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R M Muñoz

Publications and source records attributed to R M Muñoz.

26 records · Page 2Linked to original sources
PubMed1994

Analysis of the DNA-binding domain of the HSV-1 origin-binding protein.

In order to understand DNA-protein interactions at the origin of DNA replication in herpes simplex virus type 1 (HSV-1), we have undertaken an analysis of the DNA-binding domain of the origin-binding protein (OBP) and its mechanism of binding to the Oris sequence of HSV-1. Mutant DNA-binding domains were constructed, expressed in vitro, and used to test for binding by gel shift analysis. A C-terminal deletion mutant was functional in binding, thereby redefining the C-terminal boundary of the DNA-binding domain at amino acid 822. Fifteen insertion mutants were also constructed across the DNA-binding domain. Several of these mutants were unable to bind DNA. Interestingly, 4 mutants that destroy DNA binding fall within a region that has a particularly high degree of sequence similarity to the varicella zoster virus gene 51 product. A second objective was to define how the DNA-binding domain interacts with the origin. Results of gel shift analysis using contranslated proteins of different sizes suggest that the DNA-binding domain can interact with a single binding site as a monomer. Binding to the wild-type Oris template indicated that the binding domains can interact with both binding sites I and II independent of any cooperative effect mediated by the amino-termini. This suggests that the basic unit of recognition involved in OBP/Oris interactions may contain a single DNA-binding domain of OBP in association with a single binding site.

Amino Acid Sequence↗
PubMed1994

Wild-type human p53 activates the human epidermal growth factor receptor promoter.

We show that wild-type human p53 transactivates the human epidermal growth factor receptor (EGFR) promoter in vivo in a dose-dependent manner, implicating p53 in promotion of cell proliferation. This activation is sensitive to the expression of cellular oncoprotein MDM2 and human papillomavirus type 18 (HPV-18) E6 protein. The p53 response element is localized within -15 and -569 of the promoter. The EGFR promoter does not have a TATA box, and has low activity in Saos-2 cells in the absence of p53. Results from our in vivo transient transfection assays suggest that p53-binding sites, without any other known promoter element, can act as bidirectional promoters in the presence of wild-type p53. Gel retardation analyses suggest that p53 may serve to nucleate TBP on a promoter. We propose that p53 successfully nucleates the transcription complex, possibly via direct interaction with TFIID, and activates the EGFR promoter.

Base Sequence↗
PubMed1993

p53 and SV40 T antigen bind to the same region overlapping the conserved domain of the TATA-binding protein.

In this report we demonstrate that the cloned human TATA-binding protein (TBP) interacts with T antigen. TBP co-immunoprecipitates with T antigen when incubated with the T antigen-specific monoclonal antibody PAb419, and Protein-A agarose. Gel retention analysis with a radiolabeled TATA box-containing probe showed that the complex of TBP and T antigen can bind to the TATA box. Recently, p53 has also been shown to interact with TBP. Using TBP deletion mutants and co-immunoprecipitation experiments with p53 or T antigen, we show that both p53 and T antigen bind to the same region, amino acids 203-275, within the conserved C-terminal domain of TBP. Binding of p53 and T antigen to the same domain on TBP may lead to competition between the two proteins for transcriptional function.

Amino Acid Sequence↗
PubMed1993

p53 binds to the TATA-binding protein-TATA complex.

Earlier reports show that p53, both wild type and mutants, may affect transcription. Wild-type p53 activates promoters with p53-binding sites while inhibiting promoters without binding sites. Mutant p53, on the other hand, has been shown to activate transcription from specific promoters. These observations suggest that both wild-type and mutant p53 may interact with a general transcription factor(s). In this report, we have shown that the cloned TATA-binding protein (TBP) from human and yeast interacts with human p53. TBP co-immunoprecipitates with wild-type or mutant human p53 when incubated with the p53-specific monoclonal antibody and Protein A-agarose. Wild-type murine p53 has also been found to interact with human TBP. Protein blot assays have demonstrated that the interaction between p53 and human TBP is direct. By gel retention analysis, we have shown that the complex of TBP and p53 (both wild type and mutant) can bind to the TATA box. The similar qualitative binding capability of wild-type and mutant p53 with human TBP and the similarity of the two complexes in binding to the TATA box suggest that the functional discrimination between wild-type and mutant p53 may not lie in their ability to bind TBP. The nature of the p53.TBP or p53.TBP.TATA complex may determine the success of transcription.

Animals↗
PubMed1993

The tumor suppressor p53 and the oncoprotein simian virus 40 T antigen bind to overlapping domains on the MDM2 protein.

The oncogene mdm2 has been found to be amplified in human sarcomas, and the gene product binds to the tumor suppressor p53. In this report, we describe the dissection of the MDM2-binding domain on p53 as well as the p53-binding domain on MDM2. We also demonstrate that the oncoprotein simian virus 40 T antigen binds to the product of cellular oncogene mdm2. We have constructed several N- and C-terminal deletion mutants of p53 and MDM2, expressed them in vitro, and assayed their in vitro association capability. The N-terminal boundary of the p53-binding domain on MDM2 is between amino acids 1 and 58, while the C-terminal boundary is between amino acids 221 and 155. T antigen binds to an overlapping domain on the MDM2 protein. On the other hand, the MDM2-binding domain of p53 is defined by amino acids 1 and 159 at the N terminus. At the C terminus, binding is progressively reduced as amino acids 327 to 145 are deleted. We determined the effect of human MDM2 on the transactivation ability of wild-type human p53 in the Saos-2 osteosarcoma cell line, which does not have any endogenous p53. Human MDM2 inhibited the ability of human p53 to transactivate the promoter with p53-binding sites. Thus, human MDM2 protein, like the murine protein, can inactivate the transactivation ability of human p53. Interestingly, both the transactivation domain and the MDM2-binding domain of p53 are situated near the N terminus. We further show that deletion of the N-terminal 58 amino acids of MDM2, which eliminates p53 binding, also abolishes the capability of inactivating p53-mediated transactivation. This finding suggests a correlation of in vitro p53-MDM2 binding with MDM2's ability in vivo to interfere with p53-mediated transactivation.

Amino Acid Sequence↗
PubMedDate not supplied

Perinatal delta(9)-tetrahydrocannabinol exposure alters the responsiveness of hypothalamic dopaminergic neurons to dopamine-acting drugs in adult rats.

We have recently reported that perinatal cannabinoid exposure altered the normal development of dopaminergic neurons in the medial basal hypothalamus at early postnatal and peripubertal ages. Most of these effects tended to disappear in adulthood, although we suspect the existence of a persistent, but possibly silent, alteration in the adult activity of these neurons. To further explore this possibility, we evaluated the responsiveness of these neurons to pharmacological challenges with a variety of dopaminergic drugs administered to adult male and female rats that had been exposed to delta(9)-tetrahydrocannabinol (delta(9)-THC) or vehicle during the perinatal period. In the first experiment, we evaluated the sensitivity of hypothalamic dopaminergic neurons to amphetamine (AMPH), which causes enhancement of dopaminergic activity by a variety of mechanisms. The most interesting observation was that both adult males and females, when perinatally exposed to delta(9)-THC, showed a more marked AMPH-induced decrease in the production of L-3,4-dihydroxyphenylacetic acid (DOPAC), the main intraneuronal metabolite of dopamine (DA), although this did not affect the prolactin (PRL) release. In the second experiment, we evaluated the in vivo synthesis of DA by analyzing the magnitude of L-3,4-dihydroxyphenylalanine (L-DOPA) accumulation caused by the blockade of L-DOPA decarboxylase with NSD 1015. As expected, NSD 1015 increased L-DOPA accumulation and decreased DOPAC production, with a parallel increase in PRL release, all of similar magnitude in both delta(9)-THC- and oil-exposed adult animals. In the last experiment, we tested the magnitude of the increase in PRL release produced by the administration of either SKF 38393, a specific D1 agonist, or sulpiride, a specific D2 antagonist. Both compounds increased plasma PRL levels in adult animals of both sexes, the effects in females being significantly more marked. The perinatal exposure to delta(9)-THC also modified the degree of increase in plasma PRL levels induced by both compounds, with opposite responses as a function of sex. Thus, delta(9)-THC-exposed females responded more intensely to SKF 38393 and, particularly, to sulpiride than oil-exposed females, whereas delta(9)-THC-exposed males responded to SKF 38393 lesser than oil-exposed males, although both responded equally to sulpiride. In summary, our results are consistent with the possible existence of subtle changes in the activity of hypothalamic dopaminergic neurons in adulthood caused by the exposure to delta(9)-THC during perinatal development. These silent changes could be revealed after the administration of drugs such as: (i) AMPH, whose effect producing a decreased DOPAC accumulation was more marked in delta(9)-THC-exposed males and females; and (ii) SKF 38393 and sulpiride, whose stimulatory effects on PRL secretion were of different magnitude in delta(9)-THC-exposed animals, with an evident sexual dimorphism in the response. The neurochemical basis for these differences remains to be determined.

2,3,4,5-Tetrahydro-7,8-dihydroxy-1-phenyl-1H-3-ben↗
PubMedDate not supplied

[Effects of fiber administration in the prevention of gallstones in obese patients on a reducing diet. A clinical trial].

Nearly 30% of the obese patients treated with hypoenergetic diets for weight reduction develop gallstone disease (GD). Until the present time, the use of ursodeoxycholic acid (UDA) is the only available therapeutic measure to avoid the development of GD. Dietary fiber induce a bile acid synthesis. A double-blind clinical trial was conducted to compare the effect of rational diet plus UDA vs a rational diet supplemented with Psyllium plantago (Pp) for the prevention of GD in obese subjects undergoing a weight-reduction diet. Patients with a body mass index (BMI = weight in Kg/square height in m) of 30 Kg/m2 or more and with normal gallbladder and biliary tree ultrasound (GBUS) were included. Weight-reduction diets were individually calculated for each patient according to their energy expenditure (EE). Patients were randomly and blindly assigned either to group I (diet + 750 mg UDA + fiber placebo) or group II (diet + 15 g Pp+ UDA placebo). An anthropometric evaluation was performed to each patient before and after the two-month treatment, as well as resting EE by indirect calorimetry, GBUS and endoscopy for the determination of cholesterol crystals in duodenal bile. Weight reduction was similar in both groups (group I = 6 +/- 2 Kg vs group II = 6 +/- 3 Kg). GD development was observed in one patient of group I (5.5%) and two patients of group II (p > 0.05). All patients with GD lost a minimum of 4 Kg during the study period. GD development did not correlate with the presence of crystals in the duodenal bile at the beginning of the study. Our results suggest a beneficial effect of a rational diet with fiber supplementation to prevent GD development in obese patients included in a weight reduction program.

Adult↗