RESUMEN
Cell therapies utilizing mesenchymal stem cells (MSCs) could overcome limitations of traditional treatments for reconstructing craniofacial tissues. This large-scale study explored a standardized methodology for the isolation and clinical-scale expansion of alveolar bone marrow-derived MSCs (aBMSCs). We harvested 103 alveolar bone marrow samples from 45 patients using 1 of 3 standardized methodologies. Following aBMSC isolation, cells were characterized through cell-surface marker expression and lineage-specific differentiation. Long-term cultures (> 50 population doublings [PDs]) were evaluated for transformational changes through senescence, gene expression, and karyotyping. Finally, aBMSC bone-forming potential was determined in vivo. More than 0.5 cc of bone marrow was needed to predictably isolate aBMSCs, and, regardless of methodology for harvest, cell-surface marker expression of CD73, CD90, CD105, and Stro-1 was similar for aBMSCs, being 89.8%, 98.8%, 93.8%, and 3.2%, respectively; all cells were negative for CD11b, CD19, and CD45. aBMSCs exhibited multipotency, and karyotypes were normal up to 30 PDs, with significant cell senescence beginning following 35 PDs. Additionally, aBMSCs induced ectopic bone formation following subcutaneous transplantation into mice. These findings demonstrate a predictable approach for the isolation and safe clinical-scale expansion of aBMSCs, and thus, their clinical use could be considered for craniofacial regenerative therapies.
Asunto(s)
Proceso Alveolar/citología , Técnicas de Cultivo de Célula/normas , Separación Celular/normas , Células Madre Mesenquimatosas/citología , 5'-Nucleotidasa/análisis , Animales , Antígenos CD/análisis , Antígenos CD19/análisis , Antígenos de Superficie/análisis , Biomarcadores/análisis , Antígeno CD11b/análisis , Diferenciación Celular/fisiología , Linaje de la Célula , Senescencia Celular/fisiología , Endoglina , Estudios de Factibilidad , Proteínas Ligadas a GPI/análisis , Expresión Génica/genética , Humanos , Cariotipificación , Antígenos Comunes de Leucocito/análisis , Trasplante de Células Madre Mesenquimatosas/métodos , Ratones , Células Madre Multipotentes/citología , Osteogénesis/fisiología , Receptores de Superficie Celular/análisis , Seguridad , Tejido Subcutáneo/cirugía , Telómero/química , Antígenos Thy-1/análisis , Factores de Tiempo , Andamios del TejidoRESUMEN
Recently, extracted teeth have been identified as a viable source of stem cells for tissue regenerative approaches. Current expansion of these cells requires incorporation of animal sera; yet, a fundamental issue underlying cell cultivation methods for cell therapy regards concerns in using animal sera. In this study, we investigated the development of a chemically defined, serum-free media (K-M) for the expansion of human periodontal ligament stem cells (PDLSCs) and human stem cells from exfoliated deciduous teeth (SHEDs). Proliferation assays were performed comparing cells in serum-containing media (FBS-M) with cells cultured in four different serum-free medium and these demonstrated that in these medium, the cell proliferation of both cell types was significantly less than the proliferation of cells in FBS-M. Additional proliferation assays were performed using pre-coated fibronectin (FN) tissue culture plates and of the four serum-free medium, only K-M enabled PDLSCs and SHEDs to proliferate at higher rates than cells cultured in FBS-M. Next, alkaline phosphatase activity showed that PDLSCs and SHEDs exhibited similar osteogenic potential whether cultured in K-M or FBS-M, and, additionally, cells retained their multipotency in K-M as seen by expression of chondrogenic and adipogenic genes, and positive Von Kossa, Alcian blue, and Oil Red O staining. Finally, differential expression of 84 stem cell associated genes revealed that for most genes, PDLSCs and SHEDs did not differ in their expression regardless of whether cultured in K-M or FBS-M. Taken together, the data suggest that K-M can support the expansion of PDLSCs and SHEDs and maintenance of their multipotency.
Asunto(s)
Técnicas de Cultivo de Célula , Medio de Cultivo Libre de Suero , Ligamento Periodontal/citología , Células Madre/citología , Diente Primario/citología , Proliferación Celular , Regulación de la Expresión Génica/fisiología , Humanos , Osteogénesis , Células Madre/fisiologíaRESUMEN
Bacterial overgrowth in the stomach may occur under conditions of diminished or absent acid secretion. Under these conditions, secretion of the hormone gastrin is elevated. Alternatively, bacterial factors may directly stimulate gastrin. Consistent with this hypothesis, we found that mice colonized for 2 months with a mixed bacterial culture of opportunistic pathogens showed an increase in serum gastrin. To examine regulation of gene expression by bacterial proteins, stable transformants of AGS cells expressing gastrin or interleukin-8 (IL-8) promoters were cocultured with live organisms. Both whole-cell sonicates and a heat-stable fraction were also coincubated with the cells. A level of 10(8) organisms per ml stimulated both the gastrin and IL-8 promoters. Heat-stable proteins prepared from these bacterial sonicates stimulated the promoter significantly more than the live organism or unheated sonicates. A 38-kDa heat-stable protein stimulating the gastrin and IL-8 promoters was cloned and found to be an OmpA-related protein. Immunoblotting using antibody to the OmpA-like protein identified an Acinetobacter sp. as the bacterial species that expressed this protein and colonized the mouse stomach. Moreover, reintubation of mice with a pure culture of the Acinetobacter sp. caused gastritis. We conclude that bacterial colonization of the stomach may increase serum gastrin levels in part through the ability of the bacteria to produce OmpA-like proteins that directly stimulate gastrin and IL-8 gene expression. These results implicate OmpA-secreting bacteria in the activation of gastrin gene expression and raise the possibility that a variety of organisms may contribute to the increase in serum gastrin and subsequent epithelial cell proliferation in the hypochlorhydric stomach.
Asunto(s)
Acinetobacter/patogenicidad , Proteínas de la Membrana Bacteriana Externa/farmacología , Gastrinas/biosíntesis , Gastritis/etiología , Interleucina-8/biosíntesis , Estómago/microbiología , Acinetobacter/genética , Secuencia de Aminoácidos , Animales , Proteínas de la Membrana Bacteriana Externa/genética , Secuencia de Bases , Clonación Molecular , Técnicas de Cocultivo , Gastrinas/genética , Genes Bacterianos , Humanos , Interleucina-8/genética , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Moraxella catarrhalis/genética , Regiones Promotoras Genéticas , Proteínas Recombinantes/farmacología , Homología de Secuencia de Aminoácido , Estómago/citologíaAsunto(s)
Cromosomas Humanos Par 3 , Proteínas de Unión al ADN/genética , Gastrinas/genética , Regulación de la Expresión Génica , Factores de Transcripción , Animales , Mapeo Cromosómico , Clonación Molecular , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/fisiología , Humanos , Mutación , Ratas , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Transcripción GenéticaRESUMEN
ZBP-89 is a Krüppel-type zinc finger transcription factor that binds to GC-rich sequences. Overexpression of this factor prevents EGF induction of the gastrin promoter; therefore, we postulated that ZBP-89 may modulate cellular proliferation. To test this hypothesis, ZBP-89 was overexpressed in immortalized (GH4) and malignant (AGS) cell lines. Growth parameters, e.g., 3H-thymidine, BrdU labeling, flow cytometry and ornithine decarboxylase promoter activity were analyzed. The results show that DNA synthesis is inhibited and progression to S phase is blocked in GH4 cells. Collectively, these studies demonstrate that ZBP-89 inhibits cellular proliferation at least in part through its ability to bind and repress ornithine deacarboxlyase promoter activity.
Asunto(s)
División Celular/fisiología , Factores de Transcripción/fisiología , Dedos de Zinc/fisiología , Adenocarcinoma/patología , Unión Competitiva , Línea Celular , Humanos , Ornitina Descarboxilasa/genética , Regiones Promotoras Genéticas , Factor de Transcripción Sp1/metabolismo , Neoplasias Gástricas/patología , Factores de Transcripción/metabolismo , Células Tumorales CultivadasRESUMEN
The gene for type 1 neurofibromatosis (NF1) is most highly expressed in brain and spinal cord, although low levels of mRNA can be found in nearly all tissues. As a first step in investigating the regulation of NF1 gene expression, we have cloned and sequenced the promoter regions of the human and mouse NF1 genes and mapped the transcriptional start sites in both species. We report here that the 5' ends of the human and murine NF1 genes are highly conserved. While no discernable TATA or CCAAT box sequences are seen, transcription initiates at identical sites in both species, 484 nucleotides upstream of the ATG initiation codon in the human gene. The human and mouse NF1 genes share particularly high sequence homology (95%) between nucleotides -33 and +261 and contain several perfectly conserved transcription factor binding site motifs, including a cAMP response element, several AP2 consensus binding sites, and a serum response element. The high conservation of these sequences indicates that they are likely to be significant in the regulation of NF1 gene expression.
Asunto(s)
ADN/genética , Genes de Neurofibromatosis 1 , Hominidae/genética , Ratones/genética , Regiones Promotoras Genéticas , Animales , Secuencia de Bases , Sitios de Unión , Encéfalo/metabolismo , Clonación Molecular , Secuencia Conservada , ADN/metabolismo , Expresión Génica , Humanos , Datos de Secuencia Molecular , Neurofibromatosis 1/genética , Homología de Secuencia de Ácido Nucleico , Médula Espinal/metabolismo , TATA Box , Factores de Transcripción/metabolismoRESUMEN
The pericentric inversion of chromosome 16 [inv(16)(p13q22)] is a characteristic karyotypic abnormality associated with acute myeloid leukemia, most commonly of the M4Eo subtype. The 16p and 16q breakpoints were pinpointed by yeast artificial chromosome and cosmid cloning, and the two genes involved in this inversion were identified. On 16q the inversion occurred near the end of the coding region for CBF beta, also known as PEBP2 beta, a subunit of a heterodimeric transcription factor regulating genes expressed in T cells; on 16p a smooth muscle myosin heavy chain (SMMHC) gene (MYH11) was interrupted. In six of six inv(16) patient samples tested, an in-frame fusion messenger RNA was demonstrated that connected the first 165 amino acids of CBF beta with the tail region of SMMHC. The repeated coiled coil of SMMHC may result in dimerization of the CBF beta fusion protein, which in turn would lead to alterations in transcriptional regulation and contribute to leukemic transformation.
Asunto(s)
Inversión Cromosómica , Cromosomas Humanos Par 16 , Proteínas de Unión al ADN/genética , Leucemia Mielomonocítica Aguda/genética , Miosinas/genética , Proteínas de Neoplasias , Factores de Transcripción/genética , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , Subunidad alfa 1 del Factor de Unión al Sitio Principal , Subunidad beta del Factor de Unión al Sitio Principal , Factores de Unión al Sitio Principal , Cósmidos , Humanos , Hibridación Fluorescente in Situ , Datos de Secuencia Molecular , Músculo Liso/química , Reacción en Cadena de la Polimerasa , Multimerización de Proteína , Mapeo Restrictivo , Factor de Transcripción AP-2RESUMEN
Neurofibromatosis type 1 (NF1) is a common autosomal dominant disorder characterized by progressive and variable involvement of tissues predominantly derived from the neural crest and a predisposition toward malignancies. The NF1 gene encodes neurofibromin, a GTPase-activating protein containing a GAP-related domain (NF1-GRD) that is capable of down-regulating ras by stimulating its intrinsic GTPase activity. We report a homozygous deletion of most of NF1 in one of eight malignant melanoma cell lines leading to loss of detectable mRNA and protein, as well as the apparent absence of protein and mRNA in another melanoma. This data suggests that NF1 can function as a tumour suppressor gene in the development or progression of malignant melanoma.
Asunto(s)
Genes de Neurofibromatosis 1 , Melanoma/genética , Mapeo Cromosómico , Cromosomas Humanos Par 17 , ADN de Neoplasias/genética , Eliminación de Gen , Genes Supresores de Tumor , Humanos , Mutación , Neurofibromina 1 , Proteínas/genética , Transcripción Genética , Células Tumorales CultivadasRESUMEN
Tissue- and developmental stage-specific expression of the human beta-like globin genes is regulated by a combination of ubiquitous and erythroid-restricted trans factors that bind to cis elements near each of the five active genes. Additional interactions of these cis and trans factors with sequences located in the far 5' end of the cluster occur by as yet obscure mechanisms. Because of the complexity of this regulatory puzzle, precise identification of the determinants that control hemoglobin switching has proven difficult. Phylogenetic footprinting is an evolutionary approach to this problem which is based on the supposition that the basic mechanisms of switching are conserved throughout mammalian phylogeny. Alignment of the 5' flanking regions of the gamma genes of several species allows the identification of footprints of 100% conserved sequence. We have now tested oligomers spanning 13 such phylogenetic footprints and find that 12 are bound by nuclear proteins. One conserved element located at -1086 from the gamma genes exhibits repressor activity in transient transfection studies. The protein that binds this element, CSBP-1 (conserved sequence-binding protein 1), also binds at three sites within a silencer element upstream from the epsilon globin gene. Further analysis reveals that the CSBP-1 binding activity is identical to that of a recently cloned zinc finger protein that has been shown to act as a repressor in other systems. The binding of CSPB-1 to silencer sequences in the epsilon and gamma globin genes may be important in the stage-specific silencing of these genes.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Globinas/genética , Proteínas Nucleares/metabolismo , Secuencias Reguladoras de Ácidos Nucleicos , Animales , Secuencia de Bases , Clonación Molecular , Secuencia Conservada , ADN , Factores de Unión al ADN Específico de las Células Eritroides , Humanos , Leucemia Eritroblástica Aguda , Ratones , Datos de Secuencia Molecular , Filogenia , Regiones Promotoras Genéticas , Homología de Secuencia de Ácido Nucleico , Factores de Transcripción/metabolismo , Células Tumorales Cultivadas , Dedos de ZincRESUMEN
Hypoxanthine--guanine phosphoribosyltransferase (HPRT) is a purine salvage enzyme that catalyzes the conversion of hypoxanthine to inosine monophosphate and guanine to guanosine monophosphate. Previous studies of mutant HPRT proteins analyzed at the molecular level have shown a significant heterogeneity. This investigation further verifies this heterogeneity and identifies insertions, deletions, and point mutations. The direct sequencing of the polymerase chain reaction-amplified product of reverse-transcribed HPRT mRNA enabled the rapid identification of the mutations found in 17 previously uncharacterized cell lines derived from patients with the Lesch-Nyhan syndrome.
Asunto(s)
Hipoxantina Fosforribosiltransferasa/genética , Síndrome de Lesch-Nyhan/genética , Mutación , Línea Celular , Análisis Mutacional de ADN , Humanos , Reacción en Cadena de la PolimerasaRESUMEN
Complete hypoxanthine-guanine phosphoribosyltransferase (HPRT) deficiency causes the Lesch-Nyhan syndrome, an X-linked, purine metabolism disorder manifested by hyperuricemia, hyperuricaciduria, and neurologic dysfunction. Partial HPRT deficiency causes hyperuricemia and gout. One requirement for understanding the molecular basis of HPRT deficiency is the determination of which amino acids in this salvage enzyme are necessary for structural or catalytic competence. In this study we have used the PCR coupled with direct sequencing to determine the nucleotide and subsequent amino acid changes in 22 subjects representing 17 unrelated kindreds from the United Kingdom. These mutations were confirmed by using either RNase mapping or Southern analyses. In addition, experiments were done to determine enzyme activity and electrophoretic mobility, and predictive paradigms were used to study the impact of these amino acid substitutions on secondary structure.
Asunto(s)
Hipoxantina Fosforribosiltransferasa/genética , Síndrome de Lesch-Nyhan/genética , Mutación/genética , Deleción Cromosómica , Humanos , Hipoxantina Fosforribosiltransferasa/metabolismo , Síndrome de Lesch-Nyhan/enzimología , Mutagénesis InsercionalRESUMEN
Adenine phosphoribosyltransferase (APRT) deficiency is a genetic disorder which causes 2,8-dihydroxy-adenine urolithiasis. The estimated incidence of heterozygosity in Caucasian and Japanese populations is 1%. Mutant alleles responsible for the disease have been classified as APRT*Q0 (type I) and APRT* (type II). In our previous study, we demonstrated in APRT*J a single common base change which accounts for 70% of the Japanese mutants. The present report describes the analysis of an APRT*Q0 mutation in Japanese subjects. Two nucleotide substitutions common to all seven affected alleles from four unrelated subjects (three homozygotes and a heterozygote) were identified: G----A at nucleotide position 1453 and C----T at 1456. The G----A altered the amino acid Trp98 to a stop codon. The C----T did not alter Ala99. These point mutations were demonstrated by sequence analysis of polymerase chain reaction (PCR)-amplified genomic DNA and cDNA. The G----A change at 1453 results in the elimination of a PflMI site in the APRT gene. PflMI digests, which were used to confirm the G----A transition, can be useful in screening for this specific mutation.
Asunto(s)
Adenina Fosforribosiltransferasa/genética , Alelos , Mutación , Adenina Fosforribosiltransferasa/deficiencia , Secuencia de Aminoácidos , Secuencia de Bases , Southern Blotting , Línea Celular , Sondas de ADN , Humanos , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Mapeo RestrictivoRESUMEN
Hypoxanthine-guanine phosphoribosyltransferase (HPRT) deficiency is an inborn error of purine metabolism. Mutant HPRT gene sequences from patients deficient in enzyme activity have previously been characterized by cDNA cloning or amino acid sequencing techniques. The presence of HPRT-specific mRNA in nearly all deficient subjects, as well as the small size of the HPRT mRNA (1,400 bp), make the polymerase chain reaction (PCR) an alternative for the identification of mutations at this locus. In this report we use the PCR to identify previously undetermined mutations in HPRT mRNA from B lymphoblasts derived from 10 deficient individuals. Six of these variants contain single point mutations, three contain deletions, and one contains a single nucleotide insertion. Several of these mutations map near previously identified HPRT variants, and are located in evolutionarily conserved regions of the molecule.
Asunto(s)
Amplificación de Genes , Hipoxantina Fosforribosiltransferasa/deficiencia , Mutación , Transcripción Genética , Secuencia de Aminoácidos , ADN/análisis , Humanos , Hipoxantina Fosforribosiltransferasa/genética , Datos de Secuencia MolecularAsunto(s)
Adenina Fosforribosiltransferasa/deficiencia , Tamización de Portadores Genéticos , Mutación , Pentosiltransferasa/deficiencia , Adenina Fosforribosiltransferasa/genética , Alelos , Secuencia de Bases , Línea Celular , ADN/biosíntesis , ADN Polimerasa Dirigida por ADN , Amplificación de Genes , Humanos , Immunoblotting , Japón , Datos de Secuencia Molecular , Sondas de OligonucleótidosRESUMEN
Complete adenine phosphoribosyltransferase (APRT) deficiency causes 2,8-dihydroxyadenine urolithiasis. In previous reports, analysis of the kinetic properties of APRT from APRT-deficient Japanese subjects revealed strikingly similar abnormalities suggesting a distinct "Japanese-type" mutation. In this paper, we report studies of 11 APRT-deficient lymphoblast cell lines. Nucleotide sequence analysis of APRT genomic DNA from WR2, a Japanese-type homozygote, identified a T to C substitution in exon 5, giving rise to the substitution of threonine for methionine at position 136. RNase mapping analysis confirmed this mutation in WR2 and revealed that six other Japanese-type homozygotes carry the same mutation on at least one allele. The remaining Japanese subject, who does not express the Japanese-type phenotype, did not demonstrate this mutation. Southern blot analysis showed that all seven Japanese-type subjects were confined to one TaqI restriction fragment length polymorphism (RFLP) haplotype. These studies provide direct evidence for the nature of the mutation in the Japanese-type APRT deficiency.
Asunto(s)
Adenina Fosforribosiltransferasa/deficiencia , Alelos , Mutación , Pentosiltransferasa/deficiencia , Adenina Fosforribosiltransferasa/genética , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , Humanos , Japón/etnología , Linfocitos/enzimología , Fenotipo , Población BlancaRESUMEN
This study reports the first demonstration of specific mutations leading to human adenine phosphoribosyltransferase (APRT) deficiency. The molecular basis of the deficiency was investigated by determining the sequence of both alleles of a patient with a complete deficiency in APRT activity. A trinucleotide deletion, corresponding to phenylalanine on the deduced amino acid sequence, was confirmed on one allele. A single nucleotide insertion, immediately adjacent to the splice site at the 5' end of the fourth intervening sequence, was confirmed on the other allele. This insertion lead to aberrant splicing, as was demonstrated by the absence of exon 4 in the complementary DNA sequence and by altered RNase mapping analysis of the abnormal messenger RNA.