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1.
Arq. bras. med. vet. zootec ; 66(2): 573-578, Jan.-Apr. 2014. graf, tab
Article in Portuguese | LILACS | ID: lil-709299

ABSTRACT

O objetivo deste trabalho foi avaliar a variação do perfil proteico e do cálcio solúvel na coagulação do leite pelo etanol nas temperaturas de 4ºC, 10ºC, 15ºC e 20ºC. Amostras de leite de 61 animais foram avaliadas quanto à estabilidade ao etanol nas concentrações de 66 a 92 por cento (v/v) nas temperaturas de 4ºC, 10ºC, 15ºC e 20ºC. Três amostras, após 24 horas de armazenamento a 4ºC, foram ultracentrifugadas em quadruplicata (40.000 x g) a 4ºC e a 20ºC, respectivamente, por 60 minutos. Em seguida, o sobrenadante foi retirado e submetido à análise do cálcio solúvel pela técnica via úmida (digestão nitroperclórica) e leitura em espectrofotômetro de absorção atômica. O perfil proteico foi analisado pela técnica de eletroforese capilar empregando kit específico para determinação proteica. Os resultados mostraram uma correlação positiva entre o aumento da temperatura das amostras e a estabilidade do leite frente às diferentes concentrações de etanol. A porcentagem de cálcio solúvel no sobrenadante após ultracentrifugação foi maior nas amostras tratadas a 4ºC (P<0,05). As amostras ultracentrifugadas na temperatura de 4ºC apresentaram quantidades superiores de β-caseína no sobrenadante em comparação com as amostras tratadas a 20ºC. O abaixamento da temperatura favoreceu a migração da β-caseína e do cálcio coloidal para a fase solúvel do leite, o que possivelmente favoreceu o aumento da instabilidade das amostras no teste do etanol. Os resultados sugerem que a temperatura ideal para a realização de teste de estabilidade do leite frente ao etanol deveria ser de 21ºC...


The aim of this study was to evaluate the variation in protein profile and soluble calcium in milk coagulation by ethanol at 4ºC, 10ºC, 15ºC and 20ºC. Milk samples from 61 dairy cows were evaluated for stability of ethanol concentrations from 66 to 92 percent (v/v) at temperatures of 4°C, 10°C, 15°C and 20°C. Three samples were ultracentrifuged (40,000 x g) after 24 hours of storage at 4°C and 20°C, respectively, for 60 minutes. Their supernatants were removed and subjected to analyses of soluble calcium through nitro-perchloric digestion and atomic absorption spectrophotometry. The protein profiles were determined by capillary electrophoresis using a specific kit for protein determination. The results showed a positive correlation between the increase in temperature of the samples and the stability of milk against various concentrations of ethanol. The percentage of soluble calcium in the supernatant after centrifugation was higher in samples treated at 4°C (P<0.05). The samples ultracentrifuged at 4°C showed higher amounts of β-casein in the supernatant compared with samples stored at 20°C. The lowering of the temperature favored the migration of β-casein and colloidal calcium to the soluble phase of milk, which may also have favored the instability of milk in the ethanol test. According to the results, the milk sample temperature for the ethanol stability test should be 21ºC...


Subject(s)
Animals , Calcium/chemistry , Ethanol/adverse effects , Milk Proteins/chemistry , Ultracentrifugation , Milk/metabolism , Transition Temperature
2.
Braz. j. med. biol. res ; 44(1): 16-22, Jan. 2011. ilus, tab
Article in English | LILACS | ID: lil-571364

ABSTRACT

Alpha-thalassemia is the most common inherited disorder of hemoglobin synthesis. Genomic deletions involving the alpha-globin gene cluster on chromosome 16p13.3 are the most frequent molecular causes of the disease. Although common deletions can be detected by a single multiplex gap-PCR, the rare and novel deletions depend on more laborious techniques for their identification. The multiplex ligation-dependent probe amplification (MLPA) technique has recently been used for this purpose and was successfully used in the present study to detect the molecular alterations responsible for the alpha-thalassemic phenotypes in 8 unrelated individuals (3 males and 5 females; age, 4 months to 30 years) in whom the molecular basis of the disease could not be determined by conventional methods. A total of 44 probe pairs were used for MLPA, covering approximately 800 kb from the telomere to the MSLN gene in the 16p13.3 region. Eight deletions were detected. Four of these varied in size from 240 to 720 kb and affected a large region including the entire alpha-globin gene cluster and its upstream regulatory element (alpha-MRE), while the other four varied in size from 0.4 to 100 kb and were limited to a region containing this element. This study is the first in Brazil to use the MLPA method to determine the molecular basis of alpha-thalassemia. The variety of rearrangements identified highlights the need to investigate all cases presenting microcytosis and hypochromia, but without iron deficiency or elevated hemoglobin A2 levels and suggests that these rearrangements may be more frequent in our population than previously estimated.


Subject(s)
Adolescent , Adult , Child, Preschool , Female , Humans , Infant , Male , Young Adult , DNA Probes/genetics , Multiplex Polymerase Chain Reaction , Mutation/genetics , alpha-Globins/genetics , alpha-Thalassemia/genetics , Brazil , Genotype , Pedigree , Phenotype , Sensitivity and Specificity , alpha-Thalassemia/diagnosis
3.
Braz. j. med. biol. res ; 43(7): 634-644, July 2010. ilus, graf
Article in English | LILACS | ID: lil-550731

ABSTRACT

The effectiveness of the caspase-9-based artificial "death switch" as a safety measure for gene therapy based on the erythropoietin (Epo) hormone was tested in vitro and in vivo using the chemical inducer of dimerization, AP20187. Plasmids encoding the dimeric murine Epo, the tetracycline-controlled transactivator and inducible caspase 9 (ptet-mEpoD, ptet-tTAk and pSH1/Sn-E-Fv’-Fvls-casp9-E, respectively) were used in this study. AP20187 induced apoptosis of iCasp9-modified C2C12 myoblasts. In vivo, two groups of male C57BI/6 mice, 8-12 weeks old, were injected intramuscularly with 5 µg/50 g ptet-mEpoD and 0.5 µg/50 g ptet-tTAk. There were 20 animals in group 1 and 36 animals in group 2. Animals from group 2 were also injected with the 6 µg/50 g iCasp9 plasmid. Seventy percent of the animals showed an increase in hematocrit of more than 65 percent for more than 15 weeks. AP20187 administration significantly reduced hematocrit and plasma Epo levels in 30 percent of the animals belonging to group 2. TUNEL-positive cells were detected in the muscle of at least 50 percent of the animals treated with AP20187. Doxycycline administration was efficient in controlling Epo secretion in both groups. We conclude that inducible caspase 9 did not interfere with gene transfer, gene expression or tetracycline control and may be used as a safety mechanism for gene therapy. However, more studies are necessary to improve the efficacy of this technique, for example, the use of lentivirus vector.


Subject(s)
Animals , Male , Mice , Anemia/therapy , Caspase 9/genetics , Dimerization , Erythropoietin , Gene Expression/genetics , Genetic Therapy/methods , Tacrolimus/analogs & derivatives , Caspase 9/administration & dosage , Erythropoietin , Genetic Vectors/genetics , Hematocrit , Injections, Intramuscular , Lentivirus/genetics , Plasmids/therapeutic use , Tacrolimus/therapeutic use
4.
Braz. j. med. biol. res ; 42(9): 783-786, Sept. 2009. graf, tab
Article in English | LILACS | ID: lil-524320

ABSTRACT

The α-MRE is the major regulatory element responsible for the expression of human α-like globin genes. It is genetically polymorphic, and six different haplotypes, named A to F, have been identified in some population groups from Europe, Africa and Asia and in native Indians from two Brazilian Indian tribes. Most of the mutations that constitute the α-MRE haplotypes are located in flanking sequences of binding sites for nuclear factors. To our knowledge, there are no experimental studies evaluating whether such variability may influence the α-MRE enhancer activity. We analyzed and compared the expression of luciferase of nine constructs containing different α-MRE elements as enhancers. Genomic DNA samples from controls with A (wild-type α-MRE) and B haplotypes were used to generate C-F haplotypes by site-directed mutagenesis. In addition, three other elements containing only the G→A polymorphism at positions +130, +199, and +209, separately, were also tested. The different α-MRE elements were amplified and cloned into a plasmid containing the luciferase reporter gene and the SV40 promoter and used to transiently transfect K562 cells. A noticeable reduction in luciferase expression was observed with all constructs compared with the A haplotype. The greatest reductions occurred with the F haplotype (+96, C→A) and the isolated polymorphism +209, both located near the SP1 protein-binding sites believed not to be active in vivo. These are the first analyses of α-MRE polymorphisms on gene expression and demonstrate that these single nucleotide polymorphisms, although outside the binding sites for nuclear factors, are able to influence in vitro gene expression.


Subject(s)
Humans , Gene Expression Regulation/genetics , Globins/genetics , Haplotypes/genetics , Mutation/genetics , Polymorphism, Genetic/genetics , Regulatory Elements, Transcriptional/genetics , Luciferases/genetics
5.
Braz. j. med. biol. res ; 41(7): 571-578, July 2008. ilus, tab, graf
Article in English | LILACS | ID: lil-489516

ABSTRACT

Apoptotic protease activating factor 1 (APAF-1) has a critical role in the regulation of apoptosis. In the present study, the mRNA expression analysis of different APAF-1 transcripts (APAF-1S, APAF-1LC, APAF-1LN, and APAF-1XL) was analyzed in bone marrow samples from 37 patients with acute myeloid leukemia (newly diagnosed, with no previous treatment). APAF-1XL and APAF-1LN transcripts (with and without an extra WD-40 repeat region, respectively) were detected in all samples, although the major form expressed was APAF-1XL in 65 percent of the samples (group 1), while 35 percent of the samples expressed primarily APAF-1LN (group 2). Only 46 percent of the patients presented complete remission in response to remission induction therapy (represented by less than 5 percent marrow blasts and hematological recovery), all but 2 cases being from group 1, 21.6 percent did not attain complete remission (only 1 case from group 1), and 32.4 percent of the patients died early. Lower expression of APAF-1XL (APAF-1XL/APAF-1LN ratio <1.2) was associated with a poor response to therapy (P = 0.0005, Fisher exact test). Both groups showed similar characteristics regarding white blood cell counts, cytogenetic data or presence of gene rearrangements associated with good prognosis as AML1-ETO, CBFB-MYH11 and PML/RARA. Since it has been shown that only the isoforms with the extra WD-40 repeat region activate procaspase-9, we suggest that low procaspase-9 activation may also be involved in the deregulation of apoptosis and chemotherapy resistance in acute myeloid leukemia.


Subject(s)
Adult , Aged , Aged, 80 and over , Female , Humans , Male , Middle Aged , Young Adult , Apoptotic Protease-Activating Factor 1/genetics , Leukemia, Myeloid, Acute/genetics , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Bone Marrow Cells/chemistry , Case-Control Studies , Densitometry , DNA, Complementary/genetics , Gene Expression Regulation, Neoplastic , Leukemia, Myeloid, Acute/drug therapy , Reverse Transcriptase Polymerase Chain Reaction , RNA, Messenger/genetics , Transcription Factors , Treatment Failure , Transcription, Genetic/genetics , Biomarkers, Tumor/genetics , Young Adult
6.
Braz. j. med. biol. res ; 38(9): 1321-1324, Sept. 2005. tab
Article in English | LILACS | ID: lil-408358

ABSTRACT

Patients with chronic renal insufficiency (CRI) have reduced hemoglobin levels, mostly as a result of decreased kidney production of erythropoietin, but the relation between renal insufficiency and the magnitude of hemoglobin reduction has not been well defined. Hereditary hemochromatosis is an inherited disorder of iron metabolism. The importance of the association of hemochromatosis with treatment for anemia among patients with CRI has not been well described. We analyzed the frequency of the C282Y and H63D mutations in the HFE gene in 201 Brazilian individuals with CRI undergoing hemodialysis. The analysis of the effects of HFE mutations on iron metabolism and anemia with biochemical parameters was possible in 118 patients of this study (hemoglobin, hematocrit, ferritin levels, transferrin saturation, and serum iron). A C282Y heterozygous mutation was found in 7/201 (3.4 percent) and H63D homozygous and heterozygous mutation were found in 2/201 (1.0 percent) and 46/201 (22.9 percent), respectively. The allelic frequencies of the HFE mutations (0.017 for C282Y mutation and 0.124 for H63D mutation) did not differ between patients with CRI and healthy controls. Regarding the biochemical parameters, no differences were observed between HFE heterozygous and mutation-negative patients, although ferritin levels were not higher among patients with the H63D mutation (P = 0.08). From what we observed in our study, C282Y/H63D HFE gene mutations are not related to degrees of anemia or iron stores in CRI patients receiving intravenous iron supplementation (P > 0.10). Nevertheless, the present data suggest that the H63D mutation may have an important function as a modulating factor of iron overload in these patients.


Subject(s)
Adolescent , Adult , Aged , Aged, 80 and over , Female , Humans , Male , Middle Aged , Hemochromatosis/genetics , Histocompatibility Antigens Class I/genetics , Kidney Failure, Chronic/genetics , Membrane Proteins/genetics , Brazil , Case-Control Studies , Genotype , Hemochromatosis/blood , Kidney Failure, Chronic/blood , Kidney Failure, Chronic/therapy , Mutation/genetics , Renal Dialysis
7.
Braz. j. med. biol. res ; 36(11): 1471-1474, Nov. 2003. ilus
Article in English | LILACS | ID: lil-348293

ABSTRACT

Point mutations and small insertions or deletions in the human alpha-globin genes may produce alpha-chain structural variants and alpha-thalassemia. Mutations can be detected either by direct DNA sequencing or by screening methods, which select the mutated exon for sequencing. Although small (about 1 kb, 3 exons and 2 introns), the alpha-globin genes are duplicate (alpha2 and alpha1) and highy G-C rich, which makes them difficult to denature, reducing sequencing efficiency and causing frequent artifacts. We modified some conditions for PCR and electrophoresis in order to detect mutations in these genes employing nonradioactive single-strand conformation polymorphism (SSCP). Primers previously described by other authors for radioactive SSCP and phast-SSCP plus denaturing gradient gel electrophoresis were here combined and the resultant fragments (6 new besides 6 original per alpha-gene) submitted to silver staining SSCP. Nine structural and one thalassemic mutations were tested, under different conditions including two electrophoretic apparatus (PhastSystemÖ and GenePhorÖ, Amersham Biosciences), different polyacrylamide gel concentrations, run temperatures and denaturing agents, and entire and restriction enzyme cut fragments. One hundred percent of sensitivity was achieved with four of the new fragments formed, using the PhastSystemÖ and 20 percent gels at 15ºC, without the need of restriction enzymes. This nonradioactive PCR-SSCP approach showed to be simple, rapid and sensitive, reducing the costs involved in frequent sequencing repetitions and increasing the reliability of the results. It can be especially useful for laboratories which do not have an automated sequencer.


Subject(s)
Humans , beta-Thalassemia , Globins , beta-Thalassemia , DNA Mutational Analysis , Electrophoresis, Polyacrylamide Gel , Mass Screening , Point Mutation , Polymerase Chain Reaction , Polymorphism, Single-Stranded Conformational , Reproducibility of Results
8.
Braz. j. med. biol. res ; 36(6): 699-701, June 2003. ilus, tab
Article in English | LILACS | ID: lil-340661

ABSTRACT

We report a case in which the interaction of heterozygosis for both the beta0-IVS-II-1 (G->A) mutation and the aaa anti-3.7 allele was the probable cause for the clinical occurrence of thalassemia intermedia. The propositus, a 6-year-old Caucasian Brazilian boy of Portuguese descent, showed a moderately severe chronic anemia in spite of having the beta-thalassemia trait. Investigation of the alpha-globin gene status revealed heterozygosis for alpha-gene triplication (aaa /aa). The patient's father, also presenting mild microcytic and hypochromic anemia, had the same alpha and beta genotypes as his son, while the mother, not related to the father and hematologically normal, was also a carrier of the aaa anti-3.7 allele. The present case emphasizes the need for considering the possibility of alpha-gene triplication in beta-thalassemia heterozygotes who display an unexpected severe phenotype. The beta-thalassemia mutation found here is being described for the first time in Brazil


Subject(s)
Humans , Male , Child , Alleles , Heterozygote , Mutation , Thalassemia , beta-Thalassemia , Genotype , Polymerase Chain Reaction , Severity of Illness Index , Thalassemia
9.
Braz. j. med. biol. res ; 35(8): 873-876, Aug. 2002. ilus, tab
Article in English | LILACS | ID: lil-325540

ABSTRACT

We describe the clinical and molecular characteristics of two unrelated Brazilian families with an association of the Sicilian form of (deltaß)º-thalassemia with hemoglobin S and ß-thalassemia. Direct sequencing of the ß-globin gene showed only the hemoglobin S mutation in patient 1 and the ß-thalassemia IVS1-110 in patient 2. The other allele was deleted in both patients and PCR of DNA samples of the breakpoint region of both patients showed a band of approximately 1,150 bp, expected to be observed in the DNA of carriers of Sicilian (deltaß)º-thalassemia. The nucleotide sequence of this fragment confirmed the Sicilian deletion. There are few reports concerning the Hb S/(deltaß)º-thalassemia association and patient 2 is the first reported case of Sicilian type of (deltaß)º-thalassemia in association with ß-thalassemia documented at the molecular level


Subject(s)
Humans , Male , Female , Adult , beta-Thalassemia , Hemoglobin, Sickle , beta-Thalassemia , Brazil , DNA , Polymerase Chain Reaction
10.
Braz. j. med. biol. res ; 35(8): 921-925, Aug. 2002. ilus
Article in English | LILACS | ID: lil-325546

ABSTRACT

Hereditary spherocytosis (HS) is a common inherited anemia characterized by the presence of spherocytic red cells. Defects in several membrane protein genes have been involved in the pathogenesis of HS. ß-Spectrin-related HS seems to be common. We report here a new mutation in the ß-spectrin gene coding region in a patient with hereditary spherocytosis. The patient presented acanthocytosis and spectrin deficiency and, at the DNA level, a novel frameshift mutation leading to HS, i.e., a C deletion at codon 1392 (ß-spectrin Säo PauloII), exon 20. The mRNA encoding ß-spectrin Säo PauloII was very unstable and the mutant protein was not detected in the membrane or in other cellular compartments. It is interesting to note that frameshift mutations of the ß-spectrin gene at the 3' end allow the insertion of the mutant protein in the red cell membrane, leading to a defect in the auto-association of the spectrin dimers and consequent elliptocytosis. On the other hand, ß-spectrin Säo PauloII protein was absent in the red cell membrane, leading to spectrin deficiency, HS and the presence of acanthocytes


Subject(s)
Humans , Female , Adult , Frameshift Mutation , RNA, Messenger , Spectrin , Spherocytosis, Hereditary , Acanthocytes , Electrophoresis, Polyacrylamide Gel , Pedigree , Polymerase Chain Reaction , Reticulocytes
11.
Braz. j. med. biol. res ; 35(7): 767-773, July 2002. ilus, tab
Article in English | LILACS | ID: lil-316735

ABSTRACT

The molecular basis for RHD pseudogene or RHDpsi is a 37-bp insertion in exon 4 of RHD. This insertion, found in two-thirds of D-negative Africans, appears to introduce a stop codon at position 210. The hybrid RHD-CE-Ds, where the 3' end of exon 3 and exons 4 to 8 are derived from RHCE, is associated with the VS+V- phenotype, and leads to a D-negative phenotype in people of African origin. We determined whether Brazilian blood donors of heterogeneous ethnic origin had RHDpsi and RHD-CE-Ds. DNA from 206 blood donors were tested for RHDpsi by a multiplex PCR that detects RHD, RHDpsi and the C and c alleles of RHCE. The RHD genotype was determined by comparison of size of amplified products associated with the RHD gene in both intron 4 and exon 10/3'-UTR. VS was determined by amplification of exon 5 of RHCE, and sequencing of PCR products was used to analyze C733G (Leu245Val). Twenty-two (11 percent) of the 206 D-negative Brazilians studied had the RHDpsi, 5 (2 percent) had the RHD-CE-Ds hybrid gene associated with the VS+V- phenotype, and 179 (87 percent) entirely lacked RHD. As expected, RHD was deleted in all the 50 individuals of Caucasian descent. Among the 156 individuals of African descent, 22 (14 percent) had inactive RHD and 3 percent had the RHD-CE-Ds hybrid gene. These data confirm that the inclusion of two different multiplex PCR for RHD is essential to test the D-negative Brazilian population in order to avoid false-positive typing of polytransfused patients and fetuses


Subject(s)
Humans , Ethnicity , Oncogene Proteins, Fusion , Pseudogenes , Rh-Hr Blood-Group System , Black People/genetics , Blood Donors , Brazil , Exons , White People/genetics , Glycoproteins , Phenotype , Polymerase Chain Reaction , Polymorphism, Genetic , Sequence Analysis , Sequence Analysis, DNA
12.
Braz. j. med. biol. res ; 34(6): 759-62, Jun. 2001. tab
Article in English | LILACS | ID: lil-285849

ABSTRACT

In order to determine the contribution of alpha-thalassemia to microcytosis and hypochromia, 339 adult outpatients seen at Unicamp University Hospital (with the exception of the Clinical Hematology outpatient clinics), who showed normal hemoglobin (Hb) levels and reduced mean corpuscular volume and mean corpuscular hemoglobin, were analyzed. Ninety-eight were Blacks (28.9 percent) and 241 were Caucasians (71.1 percent). In all cases, Hb A2 and F levels were either normal or low. The most common deletional and nondeletional forms of alpha-thalassemia [-alpha3.7, -alpha4.2, --MED, -(alpha)20.5, alphaHphIalpha, alphaNcoIalpha, aaNcoI and alphaTSAUDI] were investigated by PCR and restriction enzyme analyses. A total of 169 individuals (49.9 percent) presented alpha-thalassemia: 145 (42.8 percent) were heterozygous for the -alpha3.7 deletion (-alpha3.7/aa) and 18 (5.3 percent) homozygous (-alpha3.7/-alpha3.7), 5 (1.5 percent) were heterozygous for the nondeletional form alphaHphIalpha (alphaHphIalpha/aa), and 1 (0.3 percent) was a --MED carrier (--MED/aa). Among the Blacks, 56 (57.1 percent) showed the -alpha3.7/aa genotype, whereas 12 (12.2 percent) were -alpha3.7/-alpha3.7 and 1 (1.0 percent) was an alphaHphIalpha carrier; among the Caucasians, 89 (36.9 percent) were -alpha3.7/aa, 6 (2.5 percent) had the -alpha3.7/-alpha3.7 genotype, 4 (1.7 percent) presented the nondeletional form (alphaHphIalpha/aa), and 1 (0.4 percent) was a --MED carrier. These results demonstrate that alpha-thalassemia, mainly through the -alpha3.7 deletion, is an important cause of microcytosis and hypochromia in individuals without anemia. These data are of clinical relevance since these hematological alterations are often interpreted as indicators of iron deficiency


Subject(s)
Humans , Male , Female , Adolescent , Adult , alpha-Thalassemia/epidemiology , Erythrocyte Indices , Erythrocytes, Abnormal , Hemoglobins/analysis , alpha-Thalassemia/genetics , Brazil/epidemiology , Racial Groups , Ferritins/blood , Gene Deletion , Genotype , Prevalence
13.
Braz. j. med. biol. res ; 34(6): 763-6, Jun. 2001. tab, ilus
Article in English | LILACS | ID: lil-285850

ABSTRACT

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired clonal syndrome characterized by intravascular hemolysis mediated by complement, thrombotic events and alterations in hematopoiesis. Basically, the molecular events which underlie the complexity of the syndrome consist of the absence of the glycosylphosphatidylinositol (GPI) anchor as a consequence of somatic mutations in the PIG-A gene, located on the X chromosome. The GPI group is responsible for the attachment of many proteins to the cytoplasmic membrane. Two of them, CD55 and CD59, have a major role in the inhibition of the action of complement on the cellular membrane of blood cells. The absence of GPI biosynthesis can lead to PNH. Since mutations in the PIG-A gene are always present in patients with PNH, the aim of this study was to characterize the mutations in the PIG-A gene in Brazilian patients. The analysis of the PIG-A gene was performed using DNA samples derived from bone marrow and peripheral blood. Conformation-sensitive gel electrophoresis was used for screening the mutation and sequencing methods were used to identify the mutations. Molecular analysis permitted the identification of three point mutations in three patients: one G->A transition in the 5' portion of the second intron, one T->A substitution in the second base of codon 430 (Leu430->stop), and one deletion deltaA in the third base of codon 63. This study represents the first description of mutations in the PIG-A gene in a Brazilian population.


Subject(s)
Humans , Male , Female , Adolescent , Adult , Aged , Hemoglobinuria, Paroxysmal/genetics , Mutation , X Chromosome/genetics , Base Sequence , Brazil , Glycosylphosphatidylinositols/metabolism
14.
Braz. j. med. biol. res ; 34(4): 489-92, Apr. 2001. tab
Article in English | LILACS | ID: lil-282614

ABSTRACT

Hereditary persistence of fetal hemoglobin is an uncommon, benign disorder in which the expression of gamma-globin genes persists into adult life. Several point mutations have been associated with the increased gamma-globin gene promoter activity. We evaluated the -195 (C->G) mutation by a functional in vitro assay based on the luciferase reporter gene system. The results indicated that the increased promoter activity observed in vivo could not be reproduced in vitro under the conditions employed, suggesting that other factors may be involved in the overexpression of the gamma-globin gene containing the -195 (C->G) mutation. Furthermore, this is the first time that the -195 (C->G) mutation of the Agamma-globin gene has been evaluated by in vitro gene expression


Subject(s)
Humans , Adult , Fetal Hemoglobin/genetics , Genes, Reporter , Globins/genetics , Hemoglobinopathies/genetics , In Vitro Techniques , Mutation , beta-Galactosidase/metabolism , DNA Primers , Gene Expression , Globins/metabolism , Luciferases/genetics , Luciferases/metabolism , Point Mutation , Polymerase Chain Reaction , Transfection
15.
Braz. j. med. biol. res ; 33(9): 1041-5, Sept. 2000.
Article in English | LILACS | ID: lil-267979

ABSTRACT

Seven unrelated patients with hemoglobin (Hb) H disease and 27 individuals with alpha-chain structural alterations were studied to identify the alpha-globin gene mutations present in the population of Southeast Brazil. The -alpha3.7, --MED and -(alpha)20.5 deletions were investigated by PCR, whereas non-deletional alpha-thalassemia (alphaHphalpha, alphaNcoIalpha, aaNcoI, alphaIcalpha and alphaTSaudialpha) was screened with restriction enzymes and by nested PCR. Structural alterations were identified by direct DNA sequencing. Of the seven patients with Hb H disease, all of Italian descent, two had the -(alpha)20.5/-alpha3.7 genotype, one had the --MED/-alpha3.7 genotype, one had the --MED/alphaHphalpha genotype and three showed interaction of the -alpha3.7 deletion with an unusual, unidentified form of non-deletional alpha-thalassemia [-alpha3.7/(aa)T]. Among the 27 patients with structural alterations, 15 (of Italian descent) had Hb Hasharon (alpha47Asp->His) associated with the -alpha3.7 deletion, 4 (of Italian descent) were heterozygous for Hb J-Rovigo (alpha53Ala->Asp), 4 (3 Blacks and 1 Caucasian) were heterozygous for Hb Stanleyville-II (alpha78Asn->Lys) associated with the alpha+-thalassemia, 1 (Black) was heterozygous for Hb G-Pest (alpha74Asp->Asn), 1 (Caucasian) was heterozygous for Hb Kurosaki (alpha7Lys->Glu), 1 (Caucasian) was heterozygous for Hb Westmead (alpha122His->Gln), and 1 (Caucasian) was the carrier of a novel silent variant (Hb Campinas, alpha26Ala->Val). Most of the mutations found reflected the Mediterranean and African origins of the population. Hbs G-Pest and Kurosaki, very rare, and Hb Westmead, common in southern China, were initially described in individuals of ethnic origin differing from those of the carriers reported in the present study and are the first cases to be reported in the Brazilian population


Subject(s)
Humans , Child , Adolescent , Adult , alpha-Thalassemia/genetics , Globins/genetics , Mutation/genetics , alpha-Thalassemia/blood , Black People/genetics , Brazil/ethnology , White People/genetics , Genetic Testing , Polymerase Chain Reaction
16.
Braz. j. med. biol. res ; 32(8): 953-9, Aug. 1999. tab
Article in English | LILACS | ID: lil-238963

ABSTRACT

Cytomegalovirus (CMV) is the single most important infectious agent affecting recipients of organ transplants. To evaluate the incidence and the clinical importance of CMV infection in renal transplants in Brazil, 37 patients submitted to renal allograft transplants were tested periodically for the presence of cytomegalovirus DNA in urine using the polymerase chain reaction (PCR), and for the presence of IgM and IgG antibodies against CMV by enzyme-linked immunosorbent assay (ELISA) and indirect immunofluorescence (IIF). The PCR-amplified products were detected by gel electrophoresis and confirmed by dot-blot hybridization with oligonucleotide probes. Thirty-two of the 37 patients (86.4 percent) were positive by at least one of the three methods. In six patients, PCR was the only test which detected the probable CMV infection. Ten patients had a positive result by PCR before transplantation. In general, the diagnosis was achieved earlier by PCR than by serologic tests. Active infection occurred more frequently during the first four months after transplantation. Sixteen of the 32 patients (50 percent) with active CMV infection presented clinical symptoms consistent with CMV infection. Five patients without evidence of active CMV infection by the three tests had only minor clinical manifestations during follow-up. Our results indicate that PCR is a highly sensitive procedure for the early detection of CMV infection and that CMV infection in renal transplant patients is a frequent problem in Brazil


Subject(s)
Humans , Cytomegalovirus Infections/diagnosis , Cytomegalovirus Infections/epidemiology , Cytomegalovirus/isolation & purification , Kidney Transplantation , Polymerase Chain Reaction , Postoperative Complications , Cytomegalovirus Infections/complications , Cytomegalovirus Infections/virology , Enzyme-Linked Immunosorbent Assay , Incidence , Prevalence , Prospective Studies , Serologic Tests
17.
Braz. j. med. biol. res ; 30(8): 933-40, Aug. 1997. tab, graf
Article in English | LILACS | ID: lil-197248

ABSTRACT

The use of hydroxyurea (HU) can improve the clinical course of sickle cell disease. However, several features of HU treatment remain unclear, including the predictability of drug response and determination of adequate doses, considering positive responses and minimal side effects. In order to identify adequate doses of HU for treatment of sickle cell disease, 10 patients, 8 with sickle cell anemia and 2 with Sbeta thalassemia (8SS, 2SBeta), were studied for a period of 6 to 19 months in an open label dose escalation trial (10 to 20 mg kg(-1) day(-1)). Hemoglobin (Hb), fetal hemoglobin (Hb F) and mean corpuscular volume (MCV) values and reticulocyte, neutrophil and platelet counts were performed every two weeks during the increase of the HU dose and every 4 weeks when the maximum HU dose was established. Reduction in the number of vasoocclusive episodes was also considered in order to evaluate the efficiency of the treatment. The final Hb and Hb F concentrations, and MCV values were significantly higer than the initial values, while the final reticulocyte and neutrophil counts were significantly lower. There was an improvement in the concentration of Hb (range: 0.7-2.0 g/dl) at 15 mg HU kg(-1) day(-1), but this concentration did not increase significantly when the HU dose was raised to 20 mg kg(-1) day(-1). The concentration of Hb F increased significantly (range: 1.0-18.1 per cent) when 15 mg HU was used, and continued to increase when the dose was raised to 20 mg kg(-1) day (-1). The final MCV values increased 11-28 fl (femtoliters). However, reticulocyte (range: 51-205 x 10(9)/l) and neutrophil counts (range: 9.5-1.3 x 10(9)/l) obtained at this dose were significantly lower than those obtained with 15 mg kg(-1) day(-1). All patients reported a decrease in frequency or severity of vasoocclusive episodes. These results suggest that a hydroxyurea dose of 15 mg kg(-1) day(-1) seems to be adequate for treatment of sickle cell disease in view of the minimal side effects observed and the improvement in laboratory and clinical parameters.


Subject(s)
Humans , Anemia, Sickle Cell/drug therapy , Hydroxyurea/administration & dosage , Dosage Forms , Hydroxyurea/therapeutic use , Thalassemia/drug therapy
18.
Rev. bras. genét ; 20(2): 319-21, Jun. 1997. ilus, tab
Article in English | LILACS | ID: lil-219010

ABSTRACT

O heterozigoto para a talassemia ß apresenta um típico padräo hematológico. A severidade desses parâmetros varia consideravelmente de valores claramente anormais até próximos à normalidade. Postula-se que essa variaçäo seria influenciada pelo tipo de mutaçäo de talassemia beta encontrada e que portanto seria possível suspeitar-se do tipo de alteraçäo pelos índices hematológicos. Nossos resultados demonstram que mutaçöes brandas (ß IVSI-nt 6) mostram maiores níveis de hemoglobina corpuscular média (MCH) do que as formas mais severas (ߺ39 or ß IVSI-nt 1) e que os níveis de hemoglobina A2 säo menores em mutaçöes ߧ do que em formas brandas (ß IVSI-nt 6). Entretanto, contrariando estudos anteriores, näo pudemos indicar o MCH como um discriminador entre as mutaçöes ߺ ou ß+.


Subject(s)
Humans , Adolescent , Adult , Middle Aged , beta-Thalassemia/blood , Heterozygote , Brazil , Mutation/genetics , Phenotype , Polymerase Chain Reaction
19.
Braz. j. med. biol. res ; 29(10): 1307-10, Oct. 1996. ilus, graf
Article in English | LILACS | ID: lil-186178

ABSTRACT

Chronic myeloid leukemia (CML) is a myeloproliferative disorder characterized by the presence of a reciprocal translocation between chromosomes 9 and 22 in at least 95 per cent of cases. At the molecular level, this translocation results in the activation of the ABL oncogene of chromosome 9, which becomes contiguous with the 5'end of the BCR gene on chromosome 22. The breakpoint usually occurs between exons 2 and 3 (b2-a2 rearrangement), or 3 and 4 (b3-a2 rearrangement) of the major breakpoint cluster region (M-BCR) of the BCR gene. The aim of the present study was to characterize the type of BCR-ABL transcript in 32 patients with CML using the reverse transcriptase-polymerase chaim reaction (RT-PCR) and to determine if this type of rearrangement is related to the survival of the patients. Our results confirmed that RT-PCR is more sensitive than cytogenetic analysis for identifying the Philadelphia (Ph1) chromosome (96.9 per cent vs 79.3 per cent). The frequencies of b2-a2 and b3-a2 rearrangements were 28.1 per cent and 65.7 per cent, respectively. The survival of patients presenting the b2-a2 or the b3-a2 rearrangement was not significantly different (P = 0.27750). The data suggest that the type of transcript has no prognostic value for CML patients.


Subject(s)
Adult , Aged , Female , Humans , Adolescent , Fusion Proteins, bcr-abl/genetics , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/genetics , Translocation, Genetic/genetics , Leukemia, Myelogenous, Chronic, BCR-ABL Positive/diagnosis
20.
Braz. j. med. biol. res ; 27(11): 2573-8, Nov. 1994. ilus, tab, graf
Article in English | LILACS | ID: lil-153978

ABSTRACT

Two patients receiving the same cadaver kidney graft were investigated prospectively for cytomegalovirus (CMV) infection using the polymerase chain reaction (PCR) and serologic tests (ELISA and IFI). The data indicate that a strain of CMV was probably transmitted from the same donor to both kidney recipients including one who was seropositive for CMV


Subject(s)
Humans , Male , Adolescent , Cytomegalovirus Infections/transmission , Postoperative Complications/diagnosis , Kidney Transplantation , Tissue Donors , Antibodies, Viral/blood , Base Sequence , Cytomegalovirus Infections/diagnosis , Cytomegalovirus Infections/immunology , Cytomegalovirus/immunology , Postoperative Complications/immunology , DNA, Viral/urine , Enzyme-Linked Immunosorbent Assay , Fluorescent Antibody Technique , Immunoglobulin G/blood , Immunoglobulin M/blood , Polymerase Chain Reaction
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