Biomedical subjects
Publications and source records attributed to C Figueroa.
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Localization of Ras and Ras-like proteins to the correct subcellular compartment is essential for these proteins to mediate their biological effects. Many members of the Ras superfamily (Ha-Ras, N-Ras, TC21, and RhoA) are prenylated in the cytoplasm and then transit through the endomembrane system on their way to the plasma membrane. The proteins that aid in the trafficking of the small GTPases have not been well characterized. We report here that prenylated Rab acceptor protein (PRA1), which others previously identified as a prenylation-dependent receptor for Rab proteins, also interacts with Ha-Ras, RhoA, TC21, and Rap1a. The interaction of these small GTPases with PRA1 requires their post-translational modification by prenylation. The prenylation-dependent association of PRA1 with multiple GTPases is conserved in evolution; the yeast PRA1 protein associates with both Ha-Ras and RhoA. Earlier studies reported the presence of PRA1 in the Golgi, and we show here that PRA1 co-localizes with Ha-Ras and RhoA in the Golgi compartment. We suggest that PRA1 acts as an escort protein for small GTPases by binding to the hydrophobic isoprenoid moieties of the small GTPases and facilitates their trafficking through the endomembrane system.
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B-Raf contains multiple Akt consensus sites located within its amino-terminal regulatory domain. One site, Ser(364), is conserved with c-Raf but two additional sites, Ser(428) and Thr(439), are unique to B-Raf. We have investigated the role of both the conserved and unique phosphorylation sites in the regulation of B-Raf activity in vitro and in vivo. We show that phosphorylation of B-Raf by Akt occurs at multiple residues within its amino-terminal regulatory domain, at both the conserved and unique phosphorylation sites. The alteration of the serine residues within the Akt consensus sites to alanines results in a progressive increase in enzymatic activity in vitro and in vivo. Furthermore, expression of Akt inhibits epidermal growth factor-induced B-Raf activity and inhibition of Akt with LY294002 up-regulates B-Raf activity, suggesting that Akt negatively regulates B-Raf in vivo. Our results demonstrate that B-Raf activity can be negatively regulated by Akt through phosphorylation in the amino-terminal regulatory domain of B-Raf. This cross-talk between the B-Raf and Akt serine/threonine kinases is likely to play an important role in modulating the signaling specificity of the Ras/Raf pathway and in promoting biological outcome.
The activity of allopurinol-loaded polyethylcyanoacrylate nanoparticles against Trypanosoma cruzi was compared to that of free allopurinol using in vitro cultures of epimastigotes. Ethylcyanoacrylate nanoparticles were prepared by an emulsion polymerization process, and formulations containing different concentrations of allopurinol, polyethylcyanoacrylate and surfactants were investigated and analyzed in size and amount of drug entrapped. The nanoparticles obtained were less than 200 nm in size, as measured by electron microscopy and cytometry. The peak amount of allopurinol entrapped in the nanoparticles was 62.8+/-1.9 microg mg(-1) of nanoparticles using 400 microl of polyethylcyanoacrylate, 200 microl of surfactant (Tween 20) and 20 mg of allopurinol in 50 ml of polymerization medium and the association efficiency was 100.7%. After 6 h of incubation at pH 7.4 the release of allopurinol from the nanoparticles was 7.4%, while at pH 1.2 only 3.1% was released after 4-6 h (t=42.8, P<0.0001). The in vitro studies, using cultures of T. cruzi epimastigotes, demonstrated considerable increases in the trypanocidal activity of the allopurinol-loaded nanoparticles in comparison with a standard solution of allopurinol (91.5 vs. 45.9%) at an allopurinol concentration of 16.7 microg ml(-1). In addition, it was shown that the unloaded nanoparticles, by mechanisms not completely elucidated, had a trypanocidal activity similar to that of standard solutions of allopurinol. To study cytotoxicity, increasing concentrations of unloaded nanoparticles were incubated on vero-line cell cultures. The concentration that killed 50% cells was 200 microg ml(-1), four times higher than that necessary to kill 50% of T. cruzi. It is concluded that the polyethylcyanoacrylate nanoparticles constitute a good carrier of drugs against the T. cruzi. The allopurinol loaded-nanoparticles significantly increased the trypanocidal activity in comparison to the free drug.
1. In the rat balloon injury model, angiotensin-converting enzyme (ACE) inhibitors prevent vascular remodelling by inhibiting angiotensin II generation and kinin breakdown. We investigated if ACE inhibition also prevents the structural vascular responses to disruption of carotid artery blood flow and if kinin potentiation plays a role in such a protection. 2. Morphometric analysis of the structural alterations caused by ligation of the left carotid artery was performed 14 days after surgery in J129Sv wild-type mice (B(2)(+/+)) drinking normal tap water or water containing captopril (120 mg kg(-1) per day). In addition, the effect of captopril on vascular remodelling was tested in B(2)(+/+) given the bradykinin (BK) B(1) receptor antagonist des-Arg(9)-[Leu(8)]-BK (DALBK, 50 nmol kg(-1) per day, intraperitoneally) or the BK B(2) receptor antagonist D-Arg, [Hyp(3),Thi(5)D-Tic(7),Oic(8)]-BK (icatibant, 1 micromol kg(-1) per day, intraperitoneally), and in B(2) receptor gene knockout mice (B(2)(-/-)). 3. Interruption of blood flow resulted in carotid artery intimal hyperplasia and media thickening in untreated B(2)(+/+), these responses being partially suppressed by captopril. The inhibition of intimal thickening exerted by captopril was reduced in B(2)(+/+) given DALBK or icatibant (P<0.05 for both comparisons) as well as in B(2)(-/-) (P<0.05). Neither antagonism of kinin receptors nor disruption of the B(2) receptor gene altered the suppressive effect of captopril on media thickening. The protection of vascular wall structure was independent of the reduction in blood pressure by captopril. 4. These results demonstrate that kinins participate in the inhibitory effect of captopril on intimal hyperplasia via B(1) and B(2) receptor signalling. Our findings may have important implications in treating vascular remodelling evoked by altered shear stress conditions.
OBJECTIVE: The aim of this study was to evaluate the effect of Helicobacter pylori (H. pylori) eradication on the natural history of duodenal ulcer disease and the reinfection rate after treatment in a developing country. METHODS: A total of 111 H. pylori-infected patients with duodenal ulcer were treated with either omeprazole or famotidine plus two antibiotics for 2 wk. Those failed to respond to treatment were retreated with bismuth-based triple therapy. RESULTS: The radication rate was 76% (95% CI: 67-83%). Eventually, H. pylori was eradicated in 96 of the 111 patients (86%), who were followed-up clinically and endoscopically for a mean of 37.2 months. The cumulative reinfection rate after eradication (Kaplan-Meier) was 8%+/-3% in yr 1, 11%+/-4% in yr 2, and 13%+/-4% in yr 3. Nine of the 12 reinfections occurred during yr 1. Recurrence of duodenal ulcer was detected in five patients (5.2%), all of them during yr 1 of follow-up. Histologically, gastritis scores (according to the Sydney system) improved significantly after eradication. CONCLUSIONS: In a high prevalence setting, H. pylori eradication and early reinfection rates after treatment are similar to rates observed in a low prevalence environment, whereas the late reinfection rate seems to be higher. However, up to 3 yr after treatment, most treated patients are free of H. pylori infection and/or ulcer activity. Even longer follow-up studies are necessary to determine whether specific retreatment policies are necessary to maintain long term eradication in developing countries.
Peroxisomes are essential subcellular organelles, which appear to be derived from pre-existing organelles. This biogenetic mechanism assumes the presence of peroxisomes in either or both mammalian gametes (sperms and/or oocytes). In order to test the presence and subcellular localization of peroxisomal proteins in rat sperms and oocytes, the authors carried out fractionation and immunofluorescence experiments. The results showed that rat oocytes contain peroxisome-like structures, which were detected by indirect immunofluorescence, using an antisera against total peroxisomal proteins. In contrast, such structures were not detected in rat sperms, which appear to contain catalase localized in the cell cytosol. The results reported herein show evidence for the first time of the presence of peroxisome-like structures in mammalian oocytes, and provide evidence for the peroxisome biogenesis hypothesis, by division of pre-existing organelles.
BACKGROUND: In Chile, cerebrovascular diseases are the fifth cause of death among men and the third cause among women. AIM: To assess the clinical features and management of patients with cerebrovascular disease admitted to a public hospital during 1997. PATIENTS AND METHODS: A retrospective analysis of clinical records of patients discharged with a diagnosis of cerebrovascular disease. Those records in which there was discordance between the discharge diagnosis and the clinical picture were not considered in the analysis. RESULTS: Of the 563 discharges from the hospital with the diagnosis of cerebrovascular disease, 487 records were located and 450 were considered in the analysis. Fifty four percent of patients were male and ages ranged from 17 to 96 years old. Fifty-one percent of patients had an ischemic stroke, 34% a cerebral hemorrhage, 12% a subarachnoidal hemorrhage and 3% a transient ischemic attack. There was a history of hypertension in 64% patients and 20% had an adequate treatment. Eighteen percent were diabetics, 34% had a heart disease and 20% had a previous episode of stroke. Mean hospital stay was 6.3 days in the emergency room and 11 days in the neurology ward. Hospital infections appeared in 21% of patients (respiratory in 68% and urinary in 22%), lethality was 30.5% and a CAT scan was done in 94%. At the moment of admission, 10% of patients had an evolution of less than 2 hours, 27% had an evolution between 2 and 6 hours and nine cases were potential eligible for thrombolysis. CONCLUSION: This is a picture of the local features of patients with cerebrovascular diseases that can be used as a reference for future studies.
Major histocompatibility complex (MHC) class II deficiency is a rare form of primary combined immunodeficiency that can only be corrected by stem cell transplantation. We report a 4(1/2)-year-old girl with MHC class II deficiency who underwent a related CBT due to graft failure following T cell-depleted non-identical BMT. The patient is alive and well 2 years after the second transplant. A sustained hematopoietic engraftment and a progressive immune recovery have been detected. We conclude that cord blood may be an effective source of hematopoietic stem cells for patients with immuno- deficiency disorders including diseases with a high rate of graft failure.
OBJECTIVE: The clinical usefulness of preoperative localization and intraoperative PTH assay (QPTH) in primary hyperparathyroidism have been established. However, without the use of QPTH, the parathyroidectomy failure rate remains 5% to 10% in large reported series and is probably much higher in the hands of less experienced parathyroid surgeons. Persistent hypercalcemia requires another surgical procedure. The authors compared the outcomes in 50 consecutive patients undergoing more difficult secondary parathyroidectomy with and without the adjunctive support of QPTH. METHODS: Two groups of similar patients underwent reoperative parathyroidectomy for failed surgery or recurrent disease. The successful return to normocalcemia in group I, with QPTH used to localize and confirm complete excision of all hyperfunctioning glands, was compared with group II, who did not have this intraoperative adjunct. RESULTS: In 31/33 patients in group I, calcium levels returned to normal. With good preoperative localization studies, 17 patients underwent successful straightforward parathyroidectomies as predicted by QPTH. In the other 14 patients, QPTH assay proved extremely beneficial by facilitating localization with differential venous sampling; measuring the increase in hormone secretion after massage of specific areas; recognizing suspicious nonparathyroid tissue excised without a decrease in hormone levels, avoiding frozen-section delay; and correctly identifying the excision of abnormal tissue despite false-positive/false-negative sestamibi scans. In group II, who underwent surgery before QPTH was available, 4 of 17 patients (24%) remained hypercalcemic after extensive reexploration. CONCLUSION: With the intraoperative hormone assay used to facilitate localization and confirm excision of all hyperfunctioning tissue, the success rate of reoperative parathyroidectomy has improved from 76% to 94%.
BACKGROUND: Gallbladder cancer is frequent in Chile and there is sparse information about the association between this type of cancer and the presence of bacteria in the gallbladder bile. AIM: To determine the presence of aerobic bacteria in gallbladder bile in patients with and without gallbladder cancer. MATERIAL AND METHODS: A microbiological analysis of bile and pathological study was performed in 608 gallbladders, obtained during to cholecystectomies performed to 513 women and 95 men aged 44 years old as a mean. RESULTS: Pathological study showed a chronic cholecystitis in 468 cases (77%), an acute cholecystitis in 140 (33%), cancer in 24 (3.9%) and dysplasia in 5 cases (0.8%). A positive culture was obtained in 22.5% of women and 28.5% of males. Twenty seven percent of women over 30 years old had positive cultures compared with 10% of younger women (p < 0.001). Thirty two percent of acute cholecystitis had positive cultures, compared with 24% of chronic cholecystitis (p = 0.03). E Coli was isolated in 51% of positive cases, Streptococci-Enterococci in 24%, Enterobacter sp in 9%, Klebsiella and Proteus in lower proportions. Salmonella sp was isolated in 4 cases, being all women with chronic cholecystitis. Thirteen of 29 cases with cancer or dysplasia had positive cultures (45%), compared with 25% of patients with inflammatory gallbladder diseases (p = 0.02). Streptococci-Enterococci were isolated in 7 cases and Enterobacter sp in three. CONCLUSIONS: The presence of Salmonella sp in gallbladder bile was not frequent in the studied patients. Its role in the pathogenesis of gallbladder cancer must be reassessed.
Severe aplastic anemia is a hematological disease with a high mortality rate, for which bone marrow transplantation is the treatment of choice, specially in children and young adults. The number of transfusions undergone before the transplant is the most important factor to predict the possibility of graft failure. Twenty patients with severe aplastic anemia, most of them already multiple transfused, were transplanted utilizing cyclophosphamide combined with antilymphocyte globulin as a conditioning regiment. All the evaluable patients engrafted and there were no episodes of graft failure. Three patients died, and 17 (85%) are alive with hematopoietic recovery at a median of 27.7 months post-transplant. Bone marrow transplantation was an excellent therapeutic option in this series of patients with severe aplastic anemia and the conditioning regiment appeared to be sufficiently myeloablative and immunosuppressive to avoid early or late graft failure.
To understand the regulation of rat uterine kallikrein, we evaluated its variations in animals that had been ovariectomized and supplemented with estradiol or progesterone, in pseudopregnant animals intraluminally oil-stimulated or unstimulated, and in unilaterally pregnant animals. The content of kallikrein, determined by an RIA highly specific for rK1 (true tissue kallikrein), rose in ovariectomized rats with estradiol supplementation (0.28 +/- 0.03 to 0.44 +/- 0.05 ng/mg) and decreased with progesterone (0.13 +/- 0.02 ng/mg; n = 15; p < 0.001). Kallikrein content rose from Day 1 of pseudopregnancy (PP1) to a maximum on PP7 (0.18 +/- 0.01 to 0.39 +/- 0.04 ng/mg protein; n = 36; p < 0.001). On PP7 with unilateral oil intraluminal stimulation, the decidualized horn had higher kallikrein content than did the contralateral (0.98 +/- 0.09 vs. 0.35 +/- 0.05 ng/mg protein; n = 7; p < 0.001). Immunocytochemistry revealed that mainly rK1 is localized in the luminal and glandular epithelium, and it increased in the stimulated horn. In the unilaterally pregnant rat on Day 7, the fertile horn had a higher kallikrein content than its contralateral control (0.71 +/- 0.07 vs. 0.37 +/- 0.03 ng/mg protein, p < 0.001; n = 8), as well as a higher kininogenase activity (239 +/- 34.3 vs. 83.5 +/- 7.9 ng bradykinin(BK)/h per horn, p < 0.003; and 945 +/- 90 vs. 585 +/- 40 ng BK/h per gram tissue, p < 0.002; n = 6). These results indicate that estrogen stimulates, whereas progesterone inhibits, kallikrein production, and that hormonal regulation is overridden by intraluminal stimulation, thus associating the enzyme with decidualization.
OBJECTIVES: To compare the diagnostic accuracy of the most widely available tests for diagnosis of Helicobacter pylori infection after antibiotic treatment. METHODS: A total of 59 H. pylori-positive, duodenal ulcer patients (mean age, 40.7 +/- 11.7 yr; 40 male and 19 female) were treated for 2 wk with either amoxicillin-metronidazole (n = 36) or omeprazole-amoxicillin-tinidazole (n = 23), and after 4 wk, were tested for H. pylori infection by [14C]urea breath test (UBT), serum IgG antibody level, and multiple antral biopsies for rapid urease testing, histology, Warthin-Starry stain, and polymerase chain reaction to detect H. pylori DNA. Infection status was established by a concordance of test results. RESULTS: H. pylori was eradicated in 47 patients (80%). UBT and rapid urease testing had the best sensitivity and specificity, although not statistically different to Warthin-Starry stain and polymerase chain reaction. Serology and histology had little diagnostic value in this setting due to high proportion of false-positive results. CONCLUSIONS: Noninvasive UBT is as accurate in predicting H. pylori status after antibiotic treatment as rapid urease testing and Warthin-Starry stain. Especially for duodenal ulcer patients, UBT could be considered the gold standard to confirm eradication of H. pylori.
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The gene for spinocerebellar ataxia type 2 (SCA2) has been mapped to 12q24.1. A 1.1-megabase contig in the candidate region was assembled in P1 artificial chromosome and bacterial artificial chromosome clones. Using this contig, we identified a CAG trinucleotide repeat with CAA interruptions that was expanded in patients with SCA2. In contrast to other unstable trinucleotide repeats, this CAG repeat was not highly polymorphic in normal individuals. In SCA2 patients, the repeat was perfect and expanded to 36-52 repeats. The most common disease allele contained (CAG)37, one of the shortest expansions seen in a CAG expansion syndrome. The repeat occurs in the 5'-coding region of SCA2 which is a member of a novel gene family.
Various proteases have been shown to be present in malignant breast tissue. Although the question of the involvement of tissue kallikrein, a serine protease, in the pathophysiology of tumours has been raised, the presence of this enzyme in human breast carcinoma has so far not been examined. In the present study, both neoplastic and normal human breast are scanned by immunocytochemistry for the presence and cellular localization of tissue kallikrein. In the healthy breast, tissue kallikrein was observed as a deposit of immunoreactive material that localized in the apical portion of duct cells. In the malignant breast tumours surveyed, the enzyme was observed only in ductal carcinomas, whereas lobular carcinomas were devoid of immunostaining. In ductal carcinomas, the immunoreactivity for tissue kallikrein appeared to be associated with gradations of malignancy, being absent in dedifferentiated tumours. The presence of tissue kallikrein in malignant breast tumours poses the question of the role of this enzyme in malignant breast tissue. The enzyme may participate within the tissue either in proteolytic processes (it has been shown to activate procollagenase) or by enhancing vascularity or mitogenicity by the generation of kinins.