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Biomedical subjects

J Costa

Publications and source records attributed to J Costa.

At least 91 records · Page 5Linked to original sources
PubMed1999

Crystal structure of cardosin A, a glycosylated and Arg-Gly-Asp-containing aspartic proteinase from the flowers of Cynara cardunculus L.

Aspartic proteinases (AP) have been widely studied within the living world, but so far no plant AP have been structurally characterized. The refined cardosin A crystallographic structure includes two molecules, built up by two glycosylated peptide chains (31 and 15 kDa each). The fold of cardosin A is typical within the AP family. The glycosyl content is described by 19 sugar rings attached to Asn-67 and Asn-257. They are localized on the molecular surface away from the conserved active site and show a new glycan of the plant complex type. A hydrogen bond between Gln-126 and Manbeta4 renders the monosaccharide oxygen O-2 sterically inaccessible to accept a xylosyl residue, therefore explaining the new type of the identified plant glycan. The Arg-Gly-Asp sequence, which has been shown to be involved in recognition of a putative cardosin A receptor, was found in a loop between two beta-strands on the molecular surface opposite the active site cleft. Based on the crystal structure, a possible mechanism whereby cardosin A might be orientated at the cell surface of the style to interact with its putative receptor from pollen is proposed. The biological implications of these findings are also discussed.

Amino Acid Sequence↗
PubMed1999

Crystal structure of plant aspartic proteinase prophytepsin: inactivation and vacuolar targeting.

We determined at 2.3 A resolution the crystal structure of prophytepsin, a zymogen of a barley vacuolar aspartic proteinase. In addition to the classical pepsin-like bilobal main body of phytepsin, we also traced most of the propeptide, as well as an independent plant-specific domain, never before described in structural terms. The structure revealed that, in addition to the propeptide, 13 N-terminal residues of the mature phytepsin are essential for inactivation of the enzyme. Comparison of the plant-specific domain with NK-lysin indicates that these two saposin-like structures are closely related, suggesting that all saposins and saposin-like domains share a common topology. Structural analysis of prophytepsin led to the identification of a putative membrane receptor-binding site involved in Golgi-mediated transport to vacuoles.

Amino Acid Sequence↗
PubMed1999

Comparative study of a modified competitive RT-PCR and Amplicor HCV monitor assays for quantitation of hepatitis C virus RNA in serum.

A modified competitive RT-PCR (mcRT-PCR) to measure HCV RNA in serum and the Amplicor HCV Monitor assay were compared. For mcRT-PCR, the RNA extracted was retrotranscribed and coamplified in one step with a known amount of a DNA internal control (IC). Digoxigenin-labeled amplified products were hybridized to specific HCV DNA and IC-DNA probes and quantified by colorimetry. HCV RNA concentration was calculated by plotting the ratio of HCV/IC ODs against a calibration curve. Multiple samples were analyzed in the same round and tedious titration of each sample with a competitor was unnecessary. The mcRT-PCR assay was linear from 6 x 10(3) to 6 x 10(7) copies/ml, whereas Amplicor was linear up to 1-2 x 10(6) copies/ml. HCV RNA was measured in samples from 75 carriers. There was agreement between both methods in type 1 infections but not in type 2 or type 3 infections, in which the values measured by Amplicor were, on average, 15 times lower than those measured by the mcRT-PCR. HCV RNA measured by Amplicor was higher in type 1 infections than in type 2 or 3 infections, but no differences were found when viral load was assessed by mcRT-PCR. The binding efficiency of the Amplicor-probe was greater for type 1 than for types 2 or 3, suggesting Amplicor underestimates the viral load in the latter types. In contrast, the mcRT-PCR is not affected by genotype-related variation of HCV. This study suggests that mcRT-PCR assay is reliable for sensitive and accurate measurement of HCV RNA over a broad range of values independently of the HCV genotype.

Genotype↗
PubMed1999

Prevalence and mother-to-infant transmission of hepatitis viruses B, C, and E in Southern Tanzania.

Hepatitis B and C markers were tested in 980 pregnant women, in the infants born to infected mothers, and in a random sample of 42 and 50, respectively, children born to uninfected mothers in Tanzania. Sixty-two women (6.3%) were positive for HBsAg and 15 (24%) were HBeAg-seropositive. Anti-HCV was detected in 49 women (5%), 15 (31%) of whom had detectable viremia. HCV RNA serum levels were low and only genotype 4 was identified. Sixty-six women (6.7%) were positive for anti-HIV, six of whom were coinfected with HBV and one with HCV. Anti-HEV was negative in the 180 women tested. At 8 months of age, HBsAg was detected in 8% and 2% of children born to HBV-infected and noninfected mothers, respectively (P = 0.2). Corresponding figures at 18 months of age were 31% and 21% (P = 0.3). When tested at 2 months of age, HCV RNA was not detected in any of the 43 children born to anti-HCV-positive mothers nor in any of 50 children born to anti-HCV-negative mothers. At 18 months, only one child, born to an anti-HCV-positive mother, had detectable HCV RNA. None of the infants born to women with HIV coinfection were infected with hepatitis viruses. This study suggests that exposure to HEV does not occur in southern Tanzania. The prevalence of current HBV infection in pregnant women from rural Tanzania is lower than in other sub-Saharan areas. In early childhood, HBV infection appears to occur by horizontal rather than maternofilial mechanisms of transmission. The prevalence of HCV infection is similar to that in other African countries. The results of this study show for the first time in Africa that mother-to-infant transmission does not play a significant role in the acquisition of HCV infection.

Adolescent↗
PubMed1999

Relationship of the genomic complexity of hepatitis C virus with liver disease severity and response to interferon in patients with chronic HCV genotype 1b infection [correction of interferon].

In patients with chronic hepatitis C, the influence of the genetic heterogeneity of the hepatitis C virus (HCV) on the progression of liver disease and on the responsiveness to interferon therapy is a matter of controversy. In this study we evaluated the genetic complexity of HCV by single-strand conformation polymorphism (SSCP) analysis of amplicons from the hypervariable region 1 (HVR1) in 168 patients with chronic genotype 1b HCV infection, of whom 122 received a single course of interferon therapy (3 MU, three times weekly for 6 months). No correlation was observed between the degree of genetic complexity of HCV (indicated by the number of bands in the SSCP assay) and patient age, serum alanine aminotransferase activity, or serum HCV-RNA concentration, measured by competitive polymerase chain reaction. HCV genomic complexity was not related to gender nor to the presumed source of infection. The number of SSCP bands detected in serum samples from patients with chronic hepatitis, either mild (8.1 +/- 3.9), moderate (8.0 +/- 3.3), or severe (9.2 +/- 3.3), and in patients with liver cirrhosis, either compensated (8.0 +/- 2.9), decompensated (6.3 +/- 2.9), or with superimposed hepatocellular carcinoma (9.5 +/- 2.9), was similar. The number of SSCP bands detected in patients with sustained response (7.5 +/- 3. 9), transient response (8.3 +/- 2.9), or no response (8.2 +/- 3.6) to interferon administration was similar as well. These observations suggest that the genetic complexity of hypervariable region (HVR1) of HCV, as estimated by SSCP analysis, is not related to the severity of liver injury nor to the type of response to interferon therapy. Thus, information offered by SSCP analysis of HVR1 of HCV in chronic HCV genotype 1b infection does not appear to be useful in the clinical management of these patients. (HEPATOLOGY 1999;29:897-903.)

Adult↗
PubMed1999

Gastropyeloplasty: a swine model.

OBJECTIVES: Struvite calculus formation requires an alkaline urinary pH. An acidic urinary pH will dissolve struvite calculi and prevent recurrent struvite stone formation. Long-term urinary acidification has been unsuccessful. We sought to determine whether a gastric patch (with viable parietal cells) anastomosed to the renal pelvis could create an acidic urinary milieu. METHODS: A vascularized stomach patch (from the greater curvature) was anastomosed to the left renal pelvis in 15 female pigs. The right kidney was used as a control. The first 6 pigs were used to refine the surgical technique. The remaining 9 pigs were subjected to a formal gastropyeloplasty and followed up for 4 weeks. Urine was collected before and after stimulation with pentagastrin. Urine pH was measured from both kidneys in response to gastrin stimulation and oral intake. The kidneys, ureters, and bladders were examined for gross changes and histologic review. RESULTS: The 9 test animals had more acidic urine in the control kidney than in the gastropyeloplasty kidney. Pentagastrin had no significant impact on urinary pH. Hydroureteronephrosis and a concentrating defect were noted in the treated kidney. Histologic review revealed smooth muscle hyperplasia of the left ureter and viable parietal cells in the stomach patch. CONCLUSIONS: An animal model was developed to transfer a gastric patch to the renal pelvis. Hydronephrosis and ureteral dilation were associated with this gastric patch. We were unable to acidify the urine despite viable parietal cells in the transposed stomach segment. Further refinements of this concept may be successful in acidifying urine in the hope of preventing recurrent struvite nephrolithiasis.

Anastomosis, Surgical↗
PubMed1999

Efficacy and tolerance of interferon-alpha(2b) in the treatment of chronic hepatitis C virus infection in haemodialysis patients. Pre- and post-renal transplantation assessment.

BACKGROUND: Hepatitis C virus (HCV) infection represents an important problem for the dialysis population due to its high prevalence and the long-term development of chronic liver disease, particularly following renal transplantation. METHODS: In order to assess the efficacy and tolerance of interferon (IFN) in the treatment of chronic hepatitis C in haemodialysis (HD) patients and their clinical course following renal transplantation, a multicentre, randomized, open-label study was conducted to compare IFN therapy vs a control group. RESULTS: Nineteen HCV RNA-positive patients received 3 x 10(6) U of IFN s.c., three times a week (post-HD), and 17 HCV RNA-positive patients were assigned to the control group. Tolerance to IFN therapy was good in nine patients, while treatment was discontinued in the other 10 due to the occurrence of side effects. HCV RNA was negative at the end of treatment in 14 out of 19 patients (74%) receiving IFN and in one patient (5%) in the control group. Six out of the 14 patients who initially responded to IFN therapy had a virological relapse (43%). Eight patients (42%) remained HCV RNA-negative, three of them until the day that renal transplantation (RT) was performed (7, 12 and 27 months, respectively), as did five patients on HD during the follow-up (27+/-5 months). Eight out of the nine patients (89%) who completed therapy were HCV RNA-negative at the end of treatment, and seven of them (78%) remained HCV RNA-negative during the follow-up on dialysis (21+/-8 months). Mean transaminase (ALT) values were significantly decreased following IFN therapy, while no changes were observed during the follow-up period in the control group. Fifteen patients (10 in the treatment group and five in the control group) underwent RT. Three patients in the treatment group were HCV RNA-negative at RT, and one of them had a virological relapse 20 months after RT, while the other two remained HCV RNA-negative at 3 months and 24 months after RT, respectively. In contrast to the control group, transaminase (ALT) remained within normal limits in all patients in the treatment group. Finally, during the post-RT follow-up, the transaminase mean values were significantly lower in treated patients vs patients in the control group (P<0.05). CONCLUSIONS: It is concluded that the biochemical and virological response to IFN therapy is good in HD patients. In addition, IFN therapy appears to exert a beneficial effect on the course of liver disease following RT, regardless of the virological response. Despite the fact that IFN therapy was discontinued in 10 out of the 19 patients due to the occurrence of side effects, these disappeared following discontinuation of therapy. Therefore, IFN therapy is advisable for HCV-infected dialysis patients who are candidates for RT.

Adult↗
PubMed1999

Analysis of K-ras, N-ras, H-ras, and p53 in lung neuroendocrine neoplasms.

This study screened 11 samples of typical carcinoid (TC), 4 samples of atypical carcinoid (AC), 1 sample of large cell neuroendocrine carcinoma (LCNEC), and four metastases for point mutations in exons 5 to 8 of the p53 gene, and exons 1 and 2 of the K-ras. H-ras, and N-ras genes using polymerase chain reaction (PCR)-single-strand conformation polymorphism (SSCP) and direct sequencing and by immunohistochemistry for p53. Exon 1 of K-ras was mutated in two samples of low-grade AC and a metastasis from one of these tumors (GAT12 and AGT12, respectively). No mutations in N-ras or H-ras were found. Mutations in exons 5 and 8 of the p53 gene were identified in a high-grade AC and a LCNEC. Positive immunostaining for p53 was present in three samples, with only one genotypic mutation shown (LCNEC). In conclusion, point mutations of the p53 gene were infrequent in these pulmonary neuroendocrine tumors, did not correlate in all samples with immunostaining, and were associated with the higher-grade tumors. Second, the presence of K-ras mutations seems to be associated with the higher-grade carcinomas. Third, N-ras and H-ras mutations were not found with these pulmonary neuroendocrine tumors.

Adult↗
PubMed1999

Effect of clozapine and adjunctive high-dose glycine in treatment-resistant schizophrenia.

OBJECTIVE: The focus of this study was the systematic evaluation of the clinical effects of glycine as an adjunct to the atypical antipsychotic clozapine in the treatment of schizophrenia. METHOD: In a double-blind, placebo-controlled study, 19 patients with chronic, treatment-resistant schizophrenia who were maintained on optimal doses of clozapine (400-1200 mg/day) were administered either 30 g/day of glycine (N=9) or placebo (N=10) for 12 weeks. Clinical evaluations with the Brief Psychiatric Rating Scale, the Scale for the Assessment of Negative Symptoms, and the Simpson-Angus movement scale were completed biweekly. RESULTS: The use of glycine as an adjunct to clozapine was not effective in decreasing positive or negative symptoms. In contrast, the patients treated with clozapine without glycine had a 35% reduction in positive symptoms. CONCLUSIONS: These preliminary data suggest that glycine may interfere with the antipsychotic efficacy of atypical neuroleptics such as clozapine.

Adult↗
PubMed1999

Tolerability and safety of 0.1% diclofenac plus 0.3% tobramycin fixed-dose ophthalmic solution: a randomized, comparative, controlled study in healthy volunteers.

The purpose of this double-blind, observer-masked, randomized, crossover trial was to compare the tolerability and safety of a fixed-dose ophthalmic solution of 0.3% tobramycin plus 0.1% diclofenac versus Tobrex (tobramycin sulfate ophth) and Voltaren (diclofenac sodium). Control treatments included a saline solution and a control solution of 0.3% tobramycin prepared by Alcon Cusí. Ten healthy volunteers received three consecutive instillations of 1 drop of a given ophthalmic solution at 08:00, 11:00 and 14:00 h to the same eye; after a washout period of 18 h, the next ophthalmic solution was tested according to a randomized sequence. Occurrence, intensity, and duration of ocular irritation and conjunctival hyperemia at baseline and after the three instillations were recorded. Slit lamp biomicroscopy examination, measurement of intraocular pressure (IOP) changes, visual acuity, and examination of the fundus of the eye were performed after each third instillation by an ophthalmologist. Side effect incidence and patient and investigator opinions were also recorded. Results showed that Voltaren instillation induced statistically significant ocular irritation (p = 0.0077); the remaining ophthalmic solutions tested caused no ocular irritation (Physiological Saline Braun, p = 0.9808; Tobrex, p = 0.8826; control 0.3% tobramycin solution, p = 0.8327; and 0.1% diclofenac plus 0.3% tobramycin, p = 0.5399). None of the ophthalmic solutions tested caused severe conjunctival hyperemia. Analysis of the sum of conjunctival parameters of both eyes for all ophthalmic solutions studied showed no statistically significant differences (p = 0.4688). Moderate superficial punctate keratitis was observed after instillation of Voltaren and of 0.1% diclofenac plus 0.3% tobramycin (1 subject each) that spontaneously resolved within 2 days. Slit lamp biomicroscopy, visual acuity and IOP values showed no statistically significant changes. No systemic side effects related to the study treatments were recorded. In conclusion, the ophthalmic solution containing 0.1% diclofenac plus 0.3% tobramycin was well tolerated under the study conditions. Its tolerability was equivalent to that of Braun physiological saline, Tobrex and a control 0.3% tobramycin solution and was better than that of Voltaren.

Adolescent↗
PubMed1999

Beta- and gamma-catenin mutations, but not E-cadherin inactivation, underlie T-cell factor/lymphoid enhancer factor transcriptional deregulation in gastric and pancreatic cancer.

Adenomatous polyposis coli (APC) mutations are present in >70% of colon cancers. The APC protein binds to beta-catenin (beta-cat), a protein first identified because of its role in E-cadherin (E-cad) cell adhesion. In some colon cancers lacking APC defects, mutations in presumptive glycogen synthase kinase 3beta phosphorylation sites near the beta-cat NH2 terminus appear to render beta-cat resistant to regulation by APC and glycogen synthase kinase 3beta. In cells with APC or beta-cat defects, beta-cat is stabilized and, in turn, binds to and activates T-cell factor (Tcf)/lymphoid enhancer factor (Lef) transcription factors. To further explore the role of APC, beta-cat, Tcf, and E-cad defects in gastrointestinal cancers, we assessed gastric and pancreatic cancers for constitutive Tcf transcriptional activity (CTTA). Two of four gastric and two of eight pancreatic cancer lines showed CTTA. One gastric and one pancreatic cancer had mutations in the NH2-terminal phosphorylation sites of beta-cat. The other gastric cancer with CTTA had a missense mutation at serine 28 of gamma-cat, a potential phosphorylation site in this beta-cat-related protein. Although E-cad is an important binding partner for beta-cat and gamma-cat, E-cad inactivation did not result in CTTA. The beta-cat and gamma-cat mutant proteins identified in our studies strongly activated Tcf transcription in vitro, whereas beta-cat mutant proteins with large NH2-terminal deletions had only modest effects on Tcf. Our results suggest a role for Tcf deregulation in gastric and pancreatic cancer, resulting from beta-cat and gamma-cat mutations in some cases and, in others, from yet to be defined defects. Furthermore, these data imply that the consequences of APC and beta-cat mutations are distinct from the effects of E-cad inactivation.

Adenomatous Polyposis Coli Protein↗
PubMed1999

[Extra-adrenal pheochromocytoma simulating acute myocardial infarction].

The authors present the case of a 65 year-old female who was admitted to the emergency room with epigastric pain, headache, palpitations, nausea, vomiting and sweating. The laboratory tests performed showed elevation of CK and CK-MB and the ECG presented sinus tachycardia. T-wave inversion and prolonged QT interval. The echocardiogram was normal. The patient was admitted to the ICU and during the standard myocardial infarction treatment (including beta-blocker) a wide range of the arterial pressure (230/140 to 70/40 mm Hg) was registered. In view of these new data, the hypothesis of pheochromocytoma and catecholamine induced myocarditis was suggested and later confirmed by high levels of plasmatic and urinary catecholamines. The abdominal echography. CT and MRI showed a large retroperitonal and para-aortic mass. The administration of phenoxybenzamine (30 mg/day) led to the normalization of the ECG and arterial pressure and the respective clinical improvement. The anatomopathological exam, after surgical removal, confirmed our hypothesis. Sixteen months after the surgical procedure the patient is assymptomatic, with normal arterial pressure and normal levels of plasmatic and urinary catecholamines.

Aged↗
PubMed1998

In vivo specificity of human alpha1,3/4-fucosyltransferases III-VII in the biosynthesis of LewisX and Sialyl LewisX motifs on complex-type N-glycans. Coexpression studies from bhk-21 cells together with human beta-trace protein.

Each of the five human alpha1,3/4-fucosyltransferases (FT3 to FT7) has been stably expressed in BHK-21 cells together with human beta-trace protein (beta-TP) as a secretory reporter glycoprotein. In order to study their in vivo properties for the transfer of peripheral Fuc onto N-linked complex-type glycans, detailed structural analysis was performed on the purified glycoprotein. All fucosyltransferases were found to peripherally fucosylate 19-52% of the diantennary beta-TP N-glycans, and all enzymes were capable of synthesizing the sialyl LewisX (sLex) motif. However, each enzyme produced its own characteristic ratio of sLex/Lex antennae as follows: FT7 (only sLex), FT3 (14:1), FT5 (3:1), FT6 (1.1:1), and FT4 (1:7). Fucose transfer onto beta-TP N-glycans was low in FT3 cells (11% of total antennae), whereas the values for FT7, FT5, FT4, and FT6 cells were 21, 25, 35, and 47%, respectively. FT3, FT4, FT5, and FT7 transfer preponderantly one Fuc per diantennary N-glycan. FT4 preferentially synthesizes di-Lex on asialo diantennary N-glycans and mono-Lex with monosialo chains. In contrast, FT6 forms mostly alpha1,3-difucosylated chains with no, one, or two NeuAc residues. FT3, FT4, and FT6 were proteolytically cleaved and released into the culture medium in significant amounts, whereas FT7 and FT5 were found to be largely resistant toward proteolysis. Studies on engineered soluble variants of FT6 indicate that these forms do not significantly contribute to the in vivo fucose transfer activity of the enzyme when expressed at activity levels comparable to those obtained for the wild-type Golgi form of FT6 in the recombinant host cells.

Amino Acid Sequence↗
PubMed1998

Transport and activation of the vacuolar aspartic proteinase phytepsin in barley (Hordeum vulgare L.).

The primary translation product of barley aspartic proteinase, phytepsin (EC 3.4.23.40), consists of a signal sequence, a propart, and mature enzyme forms. Here, we describe post-translational processing and activation of phytepsin during its transport to the vacuole in roots, as detected by using metabolic labeling and immunoprecipitation. After removal of the signal sequence, the glycosylated precursor of 53 kDa (P53) was produced and further processed to polypeptides of 31 and 15 kDa (P31 + P15) and, subsequently, to polypeptides of 26 and 9 kDa (P26 + P9), 45 min and 24 h after synthesis, respectively. The processing occurred in a late-Golgi compartment or post-Golgi compartment, because brefeldin A inhibited the processing, and P53 acquired partial endoglycosidase H resistance 30 min after synthesis, whereas P15 was completely resistant. The N-glycosylation inhibitor tunicamycin had no effect on transport, but the absence of glycans on P53 accelerated the proteolytic processing. Phytepsin was also expressed in baculovirus-infected insect cells. The recombinant prophytepsin underwent autoproteolytic activation in vitro and showed enzymatic properties similar to the enzyme purified from grains. However, a comparison of the in vitro/in vivo processing sites revealed slight differences, indicating that additional proteases are needed for the completion of the maturation in vivo.

Amino Acid Sequence↗