RESUMEN

Essentials Heat shock protein 47 (HSP47), a collagen specific chaperone is present on the platelet surface. Collagen mediated platelet function was reduced following blockade or deletion of HSP47. GPVI receptor regulated signalling was reduced in HSP47 deficient platelets. Platelet HSP47 tethers to exposed collagen thus modulating thrombosis and hemostasis. SUMMARY: Objective Heat shock protein 47 (HSP47) is an intracellular chaperone protein that is vital for collagen biosynthesis in collagen secreting cells. This protein has also been shown to be present on the surface of platelets. Given the importance of collagen and its interactions with platelets in triggering hemostasis and thrombosis, in this study we sought to characterize the role of HSP47 in these cells. Methods and Results The deletion of HSP47 in mouse platelets or its inhibition in human platelets reduced their function in response to collagen and the GPVI agonist (CRP-XL), but responses to thrombin were unaltered. In the absence of functional HSP47, the interaction of collagen with platelets was reduced, and this was associated with reduced GPVI-collagen binding, signalling and platelet activation. Thrombus formation on collagen, under arterial flow conditions, was also decreased following the inhibition or deletion of HSP47, in the presence or absence of eptifibatide, consistent with a role for HSP47 in enhancing platelet adhesion to collagen. Platelet adhesion under flow to von Willebrand factor was unaltered following HSP47 inhibition. Laser-induced thrombosis in cremaster muscle arterioles was reduced and bleeding time was prolonged in HSP47-deficient mice or following inhibition of HSP47. Conclusions Our study demonstrates the presence of HSP47 on the platelet surface, where it interacts with collagen, stabilizes platelet adhesion and increases collagen-mediated signalling and therefore thrombus formation and hemostasis.

Asunto(s)

Plaquetas/metabolismo , Proteínas Portadoras/sangre , Colágeno/sangre , Proteínas HSP70 de Choque Térmico/sangre , Hemostasis , Activación Plaquetaria , Trombosis/sangre , Animales , Plaquetas/efectos de los fármacos , Señalización del Calcio , Proteínas Portadoras/antagonistas & inhibidores , Proteínas Portadoras/genética , Modelos Animales de Enfermedad , Proteínas HSP70 de Choque Térmico/antagonistas & inhibidores , Proteínas HSP70 de Choque Térmico/deficiencia , Proteínas HSP70 de Choque Térmico/genética , Hemostasis/efectos de los fármacos , Humanos , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas Mitocondriales , Activación Plaquetaria/efectos de los fármacos , Adhesividad Plaquetaria , Inhibidores de Agregación Plaquetaria/farmacología , Glicoproteínas de Membrana Plaquetaria/metabolismo , Unión Proteica , Trombosis/genética , Trombosis/prevención & control

RESUMEN

Essentials ERp72 is a thiol isomerase enzyme. ERp72 levels increase at the platelet surface during platelet activation. We generated a humanized monoclonal antibody which blocks ERp72 enzyme activity (anti-ERp72). Anti-ERp72 inhibits platelet functional responses and thrombosis. SUMMARY: Background Within the endoplasmic reticulum, thiol isomerase enzymes modulate the formation and rearrangement of disulfide bonds in newly folded proteins entering the secretory pathway to ensure correct protein folding. In addition to their intracellular importance, thiol isomerases have been recently identified to be present on the surface of a number of cell types where they are important for cell function. Several thiol isomerases are known to be present on the resting platelet surface, including PDI, ERp5 and ERp57, and levels are increased following platelet activation. Inhibition of the catalytic activity of these enzymes results in diminished platelet function and thrombosis. Aim We previously determined that ERp72 is present at the resting platelet surface and levels increase upon platelet activation; however, its functional role on the cell surface was unclear. We aimed to investigate the role of ERp72 in platelet function and its role in thrombosis. Methods Using HuCAL technology, fully humanized Fc-null anti-ERp72 antibodies were generated. Eleven antibodies were screened for their ability to inhibit ERp72 activity and the most potent inhibitory antibody (anti-ERp72) selected for further testing in platelet functional assays. Results and conclusions Anti-ERp72 inhibited platelet aggregation, granule secretion, calcium mobilisation and integrin activation, revealing an important role for extracellular ERp72 in the regulation of platelet activation. Consistent with this, infusion of anti-ERp72 into mice protected against thrombosis.

Asunto(s)

Anticuerpos Monoclonales Humanizados/farmacología , Plaquetas/efectos de los fármacos , Fibrinolíticos/farmacología , Glicoproteínas de Membrana/antagonistas & inhibidores , Activación Plaquetaria/efectos de los fármacos , Inhibidores de Agregación Plaquetaria/farmacología , Proteína Disulfuro Isomerasas/antagonistas & inhibidores , Trombosis/prevención & control , Animales , Plaquetas/enzimología , Plaquetas/inmunología , Calcio/sangre , Modelos Animales de Enfermedad , Fibrinógeno/metabolismo , Humanos , Masculino , Glicoproteínas de Membrana/sangre , Glicoproteínas de Membrana/inmunología , Ratones Endogámicos C57BL , Adhesividad Plaquetaria/efectos de los fármacos , Agregación Plaquetaria/efectos de los fármacos , Complejo GPIIb-IIIa de Glicoproteína Plaquetaria/metabolismo , Proteína Disulfuro Isomerasas/sangre , Proteína Disulfuro Isomerasas/inmunología , Transducción de Señal/efectos de los fármacos , Trombosis/sangre , Trombosis/enzimología

RESUMEN

BACKGROUND: Thiol isomerases are a family of endoplasmic reticulum enzymes which orchestrate redox-based modifications of protein disulphide bonds. Previous studies have identified important roles for the thiol isomerases PDI and ERp5 in the regulation of normal platelet function. AIM: Recently, we demonstrated the presence of a further five thiol isomerases at the platelet surface. In this report we aim to report the role of one of these enzymes - ERp57 in the regulation of platelet function. METHODS/RESULTS: Using enzyme activity function blocking antibodies, we demonstrate a role for ERp57 in platelet aggregation, dense granule secretion, fibrinogen binding, calcium mobilisation and thrombus formation under arterial conditions. In addition to the effects of ERp57 on isolated platelets, we observe the presence of ERp57 in the developing thrombus in vivo. Furthermore the inhibition of ERp57 function was found to reduce laser-injury induced arterial thrombus formation in a murine model of thrombosis. CONCLUSIONS: These data suggest that ERp57 is important for normal platelet function and opens up the possibility that the regulation of platelet function by a range of cell surface thiol isomerases may represent a broad paradigm for the regulation of haemostasis and thrombosis.

Asunto(s)

Plaquetas/enzimología , Proteína Disulfuro Isomerasas/metabolismo , Trombosis/enzimología , Adenosina Trifosfato/metabolismo , Animales , Anticuerpos/farmacología , Plaquetas/efectos de los fármacos , Calcio/metabolismo , Modelos Animales de Enfermedad , Inhibidores Enzimáticos/farmacología , Fibrinógeno/metabolismo , Humanos , Ratones , Ratones Endogámicos C57BL , Agregación Plaquetaria , Proteína Disulfuro Isomerasas/antagonistas & inhibidores , Proteína Disulfuro Isomerasas/inmunología , Vesículas Secretoras/metabolismo , Trombosis/patología , Trombosis/prevención & control , Factores de Tiempo

Asunto(s)

Plaquetas/citología , Supervivencia Celular , Cloruros/farmacología , Compuestos Férricos/farmacología , Inhibidores de Agregación Plaquetaria/farmacología , Trombosis/patología , Animales , Humanos

RESUMEN

BACKGROUND: The plasma membrane calcium ATPase (PMCA) regulates localized signaling events in a variety of cell types, although its functional role in platelets remains undefined. OBJECTIVES: To investigate the role of PMCA in determining platelet intracellular calcium concentration ([Ca²(+) ](i) ) at rest and following agonist stimulation, and to define the corresponding effects upon different stages of platelet activation. METHODS: [Ca²(+) ](i) was continuously measured in Fura-2-loaded platelets and in vitro and in vivo functional analyses performed in the presence of the PMCA inhibitor carboxyeosin (CE). RESULTS: Concentrations of CE that selectively inhibited Ca²(+) extrusion through PMCA were established in human platelets. [Ca²(+) ](i) was elevated by CE in resting platelets, although collagen-stimulated Ca²(+) release was reduced. Impaired Ca²(+) mobilization upon agonist stimulation was accompanied by reduced dense granule secretion and impaired platelet aggregation. Platelet aggregation responses were also reduced in PMCA4(-/-) mice and in an in vivo mouse model of platelet thromboembolism. Conversely, inhibition of PMCA promoted the early and later stages of platelet activation, observed as enhanced adhesion to fibrinogen, and accelerated clot retraction. Investigations into the signaling mechanisms underlying CE-mediated inhibition of platelet aggregation implicated cGMP-independent vasodilator-stimulated phosphoprotein phosphorylation. CONCLUSIONS: Disruption of PMCA activity perturbs platelet Ca²(+) homeostasis and function in a time-dependent manner, demonstrating that PMCA differentially regulates Ca²(+) -dependent signaling events, and hence function, throughout the platelet activation process.

Asunto(s)

Plaquetas/enzimología , ATPasas Transportadoras de Calcio/metabolismo , Calcio/metabolismo , Homeostasis , Proteínas de la Membrana/metabolismo , Transducción de Señal , Animales , Plaquetas/metabolismo , Western Blotting , Masculino , Ratones , Ratones Endogámicos BALB C , Fosforilación , Agregación Plaquetaria

RESUMEN

BACKGROUND: Platelet activation by collagen depends on signals transduced by the glycoprotein (GP)VI-Fc receptor (FcR)γ-chain collagen receptor complex, which involves recruitment of phosphatidylinositol 3-kinase (PI3K) to phosphorylated tyrosines in the linker for activation of T cells (LAT). An interaction between the p85 regulatory subunit of PI3K and the scaffolding molecule Grb-2-associated binding protein-1 (Gab1), which is regulated by binding of the Src homology 2 domain-containing protein tyrosine phosphatase-2 (SHP-2) to Gab1, has been shown in other cell types to sustain PI3K activity to elicit cellular responses. Platelet endothelial cell adhesion molecule-1 (PECAM-1) functions as a negative regulator of platelet reactivity and thrombosis, at least in part by inhibiting GPVI-FcRγ-chain signaling via recruitment of SHP-2 to phosphorylated immunoreceptor tyrosine-based inhibitory motifs in PECAM-1. OBJECTIVE: To investigate the possibility that PECAM-1 regulates the formation of the Gab1-p85 signaling complexes, and the potential effect of such interactions on GPVI-mediated platelet activation in platelets. METHODS: The ability of PECAM-1 signaling to modulate the LAT signalosome was investigated with immunoblotting assays on human platelets and knockout mouse platelets. RESULTS: PECAM-1-associated SHP-2 in collagen-stimulated platelets binds to p85, which results in diminished levels of association with both Gab1 and LAT and reduced collagen-stimulated PI3K signaling. We therefore propose that PECAM-1-mediated inhibition of GPVI-dependent platelet responses result, at least in part, from recruitment of SHP-2-p85 complexes to tyrosine-phosphorylated PECAM-1, which diminishes the association of PI3K with activatory signaling molecules, such as Gab1 and LAT.

Asunto(s)

Plaquetas/metabolismo , Colágeno/metabolismo , Proteína Adaptadora GRB2/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Plaquetas/citología , Fosfatidilinositol 3-Quinasa Clase Ia/metabolismo , Humanos , Proteínas de la Membrana/metabolismo , Ratones , Fosfoproteínas/metabolismo , Fosforilación , Activación Plaquetaria , Glicoproteínas de Membrana Plaquetaria/metabolismo , Proteína Tirosina Fosfatasa no Receptora Tipo 11/metabolismo , Transducción de Señal , Tirosina/química

RESUMEN

Platelets have long been recognized to be of central importance in haemostasis, but their participation in pathological conditions such as thrombosis, atherosclerosis and inflammation is now also well established. The platelet has therefore become a key target in therapies to combat cardiovascular disease. Anti-platelet therapies are used widely, but current approaches lack efficacy in a proportion of patients, and are associated with side effects including problem bleeding. In the last decade, substantial progress has been made in understanding the regulation of platelet function, including the characterization of new ligands, platelet-specific receptors and cell signalling pathways. It is anticipated this progress will impact positively on the future innovations towards more effective and safer anti-platelet agents. In this review, the mechanisms of platelet regulation and current anti-platelet therapies are introduced, and strong, and some more speculative, potential candidate target molecules for future anti-platelet drug development are discussed.

Asunto(s)

Plaquetas/efectos de los fármacos , Enfermedades Cardiovasculares/tratamiento farmacológico , Drogas en Investigación/farmacología , Hemostasis/efectos de los fármacos , Inhibidores de Agregación Plaquetaria/farmacología , Trombosis/tratamiento farmacológico , Animales , Plaquetas/metabolismo , Enfermedades Cardiovasculares/sangre , Diseño de Fármacos , Drogas en Investigación/uso terapéutico , Fibrinolíticos/farmacología , Humanos , Inhibidores de Agregación Plaquetaria/uso terapéutico , Transducción de Señal/efectos de los fármacos , Trombosis/sangre

RESUMEN

OBJECTIVE: To describe the knowledge and beliefs on fluoridation of a community served by an unfluoridated water supply prior to consultation on implementing fluoridation. METHODS: Telephone survey of a sample of residents and businesses utilising semi-structured questionnaires. Comparisons were made among responses of ethnic and age groups. RESULTS: Residents were more conversant concerning fluoridation than business users. Knowledge and beliefs among ethnic groups differed significantly. Younger persons and those who had never been married were less knowledgeable. Pamphlets supplied by water-provider organisations or health professionals were the preferred methods of acquiring fluoridation information. The majority of respondents wished the opportunity to express their views via a referendum. Health authorities were the favoured decision-makers. CONCLUSIONS AND IMPLICATIONS: Gaps were identified in this community's knowledge on fluoridation with important differences based on ethnicity and age. Strategies to inform the public on water fluoridation need to address these subgroup differences.

Asunto(s)

Actitud Frente a la Salud/etnología , Fluoruración , Salud Pública , Abastecimiento de Agua , Adolescente , Adulto , Etnicidad , Femenino , Educación en Salud , Humanos , Conocimiento , Masculino , Persona de Mediana Edad , Nueva Zelanda , Encuestas y Cuestionarios

RESUMEN

An Arabidopsis oleosin was used as a model to study oleosin topology and targeting to oil bodies. Oleosin mRNA was in vitro translated with canine microsomes in a range of truncated forms. This allowed proteinase K mapping of the membrane topology. Oleosin maintains a conformation with a membrane-integrated hydrophobic domain flanked by N- and C-terminal domains located on the outer microsome surface. This is a unique membrane topology on the endoplasmic reticulum (ER). Three universally conserved proline residues within the "proline knot" motif of the oleosin hydrophobic domain were substituted by leucine residues. After in vitro translation, only minor differences in proteinase K protection could be observed. These differences were not apparent in soybean microsomes. No significant difference in incorporation efficiency on the ER was observed between the two oleosin forms. However, as an oleosin-beta-glucuronidase translational fusion, the proline knot variant failed to target to oil bodies in both transient embryo expression and in stably transformed seeds. Fractionation of transgenic embryos expressing oleosin-beta-glucuronidase fusions showed that the proline knot variant accumulated in the ER to similar levels compared with the native form. Therefore, the proline knot motif is not important for ER integration and the determination of topology but is required for oil body targeting. The loss of the proline knot results in an intrinsic instability in the oleosin polypeptide during trafficking.

Asunto(s)

Proteínas de Arabidopsis , Arabidopsis/metabolismo , Proteínas de Plantas/química , Secuencia de Aminoácidos , Animales , Arabidopsis/genética , Arabidopsis/ultraestructura , Secuencia de Bases , Cartilla de ADN/genética , ADN de Plantas/genética , Perros , Retículo Endoplásmico/metabolismo , Variación Genética , Técnicas In Vitro , Cuerpos de Inclusión/metabolismo , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Microsomas/metabolismo , Datos de Secuencia Molecular , Aceites de Plantas/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Modificadas Genéticamente , Prolina/química , Biosíntesis de Proteínas , Conformación Proteica , ARN Mensajero/genética , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Glycine max

RESUMEN

The importance of the two chiral centers at C-3 and C-7 in the molecular structure of jasmonic acid in plant responses was investigated. We separated methyl jasmonate (MeJA) into (3R)- and (3S)-isomers with a fixed stereochemistry at C-3, but epimerization at C-7 is possible. The four isomers of the nonepimerizable analog 7-methyl MeJA were synthesized. These six esters and their corresponding acids were tested in three bioassays: (a) senescence in sunflower (Helianthus annuus) cotyledons; (b) proteinase inhibitor II gene expression in transgenic tobacco (Nicotiana tabacum) with [beta]-glucuronidase as a biochemical reporter; and (c) seed germination in Brassica napus and wheat (Triticum aestivum). The esters and acids had similar activities in the three assays, with the ester being more effective than its acid. The (3R)-stereochemistry was critical for jasmonate activity. Although activity was reduced after substituting the C-7 proton with a methyl group, the analogs with (3R,7R)- or (3R,7S)-stereochemistry were active in some of the assays. Although the four isomers of 7-methyl MeJA were inactive or only weakly active in the senescence assay, they could overcome the senescence-promoting effect of (3R)-MeJA. The strongest antagonistic effect was observed with the (3R,7S)-isomer.

Asunto(s)

Plantas , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/aislamiento & purificación , Biotecnología/métodos , Cloroplastos/metabolismo , Enzimas/biosíntesis , Ingeniería de Proteínas/métodos , Semillas , Fracciones Subcelulares/metabolismo

RESUMEN

An Arabidopsis oleosin gene including 860 bp of promoter as well as coding sequence was fused to the reporter, ß-glucuronidase (GUS). In transient expression assays following particle bombardment, the Arabidopsis gene in both transcriptional and translational fusions with GUS was functional in a range of oilseed embryos as determined histochemically and fluorimetrically. Significantly, upon homogenization and separation into cytoplasmic and oil body fractions, the complete oleosin-GUS fusion was found to be targeted to the oil bodies in all of the heterologous species tested including canola, flax, soybean, sunflower, safflower, cotton, castor bean, coriander as well as the monocot, corn. The utilization of oleosin protein fusions for recombinant protein expression should be applicable to this wide range of oilseed crops. Additionally, the method of transient expression subcellular targeting should be useful in determining structural motifs controlling intracellular trafficking of these proteins.

RESUMEN

A number of treatments were tested for their ability to affect the synthesis of oilbody proteins in microspore-derived embryos of rapeseed (Brassica napus). Synthesis of the oilbody proteins was determined by [(35)S]methionine incorporation in vivo and sodium dodecyl sulfate-polyacrylamide gel electrophoresis of washed oilbody fractions. Oilbody proteins of approximately 19, 23, and 32 kilodaltons were found to be prominent. These proteins showed differential patterns of regulation. The 19 and 23 kilodalton proteins (oleosins) were greatly enhanced by treatments with abscisic acid, jasmonic acid, and osmotic stress imposed using sorbitol (12.5%). Synthesis of the 32 kilodalton protein was inhibited by abscisic acid and by sorbitol (12.5%), but unaffected by jasmonates. The strong promotion of synthesis of the 19 and 23 kilodalton oilbody proteins appeared to be specific as they are not seen with gibberellic acid treatment or with a stress such as heat shock. Time course experiments revealed that the abscisic acid stimulation of oleosin synthesis is quite rapid (less than 2 hours), reaching a maximum at 6 to 8 hours. The response of the oleosins to abscisic acid is found in all stages of embryogenesis, with a major increase in synthetic rates even in globular embryos on abscisic acid treatment. This suggests that these proteins may accumulate much earlier in embryogenesis than has previously been believed. The 32 kilodalton oilbody-associated protein appears different from the oleosins in several ways, including its distinct pattern of regulation and its unique property, among the oilbody proteins, of undergoing phosphorylation.

RESUMEN

A number of effects on embryogenesis of the putative phytohormone jasmonic acid (JA), and its methyl ester (MeJA), were investigated in two oilseed plants, repeseed (Brassica napus) and flax (Linum usitatissimum). Results from treatments with JA and MeJA were compared with those of a known effector of several aspects of embryogenesis, abscisic acid (ABA). Jasmonic acid was identified by gas chromatography-mass spectrometry as a naturally occurring substance in both plant species during embryo development. Both JA and MeJA can prevent precocious germination of B. napus microspore embryos and of cultured zygotic embryos of both species at an exogenous concentration of >1 micromolar. This dose-response was comparable with results obtained with ABA. Inhibitory effects were also observed on seed germination with all three growth regulators in rapeseed and flax. A number of molecular aspects of embryogenesis were also investigated. Expression of the B. napus storage protein genes (napin and cruciferin) was induced in both microspore embryos and zygotic embryos by the addition of 10 micromolar JA. The level of napin and cruciferin mRNA detected was similar to that observed when 10 micromolar ABA was applied to these embryos. For MeJA only slight increases in napin or cruciferin mRNA were observed at concentrations of 30 micromolar. Several oilbody-associated proteins were found to accumulate when the embryos were incubated with either JA or ABA in both species. The MeJA had little effect on oilbody protein synthesis. The implications of JA acting as a natural regulator of gene expression in zygotic embryogenesis are discussed.

RESUMEN

Storage protein gene expression, characteristic of mid- to late embryogenesis, was investigated in microspore embryos of rapeseed (Brassica napus). These embryos, derived from the immature male gametophyte, accumulate little or no detectable napin or cruciferin mRNA when cultured on hormone-free medium containing 13% sucrose. The addition of abscisic acid (ABA) to the medium results in an increase in detectable transcripts encoding both these polypeptides. Storage protein mRNA is induced at 1 micromolar ABA with maximum stimulation occurring between 5 and 50 micromolar. This hormone induction results in a level of storage protein mRNA that is comparable to that observed in zygotic embryos of an equivalent morphological stage. Effects similar to that of ABA are noted when 12.5% sorbitol is added to the microspore embryo medium (osmotic potential = 25.5 bars). Time course experiments, to study the induction of napin and cruciferin gene expression demonstrated that the ABA effect occurred much more rapidly than the high osmoticum effect, although after 48 hours, the levels of napin or cruciferin mRNA detected were similar in both treatments. This difference in the rates of induction is consistent with the idea that the osmotic effect may be mediated by ABA which is synthesized in response to the reduced water potential. Measurements of ABA (by gas chromatography-mass spectrometry using [(2)H(6)]ABA as an internal standard) present in microspore embryos during sorbitol treatment and in embryos treated with 10 micromolar ABA were performed to investigate this possibility. Within 2 hours of culture on high osmoticum the level of ABA increased substantially and significantly above control and reached a maximum concentration within 24 hours. This elevated concentration was maintained for 48 hours after culturing and represents a sixfold increase over control embryos. The ABA-treated embryos accumulated the hormone very quickly, but ABA concentrations returned to basal levels within 72 hours after treatment. The possibility that embryo-synthesized ABA may be a mediator of effects of osmotic stress on gene expression in Brassica embryos is discussed.

RESUMEN

Embryos derived in vitro from isolated microspores of Brassica napus L. were compared with their zygotic counterparts. Parameters investigated included storage-protein accumulation and gene expression, fattyacid composition, storage-lipid biosynthesis, and the appearance of oil-body proteins. The microspore embryos accumulate storage-protein and show increases in levels of their transcripts during the torpedo stage. These embryos were sensitive to abscisic acid (ABA) with respect to accumulation of storage-protein mRNA and oil-body proteins. Post-transcriptional regulation of cruciferin accumulation is indicated by a disparity between ABA-enhanced transcript accumulation and a less marked effect at the level of protein accumulation. To investigate storage-lipid profiles, two cultivars of Brassica napus, Reston and Topas, were used. The former accumulates major quantities of C20 (11.2%) and C22 (39.9%) fatty acids in its seeds, the latter predominantly C18 fatty acids. The higher-molecular-weight fatty acids (>C18) normally occur only in seeds and were used as biochemical markers for seed-specific metabolism in microspore embryos. Microspore embryos from Reston were found to accumulate C20 (10.6%) and C22 (31.2%) fatty acids after 35 d in culture at levels and proportions comparable to those found in seeds. Similarly, microspore embryos of Topas had a fatty-acid profile similar to that of mature Topas seed. Activities of enzymes involved in the accumulation of storage lipids (erucoyl-CoA synthetase [EC 6.2.1.3], erucoyl-CoA thioesterase [EC 3.1.2.2] and erucoyl-CoA acyltransferase [EC 2.3.1.15 or EC 2.3.1.20]) were detected in torpedostage microspore embryos. Their specific activities were higher than have been reported to date for analogous preparations from zygotic embryos of B. napus. The similarities in storage-lipid and protein composition of these embryos to their zygotic counterparts, along with their sensitivity to ABA, indicate that microspore embryos might be exploited to facilitate studies of biochemistry and gene regulation in oilseeds.

RESUMEN

The recessive hyperrecombination mutation rec46-1, isolated by ultraviolet light mutagenesis of the MAT alpha n+1 chromosome VII disomic strain LBL1 (Esposito et al. 1982), enhances the mitotic rates of spontaneous gene conversion, intergenic recombination and restitution of haploidy (due to chromosomal loss or mitotic nondisjunction) in MAT alpha n+1 chromosome VII disomic strains. The rec46-1 mutation does not prevent HO directed homothallic interconversion of mating types. MATa/MaT alpha rec46-1/rec46-1 diploids exhibit the same degree of hyperrecombinational activity as MAT alpha rec46-1 n+1 chromosome VII disomics with respect to gene conversion and intragenic recombination resulting in prototrophy. When compared to MAT alpha rec46-1 n+1 disomics however, MATa/MAT alpha rec46-1/rec46-1 diploids exhibit a ten fold reduced level of hyperrecombinational activity with respect to intergenic recombination and present no evidence of chromosomal loss or nondisjunction resulting in 2n-1 monosomic segregants. MATa/MAT alpha rec46-1/rec46-1 diploids are sporulation-deficient. The results obtained demonstrate that the REC46 gene product modulates mitotic chromosomal stability and recombination and is essential for sporulation (meiosis and ascospore formation).

Asunto(s)

Meiosis , Mitosis , Recombinación Genética , Saccharomyces cerevisiae/genética , Esporas Fúngicas , Ciclo Celular , Cromosomas/fisiología , Genes Fúngicos , Factor de Apareamiento , Mutación , Péptidos/genética

RESUMEN

Alfalfa (Medicago sativa L.) protoplasts derived from cell suspension cultures divided inefficiently in liquid culture. The onset of cell division activity occurred synchronously among the protoplasts; however, many were blocked at cytokinesis and therefore did not complete first division. Very few of the cells that began to divide continued to do so. Immobilization of protoplasts in agarose after 1 to 4 days in liquid culture overcame this inhibition of division. Continuous growth in agarose was restricted and therefore microcolonies were transferred to agar medium to complete callus development. Plating efficiencies of 2-10% were achieved within 30 days of protoplast isolation. The agarose treatment was responsible for a 5- to 30-fold improvement in plating efficiency.

RESUMEN

A number of Brassica species and cultivars were tested and found to be highly susceptible to crown gall induction by both nopaline and octopine strains of Agrobacterium tumefaciens. Only B. napus did not form tumours when inoculated with octopine strains. Seedlings of very young plants were poor hosts but efficient infection occurred after 8-10 weeks of growth. Teratomas arising on tumours in planta were relatively frequent on induction with nopaline strains. Axenically cultured tumour calli of Brassicas were very active in opine synthase activity and stably maintained this transformed phenotype; however, transformed plants could not be regenerated. These results suggest that disarmed nopaline Ti plasmid vectors are well suited for the genetic engineering of this important crop family.

Asunto(s)

Neoplasias Abdominales/patología , Neoplasias de los Tejidos Blandos/patología , Adulto , Humanos , Masculino