Effects of spinal transection on presynaptic markers for glutamatergic neurons in the rat

To evaluate the hypothesis that glutamic acid may be the neurotransmitter of descending, excitatory supraspinal pathways, the uptake and release ofl-[³H] glutamate and the levels of endogenous glutamate were measured in preparations from rat lumbar spinal cord following complete mid-thoracic transection. Following transection, the activity of the synaptosomal high-affinty glutamate uptake process was increased in both dorsal and ventral halves of lumbar cord between 1 and 14 days after transection and returned to control levels by 21 days posttransection. At 7 days, the increased activity of the uptake process forl-[³H] glutamate resulted in elevation ofV _max with no significant alteration inK _t as compared to age-matched controls. Depolarization-induced release ofl-[³H]glutamate from prelabeled slices did not differ significantly from control in the lesioned rat except at 21 days after lesion when the amount of tritium release was significantly greater in the transected preparations than in control. Amino acid analysis of the lumbar cord from control and transected rats indicated only a 10% decrease in the level of endogenous glutamate and no alterations in the concentration of GABA and glycine 7 days after lesion. These findings do not support the hypothesis that glutamate serves as a major excitatory neurotransmitter in supraspinal pathways innervating the lumbar cord of the rat.     

The effect of 5-fluorouracil on the mitotic cycle and DNA synthesis in the epithelium of mouse small intestine.

The examinations were performed on 42 mice of the Porton strain. The experimental animals were injected intraperitoneally with the dose of 75 mg of 5-fluorouracil per kg body weight. The first experimental group received injections of [3H]thymidine within 48 hours and the second group within 96 hours of the injection of 5-fluorouracil. Two mice from each group were killed at within 1, 2, 4, 8, 12, 24, and 48 hours of the [3H]thymidine injection. Calculations of the mitotic index and time of duration of individual phases of the mitotic cycle in epithelial cells of the small intestine were based on application of the autoradiographic method. These studies lead to the conclusion that 5-fluorouracil disturbs the course of metabolic processes in the cell, which are also related with the distribution of the genetic material. Histological examinations show that 5-fluorouracil produces profound morphological changes in the intestine, which affect both the intestinal epithelium and the connective tissue stroma. The autoradiographic tests revealed a considerable suppression of the mitotic activity of the epithelium of intestinal crypts. Moreover, it was shown that 5-fluorouracil inhibits the mitotic activity of the intestinal epithelium by diminishing the number of cells capable of entering into mitosis. Nevertheless, by 96 hours following introduction of a single dose of 5-fluorouracil normal morphological structure and mitotic activity of the intestinal wall cells are restored.     

Solute Transport Process in Intestinal Epithelial Cells

In rat small intestine, the active transport of organic solutes results in significant depolarization of the membrane potential measured in an epithelial cell with respect to a grounded mucosal solution and in an increase in the transepithelial potential difference. According to the analysis with an equivalent circuit model for the epithelium, the changes in emf's of mucosal and serosal membranes induced by active solute transport were calculated using the measured conductive parameters. The result indicates that the mucosal cell membrane depolarizes while the serosal cell membrane remarkably hyperpolarizes on the active solute transport. Corresponding results are derived from the calculations of emf's in a variety of intestines, using the data that have hitherto been reported. The hyperpolarization of serosal membrane induced by the active solute transport might be ascribed to activation of the serosal electrogenic sodium pump. In an attempt to determine the causative factors in mucosal membrane depolarization during active solute transport, cell water contents and ion concentrations were measured. The cell water content remarkably increased and, at the same time, intracellular monovalent ion concentrations significantly decreased with glucose transport. Net gain of glucose within the cell was estimated from the restraint of osmotic balance between intracellular and extracellular fluids. In contrast to the apparent decreases in intracellular Na⁺ and K⁺ concentrations, significant gains of Na⁺ and K⁺ occurred with glucose transport. The quantitative relationships among net gains of Na⁺, K⁺ and glucose during active glucose transport suggest that the coupling ratio between glucose and Na⁺ entry by the carrier mechanism on the mucosal membrane is approximately 1:1 and the coupling ratio between Na⁺-efflux and K⁺-influx of the serosal electrogenic sodium pump is approximately 4:3 in rat small intestine. In addition to the electrogenic ternary complex inflow across the mucosal cell membrane, the decreases in intracellular monovalent ion concentrations, the temporary formation of an osmotic pressure gradient across the cell membrane and the streaming potential induced by water inflow through negatively charged pores of the cell membrane in the course of an active solute transport in intestinal epithelial cells are apparently all possible causes of mucosal membrane depolarization.     

COMPARISON OF DNA RENEWAL IN GERM-FREE AND CONVENTIONAL MICE USING [125I]IODODEOXYURIDINE AND [3H]THYMIDINE

Germ-free (GF) and conventional (CV) C3H mice received a single injection of 1 μCi [³H]thymidine and 3 μCi [¹²⁵I]iododeoxyuridine to provide simultaneous labeling of DNA with the two precursors. Thymus, spleen, mesenteric lymph nodes, bone marrow (femora), small intestine, colon and skin were examined for total organ activity and rate of DNA renewal 1–8 days after injection. Precursor incorporation, assayed on day 1, was lower in the thymus, mesenteric lymph nodes and femora (and, to a lesser extent, in the spleen and colon) of GF mice as compared to CV animals. The opposite was observed in the small intestine and skin, i.e. total organ activity was higher in GF animals. Differences in precursor incorporation were partly due to differences in organ weights between the two groups of mice. In comparison to CV animals, DNA renewal rates were diminished in the mesenteric lymph nodes, bone marrow, colon (following a 3-day plateau) and spleen of GF mice. Little, if any, difference was observed between the two groups with respect to the rate of DNA turnover in the thymus and skin. Radioactivity of the small intestine remained constant for 2 days. Thereafter intestinal activity in GF mice declined at an initial slow rate between days 2 and 5 followed by a rapid decrease between days 5 and 8. In CV mice the first phase of activity loss was short with the rapid decline in intestinal activity beginning on day 3. From the slopes of the regression lines, the percentage thymidine reutilization was estimated. Reutilization varied from 0 to 63% in the various organs examined, with the greatest difference between GF and CV mice occurring in the mesenteric lymph nodes.     

RNA synthesis by villus and crypt cell nuclei of rat intestinal mucosa

Rat intestinal mucosa was separated by eversion and vibration to provide a sequence of fractions from predominantly villus cells to predominantly crypt cells. The proportions of these cell types in each fraction were computed from the concentrations of alkaline phosphatase (villus cells) and thymidine kinase (crypt cells) in each population. The isolated mucosal fractions varied from about 90% villus cells to 90% crypt cells. Following injection of the rats with [³H]thymidine, the nuclei were isolated from each mucosal cell fraction and the amount of radioactivity incorporated into DNA was measured as an index of crypt cell abundance. The isolated nuclei were also incubated with ribonucleoside triphosphates and the amount of RNA synthesized was measured. Nuclei labeled with [³H]thymidine were found only in fractions rich in crypt cells, whereas capacity for RNA synthesis remained very active in mucosal fractions consisting predominantly of villus cells. It is concluded that non-dividing villus cells continue to make RNA.     

Rapid Anterograde Spread of Premitotic Activity Along Degenerating Cat Sciatic Nerve

Peripheral nerve transection triggers a series of phenotypic alterations in Schwann cells distal to the site of injury. Mitosis is one of the earliest and best characterized of these responses, although the mechanism by which axonal damage triggers this critical event is unknown. This study examines the appearance and spatio-temporal spread of premitotic activity in distal stumps of transected cat tibial nerves. Premitotic activity was determined by measuring incorporation of [3H]thymidine (a marker of DNA synthesis during the S-phase of the cell cycle) into consecutive segments of desheathed tibial nerve. Incorporation of [3H]thymidine spread proximo-distally within distal nerve stumps between 3 and 4 days posttransection with an apparent velocity of at least 199 +/- 67 mm/day. This suggests that anterograde fast axonal transport may directly or indirectly be associated with the Schwann cell mitotic response to axon transection.     

Incorporation of [3H]thymidine into DNA and of [35S]sulfate into sulfatides of oligodendroglial cells during development: Effect of malnutrition

Incorporation of [³H]thymidine into DNA and of [³⁵S]sulfate into sulfatides of oligodendroglial cells isolated from brain slices incubated with the radioactive precursor was studied in normal and malnourished rats at different ages. The pattern and the values of incorporation of [³H]thymidine into DNA were similar in both groups of animals. The maximum value of incorporation was observed at 7 days of age decreasing rapidly thereafter and leveling off between 18–21 days. In both groups of animals labeling of sulfatides attained a maximum at 18 days of age, showing similar values of incorporation up to that age. However, at 21 days of age; the values corresponding to malnourished rats were found to be 40% lower in comparison to controls. The results suggest that (a) proliferation of oligodendroglial cells stops at similar ages in normal and malnourished rats, (b) expression of sulfatide synthesis by oligodendroglial cells is similar in both groups of animals up to 18 days, and (c) the starved rats seem to be unable to maintain normal synthesis of these galactolipids throughout the entire period of active myelinogenesis.     

Steroidal and growth factor regulation of [3H]thymidine incorporation by cultured endosalpingeal cells of the bovine oviduct

Summary Cultured cells from the bovine endosalpinx were used to evaluate effects of estradiol-17β, progesterone, epidermal growth factor, and insulinlike growth factors I and II on [³H]thymidine incorporation. Cells were treated with hormones and growth factors when approximately 50% confluent. After 24 h, DNA synthesis was quantified by pulsing cells with [³H]thymidine for 12 h and determining uptake into DNA. Cells prepared by mechanical dispersal incorporated more [³H]thymidine than cells dispersed with collagenase. However, hormonal responses were the same for both types of cells. As compared to plastic, cells on a Matrigel substratum exhibited lower incorporation of [³H]thymidine and were unresponsive to hormones. Estradiol-17β increased [³H]thymidine incorporation slightly at 10⁻¹⁰ mol/liter and higher. Epidermal growth factor, insulinlike growth factor-I, and insulinlike growth factor-II also stimulated [³H]thymidine incorporation. Effects of insulinlike growth factor-I were greater for cells treated with estradiol-17β. In the absence of estradiol, progesterone inhibited [³H]thymidine incorporation at 1, 10, and 100 ng/ml. When estradiol-17β was present, progesterone stimulated [³H]thymidine incorporation at 1 ng/ml and reduced incorporation at 100 ng/ml. In conclusion, [³H]thymidine incorporation by cultured oviductal endosalpingeal cells can be regulated by ovarian steroids and growth factors. These molecules may represent signals through which the ovary, embryo, and oviduct regulate oviductal growth. Work conducted while on a sabbatical leave supported by the Deutsche Forschungsgemeinschaft.     

Adipose triglyceride lipase is a TG hydrolase of the small intestine and regulates intestinal PPARα signaling

Adipose triglyceride lipase (ATGL) is the rate-limiting enzyme mediating triglyceride (TG) hydrolysis. The lack of ATGL results in TG accumulation in multiple tissues, underscoring the critical role of ATGL in maintaining lipid homeostasis. Recent evidence suggests that ATGL affects TG metabolism via activation of peroxisome proliferator-activated receptor α (PPARα). To investigate specific effects of intestinal ATGL on lipid metabolism we generated mice lacking ATGL exclusively in the intestine (ATGLiKO). We found decreased TG hydrolase activity and increased intracellular TG content in ATGLiKO small intestines. Intragastric administration of [³H]trioleate resulted in the accumulation of radioactive TG in the intestine, whereas absorption into the systemic circulation was unchanged. Intraperitoneally injected [³H]oleate also accumulated within TG in ATGLiKO intestines, indicating that ATGL mobilizes fatty acids from the systemic circulation absorbed by the basolateral side from the blood. Down-regulation of PPARα target genes suggested modulation of cholesterol absorption by intestinal ATGL. Accordingly, ATGL deficiency in the intestine resulted in delayed cholesterol absorption. Importantly, this study provides evidence that ATGL has no impact on intestinal TG absorption but hydrolyzes TGs taken up from the intestinal lumen and systemic circulation. Our data support the role of ATGL in modulating PPARα-dependent processes also in the small intestine.     

Synergistic effect of amino acids on production of lymphocyte proliferation-suppressing substances by rat intestinal epithelial cells

Conditioned culture medium of rat small intestinal epithelial cells suppressed proliferation of spleen lymphocytes stimulated with concanavalin A (approx. 10% of its control [³H]thymidine incorporation) whereas conditioned phosphate-buffered saline of the epithelial cells did not. On the other hand, conditioned saline of the epithelial cells exposed to a mixture of total 22 amino acids at their concentrations in the culture medium suppressed the proliferation (approx. 45% of its control [³H]thymidine incorporation). Neither conditioned saline of the epithelial cells exposed to other medium components nor lysates of freshly harvested epithelial cells suppressed the proliferation. Thus, amino acids synergistically stimulated intestinal epithelial cells to produce substances with the ability to suppress lymphocyte proliferation.     

Nucleic Acid Precursor Incorporation by Eimeria nieschulzi (Protozoa: Apicomplexa) and Jejunal Villus Epithelium*

Autoradiography was used to investigate incorporation of tritiated adenine, adenosine, guanosine and thymidine by Eimeria nieschulzi and rat jejunal villus epithelial cells. At 2 1/2 days postinoculation, parasitized and control tissues were incubated for 20 min in oxygenated Tyrode's solution (37 C, pH 7.5) containing 30 μCi/ml of each nucleic acid precursor. Treatment of tissues with ribonuclease revealed that E. nieschulzi incorporated label from [³H]adenine primarily into RNA while that from [³H]adenosine and [³H]guanosine was present mainly in DNA. Label from [³H]thymidine was not utilized by parasites. Host villus epithelial cells incorporated label from [³H]purines primarily into RNA. Labeled cytoplasmic RNA was significantly increased in parasitized cells after incubation in [³H]adenine. Tritiated nuclear RNA and cytoplasmic RNA were significantly decreased in parasitized cells after incubation in [³H]adenosine. Incorporation of label from [³H]guanosine was similar for parasitized and control cells. A small quantity of label from each [³H]precursor was incorporated into DNA of villus epithelial cell nuclei.     

Biosynthesis and transport of glycoproteins in the small intestinal epithelium of rats: I. Developmental change and effect of hypophysectomy

The biosynthesis and intracellular transport of glycoproteins in duodenal absorptive cells of intact rats at 6 and 24 days and hypophysectomized rats at 24 days of age were studied after 20 min intralumenal pulse-labeling of d-[³H]galactose, l-[³H]fucose, or d-[³H]mannose. Autoradiographic studies showed that the incorporation of sugars increased significantly in intact rats between 6 and 24 days. When rats were hypophysectomized at 6 days of age, the intestinal epithelium at 24 days incorporated d-[³H]galactose at a level significantly lower than that of intact rats at 24 days. Hypophysectomy also interfered with the developmental increase in d-[³H]mannose, but not in l-[³H]fucose, incorporation. Biochemical study indicated that the radioactivity in the lipid-free acid-precipitable glycoproteins in the intestine of 24-day-old intact rats at 20 min after d-[³H]galactose injection was 129% and 97% higher than that in 6-day-old rats and in 24-day-old hypophysectomized rats, respectively. The patterns of intracellular transport of newly synthesized galactosylated or fucosylated glycoproteins in all animal groups were similar; the labeled glycoproteins were initially present in the Golgi and were transported through the smooth endoplasmic reticulum to either the lateral membrane or the brush-border membrane within 60 min after the injection of labeled sugars. The proportion of labeled glycoproteins that migrated to the brush-border membrane, however, increased about twofold in the intact rats between 6 and 24 days of age at 60–240 min after d-[³H]galactose injection. Hypophysectomy interfered with developmental increase in the transport of glycoproteins from the apical cytoplasm to the brush-border membrane. It was concluded that the incorporation of monosaccharide precursors into glycoproteins and the porportion of newly synthesized galactosylated or fucosylated glycoproteins transported to the brush-border membrane increase during postnatal development. The developmental changes are regulated, at least partially, by the pituitary gland.     

Cell proliferation and apoptosis in the anterior intestine of an amphibious, euryhaline mudskipper (Periophthalmus modestus)

In order to replace the diffusive loss of water to the surrounding environment, seawater (SW)-acclimated euryhaline fishes have gastrointestinal tracts with higher ion/water flux in concert with greater permeability, and contrast that to freshwater (FW)-acclimated fish. To understand the cellular basis for these differences, we examined cell proliferation and apoptosis in the anterior intestine of mudskipper transferred from one-third SW to FW or to SW for 1 and 7 days, and those kept out of water for 1 day. The intestinal apoptosis (indicated by DNA laddering) increased during seawater acclimation. TUNEL staining detected numerous apoptotic cells over the epithelium of SW-acclimated fish. Cell proliferation ([³H]thymidine incorporation) in the FW fish was greater than those in SW 7 days after transfer. Labeling with a Proliferating cell nuclear antigen (PCNA) antibody indicated that proliferating cells were greater in number and randomly distributed in the epithelium of FW fish, whereas in SW fish they were almost entirely in the troughs of the intestinal folds. There were no changes in cell turnover in fish kept out of water. During acclimation to different salinities, modification of the cell turnover and abundance may play an important role in regulating the permeability (and transport capacity) of the gastrointestinal tract of fish.     

Transdifferentiation Potential of Ciliary and Pigment Epithelial Cells in Lower Vertebrates and Mammals

Cellular composition of the peripheral region of the eye in amphibians and mammals as well as embryonic fissure in amphibians was studied. Different distributions of proliferating cells in retinal pigment epithelium have been revealed in adult amphibians (newt, axolotl, and Xenopus). Single cells incorporated [³H]thymidine in the newt and Xenopus; 0.4% cells, in the axolotl. An embryonic fissure was observed in the eye of the axolotl. Pigment epithelial cells in the embryonic palpebral region actively proliferated: about 20% cells incorporated [³H]thymidine. Proliferating cells were also localized in the ciliary marginal zone of the retina in all studied amphibians, particularly, in the axolotl. In newborn hamsters, [³H]thymidine-labeled cells have been revealed in the pigment epithelium as well as in the outer pigmented and inner unpigmented layers of the ciliary body. Proliferative activity of the peripheral regions of the eye is due to eye growth in adult amphibians and newborn hamsters. After retinectomy, the retina is regenerated from the cells of the growth ciliary marginal zone in all amphibians, pigment epithelial cells in the newt, and pigment epithelial cells of the embryonic fissure in the axolotl. Heterogeneous composition of the pigment epithelium in the newt and axolotl reflects high transdifferentiation potential of these regions. Structural comparison of the peripheral region of the eye in amphibians and mammals demonstrate that the ciliary body of mammals containing stem cells is homologous to the ciliary marginal zone of amphibians containing multipotent cells.     

Nucleic acid synthesis in proliferating peroxisomes of rat liver as revealed by electron microscopical radioautography

Summary Thirty albino rats were fed with a diet containing 1, 2 or 4% di-(2-ethylhexyl)-phthalate (DEHP), a peroxisome-proliferating agent. Others were fed with normal diet as controls. Both groups were sacrificed at varying intervals from 3 days to 4 weeks. The livers were either removed and fixed in glutaraldehyde and osmium tetroxide or fixed in glutaraldehyde, incubated in a diaminobenzidine (DAB) medium, postfixed, embedded in Epon, and sectioned. Other tissues were incubated in Eaglés MEM containing either [³H]thymidine or [³H]uridine, fixed, embedded in Epon, sectioned, and radioautographed. Specimens were observed in a Hitachi H-700 electron microscope.The number of peroxisomes showing DAB reactivity increased in DEHP-fed animals as compared with normal controls In radioautograms of normal rats labelled with [³H]thymidine, no silver grains were, observed, whereas grains were observed over some nuclei, mitochondria and peroxisomes of DEHP-fed animals. In contrast, radioautograms of tissue labelled with [³H]uridine revealed a few grains in nuclei and mitochondria or endoplasmic reticulum of normal rats, although grains appeared in nuclei, mitochondria, endoplasmic reticulum and peroxisomes of DEHP-fed animals more frequently.From these results, it is concluded that [³H]thymidine and [³H]uridine were incorporated in the proliferating peroxisomes, suggesting that nucleic acid synthesis had taken place.     

EFFECTS OF INTESTINALLY ABSORBED THYMIDINE ON TRITIATED THYMIDINE UTILIZATION

Experimental evidence presented suggests that [³H]TdR can be rapidly and efficiently transported from the intestine to the systemic circulation. This pathway for thymidine transport may be physiologically important since administration of cold thymidine in the drinking water enhances the utilization of a parenterally injected dose of [³H]TdR in several body tissues of the mouse.     

Heart Mitochondrial TTP Synthesis and the Compartmentalization of TMP

The primary pathway of TTP synthesis in the heart requires thymidine salvage by mitochondrial thymidine kinase 2 (TK2). However, the compartmentalization of this pathway and the transport of thymidine nucleotides are not well understood. We investigated the metabolism of [³H]thymidine or [³H]TMP as precursors of [³H]TTP in isolated intact or broken mitochondria from the rat heart. The results demonstrated that [³H]thymidine was readily metabolized by the mitochondrial salvage enzymes to TTP in intact mitochondria. The equivalent addition of [³H]TMP produced far less [³H]TTP than the amount observed with [³H]thymidine as the precursor. Using zidovudine to inhibit TK2, the synthesis of [³H]TTP from [³H]TMP was effectively blocked, demonstrating that synthesis of [³H]TTP from [³H]TMP arose solely from the dephosphorysynthase pathway that includes deoxyuridine triphosphatelation of [³H]TMP to [³H]thymidine. To determine the role of the membrane in TMP metabolism, mitochondrial membranes were disrupted by freezing and thawing. In broken mitochondria, [³H]thymidine was readily converted to [³H]TMP, but further phosphorylation was prevented even though the energy charge was well maintained by addition of oligomycin A, phosphocreatine, and creatine phosphokinase. The failure to synthesize TTP in broken mitochondria was not related to a loss of membrane potential or inhibition of the electron transport chain, as confirmed by addition of carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone and potassium cyanide, respectively, in intact mitochondria. In summary, these data, taken together, suggest that the thymidine salvage pathway is compartmentalized so that TMP kinase prefers TMP synthesized by TK2 over medium TMP and that this is disrupted in broken mitochondria.     

Vitamin absorption in the in vivo intestine of normal and infected (Hymenolepis diminuta: Cestoda) rats

Uptake and serosal transfer of the vitamins thiamine, riboflavin and folic acid have been studied in vivo in normal and parasitized rats infected with Hymenolepis diminuta (Cestoda). Regional differences in intestinal uptake of all three vitamins in both uninfected and parasitized animals were not satistically significant. In the parasitized intestine mucosal uptake and serosal transfer of thiamine were significantly inhibited, with increased mucosal accumulation of the vitamin as luminal thiamine concentration increased. Apparent increased riboflavin mucosal uptake in parasitized animals, was not matched by the reduced serosal transfer, suggesting adsorption of the vitamin in the unstirred aqueous layers. Mucosal uptake of folic acid increased in the parasitized gut; serosal transfer and mucosal accumulation were not affected. These results, indicating vitamin malabsorption associated with infection by H. diminuta, are consistent with the parasite inhibiting mucosal passive transport mechanisms. This conclusion is supported by the changes in net water fluxes associated with vitamin uptake in the parasitized intestine.     

Alterations of the incorporation of [3H]thymidine in cells in culture regulated by nucleoplasmic extract

Porcine skin nucleoplasmic extract (PSNE) was shown to alter the incorporation of [³H]thymidine into DNA of selected porcine, bovine, and human cell populations in culture. PSNE stimulated incorporation of [³H]thymidine into DNA of porcine and bovine dermal cells an average of 300 and 200% of control value, respectively. When porcine and bovine epidermal cells were exposed to PSNE the treatment inhibited [³H]thymidine incorporation into DNA by an average of 48 and 45%, respectively. Similar inhibitions were observed for porcine and bovine kidney, porcine lung, and human KB cells. Thus, the effect of PSNE on the incorporation of [³H]thymidine into DNA of various cultured cells was either stimulatory to dermal cells or inhibitory to a variety of other cell types, including skin epidermal cells. The stimulatory and inhibitory effects of PSNE were abolished by heating PSNE for 5 min in boiling water before its addition to cell cultures. This suggests that macromolecular structure is important in the action of PSNE. This project was supported by a grant from the Research Advisory Board, University of Nevada, Reno, NV.