Interexaminer reliability of eight evaluative dimensions of lumbar segmental abnormality: Part II.
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Electromyography↗
Biomedical subjects
C Kent
Publications and source records attributed to C Kent.
Expression, purification, and characterization of CTP:glycerol-3-phosphate cytidylyltransferase from Bacillus subtilis.
Bacillus subtilis contains the gene for CTP:glycerol-3-phosphate cytidylyltransferase, which is involved in biosynthesis of the major teichoic acid of the B. subtilis cell wall. When this gene was expressed in Escherichia coli under the control of the T7 promoter, the glycerol-3-phosphate cytidylyltransferase accumulated to a level of about 15% of cellular protein. The expressed glycerol-3-phosphate cytidylyltransferase was purified to homogeneity by ion-exchange chromatography, gel filtration, and affinity chromatography on blue Sepharose. Approximately 47 mg of pure enzyme was obtained from a 660-ml culture. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that the subunit molecular weight of the purified enzyme was about 15,000. The molecular weight of the native enzyme was found to be 30,900 by gel filtration analysis, suggesting that the native enzyme is a homodimer. The pH optimum was very broad, from 6.5 to 9.5, and the enzyme was stable at alkaline conditions. A divalent cation, either Co2+, Mg2+, Mn2+, or Fe2+, was required for enzyme activity. Km values for CTP and glycerol 3-phosphate were 3.85 and 3.23 mM, respectively, and the Vmax was 185 units/mg of protein. Initial rate studies and product inhibition patterns indicated that the enzyme catalyzes the reaction by means of a rapid eqilibrium random order mechanism. The availability of large amounts of glycerol-3-phosphate cytidylyltransferase will facilitate enzymological and structural studies on this model cytidylyltransferase.
Amino Acid Sequence↗
Regulation of CTP:phosphocholine cytidylyltransferase in HeLa cells. Effect of oleate on phosphorylation and intracellular localization.
The mechanism by which oleate induced the translocation of CTP:phosphocholine cytidylyltransferase from the soluble to particulate fraction was studied in HeLa cells. Addition of 0.5 mM oleate caused a 10-fold stimulation of [methyl-3H]choline incorporation into phosphatidylcholine. The stimulation of phosphatidylcholine synthesis was correlated with a decrease in phosphocholine and an increase in CDP-choline, indicating that cytidylyltransferase is regulatory under these conditions. The stimulation of cytidylyltransferase activity upon oleate treatment was concomitant with the translocation of this enzyme from the soluble to particulate fraction. Immunoblot analysis revealed the soluble form as multiple slowly migrating bands, and the particulate form as a faster migrating, single band. The slowly migrating form could be converted to the fast migrating form by phosphatase treatment, suggesting that the mobility difference was due to phosphorylation. In vivo 32P labeling showed that the soluble cytidylyltransferase was highly phosphorylated and the particulate form was much less phosphorylated. The kinetics of the translocation showed that the conversion of the majority of cytidylyltransferase from the soluble to the particulate form occurred within about 15 min following addition of oleate and that the translocation showed a good correlation with dephosphorylation. In contrast, the relocation of cytidylyltransferase from the particulate to the soluble fraction after the removal of oleate occurred within 1 min, and extensive phosphorylation was not required for cytidylyltransferase to leave the membrane. The relocated, soluble cytidylyltransferase was phosphorylated at a much slower rate than that at which the enzyme left the membrane. Immunolocalization showed that the cytidylyltransferase translocated to the nuclear envelope instead of the endoplasmic reticulum. These results indicate that activation of cytidylyltransferase by dephosphorylation and translocation to the nuclear envelope may be a general mechanism occurring in many cell types. Moreover, in HeLa cells, multiple mechanism exist for regulating cytidylyltransferase activity.
Biological Transport↗
Nuclear localization of soluble CTP:phosphocholine cytidylyltransferase.
The soluble form of CTP:phosphocholine cytidylyltransferase, which has previously been assumed to be cytosolic, has been localized to the nucleus of several cell types. Indirect immunofluorescence microscopy indicated a nuclear location in HepG2, NIH-3T3, and L-cells. A comparison of the fluorescence pattern of wild-type CHO cells with a cytidylyltransferase-deficient mutant provided genetic evidence that cytidylyltransferase is nuclear in CHO cells. The enzyme is also predominantly nuclear in rat liver, as revealed by staining frozen sections of that tissue. When L-cells were fractionated by enucleation, over 95% of cytidylyltransferase activity was found in the nuclear fraction, providing biochemical evidence for a nuclear location in these cells. In light of the demonstration that the membrane-bound cytidylyltransferase in CHO cells is associated with the nuclear envelope (Watkins, J. D., and Kent, C. (1992) J. Biol. Chem. 267, 5686-5692), these results suggest that this enzyme is predominantly an intranuclear enzyme.
3T3 Cells↗
Baculovirus-mediated expression of rat liver CTP:phosphocholine cytidylyltransferase.
We describe herein the expression and purification of milligram quantities of rat liver CTP:phosphocholine cytidylyltransferase in recombinant baculovirus-infected insect cells. The enzyme was purified by incorporating modifications to a previously published procedure (P. A. Weinhold and D. A. Feldman, 1992, in "Methods in Enzymology" (E. A. Dennis and D. E. Vance, Eds.), Vol. 209, pp. 248-258, Academic Press, San Diego, CA). Like cytidylyltransferase purified from rat liver, the purified recombinant cytidylyltransferase has the same molecular weight (42 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and reacts to antibodies generated against the N and C termini of rat liver cytidylyltransferase. In addition, like the purified rat liver enzyme, the recombinant cytidylyltransferase is activated by lipids. We also expressed rat liver cytidylyltransferase as a fusion protein with glutathione-S-transferase and with a thrombin cleavage site between the two enzymes for rapid isolation of cytidylyltransferase. Thrombin cleavage was, however, incomplete and prolonged exposure to thrombin resulted in the degradation of cytidylyltransferase itself.
Animals↗
Radiographic characteristics of isolated invaginated Meckel's diverticulum.
A 3-year-old boy with a radiographic finding of an isolated invaginated Meckel's diverticulum is presented. The abnormality simulates a polypoid filling defect in the distal small bowel on barium examination. This particular manifestation of Meckel's diverticulum is very rare and has been reported only once previously. This article re-emphasizes the need to think about this possibility when a polypoid filling defect is seen in the distal small bowel. At the time laparotomy was performed, the abnormality had progressed into a triple intussusception, a rare surgical finding.
Child, Preschool↗
Effects of ACTH and aminoglutethimide on the catecholamine content and chromaffin cell morphology of the adrenal medulla of the neonatal rat.
The rat adrenal medulla is immature at birth, composed of phaeochromoblasts and undifferentiated chromaffin cells, but by 7 d postnatally morphologically distinct adrenaline-storing (A) and noradrenaline-storing (NA) cells can be distinguished in the adult proportions of approximately 80-85% A and 15-20% NA cells. Glucocorticoid hormones are known to play an important role in the initial expression and maintenance of phenylethanolamine N-methyl transferase (PNMT), the enzyme characteristic of A cells. The purpose of the study was to investigate the effects of glucocorticoids on the establishment of the A and NA cell phenotype in vivo during the first postnatal week. Neonatal rats were treated from postnatal d 1 to 7 either with ACTH to increase circulating levels of glucocorticoids or with aminoglutethimide to reduce blood glucocorticoids. On postnatal d 7 the volume fractions of A and NA cells in the adrenal medulla were estimated and the amounts of stored adrenaline and noradrenaline determined by HPLC and compared with untreated controls. Adrenaline levels were increased following ACTH treatment and there was an apparent decrease after aminoglutethimide which was not statistically significant. There was cytological evidence of the effects of ACTH and aminoglutethimide on the adrenal cortex but no resultant effect on medullary cell morphology. A cells remained predominant with NA cells making up approximately 15% of chromaffin cells, suggesting that any effects of altered glucocorticoid levels were confined to a modulation of adrenaline synthesis by a morphologically unchanged chromaffin cell population.(ABSTRACT TRUNCATED AT 250 WORDS)
Adrenocorticotropic Hormone↗
The immunolocalisation of ubiquitin carboxyl-terminal hydrolase (PGP9.5) in developing paraneurons in the rat.
Protein gene product (PGP9.5) has been detected by means of immunocytochemistry in the rat fetus in intra- and extra-adrenal chromaffin cells (E12.5), pancreatic islet cells (E12.5), anterior pituitary cells (E14.5), carotid body chief cells (E15.5), thyroid C cells (E17.5) and respiratory neuroendocrine bodies (E15.5). It was expressed early in ontogenesis coincidental with other known morphological and biochemical signs of differentiation.
Adrenal Medulla↗
Immunolocalization of membrane-associated CTP:phosphocholine cytidylyltransferase in phosphatidylcholine-deficient Chinese hamster ovary cells.
The location of CTP:phosphocholine cytidylyltransferase in Chinese hamster ovary (CHO) cells made deficient in phosphatidylcholine was determined by immunofluorescence techniques. A rabbit polyclonal antibody was raised against a synthetic peptide corresponding to the amino-terminal 17 amino acid residues of rat liver cytidylyltransferase. The antibody recognized both native and denatured cytidylyltransferase from both rat liver and CHO cells. CHO cells were treated with phospholipase C to alter the lipid composition of the plasma membrane and to elicit translocation of cytidylyltransferase from the less active soluble pool to an activated membrane fraction. Visualization of cytidylyltransferase by indirect immunofluorescence revealed staining of the nuclear envelope in phospholipase C-treated cells but not in untreated cells. CHO cells were also starved for choline and supplemented with a choline analogue to provide an alternative technique of rendering the cells phosphatidylcholine-deficient. Although this treatment should affect different cellular membranes than those affected by phospholipase C treatment, cytidylyltransferase still translocated to the nuclear envelope, as shown by indirect immunofluorescence. These results indicate that activated, membrane-bound cytidylyltransferase is associated with the nuclear membrane and suggest that the nuclear membrane may be a site of de novo phosphatidylcholine synthesis.
Amino Acid Sequence↗
Regulation of CTP:phosphocholine cytidylyltransferase activity and phosphorylation in rat hepatocytes: lack of effect of elevated cAMP levels.
Immunoprecipitation of 32P-labeled CTP:phosphocholine cytidylyltransferase from freshly isolated rat hepatocytes followed by trypsin digestion and two-dimensional peptide mapping revealed multiple phosphorylation sites. Treatment of the hepatocytes with 0.5 mM of the cAMP analog, 8-(4-chlorophenylthio)-adenosine 3':5'-monophosphate or elevation of intracellular cAMP levels by cholera toxin activated the cAMP-dependent protein kinase activity in intact cells. Despite the activation of cAMP-dependent protein kinase no change in the rate of [3H]choline incorporation into phosphatidylcholine was detected. In addition, the activity of cytidylyltransferase in total cell homogenates and its distribution between soluble and particulate fractions remained unchanged. Comparison of peptide maps of 32P-labeled cytidylyltransferase obtained from control and cholera-toxin-treated hepatocytes did not reveal any differences in the phosphorylation state of cytidylyltransferase. Furthermore, only [32P]phosphoserine residues were detected following phosphoamino acid analysis. We conclude that cytidylyltransferase activity is not altered solely by the activation of the cAMP-dependent kinase in fresh hepatocytes.
1-Methyl-3-isobutylxanthine↗
Choline/ethanolamine kinase from rat liver.
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Animals↗
Ileocolonic anastomosis: a comparison of the patency of stapled versus hand-sewn techniques.
This study compared strength of hand-sewn sutures to that of staples for end-to-end anastomoses of ileocolostomies in 20 mongrel dogs. In ten dogs, the authors used four applications of staples for the anastomoses. In the other ten, they performed single-layer anastomoses using 4-0 polypropylene sutures. Half the dogs were sacrificed on the third postoperative day; half were sacrificed on the fifth postoperative day. Bursting strengths were obtained by infusing air through a catheter inserted into the colon. Results showed the single-layer hand-sewn anastomosis was significantly stronger than the stapled anastomosis. In addition, a histologic comparison of the two closures showed no difference in inflammatory responses.
Anastomosis, Surgical↗
Regulation of CTP:phosphocholine cytidylyltransferase activity and subcellular location by phosphorylation in Chinese hamster ovary cells. The effect of phospholipase C treatment.
The phosphorylation state of cytidylyltransferase in Chinese hamster ovary (CHO) cells was correlated with its subcellular distribution and activity in vivo. Western blot analysis of soluble and particulate fractions from control and phospholipase C-treated cells revealed slower migrating forms of cytidylyltransferase present only in the soluble fraction of control cells. These were abolished by incubating the soluble fraction at 37 degrees C in the presence of 5 mM Mg2+ but persisted if 135 mM NaF was present in the incubation. CHO cells were labeled with 32Pi, and cytidylyltransferase was immunoprecipitated from soluble and particulate fractions from control and phospholipase C-treated cells. The slower migrating forms of cytidylyltransferase, present in the soluble fraction of control cells, were phosphorylated at multiple sites. Although an equivalent amount of cytidylyltransferase was immunoprecipitated from the particulate fraction of phospholipase C-treated cells, it contained little 32P. Pretreatment of the CHO cells with okadaic acid, an inhibitor of type 1 and 2A phosphatases, prevented the stimulation of cytidylyltransferase in vivo by phospholipase C. These results demonstrate that dephosphorylation of soluble cytidylyltransferase is required for the phospholipase C-mediated translocation of cytidylyltransferase in CHO cells.
Animals↗
The role of protein kinase C in the stimulation of phosphatidylcholine synthesis by phospholipase C.
The role of protein kinase C in the stimulation of phosphatidylcholine (PC) synthesis by phospholipase C was investigated. Phospholipase C treatment of Chinese hamster ovary cells (CHO) generates diacylglycerol, which is an activator of protein kinase C. The protein kinase C activator, 12-O-tetradecanoyl-phorbol-13-acetate (TPA) stimulated choline incorporation into two CHO cell lines, a wild-type cell line, WTB, and a mutant cell line, DTG 1-5-4. DTG 1-5-4 is a mutant defective in receptor-mediated endocytosis. A 3-h phospholipase C treatment resulted in the activation and translocation of CTP:phosphocholine cytidylyltransferase in both cell lines. TPA treatment, however, resulted in only a slight (20%) translocation of cytidylyltransferase in WTB; no detectable translocation of cytidylyltransferase was observed in DTG 1-5-4. A decrease in the phosphocholine pools was observed in response to TPA treatment in both cell lines, which indicated that the cytidylyltransferase step was being activated. Phospholipase C stimulated choline incorporation into PC even when protein kinase C had been down-regulated in both cell lines. It was concluded that phospholipase C does not activate PC synthesis by activating protein kinase C.
Animals↗
The immunolocalisation of the neuroendocrine specific protein PGP9.5 during neurogenesis in the rat.
We have examined the immunolocalisation of the protein gene product (PGP) 9.5 during neurogenesis in the rat embryo. PGP9.5 was first present at 11.5 days gestation (E11.5): all morphologically recognisable nerve cell bodies and fibres were immunoreactive. In routinely processed, wax-embedded tissues, using a standard immunocytochemical technique, PGP9.5 polyclonal antibody specifically demonstrated the developing nervous system and primitive adrenal chromaffin cells.
Adrenal Medulla↗
Biogenesis of the endoplasmic reticulum in activated B lymphocytes: temporal relationships between the induction of protein N-glycosylation activity and the biosynthesis of membrane protein and phospholipid.
An earlier report from this laboratory documented a substantial increase in the rates of dolichol-linked oligosaccharide intermediate synthesis and protein N-glycosylation in purified murine splenic B lymphocytes (B cells) activated by treatment with bacterial lipopolysaccharide (LPS). In this study the developmental patterns for the induction of lipid-mediated protein N-glycosylation, membrane protein, and phosphatidylcholine (PC) biosynthesis were compared during the proliferative response of B cells to LPS. By electron microscopy it could be seen that a distinct endoplasmic reticulum (ER) network began to develop by 24-48 h after exposure of the purified B cells to LPS. The rate of synthesis of membrane protein increased markedly during the first 10 h after activation, reaching a maximum at 30-40 h. The induction of protein N-glycosylation was delayed slightly relative to membrane protein synthesis, with glycoprotein synthesis increasing sharply approximately 20 h after activation. When phospholipid synthesis was monitored by measuring [CH3-3H]choline incorporation into PC, the rate of labeling increased slowly during the first 35 h, but more substantially between 35 and 90 h. The incorporation of labeled choline into PC was drastically reduced by 5'-deoxy-5'-isobutylthio-3-deazaadenosine, an inhibitor of CDP-choline synthesis, indicating that the incorporation of radiolabeled choline is primarily a measurement of the rate of de novo synthesis of PC. In vitro assays revealed that while choline kinase activity was virtually unchanged, CDP-choline synthetase activity increased gradually throughout the activation period. Diacylglycerol cholinephosphotransferase activity, an ER-associated enzyme, was present at low levels between 0 and 35 h, but increased fivefold between 35 and 90 h. On the basis of the developmental patterns for the rates of protein N-glycosylation, membrane protein insertion, and PC biosynthesis determined by metabolic labeling experiments, we tentatively conclude that all of the ER-associated membrane proteins involved in these biosynthetic processes are not induced concurrently during the activation of B cells by LPS.
Animals↗
Regulation of eukaryotic phospholipid metabolism.
Phospholipids have diverse and critical roles in cellular metabolism and function. Questions about the mechanisms of regulation of phospholipid synthesis are being investigated with a variety of systems and approaches. For example, the yeast Saccharomyces cerevisiae is an organism in which both biochemical and genetic analyses are used. Biochemical approaches have yielded considerable information on the regulatory properties of enzymes of phospholipid biosynthesis. Studies of the activity of purified phosphatidylserine synthase have suggested how that enzyme is influenced by membrane phospholipids in the cell. The enzyme that regulates mammalian phosphatidylcholine biosynthesis, CTP:phosphocholine cytidylyltransferase, is also influenced by phospholipids. In addition, the activity of this enzyme often correlates with its translocation to membranes. The location of such enzymes in the cell is of particular interest in light of the possibility that the enzymatic reactions may be efficiently coupled in vivo. Techniques to render cultured cells permeable to phosphorylated molecules indicated that the enzymes of phosphatidylcholine biosynthesis may exist in an organized compartment so that the precursors of phosphatidylcholine are efficiently channeled through the pathway. To ask how phospholipids are transported in the cell, a combined biochemical and genetic approach has been used. These studies have revealed that the phosphatidylinositol/phosphatidylcholine transfer protein, considered to mediate intracellular phospholipid transfer, is a critical component of the secretory pathway for proteins. These results have allowed formulation of a number of new questions on the regulation of phospholipid metabolism and its relationship to general membrane processes.
Animals↗
Time modulation effect of diethyldithiocarbamate (DDC) on radiosensitization by superoxide dismutase (SOD) inhibition.
Superoxide dismutase (SOD) is known to protect cells from the lethal effects of ionizing radiation by the dismutation of oxygen radicals. Diethyldithiocarbamate (DDC) is known inhibitor of SOD and may therefore be useful as a radiosensitizer. DDC however, is also a thiol radioprotector due to its ability to scavenge radiation induced free radicals. We have shown that DDC, if administered to tumours 1 hour prior to x-irradiation exerts a protective effect, whereas if administered 4 hours prior to irradiation, it radiosensitizes. This time modulation effect is not apparent after neutron irradiation where DDC protects in both situations. We have also examined the effect of DDC on the LD50/30 in mice after total body irradiation.
Animals↗