experimental results. The degree of deficiency as predicted by the model on the basis of calculated upper oxidative load boundaries, as well as of maximal methylene blue stimulation, correlates with the individual clinical manifestation of the metabolic disease. Therefore, the model allows one to judge the degree of metabolic disorder in the presence of glucose-6-phosphate dehydrogenase enzymopathies if the kinetic properties of the defect enzyme are known. Experimentally accessible parameters for an assessment of the oxidative load capacity of cells in vivo are proposed. It is pointed out that the threshold of tolerance as to energetic load is drastically reduced in the case of severe glucose-6-phosphate dehydrogenase deficiency.     

Temporal relationship between glucose 6-phosphate dehydrogenase activity and DNA-synthesis

Summary Wounds have been inflicted in the skin of guinea-pigs. Measurements were made on the basal cells 39–54 h later, at different times of the day. It has been shown that there can be two peaks of mitosis, one about mid-day and the other about 22 h. Synthesis of DNA, measured by Feulgen microdensitometry, preceded mitotic activity. Marked changes were found in glucose 6-phosphate dehydrogenase activity, measured by quantitative cytochemistry and microdensitometry. The greatest activity preceded DNA-synthesis, indicating that pentose-shunt metabolic activity is involved in biosynthetic processes required for cell proliferation.     

Histochemistry and cytochemistry of glucose-6-phosphate dehydrogenase

Histochemistry and cytochemistry of glucose-6-phosphate dehydrogenase has found many applications in biomedical research. However, up to several years ago, the methods used often appeared to be unreliable because many artefacts occurred during processing and staining of tissue sections or cells. The development of histochemical methods preventing loss or redistribution of the enzyme by using either polyvinyl alcohol as a stabilizer or a semipermeable membrane interposed between tissue section and incubation medium, has lead to progress in the topochemical localization of glucose-6-phosphate dehydrogenase. Optimization of incubation conditions has further increased the precision of histochemical methods. Precise cytochemical methods have been developed either by the use of a polyacrylamide carrier in which individual cells have been incorporated before staining or by including polyvinyl alcohol in the incubation medium. In the present text, these methods for the histochemical and cytochemical localization of glucose-6-phosphate dehydrogenase for light microscopical and electron microscopical purposes are extensively discussed along with immunocytochemical techniques. Moreover, the validity of the staining methods is considered both for the localization of glucose-6-phosphate dehydrogenase activity in cells and tissues and for cytophotometric analysis. Finally, many applications of the methods are reviewed in the fields of functional heterogeneity of tissues, early diagnosis of carcinoma, effects of xenobiotics on cellular metabolism, diagnosis of inherited glucose-6-phosphate dehydrogenase deficiency, analysis of steroid-production in reproductive organs, and quality control of oocytes of mammals. It is concluded that the use of histochemistry and cytochemistry of glucose-6-phosphate dehydrogenase is of highly significant value in the study of diseased tissues. In many cases, the first pathological change is an increase in glucose-6-phosphate dehydrogenase activity and detection of these early changes in a few cells by histochemical means only, enables prediction of other subsequent abnormal metabolic events. Analysis of glucose-6-phosphate dehydrogenase deficiency in erythrocytes has been improved as well by the development of cytochemical tools. Heterozygous deficiency can now be detected in a reliable way. Cell biological studies of development or maturation of various tissues or cells have profited from the use of histochemistry and cytochemistry of glucose-6-phosphate dehydrogenase activity.(ABSTRACT TRUNCATED AT 400 WORDS)     

Reaction of striated fibers of human skeletal muscles to long-term anti-orthostatic hypokinesia

Eight men have been kept under a strict bed rest. The lower end of the bed has been elevated by 6 degrees. During the whole effect of the antiorthostatic hypokinesia (AOH) in biopsy of the skeletal muscle tissue no gross morphological changes and essential changes in relation of the muscle fiber (MF) types have been found. On the 120th day of AOH without application of any physical loading concentration of RNA, protein metabolism, glycogen, activity of the energetic metabolism enzymes, MF size decrease. Both contractile and energetic apparatus suffers essentially. This demonstrates certain atrophic processes in both types of the MF. Physical loading against the background of AOH, on the 120th day, prevents development of the atrophic processes in MF. This is demonstrated as maintenance of certain metabolic level and less pronounced changes of ultrastructure. On the 360th day of the experiment in the group without any loading, an essential atrophy of MF, a noticeable++ decrease in metabolism are observed. In the test group against the background of AOH the changes in the biopsy fibers are less pronounced and not so uniform. Application of physical loadings contributes to the development of certain adaptive reactions; their positive effect to the skeletal muscle fiber morphology depends on the structural background, against which it acts and their intensity. The earlier the load begins to act and the higher its intensity, the more pronounced is the delay in development of profound atrophic changes.     

Dynamic simulation of red blood cell metabolism and its application to the analysis of a pathological condition

Background  

Cell simulation, which aims to predict the complex and dynamic behavior of living cells, is becoming a valuable tool. In silico models of human red blood cell (RBC) metabolism have been developed by several laboratories. An RBC model using the E-Cell simulation system has been developed. This prototype model consists of three major metabolic pathways, namely, the glycolytic pathway, the pentose phosphate pathway and the nucleotide metabolic pathway. Like the previous model by Joshi and Palsson, it also models physical effects such as osmotic balance. This model was used here to reconstruct the pathology arising from hereditary glucose-6-phosphate dehydrogenase (G6PD) deficiency, which is the most common deficiency in human RBC.     

Postnatal changes of some enzymatic activities of energy supplying metabolism in the cortex, inner and outer medulla of the rat kidney

1. In rat kidney cortex, outer and inner medulla the development of activities of seven enzymes was investigated during postnatal ontogeny (10, 20, 30, 60 and 90 days of age). The enzymes were selected in such a manner, as to characterize most of the main metabolic pathways of energy supplying metabolism: hexokinase (glucose phosphorylation, HK), glycerol-3-phosphate dehydrogenase (glycerolphosphate metabolism or shunt, GPDH), triose phosphate dehydrogenase (glycolytic carbohydrate breakdown, TPDH), lactate dehydrogenase (lactate metabolism, LDH), citrate synthase (tricarboxylic acid cycle, aerobic metabolism, CS), malate NAD dehydrogenase (tricarboxylic acid cycle, intra-extra mitochondrial hydrogen transport, MDH) and 3-hydroxyacyl-CoA-dehydrogenase (fatty acid catabolism, HOADH). 2. The renal cortex already differs metabolically from the medullar structures on the 10th day of life. It displays a high activity of aerobic breakdown of both fatty acids and carbohydrates. Its metabolic capacity further increases up to the 30th day of life. 3. The outer medullar structure is not grossly different from the inner medulla on the 10th day of life. Further it differentiates into a highly aerobic tissue mainly able to utilize carbohydrates. It can, however, to some extent, also utilize fatty acids aerobically and produce lactate from carbohydrates anaerobically. 4. The inner medullar structure is best equipped to utilize carbohydrates by anaerobic glycolysis, forming lactate. This feature is already pronounced on the 10th day of life, its capacity increases to some extent during postnatal development, being highest between the 10th and the 60th day of life.     

Variations in the kinetic behaviour of the NADPH-production systems in different tissues of the trout when fed on an amino-acid-based diet at different frequencies

We have studied the effects of feeding an amino-acid-based diet (ABD) at different frequencies upon growth and several NADPH-production systems in the rainbow trout (Oncorhynchus mykiss). The kinetic behavior of glucose 6-phosphate dehydrogenase (G6PDH), 6-phosphogluconate dehydrogenase (6PGDH), malic enzyme (ME) and NADP-linked isocitrate dehydrogenase (NADP-IDH) was followed in the liver, kidney and adipose tissue.The kinetic parameters of NADP-IDH alone remained unaltered by either ABD or changes in feeding frequency. Maximum-velocity and catalytic-efficiency values of hepatic G6PDH and ME increased significantly when fed four times a day compared to twice a day with both the control diet and ABD, although these parameters for ME were significantly lower with ABD than with the control diet at both frequencies. In the kidney the activity and catalytic efficiency of G6PDH and 6PGDH increased significantly with high-frequency feeding on ABD. The activities of these enzymes in adipose tissue were much lower than in hepatic tissue. In the liver, maximum velocity and the catalytic efficiency of G6PDH, 6PGDH and ME increased significantly with the control diet at high-frequency feeding whereas they decreased significantly with ABD, especially with high-frequency feeding. Neither the Michaelis constant nor the activity ratios varied.Both feeding frequency and free amino acid altered the activity of the most important cytosolic NADPH-production systems. The varying response to nutritional stimuli of NADP-linked enzymes in fish tissues shows that they have independent physiological and metabolic roles and that their regulatory mechanisms respond to changes in nutritional and metabolic factors.     

Growth Hormone and Prolactin Action in a Teleost Anabas testudineus (Bloch): Effect of the Time of Administration

Circadian rhythms play a very important role on metabolic process and have considerable effects on growth, especially in ectotherms. Like variation in hormone levels, the sensitivity of target cells may show diurnal or seasonal fluctuations. The aim of this study was to compare the effects of morning versus evening injections of growth hormone and prolactin on malic enzyme, glucose-6-phosphate dehydrogenase, isocitrate dehydrogenase, aspartate aminotransferase, alanine aminotransferase and Na⁺,K⁺-ATPase in a teleost Anabas testudineus. Activities of malic enzyme, glucose-6-phosphate dehydrogenase and isocitrate dehydrogenase of the two control groups themselves differ significantly at morning and evening. Early morning administration of growth hormone increases malic enzyme, glucose-6-phosphate dehydrogenase and isocitrate dehydrogenase activities while evening administration of growth hormone does not effect these enzymes. Transaminase activities were stimulated by morning and evening administration of GH and PRL. Na⁺,K⁺-ATPase activity was stimulated by morning administration and inhibited by evening treatment of both hormones. The results reveal that a given hormone may provide a different message to the target tissues at different periods of the day.     

The structural changes in the lungs and the phospholipids of the pulmonary surfactant in experimental bleomycin-induced pneumosclerosis in rats]

Light microscopy was used to study rat lungs. Some morphological and biochemical characteristics after single bleomycin intratracheal administration in a dose of 10 mg/kg were studied. Some metabolic changes in the alveolar epithelium and vascular endothelium were estimated quantitatively. The peculiar picture of acute toxic alveolitis in the early stage of the injury and the features of fibrotic alveolitis by the 30-60th day were observed. The LDH and succinate dehydrogenase activities in the alveolar epithelium increased by the 3-7th day and returned to normal by the 30th day. At the same time the PL content increased 2-fold by the 30-60th day. The lysolecithin, sphingomyelin and phosphatidylserine concentration increased on the 3-7th day and returned to normal level on the 14th day. The phosphatidylglicerol content decreased significantly by the 60th day.     

Histochemistry of oxidative enzymes in the neuroglia in course of myelination.

The histoenzymic pattern of oxidative enzymes (G-6-PDH, G-PDH, ICDH, SDH, HBDH, NADH-2:tetrazolium dehydrogenase) was investigated in the developing neuroglia of rabbit brains, with special regard to the period of myelinogenesis. The obtained results lead to following conclusions: (1) During the early period of postnatal development there is maximal oxidative enzyme activity in ependymal cells, somewhat less reactive are the undifferentiated matrix cells and the differentiating cells of the mantle layer. No distinction can be made between the response of spongio- and neuroblasts. (2) Distinctly increased oxidoreductase activity, as compared to the early period of postnatal development, is demonstrated by the differentiating cells of myelination gliosis, no prevalence being demonstrable for enzymes of the particular metabolic pathways (pentose shunt, glycolysis or Krebs cycle). (3) G-6-PDH, G-PDH and oxidoreductases acting within the citric acid cycle are demonstrable only in single cells of the interfascicular oligodendroglia of adult rabbit brains, while almost all cells exhibit appreciable activity of HBDH and NADH-2 tetrazolium dehydrogenase.     

Rhythmicity of the activity of the supraoptic and paraventricular nuclei of the hypothalamus of the C57Bl mouse

Rhythmical changes in the activity of the neurosecretory processes have been compared in the supraoptic (SON) and paraventricular (PVN) nuclei in the C57Bl mice hypothalamus during vernal equinox. Parts of the neurosecretory cells, being at stages of synthesis, excretion and accumulation of secrete, volumes of the cell nuclei and nucleoli survey as criteria of their activity. Similar feature for the rhythmic of both nuclei studied is the highest activation of the processes during day time, when mice are resting; this is demonstrated as the maximal amount of actively synthesizing cells, maximal volumes of the cell nuclei and nucleoli. The peculiarities of the rhythmic display in the activity is manifested as a greater ability of the SON cells to accumulate neurosecrete. The accumulation of the secretory material in the SON cells precedes to the period of its maximal activity (1-7 PM) characterized: by making the cells free from the secrete and by a maximal increasing the volume of the nucleoli. In the PVN intensified display of the activity is noted at early hours of the day, and the amount of the cells not containing the secrete--at 6 PM. Lack of the neurosecrete accumulation in the PVN cells speaks in favour of more steady than in the SON cells excretion of the secrete. This demonstrates a more even maintenance of neurohormones concentration in the organism.     

Histochemical studies on metabolic zonation of the liver in the trout (Salmo gairdneri)

The livers of 26 adult male and female trout were studied histochemically. G6Pase activity was always found to be heterotopically distributed with a constant maximum in the periportal area. In many cases the glycogen content and the activity of phosphorylase predominated in the periportal zone as well. Maximum activity of glucose-6-phosphate-dehydrogenase and malic enzyme, however, could be demonstrated preferentially in the perivenous area. Lactate dehydrogenase, succinate dehydrogenase, alcohol dehydrogenase, acid phosphatase and beta-glucuronidase were found equally in all liver cells. 3-Hydroxybutyrate dehydrogenase was absent. Thus, the principles of metabolic zonation have been established in trout liver, the architecture of which differs essentially from that of mammals. The course of the terminal afferent and efferent vessels is the decisive factor for the heterotopic localization of functional units rather than the tubular or plate-forming arrangement of the hepatocytes.     

Alterations in the glycolytic and glutaminolytic pathways after malignant transformation of rat liver oval cells

Oval cells are liver epithelial cells that proliferate during the early stages of hepatocarcinogenesis induced by a variety of chemicals. The oval cell lines OC/CDE 6 and OC/CDE 22 have been established in our laboratory at two time points (6 and 22 weeks) of the carcinogenic process and have been malignantly transformed by different procedures. During the transformation process, the glycolytic and glutaminolytic flux rates were consistently up-regulated and this process was accompanied by an overproportional increase in the activities of cytosolic hexokinase and 6-phosphogluconate dehydrogenase. In transformed oval cells, a strong correlation between the glycolytic flux rate and glutamine consumption as well as glutamate production was observed. Furthermore, the transport of glycolytic hydrogen, produced by the glyceraldehyde 3-phosphate dehydrogenase-catalyzed reaction, from the cytosol into the mitochondria by means of the malate-aspartate shuttle was enhanced, this being due to alterations in the activities of malate dehydrogenase and glutamate oxaloacetate transaminase. The up-regulation of the glycolytic hydrogen transport and the alterations in the glycolytic enzyme complex led to an enhanced pyruvate production at high glycolytic flux rates. Taken together, our data are further proof that a special metabolic feature (increased glycolysis and glutaminolysis) is characteristic for tumor cells and that the mechanisms by which this metabolic state is induced can be totally different. J. Cell. Physiol. 181:136–146, 1999. © 1999 Wiley-Liss, Inc.     

Streptomyces sp. is a powerful biotechnological tool for the biodegradation of HCH isomers: biochemical and molecular basis

Actinobacteria are well-known degraders of toxic materials that have the ability to tolerate and remove organochloride pesticides; thus, they are used for bioremediation. The biodegradation of organochlorines by actinobacteria has been demonstrated in pure and mixed cultures with the concomitant production of metabolic intermediates including γ-pentachlorocyclohexene (γ-PCCH); 1,3,4,6-tetrachloro-1,4-cyclohexadiene (1,4-TCDN); 1,2-dichlorobenzene (1,2-DCB), 1,3-dichlorobenzene (1,3-DCB), or 1,4-dichlorobenzene (1,4-DCB); 1,2,3-trichlorobenzene (1,2,3-TCB), 1,2,4-trichlorobenzene (1,2,4-TCB), or 1,3,5-trichlorobenzene (1,3,5-TCB); 1,3-DCB; and 1,2-DCB. Chromatography coupled to mass spectrometric detection, especially GC–MS, is typically used to determine HCH-isomer metabolites. The important enzymes involved in HCH isomer degradation metabolic pathways include hexachlorocyclohexane dehydrochlorinase (LinA), haloalkane dehalogenase (LinB), and alcohol dehydrogenase (LinC). The metabolic versatility of these enzymes is known. Advances have been made in the identification of actinobacterial haloalkane dehydrogenase, which is encoded by linB. This knowledge will permit future improvements in biodegradation processes using Actinobacteria. The enzymatic and genetic characterizations of the molecular mechanisms involved in these processes have not been fully elucidated, necessitating further studies. New advances in this area suggest promising results. The scope of this paper encompasses the following: (i) the aerobic degradation pathways of hexachlorocyclohexane (HCH) isomers; (ii) the important genes and enzymes involved in the metabolic pathways of HCH isomer degradation; and (iii) the identification and quantification of intermediate metabolites through gas chromatography coupled to mass spectrometry (GC–MS).