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P Cortes

Publications and source records attributed to P Cortes.

At least 55 records · Page 3Linked to original sources
PubMed1990

A TATA-like sequence located downstream of the transcription initiation site is required for expression of an RNA polymerase II transcribed gene.

TFIID, the TATA-binding protein, was found to stimulate transcription from the adenovirus IVa2 promoter, a promoter considered to lack the TATA motif. Remarkably, a TATA-like sequence element located downstream of the transcription start site binds TFIID and is required for TFIID-dependent transcription from the IVa2 promoter. Transcription from the IVa2 and the adjacent adenovirus major late promoter (Ad-MLP) is divergent, and the cap sites are separated by 212 nucleotides. Nevertheless, the TATA motifs of the IVa2 promoter and Ad-MLP were found to be oriented in the same direction. An initiator motif around the transcription start site is located in the IVa2 promoter, and in contrast to the TATA motifs, the IVa2-initiator is in the opposite orientation with respect to the initiator of the Ad-MLP. A model is presented in which the polar nature of the initiator governs the direction of transcription. We propose that RNA polymerase II and accessory factors recognize the initiator in an orientation-dependent fashion. The recognition of the IVa2 initiator by RNA polymerase is enhanced by the binding of TFIID to the downstream TATA motif.

Adenoviridae↗
PubMed1990

Factors involved in specific transcription by mammalian RNA polymerase II: role of transcription factors IIA, IID, and IIB during formation of a transcription-competent complex.

Human transcription factor TFIID, the TATA-binding protein, was partially purified to a form capable of associating stably with the TATA motif of the adenovirus major late promoter. Binding of the human and yeast TFIID to the TATA motif was stimulated by TFIIA. TFIIA is an integral part of a complex capable of binding other transcription factors. A complex formed with human TFIID and TFIIA (DA complex) was specifically recognized by TFIIB. We found that TFIIB activity was contained in a single polypeptide of 32 kDa and that this polypeptide participated in transcription and was capable of binding to the DA complex to form the DAB complex. Formation of the DAB complex required TFIIA, TFIID, and sequences downstream of the transcriptional start site; however, the DA complex could be formed on an oligonucleotide containing only the adenovirus major late promoter TATA motif. Using anti-TFIIB antibodies and reagents that affect the stability of a transcription-competent complex, we found that yeast and human TFIID yielded DAB complexes with different stabilities.

Base Sequence↗
PubMed1989

The adenovirus inverted terminal repeat functions as an enhancer in a cell-free system.

Two binding sites for EivF, a factor involved in transcription from the adenovirus early promoter iv (Eiv), were mapped within the adenovirus inverted terminal repeats (ITR). Consistent with the observation that EivF was required to initiate transcription from the Eiv promoter and with the demonstration that two EivF binding sites were present in the ITR, we show that the inverted terminal repeat region was able to promote transcription from the CAP site of the Eiv promoter in vitro and in an EIa-dependent fashion in vivo. The minimum sequence within the ITR capable of directing EIa-dependent transcription consists of forty nine nucleotides comprising two EivF binding sites and at least one Sp1 binding site. This 49-base pair fragment possesses the characteristics of an enhancer which is induced by EIa. The enhancer is active in HeLa cell nuclear extracts. Transcription directed by the ITR required EivF and the general transcription factors. The addition of purified Sp1 factor specifically stimulated transcription which correlates with the presence of Sp1 binding sites between the two EivF recognition sites.

Adenoviridae↗
PubMed1988

EivF, a factor required for transcription of the adenovirus EIV promoter, binds to an element involved in EIa-dependent activation and cAMP induction.

The isolation of a cellular factor that was specifically required for transcription from the promoter of the adenovirus early gene iv, an EIa-activated promoter, is described. This factor (EivF) was purified from HeLa cells using a functional transcription assay and identified as a 72,000- to 65,000-dalton protein. DNase I footprinting experiments demonstrated that purified EivF bound to the sequence 5'-GT(G/T)ACGT-3' present two times upstream of the Eiv TATA box. Nuclear extracts prepared from HeLa cells contained more than one factor capable of binding to the EivF recognition site. Previous studies have indicated that a sequence similar to the EivF-binding site was recognized by a 43,000-dalton protein and participated in the cAMP response of the somatostatin promoter. The purified and transcriptionally active EivF also bound to DNA sequence elements present in the somatostatin and alpha-gonadotropin promoters shown previously to be responsive to cAMP.

Adenoviridae↗
PubMed1988

Uracil ribonucleotide metabolism in rat and human glomerular epithelial and mesangial cells.

Uridine diphosphosugars (UDP-sugars: UDP-N-acetylglucosamine, UDP-glucose, and UDP-glucuronic acid) are essential coenzymes for the synthesis of glomerular basement membrane and mesangial matrix (GBM-MM). This study has characterized UDP-sugar metabolism in rat and human glomerular cells in tissue culture. Culture of rat mesangial cells in medium containing dialyzed fetal calf serum resulted in UTP loss (28 +/- 4 nmol.mg DNA-1.h-1); the addition of 2 microM orotate to this medium resulted in net UTP accretion (5.42 +/- 0.06 nmol.mg DNA-1.h-1). Rat mesangial cells demonstrated 16- and 29- to 46-fold greater UTP and UDP-sugar pools, respectively, than whole glomeruli. In human mesangial cells, 6-azauridine (500 microM) decreased UDP-sugar pools by 48% (P less than 0.05), whereas uridine (50 microM) produced a 2.5-fold increase. Human and rat mesangial cells had greater (1.8- to 6.1-fold) UDP-sugar pools than epithelial cells and 1.7-3.4 times greater labeled precursor incorporation into UDP-sugars. In conclusion, glomerular cells utilize both exogenous orotate and uridine for ribonucleotide synthesis, and the extracellular concentration of these precursors markedly influence the formation and cellular content of UDP-sugars. Prominent differences exist between separate glomerular cell populations in their metabolism of UDP-sugars. This may represent diverse activity of glycosylating reactions.

Adenosine Triphosphate↗
PubMed1988

Glomerular uracil nucleotide synthesis.

The biosynthesis of basement membrane material requires the sugar derivatives of uridine 5'-triphosphate (UTP) for protein glycosylation. Uridine and orotate utilization for the biosynthesis of uracil ribonucleotides was studied in isolated rat glomeruli incubated in vitro. At a 1 microM concentration total orotate utilization was 9.6 +/- 1.8 pmol.min-1.mg DNA-1 (1 mg DNA approximately 0.175 X 10(6) glomeruli), 51% of the total amount metabolized was used in ribonucleotide formation, and there was a significant UTP accretion. Except at a high initial concentration (50 microM), exogenous uridine failed to increase the UTP pool due to rapid uridine breakdown by a cytosolic phosphorylase. Inhibition of this enzyme with benzylacyclouridine resulted in increased biosynthesis and accretion of UTP, and in a 17-fold higher concentration of uridine, primarily produced from performed sources of nucleosides. Continuous addition of exogenous uridine to maintain its concentration at 1 microM resulted in a total uridine utilization of 550 +/- 30 pmol.min-1.mg DNA-1. Uridine salvage for ribonucleotide biosynthesis was only 3% of the total metabolized. In contrast to uridine, and presumably due to UTP pool compartmentation, orotate incorporation into uridine 5'-diphosphosugars was prominent. The metabolism of exogenous orotate was not decreased by the presence of large amounts of uridine and by an expanded UTP pool. It is concluded that when exogenous orotate is present, it is an important precursor for glomerular uracil ribonucleotide biosynthesis. Due to its rapid rate of catabolism, uridine cannot maintain ribonucleotide biosynthesis at a rate sufficient to result in UTP accretion unless it is provided continuously in substantial quantities.

Animals↗
PubMed1988

Glomerular uracil nucleotide synthesis: effects of diabetes and protein intake.

The biosynthesis of uridine 5'-triphosphate (UTP), uridine 5'-diphosphohexoses, and 5'-diphosphohexosamines (UDP-sugars) was studied in isolated rat glomeruli 48 h after streptozotocin-induced diabetes. Compared with control, diabetic glomeruli demonstrated an increase in the following: exogenous orotate utilization, orotate incorporation into UTP and UDP-sugars, UTP accretion rate, and UDP-sugar pool size. Since these phenomena were not associated with enhanced biosynthesis of orotate de novo, the increased glomerular UDP-sugar bioavailability in diabetes is due to enhanced utilization of exogenous orotate. Plasma concentrations of orotate and uridine were measured in control, sham operated, and unilaterally nephrectomized rats receiving 5, 20, or 60% protein diets. The concentration of pyrimidine precursors correlated directly with protein intake, with doubling at the 60% dietary protein level. In conclusion, glomerular uracil ribonucleotide biosynthesis may be modulated by the quantity of dietary protein. Because UDP-sugars are necessary for basement membrane material formation, an increase in their bioavailability may be part of the metabolic change responsible for diabetic glomerulosclerosis. Diets with high protein content could augment this metabolic alteration.

Animals↗
PubMed1987

Effect on renal function of change from high to moderate protein intake in type I diabetic patients.

The effects on renal function of moderate restriction in protein intake were studied in 14- to 20-yr-old type I diabetic patients who had no clinical renal disease or hypertension; matched normal subjects served as controls. After assessment of protein intake and renal function, studies were conducted at the completion of each of two consecutive dietary periods of 1 wk. Diets containing 3.5 and 1.5 g X kg-1 X day-1 protein were provided during the first and second periods, respectively. Baseline protein intakes were substantial in both controls (1.86 g X kg-1 X day-1) and diabetics (2.17 g X kg-1 X day-1). Baseline creatinine clearance was increased in diabetics (P = .043). At the end of the high-protein intake period, both diabetics and controls showed similar high values of glomerular filtration rate (GFR) and renal plasma flow (RPF). GFR and RPF decreased markedly (P less than .001) and to a similar degree in both groups after normal protein intake. GFR and RPF in diabetics were not higher than in controls at this point, but filtration fraction was increased in diabetics. Albumin excretion rates were similar in both groups and not influenced by renal function changes. GFR and RPF values correlated significantly with the quantity of protein intake, as estimated from the urea nitrogen appearance rate in both groups. The results suggest that the functional response to variations in protein intake is not altered in the diabetic kidney. In addition, increased renal function in diabetics may be related partly to the excessive protein content in commonly prescribed diabetic diets.(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗
PubMed1987

Relationship between renal function and metabolic alterations in early streptozocin-induced diabetes in rats.

To investigate the temporal relationship of diabetes-induced renal growth and its associated metabolic alterations to the early development of renal hyperfunction, parallel functional and metabolic studies were performed shortly after the onset of diabetes in rats. Hyperglycemia and hypoinsulinemia were evident 18 h after streptozocin injection, and significant hyperglucagonemia and acidosis were present at 36-48 h. Glomerular filtration rate (GFR), expressed per unit of body weight, first increased at 3 days of diabetes [1.35 +/- 0.07 (SE) (N = 14)] and was 18% greater than in controls [1.14 +/- 0.03 ml X min-1 X 100 g-1 (SE) (N = 38)] (P less than .005). Renal enlargement preceded GFR changes, so that GFR per unit of kidney weight was lower at 48 h in diabetics [1.31 +/- 0.06 (SE) (N = 16)] than in controls [1.54 +/- 0.04 ml X min-1 X g-1 (SE) (N = 38)] (P less than .01). Nucleotide and RNA metabolism was studied in the renal cortex after infusion of radio-labeled orotate or adenine. Rate of RNA synthesis, total cellular RNA, and the pools of ATP, UTP, and uridine 5'-diphospho-N-acetyl glucosamine were significantly increased 13-51% in 48-h diabetics. Nucleotide precursor incorporation was significantly increased only in uracil ribonucleotides. The increase in uracil ribonucleotide pool exceeded the degree of cell hypertrophy. Our studies indicate that renal hypertrophy and specific increases in uracil ribonucleotide synthesis precede functional changes in early diabetes. Renal metabolic changes may be the critical primary factors in diabetic nephropathy.

Animals↗
PubMed1986

De novo pyrimidine nucleotide biosynthesis in isolated rat glomeruli.

Uracil ribonucleotide-sugars and aminosugars are required for glomerular basement membrane (GBM) biosynthesis. Since these nucleotides are metabolic derivatives of uridine 5'-triphosphate (UTP), we have studied the cellular pools of uridine 5'-diphosphoglucose (UDPG), uridine 5'-diphosphoglucuronic acid (UDPGA), uridine 5'-diphospho-N-acetyl glucosamine (UDPAG) and UTP, and measured UTP synthesis de novo in isolated glomeruli incubated in vitro. Improved techniques for nucleotide quantitation were established and the optimal conditions for glomerular isolation and incubation determined. Substantial quantities of uracil ribonucleotide coenzymes and an active utilization of orotate for the synthesis of pyrimidine nucleotides were demonstrated. UTP synthesis and the pools of UDPG and UDPGA varied markedly with changes in the experimental conditions. The adverse effects of suboptimal conditions were more apparent in glomeruli from diabetic animals than in controls. The use of suboptimal conditions could provide misleading information on GBM metabolism in isolated glomeruli since uracil ribonucleotide coenzyme availability might be reduced.

Animals↗
PubMed1982

Effects of early diabetes on uridine diphosphosugar synthesis in the rat renal cortex.

To assess the effects of streptozotocin-induced diabetes on the substrates utilized in the formation of glycoproteins, the pools of uridine 5'-diphosphoglucose (UDPG), uridine 5'-diphosphogalactose (UDP-GAL), uridine 5'-diphosphoglucuronic acid (UDPGA), and uridine 5'-diphospho N-acetyl galactosamine (UDPA-GAL) were measured in the renal cortex of control and over a 48-hr period in diabetic rats. In control rats these pools measured: UDPG, 256 +/- 23; UDP-GAL, 75 +/- 14; UDPGA, 147 +/- 16; UDPAG, 367 +/- 23; UDPG-GAL, 131 +/- 13 nmoles/mg DNA. In diabetic rats, except for UDP-GAL, all pools were increased 41 to 68%. The incorporation of radiolabeled orotate was increased in all pools, except UDP-GAL, in diabetic rats by 41 to 77% compared to control rats. The incorporation into UDPG and UDPAG was increased even after correction for the specific radioactivity of their immediate precursor, uridine 5'-triphosphate (UTP). Expansion of the UTP pool after orotate infusion was associated with an increase in the size of the UDPG and UDPAG pools in both control and diabetic rats. Depletion of the UTP pool after adenine infusion in controls was associated with a decrease in all pools. This study demonstrates that after the induction of diabetes there is a rapid increase in the bioavailability of substrates utilized in the synthesis of glycoproteins and glycosaminoglycans. It is theorized that this increase is necessary for the augmented synthesis of basement membrane-like material in the diabetic kidney.

Animals↗
PubMed1981

Alterations in glomerular RNA in diabetic rats: roles of glucagon and insulin.

Incorporation in vivo of labeled orotate into RNA and total nucleotides was measured in isolated glomeruli and whole renal cortex. In 2-day diabetic animals, glomerular RNA was increased, and there was greater incorporation of orotate into total nucleotides and RNA as compared with controls. Insulin reversed the exaggerated incorporation at infusion rates that corrected hyperglucagonemia without reducing plasma glucose and with only minimal changes in insulin concentrations. The addition of glucagon to insulin infusions reproduced the increased incorporation observed in untreated diabetics. Similar changes occurred in renal cortex, where differences in orotate incorporation into nucleotide precursors seemed to be the main cause for alterations in RNA labeling. Isotope incorporation in glomeruli correlated positively with plasma glucagon, but not with insulin or glucose concentrations. Although in 7-month diabetic animals orotate incorporation into RNA was less than in controls, probably as a consequence of renal disease, 24-hour insulin infusion decreased it further. Our results confirm that in the diabetic kidney, abnormal uracil nucleotide metabolism and increased cellular content of RNA are demonstrable in glomeruli as in the renal cortex. These changes appear to be related directly to hyperglucagonemia.

Adenine↗
PubMed1980

Phosphoribosylpyrophosphate bioavailability in diabetic rat renal cortex in vivo.

Experimental diabetes induces increased content of RNA and UTP in the renal cortex. Studies were designed to assess the bioavailability of 5-phosphoribosyl-1-pyrophosphate (PRPP) in the diabetic renal cortex because PRPP is an important determinant of the de novo synthesis of nucleotides. The tissue bioavailability of PRPP determines the effects of orotate or adenine administration on UTP, ATP, and GTP content and on the incorporation of labeled precursors into UTP and ATP. Diabetic and control rats with chronic intravenous cannulas were infused over 2.5-24 h with orotate or adenine. Orotate administration induced greater decreases in ATP and GTP and in labeled adenine incorporation into ATP concomitant with smaller increases in UTP in controls than in diabetic animals. Adenine administration induced a greater decrease of labeled orotate incorporation into UTP and a smaller increase in ATP in controls than in diabetic animals. Prolonging the adenine infusion resulted in disappearance of these differences. The results are compatible with greater initial bioavailability of PRPP in the diabetic renal cortex than in controls but with a rate of maximal PRPP generation that is the same in both tissues.

Adenine↗
PubMed1980

Uridine triphosphate and RNA synthesis during diabetes-induced renal growth.

UTP, CTP, and RNA synthesis were studied in the renal cortex of diabetic and control rats in vivo. The incorporation of UTP into RNA (nmol/h DNA) was used as estimate of RNA synthesis rate. Two to three days after streptozotocin injection, UTP and CTP ppol size and orotate incorporation into UTP and RNA were greater in diabetic animals than in controls. In addition, RNA content and RNA synthesis rate were increased. These changes were corrected by insulin infusion. In diabetic animals, additional increases in UTP pool, RNA content, and RNA synthesis rate followed contralateral nephrectomy. This increase in RNA content was greater than in uninephrectomized controls. The changes in the diabetic renal cortex were not accompanied by increased plasma concentrations of growth hormone. The increase in RNA content in the diabetic renal cortex is probably due to increased RNA synthesis. Increased synthesis of pyrimidines and expansion of the UTP pool may make this substrate more readily available for the synthesis of UDP sugars and may facilitate the synthesis of basement membrane in diabetes.

Animals↗
PubMed1980

Glomerular pyrimidine metabolism in experimental diabetic nephropathy.

Glomerular uracil nucleotide metabolism was studied in vivo in control and diabetic rats 48 h after the injection of streptozotocin. The animals were infused for 2 h with 3H-orotate and the fraction of infused dpm incorporated into glomerular uracil nucleotides and RNA/mg of glomerular DNA was calculated. Diabetic glomeruli showed an increase in RNA/DNA compared to controls (p less than 0.05) and a greater incorporation of 3H-orotate into uracil nucleotides and RNA. These changes were reversed by insulin therapy. In separate experiments the renal cortical uracil nucleotide pool was expanded by feeding chow supplemented with 0.5% orotate to rats for 6 months. Normal animals fed orotate developed significant glomerular basement membrane thickening (p less than 0.01) when compared to age-matched controls, which was morphologically indistinguishable from that of diabetics. Orotate feeding also produced further basement membrane thickening in diabetic rats. These results suggest that early diabetes is characterized by an increase in glomerular uracil nucleotides, and that chronic expansion of the uracil nucleotide pool is associated with glomerular basement membrane thickening.

Animals↗
PubMed1979

Incorporation of exogenous precursors into uridine nucleotides and ribonucleic acid. Nucleotide compartmentation in the renal cortex in vivo.

The possibility of compartmentation of UTP in vivo was investigated in the renal cortex of unanaesthetized rats. In addition, liver and spleen were studied in order to compare tissues with different utilization of precursors for pyrimidine nucleotide synthesis. After continuous 2h infusions of [(3)H]uridine or [(3)H]orotate, their incorporation into UTP, UDP-sugars and RNA was quantified. Rates of RNA synthesis were calculated by dividing the incorporation of precursor into RNA by the average specific radioactivity of the UTP pool. Although similar RNA-synthesis rates might have been expected with the two precursors, higher rates were found with uridine than with orotate. The relative incorporation into UDP-sugars of these precursors was also different. Similar results were obtained in the liver. In the spleen, equal amounts of both precursors were incorporated into UTP, but [(3)H]orotate incorporation did not lead to labelling of RNA. To evaluate the heterogeneity of cells with respect to the metabolism of pyrimidines, precursor incorporation was studied in isolated glomeruli and by radioautography. Incorporation into glomeruli was qualitatively similar to but quantitatively different from results in the renal cortex. Although there is obvious tissue heterogeneity, compartmentation of UTP pools is the most credible explanation for the results obtained with the renal cortex and liver. Consequently RNA and UDP-sugars may originate from two different UTP pools. Tissue heterogeneity is the likely explanation for the results obtained in the spleen. Studies of synthesis of pyrimidine and RNA, particularly in relation to growth and regeneration, must take into consideration the precursor used, the apparent existence of UTP compartmentation and the degree of cellular heterogeneity.

Animals↗