Stability of enterocytes and certain enzymatic activities in suspensions of cells from the villous tip to the crypt of Lieberkühn of the mouse small intestine

A series of studies in this laboratory have focused on how an indigenous microbiota influences the activities of alkaline phosphatase, phosphodiesterase I, and thymidine kinase in the enterocytes of the upper small intestine of mice. To draw conclusions about the role of the microflora in determining levels of enzymatic activity, we found it necessary to develop a procedure by which cell suspensions could be obtained containing enterocytes isolated sequentially from the villous tip to the crypt of Lieberkühn. The procedure was modified from the one developed for rats by Weiser (J. Biol. Chem. 248:2536-2541, 1973), involved a minimum number of interfering factors (e.g., proteolytic enzymes and mechanical agitation), and worked reproducibly for mice. During development of the procedure, some variables affecting the assays of the enzymes known to be present in enterocytes were also explored. Rods to which were tied everted segments of gut were incubated in a series of tubes containing a solution of EDTA the concentration of which was changed from 1.5 to 5.0 mM, thus giving a greater yield of enterocytes at every step. The cells incubating in the chelating solution were most stable when 0.23 M sucrose was included in the EDTA solutions. Success in assaying enzymatic activities in the cell suspensions depended on (i) how the cells were isolated, (ii) the assay procedure for thymidine kinase, and (iii) whether cellular suspensions or extracts were assayed.     

Influence of indigenous microbiota on activities of alkaline phosphatase, phosphodiesterase I, and thymidine kinase in mouse enterocytes.

An indigenous microflora introduced into the gastrointestinal tracts of animals in a population of germfree mice affected in different ways three enzymes in small bowel enterocytes. Cells were obtained by techniques designed for sequentially removing enterocytes from the tip of the villus to the crypts of Lieberkühn. The specific activity of alkaline phosphatase, a component of the enterocyte microvillous membrane, did not differ in cells isolated from germfree mice and from those associated with a microflora, while that of phosphodiesterase I, also a part of the microvillous membrane, was approximately 1.5-fold greater in the suspensions from all levels of the villi in germfree mice than in those from the associated animals. By contrast, the specific activity of thymidine kinase, a cytosol enzyme, in suspensions in which the cells were isolated from the lower portion of the villi and crypts was about one-half as great in cells from germfree mice as in those from the same regions of animals with a microbiota. These results support the hypothesis that activities of certain enzymes involved in metabolism, uptake, and incorporation by enterocytes of components of dietary nuclei acids are influenced by a microflora.     

Preparation and properties of isolated epithelial intestinal cells from chicken cecum and jejunum

The isolating agents, one enzymatic (hyaluronidase) and two chemical (sodium citrate and EDTA) have been used to search for the best technique to prepare suspensions of viable cells from chicken cecum and jejunum. Viability of enterocytes was assessed in terms of cell membrane integrity (trypan blue exclusion test), metabolic activity (oxygen uptake, lactate production and ATP content) and monosaccharide cumulative capacity. Results show that: In both cecum and jejunum, membrane integrity is better in cells harvested with citrate than those isolated with hyaluronidase or EDTA; The best metabolic status was found in cecal cells isolated with citrate and in jejunal cells obtained with hyaluronidase; The capacity to support alpha-methyl-D-glucoside gradients is highest in the cells harvested with citrate. The citrate-containing isolation medium is thus considered to yield epithelial cell suspensions with the best functional conditions.     

The sex difference in the regulation of liver regeneration after partial hepatectomy in the rat

The increases in the activity of hepatic thymidylate synthetase and thymidine kinase, which catalyzes the formation of thymidylate via the de novo and salvage pathways, respectively, were significantly suppressed 24 h after 70% partial hepatectomy in female rats administered either alpha- or beta-adrenoreceptor antagonists. The injection of beta-antagonist to male or ovariectomized female rats had no effect on the activities of these enzymes. Only alpha-adrenoceptor antagonist depressed these enzymatic activities of 24-h-regenerating liver in male and ovariectomized female rats. The decrease of the activities of thymidylate synthetase and thymidine kinase was accompanied by a concomitant reduction of DNA content in 24-h-regenerating liver. It is concluded that catecholamine regulates the female rat liver regeneration through both alpha- and beta-adrenergic pathways by the inductions of thymidylate synthase and thymidine kinase, while in adult male and ovariectomized female rats, only the alpha-mediated pathway is involved.     

Lactobacilli as effectors of host functions: no influence on the activities of enzymes in enterocytes of mice

Preparations of lactobacilli are often used as dietary supplements to improve the growth and efficiency in utilizing food of animals of commercial value. We tested in an experimental model whether the effects of lactobacilli on growth of and food utilization by animals may be due to alteration of the activities of absorptive enzymes in the small bowel. Germfree mice housed in isolators under tightly controlled conditions were monoassociated with one of four strains of indigenous Lactobacillus spp. From 1 to 5 weeks later, the activity of alkaline phosphatase was assayed in homogenates of segments of the upper small intestines of the associated animals and of matched germfree controls. The specific activity of the enzyme was the same in the mice in the two groups. In other experiments, epithelial cells were isolated from the upper small intestines of mice associated with eight Lactobacillus strains (octa-associated) and from those of matched germfree mice and assayed for alkaline phosphatase, phosphodiesterase, and thymidine kinase activities. The epithelial cells were harvested sequentially from the tips of the villi toward the crypts of Lieberkühn of the intestines. In all preparations, mice of both types yielded an equivalent mass (wet weight) of cells. The protein content of the cells reflected the mass. The activities of the microvillous membrane enzymes alkaline phosphatase and phosphodiesterase and the cytosol enzyme thymidine kinase were the same whether or not the animals contained the bacteria. Therefore, any effects on animal growth and food utilization observed when lactobacilli are used as dietary supplements may not be due to a direct alteration by the bacteria of the absorptive enzymes of the host animal.(ABSTRACT TRUNCATED AT 250 WORDS)     

Lactobacilli as effectors of host functions: no influence on the activities of enzymes in enterocytes of mice.

Preparations of lactobacilli are often used as dietary supplements to improve the growth and efficiency in utilizing food of animals of commercial value. We tested in an experimental model whether the effects of lactobacilli on growth of and food utilization by animals may be due to alteration of the activities of absorptive enzymes in the small bowel. Germfree mice housed in isolators under tightly controlled conditions were monoassociated with one of four strains of indigenous Lactobacillus spp. From 1 to 5 weeks later, the activity of alkaline phosphatase was assayed in homogenates of segments of the upper small intestines of the associated animals and of matched germfree controls. The specific activity of the enzyme was the same in the mice in the two groups. In other experiments, epithelial cells were isolated from the upper small intestines of mice associated with eight Lactobacillus strains (octa-associated) and from those of matched germfree mice and assayed for alkaline phosphatase, phosphodiesterase, and thymidine kinase activities. The epithelial cells were harvested sequentially from the tips of the villi toward the crypts of Lieberkühn of the intestines. In all preparations, mice of both types yielded an equivalent mass (wet weight) of cells. The protein content of the cells reflected the mass. The activities of the microvillous membrane enzymes alkaline phosphatase and phosphodiesterase and the cytosol enzyme thymidine kinase were the same whether or not the animals contained the bacteria. Therefore, any effects on animal growth and food utilization observed when lactobacilli are used as dietary supplements may not be due to a direct alteration by the bacteria of the absorptive enzymes of the host animal.(ABSTRACT TRUNCATED AT 250 WORDS)     

Thymidine phosphorylating enzymes in the gonads of marine invertebrates

The activities of enzymes involved in the consecutive phosphorylation of thymidine were revealed in the gonad extracts of marine invertebrates. Along with thymidine kinase activity, thymidilate kinase activity was revealed in all the studied species; however, the specific activities of nucleoside and nucleotide kinases varied in different species of mollusks, sea stars and sea urchins. Thymidine and thymidilate kinases were isolated from the gonads of the scallop Mizuhopecten yessoensis and some of their enzymat properties were studied. The thymidine kinase of M. yessoensis catalyzed the phosphorylation of thymidine and deoxycytidine at a lesser rate, but didn’s use purine ribo-and deoxyribonucleosides or pyrimidine ribonucleosides as phosphate acceptors. The thymidilate kinase carried out both TMP and dCMP phosphorylation. As well as ATP, the enzymes of M. yessoensis were also able to use dATP, dGTP, GTP, UTP and CTP as donors of phosphate groups. The thymidine kinase activity was inhibited by TMP, TTP and dCTP.     

Aanlysis of dCMP deaminase and CDP reductase levels in hamster cells infected by herpes simplex virus.

Several enzymatic activities involved in the biosynthetic pathways of nucleotides, including thymidine kinase, which has been used as a biochemical marker in studies of gene transfer, are induced by herpes simplex virus (HSV). The utility of additional markers prompted us to reanalyze the effects of HSV infection on the activities of two other enzymes for which direct selective methods can be devised: dCMP deaminase and CDP reductase. For this purpose, mutant Chinese hamster (lA1) cells devoid of dCMP deaminase activity or Syrian hamster (BHK-21/C13) cells were infected by HSV type 1 or 2, and the activities of thymidine kinase, dCMP deaminase, and CDP reductase were measured in the cell extracts. The reported induction of thymidine kinase and CDP reductase by HSV was confirmed, whereas the stimulation of dCMP deaminase activity could not be observed. For both cell lines, the HSV-induced CDP reductase differed from the host enzyme by sensitivity to inhibition by both dTTP and dATP. This property should be helpful in developing a selection system for this activity.     

Isolation of Mutants of Bacteriophage T4 Unable to Induce Thymidine Kinase Activity

New mutants of T4 have been isolated by using a strain of Escherichia coli lacking thymidine kinase activity. These T4 mutants, designated tk, are able to grow on this E. coli strain under light on plates containing 5-bromodeoxyuridine and were all found to be unable to induce thymidine kinase (ATP: thymidine 5'-phosphotransferase, EC 2.7.1.21). All of these tk mutants fall into one complementation group which maps just to the right of rI on the standard T4 genetic map, far from most other genes coding for enzymes involved in pyrimidine metabolism. The tk mutants grow as well as wild-type T4, indicating that thymidine kinase is a non-essential enzyme.     

The vacuole as the lysosome of the yeast cell

Summary Vacuoles can be isolated by flotation from suspensions of lysed proto-plasts in the presence of Ficoll. The purity of the respective preparations is demonstrated by the complete absence of enzymes of the mitochondria and of the groundplasm, as well as by morphological observations. Hydrolytic enzymes (two acid proteases, p-nitrophenylacetate-esterase, RNase and leucylaminopeptidase) are present in high specific activities in isolated vacuoles. The vacuole therefore represents the lysosome of the yeast cell.Abbreviations EDTA Ethylene-diamine-tetraacetic-acid - NAD Nicotinamideadenine-dinucleotide - DIP Dichlorophenol-indophenol     

Chromosomal and enzymatic patterns provide evidence for two types of human colon cancers with abnormal nucleotide metabolism

Correlated chromosomal and enzymatic studies are reported in a series of 13 human colon cancers. Two distinct chromosomal patterns are found: one with many losses, called monosomic type and another with many gains, called trixomic type. Chromosome 18, which carries the gene for thymidylate synthase (TS) is always missing in the monosomic type, in which TS activity is low. On the contrary, TS activity is high in trysomic type on the average. The long arm of chromosome 17, which carries the gene for thymidine kinase (TK), is frequently duplicated in both monosomic and trisomic and trisomic types, in which TK activities are high. It is found that most other deletions affect chromosomes carrying genes for enzymes of the de novo pathways whereas most other gains affect chromosomes carrying for enzymes of salvage pathways for synthesis of nucleotides. The importance of this finding is discussed.     

Highly purified recombinant varicella Zoster virus thymidine kinase is a homodimer

Recombinant varicella zoster virus (VZV) thymidine kinase (TK) was isolated in a fast and gentle two-step procedure from Escherichia coli. The TK was expressed as a PreScission-cleavable fusion protein and purified by glutathione and ATP affinity chromatography, yielding homogeneous, highly pure VZV TK. The purified enzyme displays enzymatic activities with K(m) values of 0.3 +/- 0.06 microM for the natural substrate thymidine and 11.6 +/- 3.2 microM for ATP, indicating the biochemical equivalence with the viral VZV TK expressed in infected cells. Determinations of the native molecular weight by size exclusion chromatography and native polyacrylamide gel electrophoresis revealed that the pure enzyme is biologically active as a homodimer.     

DNA polymerase and thymidine kinase activities in different regions of rat brain during postnatal development: Effect of undernutrition

The effect of undernutrition on the activity of two key enzymes for DNA synthesis, namely DNA polymerase and thymidine kinase, in developing rat brain has been investigated. Both enzymatic activities in cerebral hemispheres and in brain stem are lower in undernourished animals than in controls at the 5th day after birth; succesively, from 5 to 30 days, they decrease in both groups of animals, however the decrease is less drastic in undernourished rats than in controls. At 30 days of age the specific activity of both enzymes is quite similar in the two groups of animals. In the cerebellum, DNA polymerase and thymidine kinase activities increase after 5 days of age showing a peak at around 9 days in controls and at about 13 days in undernourished animals, decreasing thereafter in both groups, although less drastically in undernourished animals, and reaching quite similar values at 30 days. The results obtained show that both enzymatic activities are impaired at 5 days and delayed thereafter, in agreement with the changes of DNA synthesis previously observed.     

Protein modification enzymes associated with the protein-synthesizing complex from rabbit reticulocytes. Protein kinase, phosphoprotein phosphatase, and acetyltransferase.

A number of protein modification activities are present in the protein-synthesizing complex isolated from rabbit reticulocytes. These enzymes are solubilized by sedimentation of the ribosomes through buffered sucrose containing 0.5 M KCl, and have been partially purified from the high salt wash fraction by chromatography on DEAE-cellulose and phosphocellulose. The ribosomal-associated enzymatic activities include cyclic AMP-regulated and cyclic nucloetide-independent protein kinase, phosphoprotein phosphatase, and acetyltransferase activities. These enzymatic activities have been shown to modify specific ribosomal and ribosomal-associated proteins. The cycli c AMP-regulated protein kinase phosphorylate the 40 S ribosomal subunit from rabbit reticulocytes. One of the cyclic nucleotide-independent protein kinase catalyzes the phosphorylation of two different factors involved in the initiation of hemoglobin synthesis. A single phosphoprotein phosphatase activity is shown to remove phosphate from 40 S ribosomal subunits. The major acetyltransferase activity associated with ribosomes acetylates a 60 S ribosomal protein.     

Inhibition of lymphocyte proliferation by liver arginase.

The activities of thymidine kinase and uridine kinase (enzymes for pyrimidine salvage pathway) in phytohemagglutinin (PHA)--prestimulated lymphocytes were inhibited by arginase in a similar pattern to the inhibition on thymidine incorporation. Further study revealed that arginase did not directly affect the activities of these enzymes in the cell-free system. Thymidine kinase and uridine kinase activities of PHA-prestimulated lymphocytes were inhibited by arginase making their activities as low as that cultured in arginine-free RPMI-1640 medium. These results suggest that arginine-depletion in the culture medium is the primary mode of action of arginase on the inhibition of mitogen-stimulated lymphocyte proliferation.     

A coordinate relationship between theGALK and theTK1 genes of the Chinese hamster

Chinese hamster cells in culture were treated with various concentrations of thymidine, 5-bromodeoxyuridine, trifluorothymidine, and 2-deoxy-D-galactose. Selection was made for deficiencies in the activities of galactokinase and thymidine kinase. Selection in the presence of thymidine, 5-bromodeoxyuridine, and trifluorothymidine was expected to produce clones deficient in thymidine kinase only, whereas those deficient in galactokinase were expected to be selected in the presence of 2-deoxy-D-galactose. However, it was found that clones growing in the presence of these inhibitors were frequently deficient in both enzymes. Or if a clone was deficient in only one, the deficiency frequently was not expected according to the selection procedure. This indicates some sort of coordinate relationship between the two gene loci, GALK and TK1, which specify galactokinase and thymidine kinase, respectively. GALK and TK1 are linked in all primates and rodents in which linkage determinations have been made. It is therefore probable that this linkage has been conserved for a long period of time. It is suggested that the apparent relationship between the two genes shown by the data presented here, as well as by others, supports the conclusion that linkage has been conserved by natural selection and is therefore not fortuitous.     

Biosynthesis and scavenging of pyrimidines by pathogenic mycobacteria

Mycobacterium microti incorporated a wide range of exogenously supplied pyrimidines into its nucleic acids. M. avium incorporated a relatively narrow range of pyrimidines but both M. avium and M. microti when recovered after growth in vivo incorporated a slightly wider range of pyrimidines than the same strains grown in vitro. M. microti and M. leprae could not take up uridine nucleotides directly but could utilize the pyrimidines by hydrolysing them to uridine and then taking up the uridine. Pyrimidine biosynthesis, judged by the ability to incorporate carbon from CO2 or aspartate into pyrimidines was readily detected in non-growing suspensions of M. microti and M. avium harvested from Dubos medium, which does not contain pyrimidines. The biosynthetic activity was diminished in mycobacteria grown in vivo when there is likely to be a source of pyrimidines which they might use. Relative activities for pyrimidine biosynthesis de novo in M. microti were 100 for cells isolated from Dubos medium, 6 for cells isolated from Dubos medium containing the pyrimidine cytidine and 11 from cells recovered after growth in mice. In contrast, relative activities for a scavenging reaction, uracil incorporation, were 100, 71 and 59, respectively. Three key enzymes in the pathway of pyrimidine biosynthesis de novo were detected in M. microti and M. avium. Two, dihydroorotate synthase and orotate phosphoribosyltransferase appeared to be constitutive in M. microti and M. avium. Aspartate transcarbamoylase activity was higher in these mycobacteria grown in vivo than in Dubos medium but it was repressed in M. microti or M. avium grown in Dubos medium in the presence of 50 microM-pyrimidine. Aspartate transcarbamoylase was strongly inhibited by the feedback inhibitors ATP, CTP and UTP. Enzymes for scavenging pyrimidines were detected at low specific activities in all mycobacteria studied. Activities of phosphoribosyltransferases, enzymes that convert bases directly to nucleotides, were not related to the ability of intact mycobacteria to take up pyrimidine bases while activities of pyrimidine nucleoside kinases were generally related to the ability of intact mycobacteria to take up nucleosides. Phosphoribosyltransferase activity for uracil, cytosine, orotic acid and--in organisms grown in Dubos medium with 50 microM-uridine-thymine, as well as kinases for uridine, deoxyuridine, cytidine and thymidine were detected in M. microti. However, M. avium only contained uracil and orotate phosphoribosyltransferase, uridine, cytidine and thymidine kinase, and additionally deoxyuridine kinase when grown axenically with 50 microM-uracil, reflecting its more limited abilities in pyrimidine scavenging.     

Homogeneous 3'----5'-exonucleases and their multienzyme complexes from the rat liver

Isolation and general properties of 3'-5' exonucleases I and II (EC 3.1.4.26), which are specific to single-stranded DNA, are described. Such enzymes, being components of replication complexes, could correct replication errors. Homogeneous exonucleases I and II consist of a single subunit with molecular mass of 50 and 40 kDa, respectively. These enzymes are located preferentially in the nuclear membrane and chromatin. They form complexes with nuclear DNA polymerases and some other proteins and are not observed practically in a free state. Molecular masses of the complexes amount from 70 to 1.500 kDa. The complexes dissociate as a result of solution hydrophobization and can be reconstituted after the decrease of hydrophobization. The heavy membrane complex form of 3'----5' exonuclease I manifests enzymatic activities of DNA polymerase alpha (EC 2.7.7.7), non-specific nucleoside triphosphatase (EC 3.1.3.2), nucleotidase (EC 3.1.3.31) and faint activity of endonuclease (EC 3.1.4.5). Complexes under study do not display activity of thymidine kinase (EC 2.7.1.21), marker protein of replitase, neither in G0 nor in S-period.     

Thymidine and thymidylate kinases from the scallop Mizuhopecten yessoensis gonads

Thymidine and thymidylate kinases were isolated from the gonads of scallop Mizuhopecten yessoensis. The enzymes were purified 537- and 100-fold, respectively, and were free of phosphatase and ATPase impurities. Ions of bivalent metals and ATP were necessary for both the nucleoside and nucleotide kinase activities; the pH optimum fall into the range of 7.5-8.5. KCl and NaCl at a concentration of up to 100 mM had no inhibiting effect on the activities of these scallop enzymes. Thymidine kinase catalyzed thymidine, and, at a lower rate, deoxycytidine phosphorylations did not utilize ribo- and deoxyribonucleosides, as well as pyrimidine ribonucleosides, as a phosphate acceptor. Thymidylate kinase phosphorylated TMP and dCMP with an efficiency of about 30%. In addition to ATP, these enzymes can also utilize with different efficiencies dATP, dGTP, GTP, UTP, and CTP as a donor of phosphate groups. Thymidine kinase activity was inhibited by TMP, TTP, and dCTP.     

Colon tumor: enzymology of the neoplastic program

The biochemical strategy of colon tumor was investigated by comparing the enzymic programs of glycolysis, pentose phosphate production and purine and pyrimidine biosynthesis and degradation in liver, normal colon mucosa and transplantable colon adenocarcinoma in the mouse. In normal colon mucosa the carbohydrate and pentose phosphate enzymes were 2- to 9-fold higher in specific activity than those in liver. Among the enzymes of CTP synthesis, CTP synthetase was the rate-limiting one in both liver and colon. In colon tumor CTP synthetase, OMP decarboxylase, uracil phosphoribosyltransferase and thymidine kinase activities increased to 927, 863, 597 and 514% of activities of normal colon. In contrast, the activity of the catabolic enzymes, dihydrothymine dehydrogenase and uridine phosphorylase, decreased to 51 and 25%. The ratios of activities of uridine kinase/uridine phosphorylase and thymidine kinase/dihydrothymine dehydrogenase were elevated 6- and 10-fold. The activity of the key purine synthetic enzyme, glutamine PRPP amidotransferase, increased 7-fold and the opposing rate-limiting enzyme of purine catabolism, xanthine oxidase, decreased to 7%. The ratio of amidotransferase/xanthine oxidase was elevated to 8, 150%. Activities of glucose-6-phosphate dehydrogenase and transaldolase did not increase, but that of pyruvate kinase was elevated to 154%. Similar enzymic programs were observed in a transplantable adenocarcinoma of the colon in the rat. The alterations in gene expression in colon tumor manifested in an integrated pattern of enzymic imbalance indicate the display of a program, a segment of which is shared with rat and human liver and kidney tumors. These alterations in gene expression should confer selective advantages to colon tumor cells. The striking increases in the activities of CTP synthetase, OMP decarboxylase, glutamine PRPP amidotransferase and thymidine kinase mark out these enzymes as potentially sensitive targets for combination chemotherapy by specific inhibitors of these enzyme activities.