Assuntos
Plaquetas , Preservação de Sangue , Temperatura Baixa , Criopreservação , Humanos , Transfusão de PlaquetasRESUMO
BACKGROUND: Cold (4°C)-stored platelets are currently under investigation for transfusion in bleeding patients. It is currently unknown how long cold-stored platelets can be stored for clinical applications. STUDY DESIGN AND METHODS: Twenty three subjects were recruited. Twenty-one subjects were available for in vivo assessment and received indium-111 radiolabeled, cold-stored platelets. We investigated 5- (n = 5), 10- (n = 6), 15- (n = 5), and 20-day-stored (n = 5) platelets and obtained samples for in vitro testing at baseline and after the designated storage time. Twenty three units were available for in vitro testing. Five- and 7-day (n = 5 each), room temperature (RT)-stored platelets served as the current clinical standard control. RESULTS: In vivo, we found a continuous decline in platelet recovery from 5 to 20 days. Platelet survival reached a low nadir after 10 days of storage. Ex vivo, we observed the maximum platelet αIIbß3 integrin response to collagen at 5 days of cold storage, and we saw a continuous decline thereafter. However, platelet integrin activation and mitochondrial membrane integrity were better preserved after 20 days at 4°C, compared to 5 days at RT. Platelet metabolic parameters suggest comparable results between 20-day cold-stored platelets and 5- or 7-day RT-stored platelets. CONCLUSION: In summary, we performed the first studies with extended, cold-stored, apheresis platelets in plasma for up to 20 days with a fresh comparator. Storing cold-stored platelets up to 20 days yields better results in vitro, but further studies in actively bleeding patients are needed to determine the best compromise between hemostatic efficacy and storage prolongation.
Assuntos
Plaquetas/fisiologia , Preservação de Sangue/métodos , Criopreservação , Humanos , Ativação Plaquetária/fisiologia , Transfusão de Plaquetas , Plaquetoferese/métodos , Fatores de TempoRESUMO
BACKGROUND: Ordinarily, whole blood (WB) is separated into components before storage. We assessed the posttransfusion viability and function of platelets (PLTs) if they were stored within WB at 4°C. STUDY DESIGN AND METHODS: Whole blood was obtained from 30 normal subjects and stored at 4°C without agitation for 12 days and for 10, 15, or 22 days with agitation. After WB storage, a PLT concentrate was prepared, and a fresh PLT sample was obtained from each donor. The stored PLTs were labeled with 111 In and the fresh with 51 Cr, and both were simultaneously transfused into their donor. Blood samples were obtained after transfusion to determine PLT recoveries and survivals. PLT samples from WB before and after storage were also assayed for PLT function and biochemistry. RESULTS: After storage for 12 days without WB rotation, poststorage PLT counts averaged only 49 ± 12% of baseline values. After storage for 10, 15, or 22 days with end-over-end WB rotation, PLT counts averaged 76 ± 14% of baseline values. Fifteen-day poststorage radiolabeled PLT recoveries averaged 27 ± 11% (49 ± 16% of fresh), and survivals averaged 1.2 ± 0.4 days (16 ± 6% of fresh). in vitro assays demonstrated marked PLT activation after any storage time, and although PLT function decreased over time, stored PLTs were still considered acceptable. CONCLUSION: These data suggest that, during rotated WB storage at 4°C for up to 15 days, PLT yields, poststorage PLT recoveries and survivals, and PLT function should be sufficient to support the short-term hemostatic needs of traumatized patients.
Assuntos
Plaquetas/citologia , Plaquetas/fisiologia , Preservação de Sangue/métodos , Hemostasia/fisiologia , Transfusão de Plaquetas/métodos , Plaquetoferese , Refrigeração , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Sobrevivência Celular , Temperatura Baixa , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Plaquetoferese/métodos , Refrigeração/métodos , Adulto JovemRESUMO
BACKGROUND: The current 5-day storage time of room temperature (22°C)-stored platelets (RSPs) severely limits platelet (PLT) availability. Extended cold (4°C)-stored PLTs (CSPs) are currently being investigated for actively bleeding patients. However, we currently do not know how to best store PLTs in the cold for extended periods of time. In this study, we investigate how storage in plasma and PLT additive solutions (PASs) affects PLT viability in vivo. STUDY DESIGN AND METHODS: Twenty normal subjects had a 2-unit hyperconcentrated apheresis PLT collection. One unit was stored at 4°C in plasma for 3 days ("control unit"), and the CSP "test" unit was stored for 10 or 15 days in plasma or 10 days in 35% plasma with either 65% Intersol or Isoplate. After storage, all units were radiolabeled and transfused into their donors. RESULTS: For 10-day storage, both the plasma and the Intersol units had significantly better PLT recoveries than the Isoplate units (24% ± 8% vs. 11% ± 3% [55% ± 11% vs. 21% ± 8% as percentage of control data], p = 0.002; and 18% ± 4% vs. 11% ± 3% [43% ± 6% vs. 21% ± 8% as percentage of control data], p = 0.004, respectively). There was a trend for lower PLT recoveries with Intersol compared to plasma (p = 0.056). PLT survivals and most in vitro measurements did not differ significantly among the units. CONCLUSIONS: While the in vitro variables suggest largely comparable results between plasma and PASs, in vivo recoveries were higher with plasma compared with both Intersol and Isoplate (p = 0.057 and p = 0.002, respectively). Whether this difference leads to clinically relevant differences in hemostatic efficacy remains to be determined.
Assuntos
Plaquetas/citologia , Preservação de Sangue/métodos , Temperatura Baixa , Plaquetoferese/métodos , Transplante Autólogo/métodos , Sobrevivência Celular , Humanos , Transfusão de Plaquetas/métodos , Soluções , Fatores de TempoRESUMO
The function of Tic40 during chloroplast protein import was investigated. Tic40 is an inner envelope membrane protein with a large hydrophilic domain located in the stroma. Arabidopsis null mutants of the atTic40 gene were very pale green and grew slowly but were not seedling lethal. Isolated mutant chloroplasts imported precursor proteins at a lower rate than wild-type chloroplasts. Mutant chloroplasts were normal in allowing binding of precursor proteins. However, during subsequent translocation across the inner membrane, fewer precursors were translocated and more precursors were released from the mutant chloroplasts. Cross-linking experiments demonstrated that Tic40 was part of the translocon complex and functioned at the same stage of import as Tic110 and Hsp93, a member of the Hsp100 family of molecular chaperones. Tertiary structure prediction and immunological studies indicated that the C-terminal portion of Tic40 contains a TPR domain followed by a domain with sequence similarity to co-chaperones Sti1p/Hop and Hip. We propose that Tic40 functions as a co-chaperone in the stromal chaperone complex that facilitates protein translocation across the inner membrane.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Cloroplastos/fisiologia , Proteínas de Membrana/metabolismo , Chaperonas Moleculares/metabolismo , Sequência de Aminoácidos , Arabidopsis/genética , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Membrana Celular/metabolismo , Cloroplastos/ultraestrutura , Humanos , Proteínas de Membrana/química , Proteínas de Membrana/genética , Chaperonas Moleculares/química , Chaperonas Moleculares/genética , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Transporte Proteico/fisiologia , Alinhamento de SequênciaRESUMO
This unit describes protocols for isolating chloroplasts from pea (Pisum sativum) and Arabidopsis thaliana for the study of nuclear-encoded plastid precursor proteins. Chloroplasts from both preparations are competent for the in vitro import of recombinant preproteins synthesized using in vitro translation systems derived from reticulocyte or wheat germ lysates. These assays can be used to test whether a particular protein is targeted to chloroplasts, for analyzing the suborganellar location of newly imported preproteins, or to study the mechanism of import itself.
Assuntos
Cloroplastos/metabolismo , Proteínas de Plantas/análise , Arabidopsis/química , Arabidopsis/metabolismo , Fracionamento Celular/métodos , Cloroplastos/química , Cloroplastos/efeitos dos fármacos , Eletroforese em Gel de Poliacrilamida , Pisum sativum/química , Pisum sativum/metabolismo , Proteínas de Plantas/metabolismo , Transporte Proteico/efeitos dos fármacosRESUMO
The import of nucleus-encoded preproteins into plastids requires the coordinated activities of membrane protein complexes that facilitate the translocation of polypeptides across the envelope double membrane. Tic20 was identified previously as a component of the import machinery of the inner envelope membrane by covalent cross-linking studies with trapped preprotein import intermediates. To investigate the role of Tic20 in preprotein import, we altered the expression of the Arabidopsis Tic20 ortholog (atTic20) by antisense expression. Several antisense lines exhibited pronounced chloroplast defects exemplified by pale leaves, reduced accumulation of plastid proteins, and significant growth defects. The severity of the phenotypes correlated directly with the reduction in levels of atTic20 expression. In vitro import studies with plastids isolated from control and antisense plants indicated that the antisense plastids are defective specifically in protein translocation across the inner envelope membrane. These data suggest that Tic20 functions as a component of the protein-conducting channel at the inner envelope membrane.
Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Cloroplastos/metabolismo , Proteínas de Membrana Transportadoras/genética , Sequência de Aminoácidos , Arabidopsis/metabolismo , Proteínas de Arabidopsis/imunologia , Proteínas de Arabidopsis/metabolismo , Transporte Biológico , Clorofila/metabolismo , Cloroplastos/ultraestrutura , DNA Antissenso/genética , Perfilação da Expressão Gênica , Immunoblotting , Proteínas de Membrana Transportadoras/imunologia , Proteínas de Membrana Transportadoras/metabolismo , Microscopia Eletrônica , Dados de Sequência Molecular , Fenótipo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas , Transporte Proteico , Homologia de Sequência de AminoácidosRESUMO
Galactolipid biosynthesis in plants is highly complex. It involves multiple pathways giving rise to different molecular species. To assess the contribution of different routes of galactolipid synthesis and the role of molecular species for growth and photosynthesis, we initiated a genetic approach of analyzing double mutants of the digalactosyldiacylglycerol (DGDG) synthase mutant dgd1 with the acyltransferase mutant, act1, and the two desaturase mutants, fad2 and fad3. The double mutants showed different degrees of growth retardation: act1,dgd1 was most severely affected and growth of fad2,dgd1 was slightly reduced, whereas fad3,dgd1 plants were very similar to dgd1. In act1,dgd1, lipid and chlorophyll content were reduced and photosynthetic capacity was affected. Molecular analysis of galactolipid content, fatty acid composition, and positional distribution suggested that the growth deficiency is not caused by changes in galactolipid composition per se. Chloroplasts of dgd1 were capable of synthesizing monogalactosyldiacylglycerol, DGDG, and tri- and tetragalactosyldiacylglycerol. Therefore, the reduced growth of act1,dgd1 and fad2,dgd1 cannot be explained by the absence of DGDG synthase activity from chloroplasts. Molecular analysis of DGDG accumulating in the mutants during phosphate deprivation suggested that similarly to the residual DGDG of dgd1, this additional lipid is synthesized in association with chloroplast membranes through a pathway independent of the mutations, act1, dgd1, fad2, and fad3. Our data imply that the severe growth defect of act1,dgd1 is caused by a reduced metabolic flux of chloroplast lipid synthesis through the eukaryotic and prokaryotic pathway as well as by the reduction of photosynthetic capacity caused by the destabilization of photosynthetic complexes.