Changes in the de novo, salvage, and degradation pathways of pyrimidine nucleotides during tobacco shoot organogenesis.

Pyrimidine nucleotide metabolism was studied in tobacco callus cultured for 21days under shoot-forming (SF) and non-shoot-forming (NSF) conditions by following the metabolic fate of orotic acid, a precursor of the de novo pathway, and uridine and uracil, intermediates of the salvage and degradation pathways respectively. Nucleic acid synthesis was also investigated by measuring the incorporation of labeled thymidine into different cellular components. Our results indicate that with respect to nucleotide metabolism, the organogenic process in tobacco can be divided in two "metabolic phases": a de novo phase followed by a salvage phase. The initial stages of meristemoid formation during tobacco organogenesis (up to day 8) are characterized by a heavy utilization of orotic acid into nucleotides and nucleic acids. Utilization of this intermediate for the de novo synthesis of nucleotides, which is limited in NSF tissue, is mainly due to the activity of orotate phosphoribosyltransferase (OPRT), which increases in tissue cultured under SF conditions. After day 8, nucleotide synthesis during shoot growth seems to be mainly due to the salvage activity of both uridine and uracil. Both intermediates are preferentially utilized in SF tissue for the formation of nucleotides and nucleic acids through the activities of their respective salvage enzymes: uridine kinase (URK), and uracil phosphoribosyltransferase (UPRT). Metabolic studies on thymidine indicate that in SF tissue maximal nucleic acid synthesis occurs at day 4, in support of the initiation of meristemoid formation. Overall these results suggest that the organogenic process in tobacco is underlined by precise fluctuations in pyrimidine metabolism which delineate structural events culminating in shoot formation.     

Modulation of in vitro thymidine incorporation into crypt cells from the murine small intestine

A method is described for the isolation of enriched populations of crypt cells from the murine small intestine. The method was developed to study the response of cells to various stimuli in vitro. The properties of the isolated cell preparations varied with the state of the intestinal mucosa of the mice from which they were isolated. Thus we could distinguish between cells from lactating and non-lactating mice. Polyamines, which are putative modulators of crypt cell division, failed to stimulate [3H]TdR incorporation in vitro. Lymphocyte culture supernatants suppressed [3H]TdR incorporation at dilutions of 1:4 to 1:64. Supernatants of 12-O-tetradecanoylphorbol-13-acetate-stimulated EL-4 cells and of mixed lymphocyte cultures failed to stimulate [3H]TdR incorporation of any dilution. Supernatants of concanavalin A-stimulated spleen cells gave less suppression of [3H]TdR incorporation than those of unstimulated spleen cells and stimulated incorporation at dilutions of 1:64 and 1:128. Phytohaemagglutinin stimulated [3H]TdR incorporation at high concentrations, whereas concanavalin A (con A) had no effect. This study shows that the isolated murine crypt cells may have the potential to provide a useful in vitro model for crypt cell responses to stimuli.     

Purine synthesis de novo and salvage in hypoxanthine phosphoribosyltransferase-deficient mice

Extreme degrees of hypoxanthine phosphoribosyltransferase (HPRT) deficiency in man are associated with gross sex-linked neurological dysfunction, gout and urinary stones (the Lesch-Nyhan or 'complete HPRT-deficiency' syndrome). The less severe degrees of enzyme deficiency (sex-linked recessive gout and/or urolithiasis or the 'partial HPRT-deficiency' syndrome) may be associated with minor neurological manifestations. Whole body purine synthesis de novo is accelerated in both these groups of patients. A strain of mice with an experimentally produced mutation at the HPRT locus showed some residual 'apparent HPRT activity' in brain, liver, testicular, splenic, kidney and ovarian tissues but not in erythrocyte haemolysates. The mutation removes exons 1 and 2 of the coding region of the gene together with the promotor and about 10 kb of upstream sequence from the gene. It is therefore possible that the observed 'apparent HPRT activity' in these mice is due to the operation of an alternative metabolic pathway. Purine synthesis de novo was markedly accelerated in their brain, testicular, splenic and kidney tissues. It was not accelerated in the liver tissue of male mice hemizygous for the mutation and the degree of acceleration in the female homozygotes only just reached statistical significance at the p = 0.02 level. This observation casts doubt on the importance of modulations in the rate of hepatic purine synthesis de novo as a mechanism for maintaining a steady supply of purines for translocation to other organs.     

Deficient BH4 production via de novo and salvage pathways regulates NO responses to cytokines in adult cardiac myocytes

Adult rat cardiac myocytes typically display a phenotypic response to cytokines manifested by low or no increases in nitric oxide (NO) production via inducible NO synthase (iNOS) that distinguishes them from other cell types. To better characterize this response, we examined the expression of tetrahydrobiopterin (BH4)-synthesizing and arginine-utilizing genes in cytokine-stimulated adult cardiac myocytes. Intracellular BH4 and 7,8-dihydrobiopterin (BH2) and NO production were quantified. Cytokines induced GTP cyclohydrolase and its feedback regulatory protein but with deficient levels of BH4 synthesis. Despite the induction of iNOS protein, cytokine-stimulated adult cardiac myocytes produced little or no increase in NO versus unstimulated cells. Western blot analysis under nonreducing conditions revealed the presence of iNOS monomers. Supplementation with sepiapterin (a precursor of BH4) increased BH4 as well as BH2, but this did not enhance NO levels or eliminate iNOS monomers. Similar findings were confirmed in vivo after treatment of rat cardiac allograft recipients with sepiapterin. It was found that expression of dihydrofolate reductase, required for full activity of the salvage pathway, was not detected in adult cardiac myocytes. Thus, adult cardiac myocytes have a limited capacity to synthesize BH4 after cytokine stimulation. The mechanisms involve posttranslational factors impairing de novo and salvage pathways. These conditions are unable to support active iNOS protein dimers necessary for NO production. These findings raise significant new questions about the prevailing understanding of how cytokines, via iNOS, cause cardiac dysfunction and injury in vivo during cardiac inflammatory disease states since cardiac myocytes are not a major source of high NO production.     

De novo synthesis of uracil nucleotides in mouse liver and intestine studied using [15N]alanine

The amount of newly synthesized uracil nucleotides in mouse liver and intestine was determined by analysis of 15N incorporation into the uracil nucleotide pool of these tissues after intraperitoneal infusion of 15N-labelled amino acids. The appearance of newly synthesized uracil nucleotides was linear with time, and essentially independent of the rate of infusion of L-[15N]alanine. Varying the amino acid used in the infusion could affect the enrichment in the uracil ring nitrogens, but had no significant effect on the calculated amount of de novo synthesis. These results demonstrate the utility of this method in measuring de novo uracil nucleotide synthesis in mouse liver and intestine in vivo. The method should be a valuable tool in the effort to understand the regulation and pharmacological manipulation of de novo uracil nucleotide synthesis.     

The de novo and salvage pathways for the synthesis of pyrimidine residues of RNA predominate in different locations within the mouse doudenal epithelium

Summary RNA synthesis was examined by radioautography in mouse doudenal epithelium using ³H-uridine as a tracer of the salvage pathway and ³H-orotic acid as a tracer of the de novo pathway. The incorporation of the two precursors was estimated by counting silver grains in light-microscopic and electron-microscopic radioautographs at successive levels of crypt and villus. With both precursors, silver grains were found over all epithelial nuclei, but in numbers varying by location. Thus, after ³H-uridine injection, the number of grains was high over nucleolus and nucleoplasm in the base of the crypt, declined gradually in the middle and top of the crypt, and was low along the villus. After ³H-orotic acid, the number of grains was fairly low throughout, but peaked over the nucleoplasm in lower villus cells. The ³H-uridine reaction over nucleolus and nucleoplasm in crypt cells was interpreted as synthesis by the salvage pathway of ribosomal RNA and heterogeneous RNA, respectively, whereas the ³H-orotic acid reaction over the nucleoplasm of some villus cells indicated that these cells synthesized heterogeneous RNA by the de novo pathway.     

Enzymes of the de novo and salvage pathways of purine synthesis in renal hypertrophy. Contrasting effects of early diabetes and unilateral nephrectomy

Evidence for differences in the mechanism of renal growth in experimental diabetes and compensatory hypertrophy after unilateral nephrectomy (UN) has been obtained from measurements of the activity of enzymes of the de novo and salvage pathways of purine synthesis in the kidneys of diabetic and UN rats and in doubly lesioned animals. In diabetes, the activity of enzymes of both pathways increased. No effect of UN on the activity of any of these enzymes was observed, nor was the effect of the double lesion greater than the effect of diabetes alone. The activity of the pentose phosphate pathway increased in diabetes but not as a result of UN. Again, the effect of the double lesion was no different from that of diabetes alone. These results indicate that the accretion of nucleic acids in diabetes involves a larger component of de novo synthesis, contrasting with UN, where depressed breakdown may play a significant role.     

Radiation-induced mitotic delay: duration, dose and cell position dependence in the crypts of the small intestine in the mouse

The cells of the proliferative compartment in the crypt of the small intestine undergo a step by step differentiation and/or maturation from stem cells to the functional cells on the villi. The consequent hierarchical organization of the proliferative cell population can be related to the actual position of cells within the crypt. The stem cells are found near the bottom of the crypt with the more mature cells occurring at increasingly higher positions. The sensitivity of proliferative cells in the crypt of small intestine to radiation-induced mitotic delay was investigated at each position within the crypt. Using the stathmokinetic method (vincristine accumulation), the following were noted. The yield of mitotic figures 3 h immediately after irradiation showed a strong cell position dependence with the cells at the base of the crypt being most inhibited and those at the top of the proliferative compartment least affected. The mitotic yields were largely unaffected for the first 15 min suggesting that there is a transition point (Tp) for radiosensitivity which is located about 15 min before metaphase for all crypt cells. Cells located less than 15 min from metaphase are unaffected while those more than 15 min from metaphase are inhibited from further cell cycle progression. After this initial delay all proliferative cells were inhibited in their progression through G2 but some recovered more quickly than others. The ratio of the time of division delay (Td) in stem cells to that in cells at the top of the proliferative compartment was about 3:1. In absolute values Td after 1.0 Gy was about 1 h and 2.8 h, for cells at the top of the crypt and at the base, respectively. After 2.5 Gy the corresponding values were less than 3 h and between 5 and 6 h for the mid-crypt and crypt base respectively. There is thus a dependence on dose for the duration of the mitotic inhibition which for the cells at the top of the crypt is similar to the widely quoted average value 1 h per Gy, but the duration depends strongly on cell position. Thus not all proliferative cells respond in the same way. The duration is shorter the closer the proliferative cells are to their last cell division in the proliferative hierarchy in the crypt and longest for cells situated where the stem cells are to be expected.     

Characterization of isolated villus and crypt cells from the small intestine of the adult mouse

Summary Suspensions of sequentially isolated villus and crypt cells were obtained in order to study certain biochemical changes associated with differentiation of epithelial cells in the small intestine of the mouse. Microscopic observation of the various cell fractions reveals that the epithelial cells detach as individual cells or small sheets of epithelium from the tip to the base of the villus, whereas cells in the crypt regions are separated as entire crypt units. The isolated cells retain their ultrastructural integrity as judged by electron microscopy. Chemical characterization of the various fractions shows that the total cellular protein content, expressed in activity per cell, remains relatively constant throughout the villus region followed by a noticeable drop in the crypt zone. On the other hand, sharp variations in values of cell DNA content are observed in the crypt zone depending on the reference of activity being used. Activity profiles of several brush border enzymes confirm the biochemical changes that occur during the migration of cells from the crypt to the villus tip, as observed in other species, with maximum activity of sucrase in the mid-villus region, of glucoamylase, trehalase, lactase and maltase in the upper third region, and of alkaline phosphatase at the villus tip. Forty-eight-hour suspension cultures of cell fractions corresponding to cells at the base of the villus and crypt zones show a moderate decrease in protein and enzyme activities to approximately 70% of their original value, with DNA content remaining stable throughout the incubation period. The use of biochemical activities as indicators of cellular integrity during cell culture is discussed.Supported by a research grant from the Medical Research Council of Canada (J.H.)     

Immunohistochemical characterization of a crypt cell-specific plasma membrane protein in rat small intestine epithelium using a monoclonal antibody

Proteins of the basolateral membrane (BLM) of small intestine epithelial cells of adult rats, in the MW ranges of 50-65 KD, 85-100 KD, and over 100 KD, were obtained as follows. After isolation of the BLM and subsequent SDS-PAGE and transblotting of the proteins on nitrocellulose sheets, the bands in these MW ranges were cut out of the nitrocellulose sheet and extracted. Balb/C mice were immunized with these protein fractions and a monoclonal antibody (MAb) was then produced. MAb SI/CC1 obtained via immunization with the 50-65 KD protein fraction shows specificity for the crypt epithelium of the small intestine. It can be used to characterize, by light and electron microscopic immunohistochemical methods, a crypt cell protein (SI/CC1-Ag) with a very specific localization. Fluorescence labeling shows that the SI/CC1-Ag can be found only in the epithelium of small intestine crypts (except for the granules in eosinophilic granulocytes). The epithelium of the colon, as well as the epithelia of other organs, could not be labeled. In the small intestine crypts, SI/CC1-Ag is found only in the Paneth cells located in the basal crypt section, and in the undifferentiated cells in the middle crypt section; it is lacking in the cells of the upper crypt section. Gold labeling shows that SI/CC1-Ag in the undifferentiated cells is localized exclusively in the basolateral PM domain. On the Paneth cells, the content of the secretory granules is labeled, along with the basolateral PM domain; the labeling sometimes present on their luminal part is probably due to passively absorbed secretion from these cells. The SI/CC1-Ag in the BLM of undifferentiated and Paneth cells is found only on Days 21-23 post partum, whereas the Paneth cell granules could be labeled as early as the Day 16 post partum. With immunodetection with SI/CC1, one band at about 55 KD is specifically labeled in the protein pattern of the isolated small intestine cell BLM. In the protein pattern of the isolated crypt cells two bands were labeled, again one at 55 KD and one at about 120 KD. These findings indicate that SI/CC1-Ag is a 55 KD protein that appears on Days 21-23 post partum in the BLM of undifferentiated cells and of Paneth cells.     

Functional Cftr in crypt epithelium of organotypic enteroid cultures from murine small intestine

Physiological studies of intact crypt epithelium have been limited by problems of accessibility in vivo and dedifferentiation in standard primary culture. Investigations of murine intestinal stem cells have recently yielded a primary intestinal culture in three-dimensional gel suspension that recapitulates crypt structure and epithelial differentiation (Sato T, Vries RG, Snippert HJ, van de Wetering M, Barker N, Stange DE, Van Es JH, Abo A, Kujala P, Peters PJ, Clevers H. Nature 459: 262-265, 2009). We investigated the utility of murine intestinal crypt cultures (termed "enteroids") for physiological studies of crypt epithelium by focusing on the transport activity of the cystic fibrosis transmembrane conductance regulator Cftr. Enteroids had multiple crypts with well-differentiated goblet and Paneth cells that degranulated on exposure to the muscarinic agonist carbachol. Modified growth medium provided a crypt proliferation rate, as measured by 5-ethynyl-2'-deoxyuridine labeling, which was similar to proliferation in vivo. Immunoblots demonstrated equivalent Cftr expression in comparisons of freshly isolated crypts with primary and passage 1 enteroids. Apparent enteroid differences in mRNA expression of other transporters were primarily associated with villous epithelial contamination of freshly isolated crypts. Microelectrode analysis revealed cAMP-stimulated membrane depolarization in enteroid epithelium from wild-type (WT) but not Cftr knockout (KO) mice. Morphological and microfluorimetric studies, respectively, demonstrated Cftr-dependent cell shrinkage and lower intracellular pH in WT enteroid epithelium in contrast to Cftr KO epithelium or WT epithelium treated with Cftr inhibitor 172. We conclude that crypt epithelium of murine enteroids exhibit Cftr expression and activity that recapitulates crypt epithelium in vivo. Enteroids provide a primary culture model that is suitable for physiological studies of regenerating crypt epithelium.     

Rising from the crypt: decreasing DNA methylation during differentiation of the small intestine

The differentiation of intestinal stem cells involves few DNA methylation changes, assayed by bisulfite sequencing, in contrast to other adult somatic stem cell hierarchies.Please see related Research article: http://genomebiology.com/2013/14/5/R50     

Regulation of purine synthesis de novo in human fibroblasts by purine nucleotides and phosphoribosylpyrophosphate

Previous studies of purine nucleotide synthesis de novo have suggested that major regulation of the rate of the pathway is affected at either the phosphoribosylpyrophosphate (PP-Rib-P) synthetase reaction or the amidophosphoribosyltransferase (amido PRT) reaction, or both. We studied control of purine synthesis de novo in cultured normal, hypoxanthine-guanine phosphoribosyltransferase (HGPRT)-deficient, and PP-Rib-P synthetase-superactive human fibroblasts by measuring concentrations and rates of synthesis of PP-Rib-P and purine nucleotide end products, proposed effectors of regulation, during inhibition of the pathway. Incubation of cells for 90 min with 0.1 mM azaserine, a glutamine antagonist which specifically blocked the pathway at the level of conversion of formylglycinamide ribotide, resulted in a 5-16% decrease in purine nucleoside triphosphate concentrations but no consistent alteration in generation of PP-Rib-P. During this treatment, however, rates of the early steps of the pathway were increased slightly (9-15%) in normal and HGPRT-deficient strains, more markedly (32-60%) in cells with catalytically superactive PP-Rib-P synthetases, and not at all in fibroblasts with purine nucleotide feedback-resistant PP-Rib-P synthetases. In contrast, glutamine deprivation, which inhibited the pathway at the amido PRT reaction, resulted in time-dependent nucleoside triphosphate pool depletion (26-43% decrease at 24 h) accompanied by increased rates of PP-Rib-P generation and, upon readdition of glutamine, substantial increments in rates of purine synthesis de novo. Enhanced PP-Rib-P generation during glutamine deprivation was greatest in cells with regulatory defects in PP-Rib-P synthetase (2-fold), but purine synthesis in these cells was stimulated only 1.4-fold control rates by glutamine readdition. Stimulation of these processes in normal and HGPRT-deficient cells and in cells with PP-Rib-P synthetase catalytic defects was, respectively: 1.5 and 2.0-fold; 1.5 and 1.7-fold; and 1.6 and 4.1-fold. These studies support the following concepts. 1) Rates of purine synthesis de novo are regulated at both the PP-Rib-P synthetase and amido PRT reactions by end products, with the latter reaction more sensitive to small changes in purine nucleotide inhibitor concentrations. 2) PP-Rib-P exerts its role as a major regulator of purine synthetic rate by virtue of its interaction with nucleotide inhibitors to determine the activity of amido PRT. 3) Activation of amido PRT by PP-Rib-P is nearly maximal at base line in fibroblasts with regulatory defects in PP-Rib-P synthetase.     

Cell migration and organization in the intestinal crypt using a lattice-free model

We present a novel class of spatial models of cell movement and arrangement applied to the two-dimensional cellular organization of the intestinal crypt. The model differs from earlier approaches in using a dynamic movement on a lattice-free cylindrical surface. Cell movement is a consequence of mitotic activity. Cells interact by viscoelastic forces. Voronoi tessellation permits simulations of individual cell boundaries. Simulations can be compared with experimental data obtained from cell scoring in sections. Simulation studies show that the model is consistent with the experimental results for the spatial distribution of labelling indices, mitotic indices and other observed phenomena using a fixed number of stem cells and a fixed number of transit cell divisions.