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1.
Article in Chinese | WPRIM | ID: wpr-928753

ABSTRACT

OBJECTIVE@#To investigate the clinical and genetic characteristics of a family with hereditary spherocytosis (HS), to clarify the cause of the disease, and to provide the basis for genetic counseling and prenatal diagnosis.@*METHODS@#The clinical data of proband and his parents were collected, and HS-related pathogenic genovariation of the proband was detected by high throughput sequencing. Suspected pathogenic mutation sites were verified by PCR-Sanger sequencing, and the fetus were conceived by a proband mother underwent prenatal diagnosis.@*RESULTS@#Clinical manifestations of the proband showed moderate anemia, mild splenomegaly, and jaundice (an indirect increase of bilirubin). The gene detection showed that the proband showed compound heterozygous mutations of SPTB gene c. 6095T > C (p.Leu2032Pro) and c. 6224A > G (p.Glu2075Gly), which was inherited from the asymptomatic mother and father, respectively. Both mutations were detected rarely in the common population. Prenatal diagnosis revealed that the fetus inherited a mutant gene of the mother.@*CONCLUSION@#The compound heterozygous mutations of SPTB genes c.6095T>C (p.Leu2032Pro) and c.6224A>G (p.Glu2075Gly) were the causes of the family disease, which provides a basis for family genetic counseling and prenatal diagnosis. This report is the first one found in the HGMD,1000G and EXAC database, which provides an addition to the mutation profile of the SPTB gene.


Subject(s)
Female , Genetic Testing , High-Throughput Nucleotide Sequencing , Humans , Infant, Newborn , Male , Mutation , Pedigree , Pregnancy , Prenatal Diagnosis , Spectrin/genetics , Spherocytosis, Hereditary/genetics
2.
Article in Chinese | WPRIM | ID: wpr-781304

ABSTRACT

OBJECTIVE@#To explore the genetic basis of a pedigree affected with hereditary spherocytosis.@*METHODS@#Peripheral blood samples were collected from 17 members of the pedigree. Genomic DNA of the proband was subjected to next generation sequencing. Candidate variant was validated by co-segregation analysis. pCAS2(c.5798+1G) and pCAS2(c.5798+1A) plasmids were constructed by homologous recombination and transfected into 293T cells. Reverse transcription PCR, TA cloning and Sanger sequencing were used to analyze the effect of candidate variant on splicing. Meanwhile, peripheral blood RNAs were extracted to analyze the effect of candidate variant on splicing in vivo.@*RESULTS@#The proband was found to carry a c.5798+1G>A variant of the SPTB gene. The variant has co-segregated with the phenotype in the pedigree. In vitro and in vivo splicing experiments confirmed that the mutation has significantly affected the splicing, resulting in shift of reading frame and produced a premature termination codon.@*CONCLUSION@#The novel c.5798+1G>A variant of the SPTB gene probably underlies the pathogenesis of hereditary spherocytosis in this pedigree.


Subject(s)
Codon, Nonsense , Genetics , Genetic Variation , HEK293 Cells , Humans , Mutation , Genetics , Pedigree , Plasmids , RNA Splicing , Spectrin , Genetics , Spherocytosis, Hereditary , Genetics , Transfection
3.
Article in Chinese | WPRIM | ID: wpr-774069

ABSTRACT

This study analyzed the clinical features of 5 children with hereditary spherocytosis (HS) and the characteristics of ANK1 and SPTB gene mutations. All 5 children were confirmed with HS by peripheral blood genetic detection. Anemia, jaundice and splenomegaly were observed in all 5 children. Three children had an increase in erythrocyte osmotic fragility. All 5 children had negative results of the Coombs test, glucose 6 phosphate dehydrogenase test, sucrose hemolysis test, acidified-serum hemolysis test and thalassemia gene test. Peripheral blood smear showed an increase in spherocyte count in one child. High-throughput sequencing revealed ANK1 gene mutations in patients 1 to 3, namely c.3398(exon29)delA, c.4306C>T and c.957(exon9)_c.961(exon9)delAATCT, among which c.3398(exon29)delA had not been reported before. Patient 4 had c.318delGExon3 mutation in the SPTB gene. Patient 5 had mutations in the SPTB and SLC4A1 genes, among which c.3484delC in the SPTB gene was a spontaneous mutation; the mutation site of the SLCA4A1 gene was inherited from the father and was a non-pathogenic gene. This study suggests that anemia, jaundice and splenomegaly are major clinical manifestations of HS children. Most children with HS do not have the typical spherocytic changes. Genetic detection may help with the accurate diagnosis of HS.


Subject(s)
Ankyrins , Genetics , High-Throughput Nucleotide Sequencing , Humans , Mutation , Spectrin , Genetics , Spherocytosis, Hereditary , Genetics
4.
Article in English | WPRIM | ID: wpr-786360

ABSTRACT

BACKGROUND: Sixty percent of infants with severe neonatal hypoxic-ischemic encephalopathy die, while most survivors have permanent disabilities. Treatment for neonatal hypoxic-ischemic encephalopathy is limited to therapeutic hypothermia, but it does not offer complete protection. Here, we investigated whether hypoxia-inducible factor (HIF) promotes cell survival and suggested neuroprotective strategies.PURPOSE: HIF-1α deficient mice have increased brain injury after neonatal hypoxia-ischemia (HI), and the role of HIF-2α in HI is not well characterized. Copper-zinc superoxide dismutase (SOD)1 overexpression is not beneficial in neonatal HI. The expression of HIF-1α and HIF-2α was measured in SOD1 overexpressing mice and compared to wild-type littermates to see if alteration in expression explains this lack of benefit.METHODS: On postnatal day 9, C57Bl/6 mice were subjected to HI, and protein expression was measured by western blotting in the ipsilateral cortex of wild-type and SOD1 overexpressing mice to quantify HIF-1α and HIF-2α. Spectrin expression was also measured to characterize the mechanism of cell death.RESULTS: HIF-1α protein expression did not significantly change after HI injury in the SOD1 overexpressing or wild-type mouse cortex. However, HIF-2α protein expression increased 30 minutes after HI injury in the wild-type and SOD1 overexpressing mouse cortex and decreased to baseline value at 24 hours after HI injury. Spectrin 145/150 expression did not significantly change after HI injury in the SOD1 overexpressing or wild-type mouse cortex. However, spectrin 120 expression increased in both wild-type and SOD1 overexpressing mouse at 4 hours after HI, which decreased by 24 hours, indicating a greater role of apoptotic cell death.CONCLUSION: HIF-1α and HIF-2α may promote cell survival in neonatal HI in a cell-specific and regional fashion. Our findings suggest that early HIF-2α upregulation precedes apoptotic cell death and limits necrotic cell death. However, the influence of SOD was not clarified; it remains an intriguing factor in neonatal HI.


Subject(s)
Animals , Blotting, Western , Brain Injuries , Cell Death , Cell Survival , Humans , Hypothermia, Induced , Hypoxia-Ischemia, Brain , Infant , Mice , Spectrin , Superoxide Dismutase , Superoxides , Survivors , Up-Regulation
5.
Article in Chinese | WPRIM | ID: wpr-775368

ABSTRACT

Drynariae Rhizoma has great significance in the clinical practice of osteoporosis treatment. Based on the perspective of integrative pharmacology, the study explored the mechanism of action of Drynariae Rhizoma in the treatment of osteoporosis. Six active components in Drynariae Rhizoma were obtained, mainly including glycosides and sterols. Taking the median of 2 times of "node connectivity" as the card value, the core node of the Chinese medicine target disease gene interaction network was selected. Based on this, three topological structural eigenvalues, such as "node connectivity" "node tightness" and "node connectivity" were calculated, thereby screening out four core targets of Drynariae Rhizoma treatment for osteoporosis, including thyroid parathyroid hormone 1 receptor (PTH1R), parathyroid hormone 2 receptor (PTH2R), calcitonin receptor gene (CALCR), and SPTBN1 gene (SPTBN1). Based on the gene ontology database GO and KEGG pathway database, the molecular function, intracellular localization, and biological reactions and pathways of proteins encoded by drug target genes were determined. Combined with enrichment calculation, it is predicted that osteoporosis may play a role in biosynthetic processes, such as circulatory system, nervous system, energy metabolism, prolactin signal pathway, GnRH signaling pathway, neurotrophic factor signaling pathway and other pathway. The conclusion of this study is certain with the existing research results, and the new target and new pathway could also be used as a theoretical basis for the further verification of osteoporosis.


Subject(s)
Drugs, Chinese Herbal , Pharmacology , Humans , Osteoporosis , Drug Therapy , Polypodiaceae , Chemistry , Receptor, Parathyroid Hormone, Type 1 , Metabolism , Receptor, Parathyroid Hormone, Type 2 , Metabolism , Receptors, Calcitonin , Metabolism , Rhizome , Chemistry , Spectrin , Metabolism
6.
Article in English | WPRIM | ID: wpr-79073

ABSTRACT

Spinocerebellar ataxias (SCAs) are autosomal dominant neurodegenerative disorders which disrupt the afferent and efferent pathways of the cerebellum that cause cerebellar ataxia. Spectrin beta non-erythrocytic 2 (SPTBN2) gene encodes the β-III spectrin protein with high expression in Purkinje cells that is involved in excitatory glutamate signaling through stabilization of the glutamate transporter, and its mutation is known to cause spinocerebellar ataxia type 5. Three years and 5 months old boy with delayed development showed leukodystrophy and cerebellar atrophy in brain magnetic resonance imaging (MRI). Diagnostic exome sequencing revealed that the patient has heterozygous mutation in SPTBN2 (p.Glu1251Gln) which is a causative genetic mutation for spinocerebellar ataxia type 5. With the patient's clinical findings, it seems reasonable to conclude that p.Glu1251Gln mutation of SPTBN2 gene caused spinocerebellar ataxia type 5 in this patient.


Subject(s)
Amino Acid Transport System X-AG , Atrophy , Brain , Cerebellar Ataxia , Cerebellum , Efferent Pathways , Exome , Glutamic Acid , Humans , Magnetic Resonance Imaging , Male , Neurodegenerative Diseases , Purkinje Cells , Spectrin , Spinocerebellar Ataxias
8.
Article in Korean | WPRIM | ID: wpr-47115

ABSTRACT

Hereditary spherocytosis is a hemolytic anemia caused by erythrocyte membrane deficiencies that lead to membrane destabilization and vesiculation. Abnormal spherocytes are trapped and destroyed in the spleen. Mutations in several genes, SPTA1, SPTB, ANK1, SLCA1 and EPB42 cause alpha-spectrin, beta-spectrin, ankyrin, band 3 or protein 4.2 protein deficiencies, respectively. The clinical severity ranged from asymptomatic to severe hemolytic anemia requiring erythrocyte transfusion. Common complications are cholelithiasis, hemolytic episodes and aplastic crises. Till now, splenectomy is considered as only curative method in this genetic disorder. However, in the future, molecular analysis will make elucidate the genotype-phenotype interactions and can innovate to modify treatment strategies.


Subject(s)
Anemia, Hemolytic , Ankyrins , Cholelithiasis , Erythrocyte Membrane , Erythrocyte Transfusion , Erythrocytes , Membranes , Protein Deficiency , Spectrin , Spherocytes , Spherocytosis, Hereditary , Spleen , Splenectomy
9.
Article in Korean | WPRIM | ID: wpr-788476

ABSTRACT

Hereditary spherocytosis is a hemolytic anemia caused by erythrocyte membrane deficiencies that lead to membrane destabilization and vesiculation. Abnormal spherocytes are trapped and destroyed in the spleen. Mutations in several genes, SPTA1, SPTB, ANK1, SLCA1 and EPB42 cause alpha-spectrin, beta-spectrin, ankyrin, band 3 or protein 4.2 protein deficiencies, respectively. The clinical severity ranged from asymptomatic to severe hemolytic anemia requiring erythrocyte transfusion. Common complications are cholelithiasis, hemolytic episodes and aplastic crises. Till now, splenectomy is considered as only curative method in this genetic disorder. However, in the future, molecular analysis will make elucidate the genotype-phenotype interactions and can innovate to modify treatment strategies.


Subject(s)
Anemia, Hemolytic , Ankyrins , Cholelithiasis , Erythrocyte Membrane , Erythrocyte Transfusion , Erythrocytes , Membranes , Protein Deficiency , Spectrin , Spherocytes , Spherocytosis, Hereditary , Spleen , Splenectomy
10.
Biomedica. 2011; 27 (Jan.-Jun.): 33-38
in English | IMEMR | ID: emr-110353

ABSTRACT

Muscular dystrophies [MD] traditionally refer to a group of genetically determined, progressive, degenerative disorders of the skeletal muscle. The most common disease manifestations being Duchenne Muscular Dystrophy [DMD] and Becker Muscular Dystrophy [BMD]. This descriptive study was carried out on 40 patients of muscular dystrophies, selected on clinical grounds and subjected to biochemical, morphological and immunohistochemical analysis. Muscle biopsies were taken from patients by an open method and formalin fixed paraffin embedded blocks were made. Haematoxylin and Eosin stain, PAS and Gomori's trichrome and immunohistochemical stains were conducted on the sections from these blocks. Dystrophin and beta - Spectrin antibodies were used for immunohistochemistry. Among the 40 cases of muscular dystrophy the above investigations were correlated with clinical findings to reach the final diagnosis in each case. In Pakistan the diagnosis of muscular dystrophies is still based on clinical grounds and CPK values only, however the present study has provided us an opportunity to combine clinical, biochemical, morphlogical and immunohistochemical evaluations of the patients with muscular dystrophies. It was concluded from this study that although muscular dystrophy can be diagnosed using clinical parameters and CPK levels, histochemistry and IHC can confirm and differentiate the various types of muscular dystrophy and make it possible to identify the female patients of DMD and BMD


Subject(s)
Humans , Male , Female , Dystrophin , Muscular Dystrophy, Duchenne/diagnosis , Creatine Kinase , Spectrin , Biopsy , Immunohistochemistry
11.
Article in Chinese | WPRIM | ID: wpr-308001

ABSTRACT

<p><b>OBJECTIVE</b>To study the single-nucleotide substitution (c.-16C to T) of the PURATROPHIN-1 gene in spinocerebellar ataxia (SCA) patients in China.</p><p><b>METHODS</b>The single-nucleotide substitution (c.-16C to T) of the PURATROPHIN-1 gene was detected by PCR, digested with EcoN I, separated on 8% polyacrylamide gel in 68 probands of autosomal dominant SCA families and 119 sporadic SCA patients, who had been excluded CAG/CAA repeat expansion at the SCA1, 2, 3, 6, 7, 17 and dentatorubral-pallidolluysian atrophy (DRPLA) loci. The results were confirmed in four patients by direct sequencing.</p><p><b>RESULTS</b>The single-nucleotide substitution (c.-16C to T) of the PURATROPHIN-1 gene was not identified in authors' cohort.</p><p><b>CONCLUSION</b>The mutation of c.-16C to T of the PURATROPHIN-1 gene might be rare in SCA patients in China.</p>


Subject(s)
Asians , Genetics , Cohort Studies , Guanine Nucleotide Exchange Factors , Genetics , Humans , Mutation , Polymerase Chain Reaction , Polymorphism, Single Nucleotide , Spectrin , Genetics , Spinocerebellar Ataxias , Genetics
12.
J Biosci ; 2007 Sep; 32(6): 1147-51
Article in English | IMSEAR | ID: sea-110949

ABSTRACT

We have shown the differential interactions of the erythroid skeletal protein spectrin with the globin subunits of adult haemoglobin (HbA); these indicate a preference for alpha-globin over that for beta-globin and intact HbA in an adenosine 5'-triphosphate (ATP)-dependent manner. The presence of Mg/ATP led to an appreciable decrease in the binding affinity of the alpha-globin chain to spectrin and the overall yield of globin-spectrin cross-linked complexes formed in the presence of hydrogen peroxide. Similar effects were also seen in the presence of 2-,3-diphosphoglycerate (2,3 DPG), the other important phosphate metabolite of erythrocytes. The binding affinity and yield of cross-linked high molecular weight complexes (HMWCs) formed under oxidative conditions were significantly higher in alpha-globin compared with intact haemoglobin, HbA and the beta-globin chain. The results of this study indicate a possible correlation of the preferential spectrin binding of the alpha-globin chain over that of the beta-globin in the haemoglobin disorder beta-thalassaemia.


Subject(s)
2,3-Diphosphoglycerate/blood , Adenosine Triphosphate/blood , Animals , Globins/metabolism , Humans , Hydrogen Peroxide/blood , Protein Subunits/blood , Sheep , Spectrin/metabolism , Thalassemia/blood
13.
Article in Chinese | WPRIM | ID: wpr-313704

ABSTRACT

<p><b>OBJECTIVE</b>To analyze the nature of the protein encoded by 8B7cDNA (1 835 bp) and to examine the localization of the protein in cells.</p><p><b>METHODS</b>The nature of the protein was analyzed using Blastn, Blastp, and TMpred. Expressions of 8B7 mRNA in tissues and cells were examined by Northern blotting. Recombinant expression vectors for localization test were constructed and transfected into COS-7 cells. Fluorescence emission in cells was examined upon a laser scan confocal microscope.</p><p><b>RESULTS</b>The protein encoded by 8B7cDNA was 363 amino acids long and contained spectrin repeats. It was completely homologous to the C-terminal 363 amino acids of Enaptin, Nasprin-1, Mynel, and Syne-1 proteins. It belonged to Spectrin super-family and was called 8B7 spectrin. Northern blotting revealed a 1.8 kb mRNA expression in human spleen and small intestine tissues. EGFP-8B7 fusion protein was observed to express on the nuclear membrane and in the cytoplasm in a reticular form in a localization assay on COS-7 cells. The position of fluorescence in COS-7 cells transfected with pEGFP-delta SR8B7 was similar to that in the cells transfected with pEGFP-8B7cDNA.</p><p><b>CONCLUSIONS</b>8 B7 spectrin is a novel member of spectrin super-family. It localizes to the nuclear membrane and the cytoplasm in a reticular form in COS-7 cells. The localization is determined by the C-terminal KASH domain in 8B7 spectrin molecule.</p>


Subject(s)
Amino Acid Sequence , Animals , Blotting, Northern , COS Cells , Chlorocebus aethiops , DNA, Complementary , Genetics , Humans , Plasmids , Genetics , RNA, Messenger , Genetics , Sequence Homology, Amino Acid , Spectrin , Chemistry , Genetics , Transfection
14.
Article in English | WPRIM | ID: wpr-190355

ABSTRACT

We previously demonstrated that the lentivirus lytic peptide 1 (LLP-1) corresponding to the carboxyl terminus of HIV-1 gp41 induced cell death in human neuronal cells. Present study was conducted to further elucidate the pathogenic mechanisms involved in HIV-1 gp41-induced neurodegeneration in AIDS patients with cognitive deficits. The effect of LLP-1 on activation of calpain-1, a calcium-activated cysteine protease, which has been implicated in neuronal degeneration and death, was monitored by the proteolysis of spectrin in rat organotypic hippocampal slice cultures. Protease specific spectrin breakdown products revealed that LLP-1 generated~150/145-kDa fragments characteristic of calpain-1 activation in hippocampus undergoing cell death as evidenced by LDH release. This spectrin cleavage pattern was further confirmed by in vitro calpain-1 proteolysis. Futhermore, calpectin and MDL28170, inhibitors of calpain activity, blocked calpain-1-mediated spectrin cleavage. Spectrin cleavage likely occurred in the absence of overt synaptic loss, as suggested by the preserved levels of synaptophysin. Among pharmacological agents tested, apocynin, NADPH oxidase inhibitor, ameliorated the LLP-1-induced spectrin. Given the role of spectrin essential for synapse stabilization, LLP-1-induced spectrin cleavage as occurs with the activation of calpain-1 may be an important effector in LLP-1mediated cell injury in hippocampus, which is primarily linked to cognitive dysfunction.


Subject(s)
Animals , Calpain , Cell Death , Cysteine Proteases , Hippocampus , HIV , HIV-1 , Humans , Lentivirus , NADPH Oxidases , Neurons , Protein Structure, Tertiary , Proteolysis , Rats , Spectrin , Synapses , Synaptophysin
15.
J Biosci ; 2005 Jun; 30(3): 317-28
Article in English | IMSEAR | ID: sea-110666

ABSTRACT

In cell membranes, local inhomogeneity in the lateral distribution of lipids and proteins is thought to exist in vivo in the form of lipid 'rafts', microdomains enriched in cholesterol and sphingolipids, and in specific classes of proteins, that appear to play specialized roles for signal transduction, cell-cell recognition, parasite or virus infection, and vesicular trafficking. These structures are operationally defined as membranes resistant to solubilization by nonionic detergents at 4 degree C (detergent-resistant membranes, DRMs). This definition appears to be necessary and sufficient, although additional manoeuvres, not always described with sufficient detail, may be needed to ensure isolation of DRMs, like mechanical homogenization, and changes in the pH and/or ionic strength of the solubilization medium. We show here for the human erythrocyte that the different conditions adopted may lead to the isolation of qualitatively and quantitatively different DRM fractions, thus contributing to the complexity of the notion itself of lipid raft. A significant portion of erythrocyte DRMs enriched in reported lipid raft markers, such as flotillin-1, flotillin-2 and GM1, is anchored to the spectrin membrane-skeleton via electrostatic interactions that can be disrupted by the simultaneous increase in pH and ionic strength of the solubilization medium.


Subject(s)
Detergents/chemistry , Erythrocyte Membrane/chemistry , Humans , Hydrogen-Ion Concentration , Membrane Microdomains/chemistry , Membrane Proteins/chemistry , Spectrin/chemistry
16.
Article in English | WPRIM | ID: wpr-727928

ABSTRACT

The kinesin proteins (KIFs) make up a large superfamily of molecular motors that transport cargo such as vesicles, protein complexes, and organelles. KIF5 is a heterotetrameric motor that conveys vesicles and plays an important role in neuronal function. Here, we used the yeast two-hybrid system to identify the neuronal protein (s) that interacts with the tail region of KIF5 and found a specific interaction with betaIII spectrin. The amino acid residues between 1394 and 1774 of betaIII spectrin were required for the interaction with KIF5C. betaIII spectrin also bound to the tail region of neuronal KIF5A and ubiquitous KIF5B but not to other kinesin family members in the yeast two-hybrid assay. In addition, these proteins showed specific interactions, confirmed by GST pull-down assay and co-immunoprecipitation. betaIII spectrin interacted with GST-KIF5 fusion proteins, but not with GST alone. An antibody to betaIII spectrin specifically co-immunoprecipitated KIF5s associated with betaIII spectrin from mouse brain extracts. These results suggest that KIF5 motor proteins transport vesicles or organelles that are coated with betaIII spectrin.


Subject(s)
Animals , Brain , Humans , Immunoprecipitation , Kinesins , Mice , Microtubules , Neurons , Organelles , Spectrin , Transport Vesicles , Two-Hybrid System Techniques
17.
Annals of Saudi Medicine. 2003; 23 (3-4): 152-157
in English | IMEMR | ID: emr-61445

ABSTRACT

Little is known about hereditary spherocytosis [HS] and hereditary elliptocytosis [HE] in the native population of Saudi Arabia, even though these conditions are seemingly common. The purpose of this study was to ascertain the protein make-up of the red cell membrane in healthy Saudis and in patients with HS and HE. Patients and Eighteen healthy Saudi subjects [13 males and 5 females], 11 patients with HS [6 males and 5 females] and 11 patients with HE [7 males and 4 females] were studied. All normal controls and patients underwent SDS-PAGE red cell membrane protein analysis in duplicate and the stained protein bands were identified and quantitated by densitometry. In normal, healthy Saudis, the mean values for seven membrane proteins [alpha spectrin, spectrin, ankyrin, band 3, protein 4.1, protein 4.2, and actin] were similar to those published for normal, healthy Americans. Of the eleven cases with HS, 7 [64%] demonstrated detectable protein abnormalities while 4 [36%] were apparently normal. The electrophoretic patterns of membrane proteins in Saudis with HS differed from those of patients with HS in other parts of the world. Of the 11 cases of HE, 7 [64%] displayed abnormalities while 4 [36%] were normal. The electrophoretic pattern of the main proteins in the membranes of red blood cells in healthy Saudis is similar to that reported from the USA. However, significant differences exist in the electrophoretic patterns between Saudi patients with HS and patients from other parts of the world


Subject(s)
Humans , Male , Female , Membrane Proteins/analysis , Spherocytosis, Hereditary , Elliptocytosis, Hereditary , Electrophoresis, Polyacrylamide Gel , Spectrin/analysis , Ankyrins/analysis , Anion Exchange Protein 1, Erythrocyte/analysis , Actins
18.
Acta bioquím. clín. latinoam ; 36(3): 317-327, sept. 2002. ilus, tab
Article in Spanish | LILACS | ID: lil-330178

ABSTRACT

Fue observada la presencia de anticuerpos naturales contra las proteínas de la membrana eritrocitaria en sueros de origen humano y animal. Se demuestran, por medio del "Western blot", las reacciones de sueros de conejos adultos contra el estroma de auto-, alo- y xeno-antígenos (humano y mono rhesus) de las membranas de los hematíes. Los sueros de conejo estaban dirigidos principalmente contra las dos espectrinas (alfa + beta) y la proteína banda 3. No hubo reacción contra estromas de tres monos rhesus. Las comparaciones entre sueros tratados y no tratados con ditiotreitol (DTT) sugieren la presencia de anticuerpos IgG. Los animales fueron clasificados de acuerdo con éstas reacciones, mediante el método de agrupamiento UPGMA. Aunque existe cierta variabilidad, probablemente debida a fenómenos estocásticos, los resultados apuntan a un reconocimiento de estructuras químicas similares entre las proteínas banda 3 y entre las espectrinas de los hematíes de conejo, mono rhesus y humanos. Esta amplia reactividad plantea el tema de cuáles serían los epitopos reconocidos por éstos anticuerpos. El presente trabajo incluye también la descripción del peso molecular de las proteínas de la membrana de los hematíes de conejo


Subject(s)
Humans , Animals , Rabbits , Autoantibodies , Erythrocyte Membrane/immunology , Spectrin , Blotting, Western , Dithiothreitol , Epitope Mapping , Macaca mulatta
19.
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
20.
Article in Korean | WPRIM | ID: wpr-35318

ABSTRACT

OBJECTIVES: Protein kinase C(PKC), a calcium and phospholipid dependent enzyme, is activated by lead in vitro at picomolar concentrations. However, the effect of lead on PKC has never been studied in a human population. The purpose of the study was to evaluate whether lead exposure was associated with PKC mediated-phosphorylation in erythrocytes among lead workers. METHODS: Two hundred and twelve lead workers were studied. To determine the levels of phosphorylation in vivo, an in vitro back phosphorylation technique was used by adding PKC and gamma-32P to preparations of erythrocyte membranes. We measured back phosphorylations of erythrocyte membrane proteins, spectrin, and 52 kDa and 48 kDa, as an indirect measure of PKC activation in vivo. RESULTS: The mean(SD) age and exposure duration was 39.1(10.0) years and 8.1(6.5) years, respectively. Tibial lead ranged from 0.8 to 290.8 microgram Pb/g bone mineral with a mean(SD) of 34.4(35.2) microgram Pb/g bone mineral. The means(SD) of back phosphorylation levels of the three proteins were 540.7(304.1), 198.6(78.2), and 247.7(83.3) photostimulated luminescence units(PSL), respectively, by phosphoimager. After adjustment for potential confounding factors, tibial lead and exposure duration were significantly and inversely associated with back phosphorylation levels. One unit of increase in tibial lead(1 microgram Pb/g bone mineral) is associated with a decline in spectrin, band 4.9 52 kDa, and band 4.9 48 kDa back phosphorylation levels by 1.4(P<0.05), 0.34(P<0.05), and 0.47(P<0.01), respectively. However, there were no associations between the back phosphorylation levels and either blood lead or ZPP levels. CONCLUSIONS: These findings suggest that the PKC activity in erythrocytes is increased by chronic lead exposure and that erythrocyte membrane protein phosphorylation may be a biomarker of lead exposure.


Subject(s)
Calcium , Erythrocyte Membrane , Erythrocytes , Humans , Luminescence , Phosphorylation , Protein Kinase C , Protein Kinases , Spectrin
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