Molecular cloning of a new guanine nucleotide-exchange protein, EF1 delta.
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Biomedical subjects
Publications and source records attributed to J Morales.
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The cdc2 protein kinase phosphorylates elongation factor-1 gamma (EF-1 gamma) during meiotic maturation of Xenopus oocytes. A synthetic peptide P2: PKKETPKKEKPA matching the cDNA-deduced sequence of EF-1 gamma was an in vitro substrate for cdc2 protein kinase and inhibited phosphorylation of EF-1 gamma. Tryptic hydrolysis of EF-1 gamma and the P2 peptide, both phosphorylated by cdc2 protein kinase, resulted in multiple partial digestion products generated by the presence of barely hydrolysable bonds. The two peptides obtained from the hydrolysis of EF-1 gamma comigrated exactly in two-dimensional separation with two of the P2 peptide hydrolysates. EF-1 gamma therefore contains one unique phosphoacceptor for cdc2 protein kinase, identified as threonine-230.
The bacterial translocation hypothesis was tested in two studies (acute and subacute) in a porcine model of hemorrhagic shock. Male pigs (30-40 kg each) under general anesthesia had their femoral vein, femoral artery, and portal vein catheterized. After stabilization (1 hour) they were bled (40% of blood volume) over 30 minutes, then maintained in the hypotensive state (MAP = 30-40 mm Hg) for 2 hours, following which, according to randomization, they entered the control group or were resuscitated with whole blood (WB group) or with lactated Ringer's solution (LR group). In the acute study, the mesenteric efferent lymphatic was also cannulated, the control group was not resuscitated, and the animals remained under general anesthesia to the end of the experiment (8.5 hours), when gut tissue was obtained for histologic study and measurement of lipid peroxidation. In the subacute study, the control group was not bled, the animals were awakened at 6.5 hours, and the portal vein catheter remained in situ until 48 hours. In both studies, samples of portal blood were obtained for culture at regular intervals and on completion, samples from mesenteric lymph nodes (MLNs) for culture were taken in the acute study, and in the subacute study samples from MLNs, spleen, and liver were obtained. In the acute study significant bacterial translocation to the MLNs and portal blood did not occur among the controls (n = 3), the LR group (n = 5), and the WB group (n = 6). Significant evidence of lipid peroxidation was found in both the LR and WB groups. Histologic assessment showed no difference among the groups.(ABSTRACT TRUNCATED AT 250 WORDS)
We have examined the possible presence of color vision anomalies in 9 individuals (17 eyes, 1 blind) with fundus findings suggesting ocular albinism using the Ishihara plates, the 28-hue Roth test, and the Davico anomaloscope. Results indicate that four of these individuals show no sign of the anomalies expected in an albino in either of the two eyes. Of the remaining cases, two are simple deuteranomals in both eyes, according to Pickford's classification criteria. The rest have protanomaly; however, in these the deviation toward red appears in both eyes in only one subject, whereas in the other two subjects it appears in only one eye, their binocular color vision being basically normal. Our study shows that a large proportion of these albinos have photophobia, pendular nystagmus, strabismus, noticeable refractive errors (astigmatism and high myopia), and poor visual acuity [usually less than 6/30 (20/100) with correction]. The measurement of contrast sensitivity function (CSF) indicates that the frequency of 12 cpd cannot be perceived, even in binocular vision.
Persons who scored higher on a measure of self-defeating personality did not believe in a just world as implied in the writings of such theorists as Reik (1941); however, they had a greater awareness of anger and feelings of mistrust and suspiciousness of others, held anger in, and were self-critical. These latter findings are consistent with conclusions regarding masochism proposed in 1991 by Glickauf-Hughes and Wells.
Microalbuminuria may be due to increased glomerular capillary pressure. In turn, this increased pressure may be associated to augmented protein intake or reduction of renal mass. 85 normal subjects who had a unilateral nephrectomy were studied. Creatinine clearance, microalbuminuria and blood pressure were measured. Hyperfiltration was evaluated by comparison of creatinine clearance before and after nephrectomy. Protein intake was evaluated by a nutritional questionnaire. Hyperfiltration was estimated as 38% and microalbuminuria was not different in patients submitted to nephrectomy (9.8 micrograms/min) compared to controls (9.7). Microalbuminuria post nephrectomy was not correlated to level of hyperfiltration, protein intake, age, blood pressure or time after surgery. These results suggest that the remaining kidney is able to double the excretion of albumin with a likely increase in glomerular filtration and intracapillary pressure.
We report the pregnancy of a 35 year old diabetic woman with a positive Australia antigen, 3 years after a successful cadaver kidney transplant. Immunosuppressive therapy with prednisone, azathioprine and cyclosporine was maintained. Pregnancy was complicated by an acute rejection episode on week 13, mild hypertension and intrahepatic cholestasis on week 22. Cesarean section and tubal ligation were performed at 32 and a half weeks. A 2020 g female infant was delivered, with Apgar scores of 8 and 9 at 1 and 5 min, respectively. Mild respiratory distress, jaundice and hypocalcemia were noted. The Australia antigen was negative. No fetal anomalies were detected. The high risk of pregnancy in a class T diabetic patient is confirmed in this case.
Mitral balloon valvulotomy has proven to be an effective method for the treatment of patients with mitral stenosis. Although several factors determining an optimal immediate result have been described, there is few information regarding the longterm follow-up as well as factors influencing late success after the procedure. In this article, we analyzed our series of 311 patients treated by mitral balloon valvulotomy who were clinically followed between 3 months and 5 years (mean 23 +/- 11 months). At least, one echo-Doppler follow-up study was obtained in 260 patients, 20 +/- 10 months after. Hemodynamic re-evaluations were performed in 63 patients after 20 +/- 11 months. Late success was defined as the patient being in functional class I-II and free of major events (death, restenosis or valve surgery). Restenosis was defined as the loss of 50% of initial gain in terms of valve area, confirmed always hemodynamically. Major events during follow-up period occurred in 19 patients (10 deaths, 8 restenosis and 11 mitral valve surgery). We performed a multivariate study using the Cox-regression model. In the analysis, all variables with or near statistic significance in the univariate analysis (Mantel-Haezel) were included. The only significant independent predictors of late success were the presence of sinus rhythm (p < 0.04) and the absence of calcium at the valve (p < 0.001). In conclusion, the best results 5 years after mitral balloon valvulotomy are observed in patients with non calcified valve and sinus rhythm.
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This study presents the findings in 33 patients with discrete subaortic stenosis who were treated by percutaneous balloon dilation and were followed up for 2 months to 6.2 years (mean 34 +/- 21 months). The mean age was 13 +/- 11 years; 10 (30%) were female and 23 (70%) male. Associated malformations were observed in nine patients (27%). All patients underwent noninvasive studies and cardiac catheterization. The mean value to membrane distance was 4.5 +/- 2 mm/m2. After balloon dilation, the pressure gradient from the left ventricle to the aorta decreased from 68 +/- 30 to 20 +/- 13 mm Hg (p less than 0.00001); there were no significant changes in the degree of aortic regurgitation. A fluttering and widely mobile remaining membrane was clearly visualized after dilation. Better immediate results were obtained in patients with a smaller baseline gradient, a larger aortic anulus and a longer valve to membrane distance. Serial follow-up echographic studies were available in 30 patients, and 18 hemodynamic reevaluations were performed in 13 patients. However, seven patients who demonstrated restenosis underwent redilation at a mean of 29 +/- 17 months after the first dilation. Redilation in six of the seven patients obtained benefits similar to those observed at the first dilation. Only one patient with unsuccessful redilation required surgery. The mean value of the last explored residual gradient (on hemodynamic or Doppler study) in the remaining 32 patients was 21 +/- 10 mm Hg. No significant changes were observed in the angiographic evolution of aortic regurgitation.(ABSTRACT TRUNCATED AT 250 WORDS)
Protein synthesis is believed to be under control of the cell cycle during meiosis and mitosis. Any relationship between substrates for cdc2 kinase and components of the protein synthetic apparatus would therefore be of prime importance. During meiosis of Xenopus laevis oocytes one of the substrates for this kinase is a p47 protein, which is complexed to two other proteins, P36 and P30. Judged from partial amino acid sequence data on P47 and P30, the P30 and P47 proteins were reported to resemble the protein synthetic elongation factors (EF) 1 beta and 1 gamma from Artemia salina (Bellé, R., Derancourt, J., Poulhe, R., Capony, J.P., Ozon, R., and Mulner-Lorillon, O. (1989) FEBS Lett. 255, 101-104). This paper shows that the complex composed of P30, P47, and P36 from Xenopus is identical to the complex of EF-1 beta, EF-1 gamma, and EF-1 delta from Artemia according to two criteria. 1) Both stimulate elongation factor 1 alpha-mediated transfer RNA binding to ribosomes and exchange of guanine nucleotides on elongation factor 1 alpha to a comparable degree. 2) Each of the three subunits of the protein complex P30.P47.P36 from Xenopus shows a structural homology with one of the corresponding subunits of EF-1 beta gamma delta from Artemia. Presumably the phosphorylation of EF-1 gamma, which associates with tubulin at least in vitro, is important in processes following the onset of meiosis which is accompanied by a rise of protein synthesis.
Thesaurin a is one of two protein components of a 42 S ribonucleoprotein particle that is very abundant in previtellogenic oocytes of Xenopus laevis. The primary function of the 42 S particle is the long-term storage of 5 S RNA and aminoacyl-tRNA. Thesaurin a is homologous to eukaryotic elongation factor 1 alpha (EF-1 alpha) and to prokaryotic elongation factor Tu (EF-Tu). Sequence comparison with EF-1 alpha and EF-Tu of different species indicates that thesaurin a is rather distantly related to all eukaryotic elongation factors. In spite of this, the secondary structure of thesaurin a, deduced from hydrophobic cluster analysis, is remarkably similar to that of EF-1 alpha and EF-Tu. The binding and catalytic properties of thesaurin a are also similar but not identical to those of EF-1 alpha. Like EF-1 alpha, purified thesaurin a binds tRNA, GDP, and GTP. Unlike EF-1 alpha, thesaurin a binds discharged tRNA more tightly than charged tRNA, and GTP more tightly than GDP. Thesaurin a also hydrolyzes GTP and catalyzes the mRNA-dependent binding of aminoacyl-tRNA to 80 S ribosomes. The functional properties of the 42 S particle are in general agreement with those of purified thesaurin a. In particular, the 42 S particle contains GTP and efficiently transfers aminoacyl-tRNA to 80 S ribosomes without addition of exogenous elongation factor.
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Elongation factor 1 alpha (EF-1 alpha) was purified to homogeneity from full-grown oocytes of Xenopus laevis. This protein is encoded by a gene previously shown to be expressed in male and female germ cells, and repressed in somatic cells. The purified protein was identified with EF-1 alpha on criteria of molecular mass, cross-reaction with antibodies raised against Artemia salina EF-1 alpha, affinity for guanine nucleotides, and ability to promote the mRNA-dependent binding of aminoacyl tRNA to 80S ribosomes.
We have purified and partially sequenced the EF-1 alpha protein from Xenopus laevis oocytes (EF-1 alpha O). We show that the two cDNA clones isolated by Coppared et al. (Coppard, N. J., K. Poulsen, H. O. Madsen, J. Frydenberg, and B. F. C. Clark. 1991. J. Cell Biol. 112:237-243) do not encode 42Sp50, as claimed by these authors, but two very similar forms of EF-1 alpha O (EF-1 alpha O and EF-1 alpha O1). 42Sp50 is the major protein component of a 42S nucleoprotein particle that is very abundant in previtellogenic oocytes of X. laevis, 42Sp50 differs from EF-1 alpha O not only by its amino acid sequence, but also by several properties already reported. In particular, 42Sp50 has a low EF-1 alpha activity. It is distributed uniformly in the cytoplasm of previtellogenic oocytes, in contrast to EF-1 alpha O which is concentrated in a small region of the cytoplasm, known as the mitochondrial mass or Balbiani body.
Endothelial cells that make up brain capillaries and constitute the blood-brain barrier become different from peripheral endothelial cells in response to inductive factors found in the nervous system. We have established a cell culture model of the blood-brain barrier by treating brain endothelial cells with a combination of astrocyte-conditioned medium and agents that elevate intracellular cAMP. These cells form high resistance tight junctions and exhibit low rates of paracellular leakage and fluid-phase endocytosis. They also undergo a dramatic structural reorganization as they form tight junctions. Results from these studies suggest modes of manipulating the permeability of the blood-brain barrier, potentially providing the basis for increasing the penetration of drugs into the central nervous system.
Bacterial translocation is proposed as an explanation for sepsis associated with hemorrhagic shock. This study attempted to document these events in a large animal model. Male swine were randomly assigned to control (n = 10) or experimental (n = 10) groups. Animals were anaesthetized, and the bladder, portal vein, and a mesenteric lymphatic vessel cannulated. Experimental animals were bled 40% of blood volume. Over the next six hours maintenance fluids were given, and cultures of portal blood and mesenteric lymph taken. Before the swine were killed, cultures were taken from portal and systemic blood, mesenteric lymph, and lymph nodes, and a portion of terminal ileum was resected for histologic study. Experimental animals experienced significant shock as demonstrated by changes in hemodynamic and biochemical variables. Cultures and histologic examination of the terminal ileum showed no significant difference between control and experimental animals. In an unresuscitated swine model, significant bacterial translocation was not demonstrated within six hours of hemorrhagic shock.