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1.
BMC Geriatr ; 21(1): 633, 2021 11 04.
Artigo em Inglês | MEDLINE | ID: mdl-34736422

RESUMO

BACKGROUND: Postoperative delirium (POD) is a common complication of older people undergoing hip fracture surgery, which negatively affects clinical- and healthcare-related outcomes. Unfortunately, POD pathophysiology is still largely unknown, despite previous studies showing that neuroinflammation, neuroendocrine dysfunction, increased reactive oxidative stress (ROS), and endothelial dysfunctions may be involved. There is also evidence that many of the pathophysiological mechanisms which are involved in delirium are involved in sarcopenia too. This article describes the protocol of a pilot study to evaluate the feasibility of a larger one that will explore the pathophysiological mechanisms correlating POD with sarcopenia. We will analyse whether various biomarkers reflecting neuroinflammation, ROS, neuroendocrine disorders, and microvasculature lesions will be simultaneously expressed in in the blood, cerebrospinal fluid (CSF), and muscles of patients developing POD. METHODS: Two centres will be involved in this study, each recruiting a convenient sample of ten older patients with hip fracture. All of them will undergo a baseline Comprehensive Geriatric Assessment, which will be used to construct a Rockwood-based Frailty Index (FI). Blood samples will be collected for each patient on the day of surgery and 1 day before. Additionally, CSF and muscle fragments will be taken and given to a biologist for subsequent analyses. The presence of POD will be assessed in each patient every morning until hospital discharge using the 4AT. Delirium subtypes and severity will be assessed using the Delirium Motor Subtype Scale-4 and the Delirium-O-Meter, respectively. We will also evaluate the patient's functional status at discharge, using the Cumulated Ambulation Score. DISCUSSION: This study will be the first to correlate biomarkers of blood, CSF, and muscle in older patients with hip fracture.


Assuntos
Delírio , Fraturas do Quadril , Idoso , Delírio/diagnóstico , Delírio/epidemiologia , Delírio/etiologia , Avaliação Geriátrica , Fraturas do Quadril/cirurgia , Humanos , Projetos Piloto , Complicações Pós-Operatórias/diagnóstico , Complicações Pós-Operatórias/epidemiologia , Complicações Pós-Operatórias/etiologia , Estudos Prospectivos
2.
Cell Death Differ ; 19(5): 827-38, 2012 May.
Artigo em Inglês | MEDLINE | ID: mdl-22095287

RESUMO

Improving stem cell therapy is a major goal for the treatment of muscle diseases, where physiological muscle regeneration is progressively exhausted. Vessel-associated stem cells, such as mesoangioblasts (MABs), appear to be the most promising cell type for the cell therapy for muscular dystrophies and have been shown to significantly contribute to restoration of muscle structure and function in different muscular dystrophy models. Here, we report that melanoma antigen-encoding gene (MAGE) protein necdin enhances muscle differentiation and regeneration by MABs. When necdin is constitutively overexpressed, it accelerates their differentiation and fusion in vitro and it increases their efficacy in reconstituting regenerating myofibres in the α-sarcoglycan dystrophic mouse. Moreover, necdin enhances survival when MABs are exposed to cytotoxic stimuli that mimic the inflammatory dystrophic environment. Taken together, these data demonstrate that overexpression of necdin may be a crucial tool to boost therapeutic applications of MABs in dystrophic muscle.


Assuntos
Sobrevivência Celular/fisiologia , Distrofia Muscular Animal/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteínas Nucleares/metabolismo , Células-Tronco/citologia , Células-Tronco/metabolismo , Animais , Apoptose/genética , Apoptose/fisiologia , Diferenciação Celular , Sobrevivência Celular/genética , Células Cultivadas , Imunoprecipitação da Cromatina , Citometria de Fluxo , Imunofluorescência , Immunoblotting , Camundongos , Camundongos Knockout , Distrofia Muscular Animal/genética , Proteínas do Tecido Nervoso/genética , Proteínas Nucleares/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sarcoglicanas/genética , Sarcoglicanas/metabolismo
3.
Genomics ; 88(5): 564-71, 2006 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-16765020

RESUMO

In Macaca mulatta, the single rDNA array is flanked by a patchwork of sequences including subregions of human Yp11.2, 4q35.2, and 10p15.3. This composite DNA region is characterized by unique or low-copy sequences, resembling a potentially transcribed region. The analysis of Cercopithecus aethiops, Presbytis cristata, and Hylobates lar suggests that this complex sequence organization could be shared by Old World monkey and lesser ape species. After the lesser apes/great apes divergence, the unique or nonduplicated DNA region underwent amplification and spreading, preferentially marking the p arm of acrocentric chromosomes bearing the rDNA. The molecular analysis of human acrocentric chromosomes revealed some extent of remodeling of the rDNA boundary: near the human NOR, a large 4q35.2 duplication partially resembles that found in MMU; conversely, infrequently represented Yp11.2 sequences totally differed from those of the macaque, and 10p15.3 sequences were lacking. Thus, although evolutionary events modified the sequence organization of the MMU rDNA boundary, its overall sequence feature and the preferential location in vicinity to the NOR have been conserved.


Assuntos
DNA Ribossômico/genética , Evolução Molecular , Macaca mulatta/genética , Animais , Cromossomos Artificiais Bacterianos/genética , Cromossomos Humanos Par 10/genética , Cromossomos Humanos Par 4/genética , Cromossomos Humanos Y/genética , Sequência Conservada , Duplicação Gênica , Genômica , Humanos , Hibridização in Situ Fluorescente , Dados de Sequência Molecular , Primatas/genética , Especificidade da Espécie
4.
Mol Biol Evol ; 21(9): 1792-9, 2004 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-15201396

RESUMO

In this article, we report studies on the evolutionary history of beta satellite repeats (BSR) in primates. In the orangutan genome, the bulk of BSR sequences was found organized as very short stretches of approximately 100 to 170 bp, embedded in a 60-kb to 80-kb duplicated DNA segment. The estimated copy number of the duplicon that carries BSR sequences ranges from 70 to 100 per orangutan haploid genome. In both macaque and gibbon, the duplicon mapped to a single chromosomal region at the boundary of the rDNA on the marker chromosome (chromosome 13 and 12, respectively). However, only in the gibbon, the duplicon comprised 100 bp of beta satellite. Thus, the ancestral copy of the duplicon appeared in Old World monkeys ( approximately 25 to approximately 35 MYA), whereas the prototype of beta satellite repeats took place in a gibbon ancestor, after apes/Old World monkeys divergence ( approximately 25 MYA). Subsequently, a burst in spreading of the duplicon that carries the beta satellite was observed in the orangutan, after lesser apes divergence from the great apes-humans lineage ( approximately 18 MYA). The analysis of the orangutan genome also indicated the existence of two variants of the duplication that differ for the length (100 or 170 bp) of beta satellite repeats. The latter organization was probably generated by nonhomologous recombination between two 100-bp repeated regions, and it likely led to the duplication of the single Sau3A site present in the 100-bp variant, which generated the prototype of Sau3A 68-bp beta satellite tandem organization. The two variants of the duplication, although with a different ratios, characterize the hominoid genomes from the orangutan to humans, preferentially involving acrocentric chromosomes. At variance to alpha satellite, which appeared before the divergence of New World and Old World monkeys, the beta satellite evolutionary history began in apes ancestor, where we have first documented a low-copy, nonduplicated BSR sequence. The first step of BSR amplification and spreading occurred, most likely, because the BSR was part of a large duplicon, which underwent a burst dispersal in great apes' ancestor after the lesser apes' branching. Then, after orangutan divergence, BSR acquired the clustered structural organization typical of satellite DNA.


Assuntos
DNA Satélite/genética , Evolução Molecular , Primatas/genética , Animais , Sequência de Bases , Cromossomos Artificiais Bacterianos/genética , Cosmídeos , Duplicação Gênica , Genoma , Humanos , Hibridização in Situ Fluorescente , Dados de Sequência Molecular , Filogenia , Pongo pygmaeus/genética , Sequências Repetitivas de Ácido Nucleico , Homologia de Sequência do Ácido Nucleico
5.
FEBS Lett ; 554(1-2): 59-66, 2003 Nov 06.
Artigo em Inglês | MEDLINE | ID: mdl-14596915

RESUMO

A cDNA clone up-regulated in hydraulic lung edema in rabbit showed high similarity with human RDH10 mRNA, which encodes a protein involved in retinoic acid metabolism. We defined the organization of the human gene, which includes a unique transcriptional start site, a coding region with six translated exons and a 3' untranslated region containing at least two used polyadenylation sites. The two poly(A) signals are responsible for the production of the 3 and 4 kb RDH10 mRNA isoforms detected in several human tissues and cell lines.


Assuntos
Oxirredutases do Álcool/genética , Componentes do Gene/genética , Transcrição Gênica , Regiões 3' não Traduzidas , Sequência de Bases , Genes/genética , Humanos , Dados de Sequência Molecular , Fases de Leitura Aberta , Especificidade de Órgãos , Isoformas de Proteínas , Sinais de Poliadenilação na Ponta 3' do RNA , Estabilidade de RNA , RNA Mensageiro/análise , RNA Mensageiro/metabolismo , Distribuição Tecidual , Sítio de Iniciação de Transcrição
6.
Gene ; 296(1-2): 21-7, 2002 Aug 21.
Artigo em Inglês | MEDLINE | ID: mdl-12383499

RESUMO

We have investigated the evolutionary history of the 4q35 paralogous region, and of a sub-family of interspersed LSau repeats. In HSA, 4q35 duplications were localized at 1q12, 3p12.3, 4q35, 10q26, 20cen, whereas duplicons and interspersed LSau repeats simultaneously labeled the p arm of acrocentric chromosomes. A multi-site localization of 4q35-like sequences was also observed in PTR, GGO, PPY, HLA (Hominoidea) and PAN (Old World monkey), thus indicating that duplications of this region have occurred extensively in the two clades, which diverged at least 25 million years ago. In HSA, PTR and PAN, 4q35-derived duplicons co-localized with rDNA, whereas in GGO and PPY this association was partially lacking. In PAN, the single- and multi-site distribution of rDNA and paralogous sequences, respectively, indicates a different timing of sequence dispersal. The sub-family of interspersed LSau repeats showed a lesser dispersal than 4q35 duplications both in man and great apes. This finding suggests that duplications and repeated sequences have undergone different expansion/contraction events during evolution. The mechanisms underlying the dispersal of paralogous regions may be further derived through studies comparing the detailed structural organization of these genomic regions in man and primates.


Assuntos
Cromossomos Humanos Par 4/genética , Evolução Molecular , Genoma Humano , Sequências Repetitivas Dispersas/genética , Animais , Sequência de Bases , Cromossomos Humanos Par 10/genética , Cromossomos Humanos Par 22/genética , Cromossomos Humanos Par 3/genética , DNA/química , DNA/genética , Duplicação Gênica , Humanos , Hibridização in Situ Fluorescente , Dados de Sequência Molecular , Análise de Sequência de DNA
7.
Hum Genet ; 108(6): 467-77, 2001 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-11499671

RESUMO

We report the molecular analysis of a 130-kb DNA region containing a junction between beta and non-beta satellite DNA from chromosome 15p. The genomic region is characterized by beta satellite blocks intermingled with variants of the D4Z4 repeat, and duplicons from 4q24 and 4q35. Besides the p-arm of acrocentric chromosomes, the duplicons showed a wide genomespread involving pericentromeric, sub-telomeric, and interstitial regions. In this regard, the paralogous sequences were characterized by a high similarity index (96%), thus indicating a recent transposition during the evolution. The acrocentrics differedwith regard to the location of the 4q24 paralogous region, since it mapped on the p-arm of chromosomes 13-15 and 21, but only on 22q11.2. Conversely, the 4q35 duplication marked the p-arm of all the acrocentrics. In different individuals, the short arm of acrocentric chromosomes revealed a great variability of sequence representation and location at p11 and/or p13 for both the 4q24 and 4q35 duplications. The studied genomic region from chromosome 15p, of which a contig of approximately 200 kb has been derived, could lead to more detailed investigations into the sequence organization and possible biological function of chromosome regions that are located close to the rDNA array.


Assuntos
Cromossomos Humanos Par 15/genética , Duplicação Gênica , Elementos Alu/genética , Centrômero , Cromossomos Humanos Par 22/genética , DNA Satélite/genética , Genoma Humano , Humanos , Hibridização in Situ Fluorescente , Dados de Sequência Molecular
8.
Gastroenterology ; 120(6): 1404-11, 2001 May.
Artigo em Inglês | MEDLINE | ID: mdl-11313310

RESUMO

BACKGROUND & AIMS: Stimulator of Fe Transport (SFT) and transferrin receptor (TfR) are proteins involved in iron transport. This study evaluated iron metabolism protein expression in duodenal biopsy specimens from controls and patients with abnormal iron metabolism. METHODS: Twelve controls, 8 patients with iron deficiency anemia, 7 with HFE-related hemochromatosis, and 6 with non-HFE-related iron overload were studied. Immunohistochemistry was performed on duodenal biopsy specimens with anti-TfR and anti-SFT antibodies which recognize a putative stimulator of Fe transport of ~80 kilodaltons. RESULTS: In controls, the putative stimulator of Fe transport was expressed in the middle and distal part of the villi in the subapical cytoplasmatic region. Its expression increased in anemics and, to a lesser degree, in HFE-related hemochromatotics, whereas it was reduced in patients with non-HFE-related iron overload. TfR expression showed a crypt-to-tip gradient in controls, but not in anemics, in whom it was uniformly overexpressed. TfR expression was intermediate in HFE-related hemochromatotics and similar to controls in non-HFE-related iron overload. CONCLUSIONS: Expression of the putative stimulator of Fe transport and TfR increases in iron deficiency. Increased expression of both proteins is present only in HFE-related hemochromatotics suggesting that other factors may be involved in determining non-HFE-related iron overload phenotype.


Assuntos
Anemia/metabolismo , Proteínas de Transporte/análise , Duodeno/química , Hemocromatose/metabolismo , Proteínas de Ligação ao Ferro , Receptores da Transferrina/análise , Enzimas de Conjugação de Ubiquitina , Adulto , Idoso , Western Blotting , Proteínas de Transporte/genética , Feminino , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Mutação , Receptores da Transferrina/genética
9.
Hum Genet ; 107(4): 304-11, 2000 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-11129329

RESUMO

High-resolution cytogenetic analysis of a large number of women with premature ovarian failure (POF) identified six patients carrying different Xq chromosome rearrangements. The patients (one familial and five sporadic cases) were negative for Turner's stigmata and experienced a variable onset of menopause. Microsatellite analysis and fluorescent in situ hybridization (FISH) were used to define the origin and precise extension of the Xq anomalies. All of the patients had a Xq chromosome deletion as the common chromosomal abnormality, which was the only event in three cases and was associated with partial Xp or 9p trisomies in the remaining three. Two of the Xq chromosome deletions were terminal with breakpoints at Xq26.2 and Xq21.2, and one interstitial with breakpoints at Xq23 and Xq28. In all three cases, the del(X)s retained Xp and Xq specific telomeric sequences. One patient carries a psu dic(X) with the deletion at Xq22.2 or Xq22.3; the other two [carrying (X;X) and (X;9) unbalanced translocations, respectively] showed terminal deletions with the breakpoint at Xq22 within the DIAPH2 gene. Furthermore, the rearranged X chromosomes were almost totally inactivated, and the extent of the Xq deletions did not correlate with the timing of POF. In agreement with previous results, these findings suggest that the deletion of a restricted Xq region may be responsible for the POF phenotype. Our analysis indicates that this region extends from approximately Xq26.2 (between markers DXS8074 and HIGMI) to Xq28 (between markers DXS 1113 and ALD) and covers approximately 22 Mb of DNA. These data may provide a starting point for the identification of the gene(s) responsible for ovarian development and folliculogenesis.


Assuntos
Deleção Cromossômica , Insuficiência Ovariana Primária/genética , Cromossomo X/genética , Adolescente , Adulto , Idade de Início , Aberrações Cromossômicas , Bandeamento Cromossômico , Cromossomos Humanos Par 9/genética , Citogenética , Mecanismo Genético de Compensação de Dose , Feminino , Humanos , Hibridização in Situ Fluorescente , Repetições de Microssatélites
10.
Gene ; 256(1-2): 43-50, 2000 Oct 03.
Artigo em Inglês | MEDLINE | ID: mdl-11054534

RESUMO

We have isolated and characterised one PAC clone (dJ233C1) containing a linkage between alphoid and non-alphoid DNA. The non-alphoid DNA was found to map at the pericentromeric region of chromosome 20, both on p and q sides, and to contain homologies with one contig (ctg176, Sanger Centre), also located in the same chromosome region. At variance with the chromosome specificity shown by the majority of non-alphoid DNA, a subset of alphoid repeats derived from the PAC yielded FISH hybridisation signals located at the centromeric region of several human chromosomes, belonging to three different suprachromosomal families. The evolutionary conservation of this boundary region was investigated by comparative FISH experiments on chromosomes from great apes. The non-alphoid DNA was found to have undergone events of expansion and transposition to different pericentromeric regions of great apes chromosomes. Alphoid sequences revealed a very wide distribution of FISH signals in the great apes. The pattern was substantially discordant with the data available in the literature, which is essentially derived from the central alphoid subset. These results add further support to the emerging opinion that the pericentromeric regions are high plastics, and that the alpha satellite junctions do not share the evolutionary history with the main subsets.


Assuntos
Cromossomos Humanos Par 20/genética , DNA Satélite/genética , DNA/genética , Evolução Molecular , Animais , Southern Blotting , Mapeamento Cromossômico , DNA/química , Feminino , Hominidae , Humanos , Hibridização in Situ Fluorescente , Dados de Sequência Molecular , Análise de Sequência de DNA
11.
FEBS Lett ; 469(2-3): 208-12, 2000 Mar 10.
Artigo em Inglês | MEDLINE | ID: mdl-10713272

RESUMO

The aim of the present study was to evaluate the effect of iron overload on gene expression in HepG2 cells by differential display. Iron-treated cells showed a 50% decrease in apolipoprotein B100 (Apo B100) and a 2- and 3-fold increase in semaphorin cd100 and aldose reductase mRNA, respectively, with parallel variations in Apo B100 and aldose reductase proteins. These effects were time-dependent. Vitamin E prevented the increase in aldose reductase expression, but had no effect on Apo B100 and semaphorin cd100. Treatment with hydrogen peroxide and 4-hydroxy-2,3-nonenal increased only aldose reductase mRNA. These data suggest that iron can affect mRNA levels by lipid peroxidation-dependent and -independent pathways.


Assuntos
Aldeído Redutase/genética , Antígenos CD , Apolipoproteínas B/genética , Sobrecarga de Ferro/metabolismo , Ferro/metabolismo , Glicoproteínas de Membrana/genética , Semaforinas , Aldeído Redutase/metabolismo , Apolipoproteínas B/metabolismo , Northern Blotting , Western Blotting , Linhagem Celular , Perfilação da Expressão Gênica , Humanos , Peroxidação de Lipídeos/fisiologia , Fígado/citologia , Glicoproteínas de Membrana/metabolismo , RNA Mensageiro/análise , Vitamina E/farmacologia
12.
Hum Reprod ; 15(1): 197-202, 2000 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-10611212

RESUMO

A total of 106 women affected by premature ovarian failure (POF) were evaluated for fragile X (FRAXA) premutation. The POF patients were classified as having a familial condition (33 women), at least one relative with early menopause (12 women), or a sporadic condition (61 women). The FRAXA premutation was only detected in patients with familial (four out of 33) or sporadic POF (two out of 61). In general, the results obtained indicated that the prevalence [six out of 106, 6%, 95% confidence interval (CI) 3-11%] of FRAXA premutation is significantly higher in women affected by POF than expected (P = 1.24x10(-3)), suggesting a phenotype consequence of the premutation alleles. This relationship is more convincingly derived from the observation in two analysed pedigrees of a co-segregation between FRAXA and POF. These findings suggest a possible involvement of premutated alleles in ovarian failure, and indicate the utility of POF families screening for FRAXA premutation in order to prevent the transmission of mental retardation syndrome.


Assuntos
Síndrome do Cromossomo X Frágil/genética , Insuficiência Ovariana Primária/genética , Adolescente , Adulto , Feminino , Humanos , Menopausa/genética , Mães , Mutação , Linhagem
13.
J Biotechnol ; 61(2): 117-28, 1998 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-9654745

RESUMO

Starting from a pool of 10(13) RNA sequences, we isolated a number of TAR RNA variants after nine rounds of selection by binding to recombinant Tat in vitro (SELEX procedure). Sequence analysis of part of the selected molecular species indicated that two TAR variants (clones A and B) were, respectively, represented five and four times. These two groups of sequences constituted approximately 25% of the total number of analyzed clones (9/34). As far as the primary and presumptive secondary structures of the wild-type TAR are concerned, the selected A and B variants showed an almost complete sequence conservation of the Tat-binding domain, but the configuration of this nucleotide region differed within the secondary structure. Despite this difference, as verified by gel retardation and filter binding assays, both the A and B variants bound Tat in vitro with an affinity that was very close to that of the wild-type TAR. Conversely, neither variant sustained Tat-mediated trans-activation in vivo when they replaced the wild-type TAR inside the long terminal repeat of HIV_1. Taken together, our results suggest that these TAR variants have lost the ability to bind cell factor(s) in vivo and may therefore represent useful decoys for the inhibition of HIV-1 replication.


Assuntos
Produtos do Gene tat/genética , Variação Genética , HIV-1/genética , RNA Viral/genética , Sequência de Bases , Sítios de Ligação/genética , Biotecnologia , Clonagem Molecular , Primers do DNA/genética , Produtos do Gene tat/metabolismo , Repetição Terminal Longa de HIV , HIV-1/metabolismo , Humanos , Técnicas In Vitro , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Reação em Cadeia da Polimerase , RNA Viral/química , RNA Viral/metabolismo , Ativação Transcricional , Transfecção , Produtos do Gene tat do Vírus da Imunodeficiência Humana
14.
Mutagenesis ; 13(2): 153-5, 1998 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-9568588

RESUMO

A series of independent tumour-derived Chinese hamster CHEF18 cell lines was analyzed for the presence of mutations in the cDNA region (exons 5-9) of the p53 gene, where the great majority of p53 mutations of human tumours accumulate. Since the gene is highly conserved among species, we used two primers designed on the basis of the human cDNA sequence to isolate the cDNA region from total RNA of Chinese hamster cells. The amplified fragments of 614 bp were digested with cleavase I endonuclease and fragment length polymorphism analysis showed that the restriction pattern of the p53 exons 5-9 region of tumour-derived cell lines was identical to that of diploid Chinese hamster CHL fibroblasts. Sequencing of the amplified fragments showed 100% homology between sequences, which demonstrated the absence of p53 mutations in the exons 5-9 cDNA region expected to have the highest mutability. Nevertheless, the antibody DO-7 recognized the presence of a stabilized p53 protein only in tumour-derived cell lines, which indicated that p53 expression correlated with transformed status.


Assuntos
Genes p53 , Mutação/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Cricetinae , Cricetulus , Análise Mutacional de DNA , DNA Complementar/isolamento & purificação , Humanos , Dados de Sequência Molecular , Células Tumorais Cultivadas , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/isolamento & purificação
16.
Cytogenet Cell Genet ; 77(3-4): 228-31, 1997.
Artigo em Inglês | MEDLINE | ID: mdl-9284922

RESUMO

The Chinese hamster tumor-derived cell line 835T2 exhibits specific karyotypic changes, including the loss and/or translocation of genetic material. To investigate whether the p53 tumor suppressor gene was involved in the exchanges, cDNA from primary Chinese hamster cells was isolated by using sense and antisense primers of the human p53 gene. The cDNA was sequenced, and the sequence was compared with the Syrian and human p53 cDNA reported sequences. The sequence homology was very elevated, demonstrating that the cloned fragment contained part of the Chinese hamster p53 gene. The corresponding genomic fragment was also cloned and used as a biotin-labeled probe for in situ hybridization on Chinese hamster chromosome spreads. Hybridization was visualized by avidin-FITC, and the assignment was done comparing the banding obtained with BamHI restriction enzyme and the location of the fluorescent signals pattern of the same metaphase. The signals revealed that the p53 gene (TP53) is localized on Chinese hamster chromosome band 2p31, which is not involved in the karyotypic changes specific to the 835T2 cell line.


Assuntos
Cricetulus/genética , Genes p53 , Animais , Sequência de Bases , Bandeamento Cromossômico , Mapeamento Cromossômico , Clonagem Molecular , Cricetinae , Primers do DNA/genética , DNA Complementar/genética , Humanos , Hibridização in Situ Fluorescente , Cariotipagem , Mesocricetus , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Homologia de Sequência de Aminoácidos , Células Tumorais Cultivadas
17.
Mutagenesis ; 11(4): 401-4, 1996 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-8671765

RESUMO

Clone CSA7 is a CHEF18 hamster cell line that shows an increased intracellular accumulation of dCTP. To localize the mutations that accumulate spontaneously in a functional gene of such a mutator phenotype, independent CSA7 mutants of the hypoxanthine-guanine phosphoribosyl transferase (hprt) gene were isolated and screened by a polymerase chain reaction-single strand conformation polymorphism technique. Sixty-two percent of mutants produced detectable changes of the strand migration profile and the mutations were preferentially localized in the exons 3 (31%) and 6 (62%). The sequencing of such exons revealed that the rate of C base incorporation was the major mutation pathway and that the A base of a GGA sequence was the preferential site of misincorporation.


Assuntos
Nucleotídeos de Desoxicitosina/metabolismo , Mutação , Animais , Sequência de Bases , Linhagem Celular , Cricetinae , Cricetulus , DNA/genética , DNA/metabolismo , Éxons , Hipoxantina Fosforribosiltransferase/genética , Dados de Sequência Molecular , Fenótipo , Reação em Cadeia da Polimerase , Polimorfismo Conformacional de Fita Simples
18.
Zentralbl Veterinarmed B ; 43(4): 201-11, 1996 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-8767766

RESUMO

Southern-blot hybridization and partial sequencing of the pol and env genes were used to characterize BLV-integrated provirus of seropositive cattle from two dairy herds in northern Italy. Comparison of the data obtained with those of previously characterized BLV strains from other geographic areas (Australia, Belgium, Japan and USA) revealed the presence of a viral variant (BLV-12), which showed both conserved and unique features. Regarding the gp51 envelope glycoprotein, the BLV-12 variant showed: 1. A high extent of conservation, which included potential glycosylation sites and cysteine residues; 2. Three unique amino acid residues not present in any of the other BLV strains analysed; and 3. Some variability at the level of one (G) of the three (F, G and H) conformational epitopes, which is probably important in the process of infection. These results agree with the suggestion that the sequence variability of the gp51 glycoprotein preferentially involves structures whose change is thought to underlie the phenomenon of escape from immune surveillance.


Assuntos
DNA Viral/análise , Genes env/genética , Genes pol/genética , Vírus da Leucemia Bovina/genética , Provírus/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Bovinos , DNA Viral/química , Produtos do Gene env/química , Produtos do Gene env/genética , Produtos do Gene pol/química , Produtos do Gene pol/genética , Variação Genética , Dados de Sequência Molecular
19.
Hum Immunol ; 46(2): 69-81, 1996 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-8727205

RESUMO

Only a few monoclonal antibodies are available with a restricted specificity to HLA-C products. In the present report, we demonstrate that antibody L31, previously shown to react with beta 2m-less (free) class I MHC heavy chains, binds to an epitope (residues 66-68 of the alpha 1 domain alpha helix) present on all the HLA-C alleles corresponding to the accepted (CW1 through CW8) serologic specificities, and on a few HLA-B heavy chains sharing with HLA-C an aromatic residue at position 67. Extensive IEF blot testing of HLA homozygous, EBV-transformed B-lymphoid cells indicates that HLA-C molecules are present at significantly lower levels than HLA-B polypeptides not only at cell surface, as previously demonstrated, but also in total cellular extracts. Testing of metabolically labeled HLA-CW1, -CW5, and -CW6 transfectants and HLA homozygous lymphoid cells, particularly HLA-CW1-expressing cells, demonstrates that the L31 epitope is present on a subpopulation of naturally occurring HLA-C molecules distinct from that identified by antibody W6/32 to beta 2m-associated heavy chains. Pulse-chase experiments demonstrate that this epitope is transiently made available to antibody binding at early biosynthetic stages, but becomes hidden upon assembly with beta 2m. Thus, free HLA-C and other Y/F67+ heavy chains are characterized by distinctive antibody binding features in a region (residues 66-68) included in a previously identified HLA-C restricted motif, which has been suggested to be the primary cause of distinctive features of the antigen-binding groove, low affinity for endogenous peptide antigens and beta 2m, and preferential uptake of exogenous peptides, possibly of viral origin. We also show that HLA-CW1 heavy chains, both free and beta 2m associated, acquire sialilation. Free HLA-CW1 heavy chains are expressed at the cell surface even when unsialilated, albeit at low levels.


Assuntos
Epitopos/imunologia , Antígenos HLA-C/imunologia , Estrutura Secundária de Proteína , Microglobulina beta-2/deficiência , Anticorpos Monoclonais/imunologia , Linfócitos B/imunologia , Linhagem Celular Transformada , Mapeamento de Epitopos/métodos , Antígenos HLA-C/biossíntese , Humanos , Focalização Isoelétrica/métodos , Microglobulina beta-2/imunologia
20.
Immunogenetics ; 43(5): 289-95, 1996.
Artigo em Inglês | MEDLINE | ID: mdl-9110932

RESUMO

Epitope mapping of HLA-Cw4 indicates that the two monoclonal antibodies (mAbs) L31 and M38, specific for beta 2-microglobulin (beta2m)-free HLA-C heavy chains, react preferentially with the KYK motif, located in the binding groove (alpha1 domain). Transfection of HLA-Cw4 cDNA into a neuroblastoma cell line, which normally expresses negligible HLA class I, resulted in the constitutive surface expression of molecules displaying different reactivities with the two mAbs. This cellular system was used to determine whether L31 and M38 recognize distinct conformations of beta2m-free HLA-C proteins, and to investigate their mechanism of expression. Interferon-gamma greatly enhanced the expression of L31-reactive free chains, while abolishing that of M38-reactive molecules. The cytokine-induced expression of L31-reactive molecules was inhibited by anti-sense oligonucleotides specific for beta2m mRNA, while constitutive expression of L31-reactive molecules was only partially affected. Exogenous beta2m resulted in a reduction of constitutive L31 reactivity, and in a concomitant increase of M38 reactivity. These results indicate that: 1) at the cell surface, L31 and M38 react with two distinct conformations of HLA-Cw4 beta2m-free heavy chains, of which the L31-reactive conformation is the least folded; 2) the expression of both conformers can be modulated by endogenous or exogenous beta2m; and (3) L31-reactive molecules exposed at the cell surface are likely to derive from the dissociation of empty HLA-Cw4/beta2m complexes.


Assuntos
Antígenos HLA-C/química , Anticorpos Monoclonais , Sequência de Bases , Mapeamento de Epitopos , Citometria de Fluxo , Dados de Sequência Molecular , Neuroblastoma/imunologia , Conformação Proteica , Proteínas Recombinantes , Transfecção , Células Tumorais Cultivadas
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