Assuntos
Criopreservação/métodos , Criopreservação/normas , Células-Tronco Hematopoéticas/citologia , Gestão da Qualidade Total/métodos , Gestão da Qualidade Total/normas , Feminino , Transplante de Células-Tronco Hematopoéticas/métodos , Transplante de Células-Tronco Hematopoéticas/normas , Humanos , Masculino , Controle de Qualidade , Transplante HomólogoRESUMO
AIMS: It is important to have clinically relevant large animal models, especially nonhuman primates, to improve the efficacy of islet isolation and transplantation prior to clinical trials. The aim of this study was to improve the efficacy of islet isolation by analyzing large-scale nonhuman primate islet isolations. METHODS: Sixty-one islet isolations were evaluated using nonhuman primates. An automated isolation method was scaled down for islet isolation. Islet yields of prepurification, postpurification, and postculture, purity of islets, viability of islets, and functionality with glucose stimulation test were assessed. Initially, we analyzed relationships between endpoints then analyzed additional factors for successful islet isolation. Those factors included donor characteristics, the two-layer method (TLM) of pancreas preservation, trypsin inhibition during digestion, and digestion and collection time. RESULTS: Prepurification islet yields were strongly correlated with postpurification yields and postculture yields. It weakly but significantly correlated with purity, viability, and functionality. The average prepurification yield was 16,267 IE/g with each case divided into either above-average (high-yield group) or below-average groups (low-yield group). In 8 cases, TLM and trypsin inhibition were used and all cases belonged to the high-yield group. There were no significant differences between high- and low-yield groups in terms of donor age, body weight, pancreas weight, and cold ischemic time. The high-yield group had significantly longer digestion times and shorter collection times. CONCLUSIONS: TLM, trypsin inhibition, complete digestion, and quick collections were key for successful islet isolation. Analysis of nonhuman primate islet isolation techniques provided useful information, which should help to improve clinical islet transplantation.
Assuntos
Ilhotas Pancreáticas/citologia , Animais , Técnicas de Cultura de Células/métodos , Separação Celular/métodos , Isquemia , Macaca nemestrina , Modelos Animais , Tamanho do Órgão , Pâncreas/anatomia & histologia , Análise de RegressãoRESUMO
BACKGROUND: Research-grade pancreata preserved by the two-layer method (TLM) compared to organs stored with University of Wisconsin (UW) solution prior to islet isolation result in significantly better islet yields. However, it is unknown whether the TLM improves islet yields from pancreata that meet the criteria for the selection of clinical-grade organs. METHODS: Six clinical-grade pancreata were preserved for 4.8 +/- 0.5 hour in UW and three clinical-grade pancreata were preserved by the TLM for 11.7 +/- 2.0 hour. The local team procured all pancreata. All donors were hemodynamically stable without norepinephrine usage and length of hospitalization was less than 96 hour. Causes of death were either head trauma or cerebrovascular accident. Islets were isolated and evaluated according to the Edmonton protocol. RESULTS: The TLM as compared to UW resulted in a significant increase in islet yields (3415 +/- 227 vs 2006 +/- 337 IE/g pancreas, P <.03). The quality of islets as assessed by visual score was significantly better in the TLM group (8.7 +/- 0.2 vs 7.3 +/- 0.3, P <.02) but other parameters (viability, survival rate after culture, insulin content, stimulation index) were similar between the two groups. We transplanted all three islet preparations in the TLM group but only two of six preparations from the UW group. CONCLUSION: Compared to UW, exposure of pancreata to the TLM resulted in greater islet yields and extended preservation times with clinical grade pancreas. Pancreata should be preserved by the TLM prior to islet isolation even for donors that meet clinical grade organ selection criteria. The Human Islet Transplantation in Seattle (HITS) Consortium is supported in part by a grant from the Juvenile Diabetes Research Foundation International. The HITS consortium is an islet transplant program involving the University of Washington, Pacific Northwest Research Institute, the Puget Sound Blood Center, Fred Hutchinson Cancer Research Center, Swedish Hospital, and the Virginia Mason Research Center.
Assuntos
Adenosina , Alopurinol , Fluorocarbonos , Glutationa , Insulina , Transplante das Ilhotas Pancreáticas/métodos , Ilhotas Pancreáticas/citologia , Soluções para Preservação de Órgãos , Rafinose , Técnicas de Cultura de Células/métodos , HumanosRESUMO
BACKGROUND: Appropriate donor selection is one of the keys for successful human islet isolation. Previous studies identified several critical donor factors; however, significant improvements in current human islet isolation protocols make reevaluation of donor factors necessary. STUDY DESIGN: Review was performed on 31 human islet isolations. Islet isolations were conducted using the standard automated islet isolation method with three protocol revisions that included the two-layer method (TLM) of pancreas preservation prior to islet isolation, usage of purified collagenase mixture Liberase, and continuous density gradient for islet purification. Factors leading to successful isolations (islet yield > 100,000 IE and static incubation stimulation index greater than 2.0) were analyzed. The impacts of various risk factors were also examined. RESULTS: Donors in the successful islet isolation group had a significantly lower incidence of elevated peak transaminases and creatinine levels, lower usage of norepinephrine or cardiac arrest, less prolonged hospitalization (> 96 hours), and less prolonged preservation time of donor pancreata (>25 hours). The TLM extended acceptable preservation time of donor pancreata from 8 to 25 hours. When donors had no risk factor, the success rate was 14/16 (87.5%). In sharp contrast, when donors had two or more risk factors, the success rate was 0/7 (0%; P <.001). CONCLUSION: Risk factors for human islet isolation with the current islet isolation protocol were identified. The decision to process pancreata based on review of donor factors should improve the consistency of human islet isolations and transplantation for curing type 1 diabetes.
Assuntos
Técnicas de Cultura de Células/métodos , Ilhotas Pancreáticas/citologia , Doadores de Tecidos/estatística & dados numéricos , Adulto , Separação Celular/métodos , Colagenases , Feminino , Humanos , Masculino , Reprodutibilidade dos Testes , Estudos RetrospectivosRESUMO
Escherichia coli DNA polymerase III holoenzyme in the presence of ATP and E. coli single-stranded DNA-binding protein forms an initiation complex on a primed template capable of rapid and highly processive DNA replication. DNase I digestion of initiation complexes demonstrated that holoenzyme protected 27-30 nucleotides of primer. Like the formation of initiation complexes, this protection required both ATP and E. coli single-stranded DNA-binding protein. Initiation complexes assembled with core DNA polymerase III (alpha, epsilon, and theta subunits), gamma-complex (gamma, delta, delta', chi, and omega) and the beta subunit produced a footprint identical to that formed with intact holoenzyme, indicating that initiation complexes formed with reconstituted enzyme and those formed with holoenzyme were equivalent. The presence of the tau subunit in reconstituted initiation complexes did not alter the DNase I footprint. Preinitiation complexes (gamma-complex plus beta subunit) assembled onto primer-template in an ATP-dependent reaction protected a larger region of the primer than did holoenzyme. The addition of core DNA polymerase III to preintiation complexes restored the 30-nucleotide footprint observed with intact holoenzyme. These results suggest that holoenzyme subunits rearrange during initiation complex formation.
Assuntos
DNA Polimerase III/metabolismo , Primers do DNA/metabolismo , Escherichia coli/enzimologia , Trifosfato de Adenosina/metabolismo , Primers do DNA/química , DNA de Cadeia Simples/metabolismo , Proteínas de Ligação a DNA/metabolismo , Desoxirribonuclease I/metabolismo , Hidrólise , Conformação de Ácido NucleicoRESUMO
BACKGROUND: Because of the limitation of cell numbers associated with cord blood harvests, there is a need to determine the efficacy of using ex vivo-expanded cord blood cells in a transplantation setting. In this study, limiting-dilution analysis was used in nonobese diabetic mice with severe combined immunodeficiency (NOD/SCID) to compare the engraftment potential of progeny cells expressing the CD34+ phenotype after expansion with that of uncultured CD34+ cells. STUDY DESIGN AND METHODS: Cord blood CD34+ cells were cultured in Iscove's modified Dulbecco medium supplemented with 10-percent fetal calf serum (FCS) and IL-6, SCF, megakaryocyte growth and development factor, and Flt3 ligand. The resulting ex vivo-expanded products were assessed for total numbers of nucleated cells, CD34+ cells, and CFUs and long-term culture-initiating cell activity. The engraftment potentials of cultured progeny CD34+ cells and uncultured CD34+ cells were determined by using NOD/SCID mice. RESULTS: After 14 days of culture, total nucleated cell counts increased over input values by 180 +/- 59-fold, CD34+ cell numbers by 44 +/- 13-fold, CFU activity by 23 +/- 5-fold, and long-term culture-initiating cell activity by 20 +/- 6-fold (mean +/- SD; n = 6). The frequency of SCID-repopulating cells (SRC) in mice transplanted with uncultured products was 1 per 20,000 CD34+ cells (95% CI, 1:10,000-1:38,000) and that in mice receiving ex vivo-expanded products was 1 per 418,000 progeny CD34+ cells (95% CI, 1:158,000-1:1,100,000). Taken together, these data indicated that, after 2 weeks of culture, there was a modest twofold increase in the total number of SRCs. However, the levels of human CD45 cell engraftment in NOD/SCID recipients of progeny CD34+ cells were significantly lower than those in mice receiving equivalent numbers of uncultured CD34+ cells (p<0.05). CONCLUSION: Umbilical cord blood progeny cells retaining a CD34+ phenotype after ex vivo expansion have less engraftment potential than do unexpanded CD34+ cells.
Assuntos
Antígenos CD34/sangue , Antígenos CD34/genética , Sangue Fetal/citologia , Sangue Fetal/imunologia , Transplante de Células-Tronco Hematopoéticas , Animais , Células Cultivadas , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , FenótipoRESUMO
The proliferation kinetics and clonogenic activity of CD34+/38hi (CD38hi) and CD34+/38lo (CD38lo) human marrow cells were measured before and after culturing the cells in vitro over a 6-day period in serum-deprived medium containing recombinant growth factors (interleukin-1 [IL-1], IL-3, IL-6, granulocyte colony-stimulating factor [G-CSF], granulocyte-macrophage [GM]-CSF, kit ligand, and erythropoietin). Before in vitro culture, 3% +/- 3% of the CD38lo and 13% +/- 2% of the CD38hi cells were in the S-phase of the cell cycle. The clonogenic activity of CD38hi cells was twofold greater than that of the CD38lo cells, as measured by colony-forming units (CFU) in short-term assays. However, CD38hi cells contained fewer pre-CFU than did the CD38lo cells, generating only 3 +/- 2 colonies per 1,000 cells after 4 weeks of culture on competent stromal layers, compared with 107 +/- 46 colonies per 1,000 cells from the CD38lo population. CD38hi and CD38lo cells exhibited distinctly different responses when cultured in serum-deprived medium supplemented with recombinant growth factors. After culturing cells for 24 hours, CD38lo cells essentially remained a noncycling population with only 5.1% +/- 3.0% of the cells cycling, whereas 44.2% +/- 6.9% of the CD38hi cells were in DNA synthesis. Gradually CD38lo cells were recruited into cycle, such that by 72 hours, approximately 28% of the CD38lo cells were in S-phase. However, during 6 days of culture, the percentage of cycling CD38lo cells never exceeded the proliferative response observed for CD38hi cells. Phenotype analysis conducted at day 6 indicated that 86% of the CD38hi population were no longer phenotypically CD34+/38hi, while 60% of CD38lo cells maintained a CD34+/38lo phenotype. Long-term cultures initiated with 6-day in vitro-expanded CD38lo cells showed approximately a twofold decrease in clonogenic activity attributable to a loss of erythroid precursors and a decrease in GM colonies. Thus, a proportion of CD38lo cells capable of generating CFU was maintained even after exposure to growth factors.
Assuntos
Antígenos CD/análise , Antígenos de Diferenciação/análise , Células da Medula Óssea , Citocinas/farmacologia , Células-Tronco Hematopoéticas/efeitos dos fármacos , ADP-Ribosil Ciclase , ADP-Ribosil Ciclase 1 , Antígenos CD34 , Ciclo Celular , Divisão Celular/efeitos dos fármacos , Separação Celular , Células Cultivadas , Células-Tronco Hematopoéticas/fisiologia , Humanos , Glicoproteínas de MembranaRESUMO
A comparison of the 3'----5' proofreading properties between Escherichia coli DNA polymerase III holoenzyme and DNA polymerase III' was conducted. This study indicated that the influence of the holoenzyme auxiliary subunits on the proofreading exonuclease parallels their effect on the elongation reaction. At physiological ionic strengths the auxiliary subunits markedly stimulated the exonuclease rate in an ATP-dependent reaction, while the exonuclease rate of DNA polymerase III' was not affected by ATP. E. coli single-stranded DNA binding protein stimulated the 3'----5' exonuclease activity of holoenzyme and inhibited DNA polymerase III'. Similarly, the auxiliary subunits and ATP converted the proofreading activity to a highly processive exonuclease. Our observation, that the exonuclease activity of the DNA polymerase III holoenzyme responded to ATP, salt, and E. coli single-stranded DNA-binding protein like the elongation activity, is consistent with the polymerase and exonuclease subunits acting within the same complex in a coordinated reaction.
Assuntos
DNA Polimerase III/metabolismo , Autorradiografia , Escherichia coli/enzimologia , Concentração OsmolarRESUMO
BACKGROUND: The prolonged periods of pancytopenia associated with cord blood transplants suggest that in some cases cell numbers may be limiting. The possibility that limiting cell numbers may be overcome and prolonged periods of pancytopenia abrogated by the transplantation of human umbilical cord blood cells expanded ex vivo has led to efforts to define optimal culture conditions for these cells. STUDY DESIGN AND METHODS: Cord blood CD34+ cells were cultured with three cytokine combinations: SCF+G-CSF+GM-CSF+MGDF (SGGM); IL-6+ SCF+MGDF+Flt3-ligand (6SMF); and IL-1+IL-3+IL-6+G-CSF+GM-CSF+SCF+Epo (GFmix). Serum effects, inoculum concentration (cells/mL) seeding density (cell/cm(2)) and accessory cell effects on the expansion of CD34+ cells were determined. RESULTS: Cellular outputs were significantly higher with fetal calf serum (FCS) than with cord blood serum (CBS) or adult group AB serum (ABS) in the presence of 6SMF, however, CBS was as effective as FCS. The best seeding concentrations varied for each of the cytokine combinations, and inoculum densities exceeding 1000 cells per cm(2) proved detrimental for cultures containing GFmix and SGGM. Accessory cell studies indicated that populations expressing the CD33 antigen inhibited the expansion of purified CD34+ cells in the presence of GFmix or SGGM, but not in the presence of 6SMF. CONCLUSION: Serum supplement, inoculum cell concentration, seeding densities, and accessory cell effects are dependent upon the cytokine combination selected to expand cord blood HPCs ex vivo. Thus, each of these measures should be assessed to establish reproducible and reliable conditions for the selection of different cytokine combinations to culture cord blood HPCs.
Assuntos
Citocinas/farmacologia , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/citologia , Adulto , Antígenos CD34/sangue , Sangue , Diferenciação Celular , Células Cultivadas , Combinação de Medicamentos , Sangue Fetal/citologia , Sangue Fetal/imunologia , HumanosRESUMO
The effect of different expansion protocols on the expression levels of CD49dw/CD29 (VLA-4), CD11a/CD18 (LFA-1), CD31 (PECAM-1), CD44, and CD34 was determined after cord blood CD34+ cells were cultured for defined periods with the following: 1) A growth factor mix (GFmix) containing interleukin (IL)-1, IL-3, IL-6, kit ligand (KL), G-CSF, GM-CSF, and erythropoietin (Epo); 2) IL-3 + KL; and 3) HS-5 (a human stromal cell line supernatant) + KL. Before culturing, cord blood CD34+ cells (> 95% purity) were 94 +/- 5% CD31+, 98 +/- 1% CD44+, 66 +/- 29% VLA-4+, and 68 +/- 18% LFA-1+ (mean +/- SEM). Immunophenotyping and morphological examination of pre- and post-cultured cells indicated that GFmix preferentially supported erythroid development, while IL-3+KL and HS-5+KL preferentially supported myeloid development. Similar to what other investigators have reported, there was an absolute increase in CD34+ cell numbers as well as clonogenic precursors with ex vivo expansion. However, the majority of clonogenic precursors post-expansion expressed CD34 antigen at reduced levels. Examination of adhesion molecules indicated that a majority of cells cultured with GFmix expressed PECAM-1 and LFA-1 at undetectable levels, but PECAM-1 and LFA-1 levels remained essentially unchanged when cells were cultured with IL-3+KL and HS-5+KL. Overall VLA-4 expression levels slightly increased and CD44 expression levels were more heterogeneous with ex vivo expansion. Nevertheless, LFA-1, VLA-4, PECAM-1, and CD44 expression levels remained essentially unchanged on cultured progeny retaining a CD34 phenotype, independent of the culture system used. Together these results indicate that differential modulation of adhesion markers occur with different culture conditions, yet adhesion receptor expression levels on progeny cells retaining a CD34 phenotype are essentially maintained independent of the culture conditions. And although there is an absolute increase in CD34+ cells after ex vivo expansion, a majority of clonogenic precursors have reduced levels of CD34 antigen.
Assuntos
Antígenos CD34/sangue , Moléculas de Adesão Celular/biossíntese , Moléculas de Adesão Celular/efeitos dos fármacos , Sangue Fetal/citologia , Sangue Fetal/imunologia , Células-Tronco Hematopoéticas/efeitos dos fármacos , Células-Tronco Hematopoéticas/metabolismo , Antígenos CD/biossíntese , Antígenos CD/efeitos dos fármacos , Antígenos CD34/biossíntese , Antígenos CD34/efeitos dos fármacos , Biomarcadores/sangue , Diferenciação Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Divisão Celular/fisiologia , Linhagem da Célula , Células Cultivadas , Ensaio de Unidades Formadoras de Colônias , Citocinas/farmacologia , Células Precursoras Eritroides/efeitos dos fármacos , Eritropoetina/farmacologia , Feminino , Sangue Fetal/efeitos dos fármacos , Fator Estimulador de Colônias de Granulócitos/farmacologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Células-Tronco Hematopoéticas/química , Humanos , Receptores de Hialuronatos/sangue , Receptores de Hialuronatos/efeitos dos fármacos , Integrina alfa4beta1 , Integrinas/sangue , Integrinas/efeitos dos fármacos , Interleucina-1/farmacologia , Interleucina-3/farmacologia , Interleucina-6/farmacologia , Antígeno-1 Associado à Função Linfocitária/sangue , Antígeno-1 Associado à Função Linfocitária/efeitos dos fármacos , Molécula-1 de Adesão Celular Endotelial a Plaquetas/sangue , Molécula-1 de Adesão Celular Endotelial a Plaquetas/efeitos dos fármacos , Receptores de Retorno de Linfócitos/sangue , Receptores de Retorno de Linfócitos/efeitos dos fármacos , Fator de Células-Tronco/farmacologiaRESUMO
Escherichia coli DNA polymerase III holoenzyme forms a stable initiation complex with RNA-primed template in the presence of ATP. To determine the linear arrangement of the holoenzyme subunits along the primer-template duplex region, we cross-linked holoenzyme to a series of photo-reactive primers. Site-specific photo-cross-linking revealed that the alpha, beta, and gamma subunits formed ATP-dependent contacts with the primer-template. The alpha-polymerase catalytic subunit covalently attached to nucleotide positions -3, -9, and -13 upstream of the primer terminus, with the most efficient adduct formation occurring at position -9. The gamma subunit contacted the primer at positions -13, -18, and -22, with the strongest gamma-primer interactions occurring at position -18. The beta subunit predominated in cross-linking at position -22. Thus, within the initiation complex, alpha contacts roughly the first 13 nucleotides upstream of the 3'-primer terminus followed by gamma at -18 and beta at -22, and the gamma subunit remains a part of the initiation complex, bridging the alpha and beta subunits. Analyses of the interaction of photo-activatible primer-templates with the preinitiation complex proteins (gamma-complex (gamma-delta-delta'-chi-psi) and beta subunit) revealed the gamma subunit within the preinitiation complex covalently attached to primer at position -3. However, addition of core DNA polymerase III to preinitiation complex, fully reconstituting holoenzyme resulted in replacement of gamma by alpha at the primer terminus. These data indicate that assembly of holoenzyme onto a primer-template can occur in distinct stages and results in a structural rearrangement during initiation complex formation.
Assuntos
DNA Polimerase III/química , DNA Polimerase III/metabolismo , Primers do DNA/metabolismo , Escherichia coli/enzimologia , Trifosfato de Adenosina/metabolismo , Sequência de Bases , Adutos de DNA/análise , Primers do DNA/síntese química , Indicadores e Reagentes , Substâncias Macromoleculares , Dados de Sequência Molecular , Sondas de Oligonucleotídeos , Ligação Proteica , Moldes GenéticosRESUMO
The DNA polymerase III holoenzyme of Escherichia coli contains a potent 3'----5' exonuclease that removes the terminal nucleotide from a synthetic deoxyoligonucleotide primer with a half-life of approximately 2 s. Degradation of primers could not be effectively prevented by permitting the holoenzyme to "idle" at the primer terminus in the presence of limited deoxynucleoside triphosphates. To further characterize this exonuclease and to develop stable primers to facilitate experimental manipulations, we synthesized a series of twelve 25-mer oligonucleotides that differed only in the two 3'-terminal residues. The penultimate position contained either a CMP or a dCMP residue, while at the terminal position either AMP, dAMP, 2',3'-dideoxyAMP, cordycepin (3'-dAMP), dAMP alpha S, or 2',3'-dideoxyAMP alpha S was incorporated. No single change at either the 3'-penultimate or 3'-terminal positions resulted in a decrease in the exonuclease rate greater than 10-fold; however, combined changes at these two sites resulted in a strong synergistic effect. Placing a ribonucleotide at the penultimate position coupled by a phosphorothioate linkage to a terminal 2',3'-dideoxynucleotide reduced the rate of exonucleolytic activity almost 30,000-fold (half-life approximately 16 h). If only the ribonucleotide and phosphorothioate substitutions were made, a primer capable of being efficiently elongated was generated that exhibited a 500-fold increase in stability (half-life = 40 min). The elemental effect observed by substituting a nonbridging oxygen in the terminal phosphodiester bond for sulfur increased from 1.5 to 200 as other substitutions were made that decreased the exonuclease rate. This was consistent with a change in the rate-limiting step of the exonuclease reaction from a conformational change to the chemical step where the covalent bond is cleaved. At least part of this effect appears to be due to perturbations within the enzyme's active site and not solely due to changes in electrophilicity.
Assuntos
DNA Polimerase III/metabolismo , Desoxirribonucleotídeos/farmacologia , Escherichia coli/enzimologia , Exonucleases/metabolismo , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Hidrólise , Cinética , Conformação Proteica , Moldes Genéticos , Transcrição GênicaRESUMO
BACKGROUND: The residual blood left in the placenta, previously considered a biologic waste, contains sufficient hematopoietic stem and progenitor cells to consistently engraft at least a small recipient. Over the past several years, more than 500 HLA-matched, related and unrelated, allogeneic cord blood transplants have been performed. Consequently, public and private cord blood banks are being developed to meet future demands. Thus, the definition of a suitable and effective cord blood component needs to be critically defined. In February 1997, the US Food and Drug Administration (FDA) proposed that cord blood banks should operate under an Investigational New Drug (IND) license. STUDY DESIGN AND METHODS: Standard operating procedures were designed using standards from the Foundation for Accreditation of Hematopoietic and Cellular Therapy, the American Association of Blood Banks, and the National Marrow Donor Program and in accordance with current good manufacturing practices. The standard operating procedures were field-tested and submitted to the FDA. RESULTS: Issues of the utmost concern to the FDA dealt with transplant recipient outcome data collection, donor recruitment, sample tracking, the use of unlicensed materials, and the reporting of positive infectious disease results. After three attempts, an IND application was approved. CONCLUSIONS: To obtain approval of an IND application, cord blood banks need a set of standard operating procedures that describe cord blood collection, processing, freezing, and storage. Issues relating to potential cord blood recipient identification, cord blood shipping, and reporting of transplant recipient outcomes are also needed. The IND process provides an opportunity for outside reviewers to make suggestions that may be included in the standard operating procedures.
Assuntos
Bancos de Sangue , Sangue Fetal , Aplicação de Novas Drogas em Teste , Doadores de Sangue , Coleta de Amostras Sanguíneas , Transplante de Células-Tronco Hematopoéticas , Humanos , Recém-NascidoRESUMO
Replication forks formed during rolling-circle DNA synthesis supported by a tailed form II DNA substrate in the presence of the primosome, the single-stranded DNA binding protein, and the DNA polymerase III holoenzyme (Pol III HE) that had been reconstituted from the purified subunits, beta, tau, and the gamma.delta complex, at limiting (with respect to nucleotide incorporation) concentrations of the Pol III core (alpha, epsilon, and theta) produced aberrantly small Okazaki fragments, while the synthesis of the leading strand was unperturbed. These small Okazaki fragments were not arrayed in tandem along the lagging-strand DNA template, but were separated by large gaps. Similarly structured synthetic products were not manufactured by replication forks reconstituted with higher, saturating concentrations of the Pol III core. Replication forks producing these small fragments could respond, by modulating the size of the Okazaki fragments produced, to variations in the concentration of NTPs or the primase, conditions that affect the frequency of priming on the lagging strand, but not to variation in the concentration of dNTPs, conditions that affect the frequency of utilization of the primers. Significantly longer Okazaki fragments (greater than 7 kilobases) could be produced in the presence of a limiting amount of Pol III core at low concentrations of the primase. These observations indicated that the production of small Okazaki fragments was not a result of a debilitated lagging-strand Pol III core, but rather a function of the time available for nascent strand synthesis during the cycle of events that are required for the manufacture of an Okazaki fragment and that it was the association of primase with the replication fork that keyed this cycle.
Assuntos
Replicação do DNA , DNA Bacteriano/biossíntese , Escherichia coli/genética , RNA Nucleotidiltransferases/metabolismo , DNA/biossíntese , DNA Primase , DNA Bacteriano/metabolismo , Desoxirribonucleosídeos/metabolismo , Eletroforese em Gel de Ágar , Ribonucleosídeos/metabolismoRESUMO
The Philadelphia chromosome (Ph) is found in both chronic myeloid leukemia (CML) and acute lymphoblastic leukemia (ALL). The Ph translocation, t(9;22)(q34;q11), can disrupt the BCR gene on chromosome 22 in one to two areas called the major (Mbcr1) and minor (mbcr1) breakpoint cluster regions. In CML the breakpoint has been mapped almost exclusively to Mbcr1, whereas in Ph positive ALL both Mbcr1 and the upstream mbcr1 breakpoints have been described. In this communication we describe an unusual patient with typical chronic phase Ph positive CML and evidence of the uncharacteristic mbcr1 breakpoint, predicting expression of the ALL-type p190 fusion protein. Fluorescence in situ hybridization demonstrated BCR gene rearrangement, the reverse transcription polymerase chain reaction detected the BCR-ABL fusion mRNA characteristic of the mbcr1 breakpoint, and failed to detect BCR-ABL mRNA characteristic of the Mbcr1 breakpoint. Southern blot analysis revealed no rearrangement in Mbcr1, and direct sequencing of the PCR product confirmed it to be the ALL-type mbcr1 fusion mRNA with the first exon of the BCR gene fused to ABL exon a2. This case differs from the previously reported cases of "p190" CML in that the patient presented without abnormal hematopoietic features other than those found in typical CML and provides further evidence that the p190 mRNA is not sufficient to cause an acute rather than chronic leukemia.
Assuntos
Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Cromossomo Filadélfia , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , RNA Mensageiro/metabolismo , Sequência de Bases , Proteínas de Fusão bcr-abl/genética , Rearranjo Gênico , Humanos , Masculino , Pessoa de Meia-Idade , Sondas Moleculares/genética , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Células-Tronco/fisiologia , Transcrição GênicaRESUMO
Fluorescence in situ hybridization (FISH) was used to discriminate between benign and malignant cells in sorted populations of chronic myelogenous leukemia (CML) marrow. FISH has the advantage of allowing for a cell by cell analysis of the breakpoint cluster region (BCR) gene rearrangement immediately after flow sorting in nondividing G0/G1 cells that are potentially transcriptionally inactive. We initially selected CD34+ cells with very low expression of the activation antigen CD38 as a candidate phenotype for an immature and hypothetically more benign cell population, but found no enrichment for Ph negativity in that subtype. In five CML samples, 55% +/- 3.3% (mean +/- SE) of CD34+/CD38hi cells had the BCR gene rearrangement, similar to 57% +/- 3.7% seen in the CD34+/CD38lo population. In contrast, subsequent experiments (n = 4) determined that the CD34+/HLA-DRlo population in CML marrow does contain an increased proportion of benign cells: 15% +/- 1% of the CD34+/DRlo cells were BCR rearranged, compared with 52% +/- 5.8% of the CD34+/DRhi cells (P = .001). Our results indicate that benign progenitors in CML are enriched within the CD34+ cells with low DR antigen expression, but not low CD38 expression. One possible interpretation of these observations is that low CD38 antigen expression is not as useful as low HLA-DR expression for isolating immature cells.