Proliferation and differentiation of androgenetic cells in fetal mouse chimeras.

The properties of androgenetic cells and their ability to proliferate and differentiate were examined in post-midgestation chimeras. In several tissues, namely the brain, cardiac muscle, skeletal muscle and intestinal epithelium, the rate of proliferation of androgenetic cells was higher than that of normal cells in day 13 embryos. This higher rate of proliferation was however less pronounced by day 17-18 of development. It is possible that IGF2, a major growth factor regulating fetal growth, could play a role in the increased proliferation of androgenetic cells. Igf2 is also an imprinted gene that is expressed only when inherited paternally. Indeed, in the smooth muscle, cartilage and intestinal epithelium, patches of androgenetic (ag) cells exhibited higher levels of IGF2 mRNA than neighbouring wild-type cells. Surprisingly, we also detected expression of Igf2 in ag cells of ectodermal origin; this gene is not normally expressed in this lineage. This expression was observed in the brain, epidermis and in the epithelium of the tongue. We attempted to confirm the identity and differentiation status of ag cells with the help of cell-type specific antibodies and lectins. Evidence for only one of the cell types analysed, i.e. the goblet cells of the gut, suggests a delay or aberrant differentiation of ag cells.

[Latent antioxidant deficiency in the East German population--causes and clinical significance. II]. / Latenter Antioxidantienmangel in der DDR-Population--Uraschen und klinische Bedeutung. II. Mitteilung.

The hitherto yielded results of the research groups for arteriosclerosis and trace elements of the Medical Clinic of Leipzig University were summarized and valuated. The most important results were than in 800 healthy persons of three counties of the GDR who were examined depending on sex and age a serum selenium deficiency with regional differences was stated and this deficiency could be increased by an antiatherogenic mode of nutrition (n = 94, p less than 0.001). A consecutive depletion of vitamin E out of normal values was to be explained as a reference of an insufficient status of antioxidants. Analyses on nearly 1,000 foodstuffs and sorts of coffee, tea, tobacco, alcohol, etc. showed as cause a high-degree depletion of vegetable foodstuffs in selenium. The established daily minimum uptake of selenium of 12 to 15 micrograms by cooked foods was covered by above all cholesterol-rich animal foodstuffs and was further lowered by their withdrawal. In a double blind study first results of a supplementation of sodium selenite referred to a favourable influence on the immune status. After explanation of the reasons which may lead to an additional insufficiency of antioxidants was postulated that the protection from activated oxygen species is indicated both for the primary prevention of autoimmunigenic, atherogenic and cancerogenic diseases and for the curative medicine by a supplementation of selenium.

[The importance of the trace element selenium in internal diseases]. / Zur Bedeutung des Spurenelementes Selen bei inneren Krankheiten.

Issuing from the existence of selenium and its interactions with other elements its essentiality in biochemical processes of man was explained. In correspondence with other research teams also own results speak for a relative selenium deficiency in the Middle-European region. Alterations of the cell integrity caused by selenium deficiency may play a role in the etiopathogenesis and perpetuation of chronic cardiovascular diseases as well as of disturbances of the immunoregulation. Recent epidemiological studies and clinical experimental investigations show potential tendencies for prophylaxis and therapy of diseases which are remarkable in health policy.

[Results of a long-term study of arteriosclerotic circulatory disorders with polyene fatty acid therapy]. / Ergebnisse einer Langzeitstudie arteriosklerotischer Durchblutungsstörungen unter Polyenfettsäurentherapie.

The antiatherogenic oil diet as one factor of therapy causes the hope of favourable effects in the complex primary prophylaxis. By means of this alone, however, a regression of a clinically significant arteriosclerosis cannot be achieved within 3 years, through biochemically unequivocal "antiatherogenic" effects are to be proved. Linseed-oil, fish and fish oil seem to be particularly suited to cause these effects desired.

Defective ornithine metabolism in cultured skin fibroblasts from patients with the syndrome of hyperornithinemia, hyperammonemia and homocitrullinuria.

The syndrome of hyperornithinemia, hyperammonemia, and homocitrullinuria (HHH) is a metabolic disorder resulting in protein intolerance and mental retardation. The primary metabolic defect has yet to be determined. We studied some aspects of ornithine metabolism in cultured skin fibroblasts from two patients from two patients with the HHH syndrome. The fibroblasts failed to incorporate 14C-label from ornithine into protein, a defect also observed in fibroblasts from patients with gyrate atrophy of the choroid and retina and a deficiency of ornithine aminotransferase activity. The defect can be corrected in heterokaryons formed between these two types of fibroblasts. These findings indicate that fibroblasts are suitable for further studying the underlying metabolic defect in HHH syndrome. The combination of the ornithine incorporation assay and genetic complementation analysis provide a confirmatory test for the diagnosis of this syndrome.

The ontogeny of thymidine kinase in tissues of man and rat.

Thymidine kinase activities, virtually all soluble in rat lung, liver, and small intestine, decreased abruptly late in gestation or immediately after birth. An injection of thyroxine delayed the fetal but not the neonatal changes in liver activity. An injection of cortisol decreased hepatic and pulmonary thymidine kinase activities in both fetal and neonatal rats but had little effect on the intestinal enzyme. Premature extrauterinization led to an earlier occurrence of the quantitative changes in thymidine kinase activity usually seen at term. Birth-associated changes included a rapid transitory increase in the hepatic enzyme and the virtual loss of intestinal thymidine kinase activity. In human tissues, the soluble thymidine kinase in liver remained high between the 11th and 22nd wk of gestation whereas the particulte enzyme, the predominant form in adult liver, rose in the second half of gestation and reached adult levels at birth. In human lung, the soluble enzyme started to decrease by the 16th gestational wk, whereas the particulate thymidine kinase reached the higher adult levels late in gestation. Thymidine kinase in adult human tissues was predominantly particulate.

Enzyme activities in human fetal and neoplastic tissues.

The concentrations of ten or 12 enzymes involved in the metabolism of DNA, collagen, amino acids, or glucose have been determined in variants of human intestinal and pulmonary tissues. In comparison to nonneoplastic adult colon, normal fetal colon had elevated concentrations of thymidine kinase, peptidyl proline hydroxylase, phosphoserine phosphatase, ornithine transcarbamylase, gamma-glutamyl transpeptidase, and ornithine aminotransferase. Raised activities of the first five of these enzymes, and of hexokinase, glucose-6-phosphate dehydrogenase, and pyrroline-5-carboxylate reductase distinguishes neoplastic from nonneoplastic sections of adult colon. Study of a wide range of pulmonary specimens permitted comparisons of different types of tumors, and revealed some subtle differences between lungs of noncancer patients and nonneoplastic portions of host lungs. The concentrations of eight previously identified enzymic indicators were less in moderately or well differentiated than in poorly differentiated pulmonary adenocarcinomas. The latter differed from epidermoid carcinomas (also poorly differentiated) by containing lower concentrations of thymidine kinase (both soluble and particulate) and hexokinase.

Enzyme pathology of the liver in patients with and without nonhepatic cancer.

Preliminary studies of 13 enzymes subserving various metabolic pathways were undertaken in tumor-free liver biopsy samples from cancer patients and control subjects. The observations indicate that as a result of nonhepatic neoplasms, with (7 cases) or without (6 cases) hepatic involvement, the biochemical composition of the liver becomes partially undifferentiated. Quantification of appropriate enzymes in histologically normal liver samples could thus distinguish clearly between cancer hosts and controls. The best discriminators include two hepatic enzymes whose concentrations were decreased to less than 30% of normal (soluble glutamate dehydrogenase and the cold stable pyrroline-5-carboxylate reductase) and three for which it was elevated two to four-fold (hexokinase, peptidyl proline hydroxylase and thymidine kinase) in response to distant neoplasms. The same alterations in hepatic enzyme pattern were not seen in any cancer-free patients with or without morphologic liver damage.

Relative activities of thymidylate synthetase and thymidine kinase in rat tissues.

The activities of thymidylate synthetase and thymidine kinase were compared in tissues of normal (adult and developing), cortisol-injected, and tumor-bearing rats. The purpose of the study was to determine whether the activities of these two enzymes, which catalyze reactions leading to the same metabolic intermediate, changed proportionately, reciprocally, or independently under different physiological conditions. Both enzymes had high activities in fetal tissues. Thymidine kinase concentrations decreased shortly before or immediately after birth; in several tissues, transient postnatal peaks in thymidine kinase activities appeared within the first 3 weeks after birth. Thymidylate synthetase activities declined gradually after parturition and showed no significant postnatal rises. In sucklings given injections of cortisol, thymidine kinase activities were reduced substantially in eight tissues while thymidlyate synthetase decreased only in lung and thymus of 11-day-old rats. In tumor-bearing rats, thymidine kinase activity increased dramatically in spleen, whereas thymidylate synthetase activities only doubled. In host liver, rises in thymidine kinase activities were not always matched by increases in thymidlyate synthetase. In the tumors, both activities were higher than in most normal adult tissues. Despite the differential sensitivities of the two enzymes to cortisol and tumor bearing, thymidylate synthetase and thymidine kinase were closely correlated in tissues of untreated animals. The Spearman rank correlation coefficient for 112 tissues was 0.895, while the correlation coefficient between the standard scores of the activities was 0.839. The activities of the two enzymes did not appear to be reciprocal or compensatory during normal differentiation or during dedifferentiation associated with tumor bearing, but their potentials for activity were independent of each other.

Uridine kinase activities in developing, adult and neoplastic rat tissues.

Uridine kinase activities were found chiefly in the soluble fractions of rat tissues. In normal adults the activities ranged from 13 munits/g in skeletal muscle to 178 munits/g in colon. Enzyme activities in several rat neoplasms were significantly higher (e.g. in a fibrosarcoma, mammary carcinoma, renal carcinoma, pancreatic carcinoma and lymphocytic lymphoma, but not in a fast-growing Morris hepatoma). The activities were not related to tumour growth rates or sizes. In normal foetal liver, lung, brain, heart and kidney, uridine kinase concentrations equalled or exceeded those in the adult homologous tissue, but maximal activities in liver were reached 3--5 days post partum. In suckling rats the intestinal activity decreased substantially immediately after birth and normally did not rise again until late in the third postnatal week. Premature upsurges could be evoked by an injection of cortisol or by starvation of the pups overnight. Pancreatic activity was absent from 1-day-old rats, and only about 5% of the adult activity was reached by day 20; adult activities were attained rapidly after weaning. In pancreas, precocious formation or uridine kinase was elicited by overnight starvation of 2-week-old rats.

Effects of cortisol or starvation on the activities of four enzymes in small intestine and liver of the rat during development.

The small intestine of the rat, like the liver, is a tissue with high activities of arginase, ornithine aminotransferase, and pyrroline-5-carbozylate reductase. These enzymes are thought to catalyse sequential steps in the synthesis of proline. We have compared the effect of cortisol or brief starvation on the activities of these enzymes and of soluble alanine aminotransrerase in the small intestine and liver during development. In the intestine, cortisol accelerated the increase in arginase activity, reversed the normal 2-week-long post-natal decline in that of pyrroline-5-carboxylate reductase, and delayed the normal decrease, in the third week, of ornithine aminotransferase activity. Starvation of neonates for 18 h raised the activity of arginase slightly, that of pyrroline-5-carboxylate reductase significantly, and had no effect on ornithine aminotransferase activity. Cortisol did not alter the hepatic activities of pyrroline-5-carboxylate reductase in neonates but induced premature rises in the activities of arginase and ornithine aminotransferase. Short starvation did not affect the hepatic activities of any of these enzymes. Alanine aminotransferase activity in both tissues was enhanced by cortisol but not by starvation. Thus in intestine, cortisol elicited some changes in the activity of three functionally related and one unrelated enzyme while starvation evoked changes only in pyrroline-5-carboxylate reductase. Neither stimulus appears to be specific for a metabolic pathway or to trigger a coordinated onset of proline synthesis from arginine.

Human colon tumors: enzymic and histological characteristics.

In samples of colonic adenocarcinomas, the mean activities of thymidine kinase, glucose-6-phosphate dehydrogenase, phosphoserine phosphatase and pyrroline-5-carboxylate reductase were several fold higher than those of nonneoplastic colon. The presence of considerable, cold labile pyrroline-5-carboxylate reductase activity provided an additional criterion for distinguishing tumors from the control tissue. Deviations from the pattern of enzymes in normal colon were much more pronounced in the five moderately well-differentiated than in the single well-differentiated adenocarcinoma.

Tissue enzyme changes in parabiotic rats with subcutaneous lymphoma or fibrosarcoma.

A solid lymphoma implanted into normal inbred Kx rats and one partner of parabiotic pairs caused appreciable decreases in hepatic ornithine aminotransferase and arginase about a week earlier (4-6 days after implantation) in single hosts than in parabiotic hosts. By 18-21 days significant decreases in both enzymes were apparent in the host partner also. The hepatic thymidine kinase showed a fivefold elevation in single hosts 4 days after implantation; by 14 days in its levels were about 200 times above normal and had also risen in the parabiotic hosts (20-fold) and host partners (fourfold). Implantation of fibrosarcoma caused qualititatively similar but generally less pronounced changes in these three enzymes in livers of single hosts, parabiotic hosts, and host partners. The splenic thymidine kinase 14 days after implantation was increased from control levels of about 3 U/g to 50.6, 44.8, and 13.5 U/g in single hosts, parabiotic hosts, and host partners, respectively. At later stages, 17-20 days after implantation of the lymphoma, appreciable amounts of thymidine kinase appeared in the plasma: The units of activity per milliliter were 6.2 in single hosts, 0.79 in parabiotic hosts, and 0.55 in host partners (control less than 0.05). Our observations on the changes in hepatic and splenic enzymes in parabiotic rats suggest that effects of neoplasms on distant host tissues are mediated by humoral factors. The less pronounced responses in parabiotic than in single hosts indicate that the tumor-free partner affords some "protection" against these systemic effects.

Enzymes metabolizing delta1-pyrroline-5-carboxylate in rat tissues.

The direction and capacity for the metabolism of delta1-pyrroline-5-carboxylate in a number of rat tissues ere investigated by measuring the activities of delta1-pyrroline-5-carboxylate reductase, delta1-pyrroline-5-carboxylate dehydrogenase and proline oxidase. Each of these enzymes catalyzed unidirectional reactions in which delta1-pyrroline-5-carboxylate was either the substrate or product. Delta1-Pyrroline-5-carboxylate reductase activities that were much higher than any previously reported were obtained by avoiding its inactivation in the cold. delta1-Pyrroline-5-carboxylate dehydrogenase, previously said to act on both D- and L-isomers of delta1-pyrroline-5-carboxylate, acted only on the L-isomer. Proline oxidase could not be measured in two adult tissues, in which an inhibitor appeared after birth. The activity of delta1-pyrroline-5-carboxylate reductase significantly paralleled that of ornithine aminotransferase in 23 tissues, showing a widespread potential for proline synthesis from ornithine. An independently distributed potential in fewer tissues for proline degradation to alpha-oxoglutarate was shown by the significantly similar tissue distributions of proline oxidase. Delta1-pyrroline-5-carboxylate dehydrogenase and glutamate dehydrogenase. Reverse metabolism of glutamate or proline to ornithine would be atypical in rat tissues with these distributions of unidirectional enzyme reactions.

The undifferentiated enzymic composition of human fetal lung and pulmonary tumors.

The concentrations of 16 to 21 enzymes, representing various metabolic pathways, have been determined in human adult, fetal, and neoplastic lung. At midgestation, 12 enzymes (among them, several that metabolize amino acids) were above their adult values while 3 other enzymes were still at low concentrations. These signs of biochemical immaturity are contrasted and compared with those in fetal human liver and rat lung. The enzymic composition of the 11 human pulmonary tumors studied resembled that of the normal fetal lungs closely; the same 12 enzymes were elevated and the same 2 were decreased (compared to nonneoplastic adult lung) in both. The characteristic abnormality in the overall pattern of enzymes, in the concentrations of individual ones, and in the quality of pyrroline-5-carboxylate reductase was clearly evident in both primary and metastatic tumors. The mean concentrations of 10 enzymes in the tumors were significantly different (higher or lower) from those in the control lungs (p less than 0.001 to less than 0.05). The best markers of neoplasticity were thymidine kinase, peptidyl proline hydroxylase, phosphoserine phosphatase, hexokinase, and pyrroline-5-carboxylate reductase. The results demonstrate that quantification of a small battery of enzymes, none of them tissue specific, can distinguish adult human lung from its neoplasms.

The effect of lymphoma and other neoplasms on hepatic and plasma enzymes of the host rat.

Considerable thymidine kinase and pyrroline-5-carboxylate reductase activities were found in the plasma of rats bearing a transplanted lymphoma; neither activity was detected in plasma of hosts carrying hepatic, renal, mammary, or submaxillary gland tumors. All host livers exhibited signs of biochemical immaturity as indicated by the appropriate increases or decreases in the concentrations of the nine enzymes measured. The extent and time schedule of the changes in host liver varied with the enzyme and with the tumor that caused them. The hepatic concentrations of ornithine aminotransferase, arginase, pyrroline-5-carboxylate reductase, and glucokinase (all diminished), and of peptidyl proline hydroxylase and hexokinase (increased) were sensitive indicators of tumor growth in general. The concentration of ornithine aminotransferase decreased before the tumors became palpable. At more advanced stages, the high hepatic thymidine kinase activity distinguished the presence of hepatoma and lymphoma from those of all other equally fast-growing tumors. However, only in lymphoma-bearing rats did a fivefold elevation of hepatic thymidine kinase occur as early as 4 days after implantation. Additional observations on the lymphoma itself, on blood cells, and on the involuting thymus of normal rats indicate that the striking systemic effects of this tumor cannot be explained by a release of enzymes from the thymus or by the increased number of lymphoma cells present in blood or liver.

Glutamate dehydrogenase, alanine aminotransferase, thymidine kinase, and arginase in fetal and adult human and rat liver.

In fetal livers of both man and rat thymidine kinase activity was 12 times higher than in the adult, glutamate dehydrogenase and arginase were present at 20-50% of their adult values, whereas alanine aminotransferase activity was only an insignificant fraction of that in the adult. Although the developmental changes for the four enzymes were quantitatively similar in both species, qualitatively there were some significant differences. In adult human liver, glutamate dehydrogenase activity was distributed almost equally between the cytosol and particles; the concentration of only the soluble enzyme increased after birth. In rat liver, glutamate dehydrogenase remained exclusively a particulate enzyme. The soluble hepatic alanine aminotransferase activity rose in both species after birth (from less than 2 U/g to 41-57 U/g, respectively). Thymidine kinase was wholly soluble in the fetal livers; only in adult human liver was additional activity (at least 50% of the total) found in the particles. Arginase isozymes, identical and apparently the same single isozyme in fetal and adult rat liver, show an ontogenetic change in man. A shift from a single form, common to human fetal liver and fetal kidney, to at least two variants in adult human liver, indicates another complexity of the fully differentiated liver in man.

Enzymes of ornithine metabolism in adult and developing rat intestine.

The levels of 11 enzymes, most of them involved in the metabolism of ornithine, were measured in whole upper intestine, or in duodenum, small intestine and colon of adult rats. The developmental formations in small intestine of arginase, ornithine aminotransferase, and ornithine transcarbamylase were compared with those in liver. Changes with age (late gestation of adult) of the intestinal activities of pyrroline-5-carboxylate reductase, proline oxidase and glutamyl transpeptidase are also described. The results suggest that the proximal part of the intestine is well endowed with enzymes involved in the conversion of ornithine to proline as well as to citrulline. Fetal intestine is rich in proline oxidase and pyrroline-5-carboxylate reductase. The peak levels of ornithine aminotransferase found in intestine in the first 3 postnatal weeks were higher than seen in any other rat tissue. Some of the properties of arginase, ornithine aminotransferase and pyrroline-5-carboxylate reductase in small intestine were compared with those in liver. Isozymes of arginase in small intestine differed from those in liver; the kinetic properties of ornithine aminotransferase were similar in the two tissues. In intestine of 14-day-old rats, the ornithine aminotransferase reaction was reversible, forming ornithine from pyrroline-5-carboxylate. The intestinal pyrroline-5-carboxylate reductase was cold-labile as was the hepatic enzyme in rat.