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Biomedical subjects

C Kent

Publications and source records attributed to C Kent.

At least 73 records · Page 4Linked to original sources
PubMed1994

Expression of wild-type and mutant rat liver CTP: phosphocholine cytidylyltransferase in a cytidylyltransferase-deficient Chinese hamster ovary cell line.

The strain 58 Chinese hamster ovary (CHO) mutant defective in CTP:phosphocholine cytidylyltransferase was characterized as an expression system for exogenous cytidylyltransferase. Strain 58 cells express less than 5% of the wild-type level of cytidylyltransferase protein at the permissive temperature even though the steady-state messenger RNA levels were found to be similar to those in the parental CHO-K1 cell line. A point mutation from arginine to histidine at amino acid 140 was identified in the strain 58 protein. Rat liver cytidylyltransferase was stably expressed in strain 58 cells and shown to be active, targeted to the nucleus, phosphorylated, and activated by methylethanolamine supplementation or phospholipase C treatment. Thus, the mechanisms by which cytidylyltransferase is processed and regulated in CHO-K1 cells are intact in strain 58 cells. The heterologously expressed protein complemented the strain 58 defects in both temperature-sensitive growth and phosphatidylcholine biosynthesis, consistent with a single lesion in the structural gene for cytidylyltransferase being responsible for both phenomena. Overexpression of cytidylyltransferase activity at levels up to eightfold higher than those in CHO-K1 cells did not appreciably affect phosphatidylcholine metabolism. A putative casein kinase II phosphorylation site was altered by site-directed mutagenesis and expressed in the strain 58 cells. Alteration of this site did not affect expression and regulation of cytidylyltransferase activity.

Amino Acid Sequence↗
PubMed1994

Identification of phosphorylation sites in rat liver CTP: phosphocholine cytidylyltransferase.

CTP:phosphocholine cytidylyltransferase (CT) is an important regulatory enzyme in phosphatidylcholine biosynthesis. The enzyme exists as a soluble, inactive form that is highly phosphorylated; activation of the enzyme is accompanied by dephosphorylation and translocation to the membrane. We have used a recombinant baculovirus clone to obtain CT labeled in vivo with 32PO4. The tryptic phosphopeptide pattern of the baculovirus-expressed CT was the same as for CT expressed in mammalian cells, indicating that insect cells modify the same phosphorylation sites as do mammalian cells. 32PO4-labeled, baculovirus-expressed CT was digested with trypsin and the peptides purified by reversed-phase high performance liquid chromatography. Phosphoamino acid analysis of the complete protein as well as individual peptides revealed that only serine residues were phosphorylated. Sequence analysis of purified radioactive peptides revealed that phosphorylation of CT was confined to the carboxyl-terminal region and that all or nearly all Ser residues from Ser315 to the carboxyl terminus were labeled. Ser315, Ser319, Ser329, Ser323, Ser331, Ser343, and Ser347 all reside in potential sites for proline-directed kinases. Two other phosphorylated serine residues, Ser315 and Ser333, are found within protein kinase C consensus phosphorylation sites. Ser321, Ser322, Ser333, Ser345, Ser346, Ser350, Ser352, and Ser362 were also found to be phosphorylated. Serine362 resides within a putative casein kinase II phosphorylation site, and there are five potential sites for phosphorylation by glycogen synthase kinase 3. Identification of these sites will allow investigations that focus on the establishment of the physiological function of phosphorylation at each site.

Amino Acid Sequence↗
PubMed1994

Differential expression of GAP-43 mRNA in adult central cholinergic neuronal populations.

The present study addresses a question of differential expression for a 'plasticity' gene within neurons identified by neurotransmitter type. A method combining immunohistochemical localization of choline acetyltransferase (ChAT) with in situ hybridization histochemistry (ISHH) in the same brain sections was used to quantitate the levels of mRNA for the growth-associated protein GAP-43 (neuromodulin) in rat central cholinergic neuronal populations. We found that many cholinergic neurons in the adult rat brain express levels of GAP-43 mRNA comparable to other brain regions noted for their expression of this plasticity gene. GAP-43 expression in cholinergic cell groups appeared to be highly heterogeneous; this was often true even for cholinergic neurons within the same brain region. A dorsal-ventral gradient in GAP-43 mRNA levels was evident in the rostral basal forebrain cholinergic groups; i.e., medial septum, nucleus basalis magnocellularis and the vertical limb of the diagonal band expressed intermediate levels, while the horizontal limb of the diagonal band and the substantia innominata expressed higher levels of GAP-43 mRNA. Cholinergic interneurons of the striatum were grouped in several populations according to mRNA levels, from very low to very high expression. Similarly, GAP-43 mRNA levels in the cholinergic neurons of the nucleus basalis magnocellularis/substantia innominata were quite variable. The expression of GAP-43 mRNA in brainstem cholinergic groups (laterodorsal tegmental and pedunculopontine nuclei) was in nearly uniform populations of somewhat lower levels. While the expression of GAP-43 mRNA in cholinergic neurons was heterogeneous, virtually every ChAT-positive neuron sampled contained GAP-43 mRNA at levels significantly over background.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetylcholine↗
PubMed1994

Complex branch renovascular disease: management options and late results.

PURPOSE: The purpose of this report is to review management options and late results of complex renovascular disease managed over the last 22 years. METHODS: Complex branch renal artery disease in 84 kidneys was repaired during 75 operations performed in 68 consecutive patients. There were 61 females (90%) and 7 males (10%) whose predominant pathologic diagnosis was fibromuscular dysplasia manifesting as either renovascular hypertension or aneurysmal degeneration. These patients underwent 15 in situ, 52 ex vivo, and 8 combined reconstructions. In situ repair primarily with use of the bifurcated internal iliac artery autograft was used for primary lesions of the proximal renal artery bifurcation (two branches). Ex vivo repairs, primarily with use of the multibranch internal iliac autograft and hypothermic perfusion preservation, were used for all other patterns of distal renal artery branch disease and reoperative problems. RESULTS: Renovascular reconstruction was successful in salvaging 83 of 84 kidneys (98.8%) in 67 of 68 patients. There were no operative deaths. Two reconstructions thrombosed in the early postoperative period. One was due to severe aortic disease, the other to branch artery dissection after a failed balloon angioplasty. Both patients continued to have hypertension. Before hospital discharge 65 patients had 81 renal revascularizations proven patent by arteriography. Their renal function was assessed and blood pressure was determined in a follow-up extending to 20 years (mean 7.5 years, median 7.9 years). Late arteriograms were obtained in 30 patients (46%) an average of 52 months after operation (range 6 months to 18 years). They demonstrate stable renal artery repair with no evidence of late graft failure in each. Hypertension was cured or improved in 51 of 53 patients (96%) with a proven patent reconstruction. Aneurysms were successfully repaired in 11 patients. Renal function was improved in four patients with ex vivo repairs, unchanged in 59 patients (15 in situ, 44 ex vivo), and persistently worse in only three patients, all of whom had in situ repairs. CONCLUSION: The branched arterial autograft allows the restoration of normal renal arterial anatomy and function when inserted to replace complex distal renovascular disease. This provides a durable repair, essential for younger patients affected by this pattern of disease who anticipate a normal life span after renovascular repair. Successful long-term correction of diastolic hypertension and aneurysmal disease was accomplished without significant morbidity.

Adolescent↗