ISSN:1066-5234    

DOI:10.1111/j.1550-7408.1995.tb01590.x

出版商:Blackwell Publishing Ltd    

年代:1995

数据来源: WILEY

ISSN:1066-5234    

DOI:10.1111/j.1550-7408.1995.tb01564.x

出版商:Blackwell Publishing Ltd    

年代:1995

数据来源: WILEY

摘要:

ABSTRACT.Dictyostelium discoideumhas a well characterized life cycle where unicellular growth and multicellular development are separated events. Development is dependent upon signal transduction mediated by cell surface, cAMP receptor/G protein linkages. Secreted cAMP acts extracellularly as a primary signal and chemoattractant. There are 4 genes for the distinct cAMP receptor subtypes, CAR1, CAR2, CAR3 and CAR4. These subtypes are expressed with temporally and spatially specific patterns and cells carrying null mutations for each gene have distinct developmental phenotypes. These results indicate an essential role for cAMP signalling throughoutDictyosteliumdevelopment to regulate such diverse pathways as cell motility, aggregation (multicellularity), cytodifferentiation, pattern formation and cell type‐specific gene expressio

ISSN:1066-5234    

DOI:10.1111/j.1550-7408.1995.tb01565.x

出版商:Blackwell Publishing Ltd    

年代:1995

数据来源: WILEY

摘要:

ABSTRACT.We have previously reported that a β‐endorphin‐like substance inhibits phagocytosis inTetrahymenaperhaps by a mu‐like opioid receptor. We now report a further characterization of the elements involved in the signal transduction mechanism of this opioid. Affinity chromatography followed by immunoblots of both intracellular extracts and extracellular medium reveal the presence of two main proteins of 64 and 75 kDa. These molecular weights are much higher than that of any known opioid peptide or precursor protein and suggest that we may be dealing with either a novel opioid or with proteins that by chance cross‐react with anti‐β‐endorphin antibody. Nevertheless, when the biological activity of these proteins was tested it was found that they had an effect similar to that of mammalian β‐endorphin, namely inhibition of phagocytosis by a naloxone‐reversible mechanism. We have probed a size‐selectedTetrahymenalibrary with a pro‐opiomelanocortin probe and have obtained several positive clones; the sequencing of their inserts should establish whether we are dealing with a bona fide member of the opioid family. Another aspect we have been studying is the G‐proteins which appear to be involved in the modulation of phagocytosis. We have found, by means of Western blotting (using an antibody against the conserved GTP‐binding region of the α‐subunit), two bands of 51 and 59 kDa; no α‐subunit of 59 kDa had been reported previously and may represent a novel G‐protein. In spite of these differences, the opioid signal transduction mechanism appears to remarkably resemble that

ISSN:1066-5234    

DOI:10.1111/j.1550-7408.1995.tb01566.x

出版商:Blackwell Publishing Ltd    

年代:1995

数据来源: WILEY

摘要:

ABSTRACT.For long, our knowledge of the biology of ciliate pheromones has long relied solely upon the study of the two structurally unrelated “gamones” identified in culture filtrates of aBlepharismaspecies. However, the characterization of a number of polypeptide pheromones secreted byEuplotes raikoviandE. octocarinatushas now established that structural relationships of homology usually link these molecules, which is consistent with the genetic basis of the mating type systems evolved by these species. In this context, our growing appreciation of the conserved and variable elements of the pheromone architecture should foster progress in the understanding of pheromone‐receptor interactions and thus, provide important clues into pheromone mechanisms of a

ISSN:1066-5234    

DOI:10.1111/j.1550-7408.1995.tb01567.x

出版商:Blackwell Publishing Ltd    

年代:1995

数据来源: WILEY

摘要:

ABSTRACT.Four methods are commonly used to study cell cycle processes inParamecium tetraurelia.These include stage frequency analysis in asynchronous cultures, hand selection of synchronous dividing cells, selection of newly divided cells by elutriation centrifugation, and the sister cell method. We have compared the timing and resolution of stages of oral morphogenesis and micronuclear mitosis with each method. The temporal resolution obtainable with the sister cell method was inadequate to position the timing of morphogenesis stages within the cell cycle. Both the asynchronous method and the hand‐selected synchronous samples methods are prone to bias. Elutriation centrifuge synchronization provides large samples with resolution comparable to that of hand selected samples. The elutriation method is the least prone to bias when<5% of the parent culture ofParameciumis selecte

ISSN:1066-5234    

DOI:10.1111/j.1550-7408.1995.tb01568.x

出版商:Blackwell Publishing Ltd    

年代:1995

数据来源: WILEY

摘要:

ABSTRACT.SixLeishmaniaspecies were studied comparatively, in order to determine the influence of temperature “in vitro” on differentiation, infectivily and protein synthesis. Differentiation ocurred in a heterogeneous manner, even in species that produce similar clinical manifestations. Thus, no association could be found between thermosensitivity and disease. The association between expression of proteins and increasing temperatures was analyzed at 34° C by polyacrylamide gel electrophoresis with sodium dodecyl sulphate (SDS‐PAGE), using different incubation times, and employing a technique involving metabolic incorporation of [35S]‐methionine. Protein synthesis was very similar in all the New World species apart fromL. amazonensis, which expressed a protein of approximately 80 kDa when incubated at 34° C for 2 hours. All the tested species had in common the expression of a 70 kDa protein. Differences, however, were observed in relation to the time interval for protein expression. inL. chagasi, synthesis was detected after 30 minutes of incubation at 34° C, whileL. braziliensisrequired 1 hour at the same temperature. The “in vivo” and “in vitro” infectivity of the differentiated forms was also analyzed, but no significant differe

ISSN:1066-5234    

DOI:10.1111/j.1550-7408.1995.tb01569.x

出版商:Blackwell Publishing Ltd    

年代:1995

数据来源: WILEY

摘要:

ABSTRACT.To investigate in more detail the structure of the high molecular weight rhoptry protein complex ofPlasmodium falciparum,Rhop‐H (140/130/110 kd), the complex was affinity purified from parasite extracts using rhoptry protein specific antisera prepared against Rhop‐H proteins bound to and eluted from Balb/c mouse erythrocytes, using 0.5 M NaCl. The individual proteins (140 kd/Rhop‐1, 130 kd/Rhop‐2, and 110 kd/Rhop‐3) were separated, electroeluted, and monospecific polyclonal antisera prepared against the individual proteins, and against the affinity purified complex. Immunofluorescence assays and immunoelectron microscopic studies were performed to verify the subcellular localization of the Rhop‐H epitopes. Immunoblotting and immunoprecipitation assays were also performed. We report novel findings regarding the localization of the rhoptry proteins to an electron lucent compartment in the neck of the rhoptries. Analysis of the amino acid composition of the individually purified Rhop‐H proteins demonstrated a predominance of negatively charged (E, D) as well as hydrophobic residues (L, A, P, S) in the three proteins. The percentage of negatively charged residues was high for all three proteins. Similarities in amino acid composition for the three proteins supports the previous data demonstrating shared properties such as erythrocyte and liposome binding, for the three proteins. Results of antibody characterizations using rhoptry protein specific antisera demonstrate the immunodominance of the R

ISSN:1066-5234    

DOI:10.1111/j.1550-7408.1995.tb01570.x

出版商:Blackwell Publishing Ltd    

年代:1995

数据来源: WILEY

摘要:

ABSTRACT.SevenPhytomonas‐specific monoclonal antibodies produced againstPhytomonas serpensandPhytomonas françaiwere further characterised in order to identify and localise their target antigens. Four monoclonal antibodies recognized carbohydrate surface epitopes, in three of the cases associated with surface glycoproteins with apparent molecular weight of 80 kDa. One monoclonal antibody apparently bound to a surface/internal protein epitope, whereas the two others recognized intra‐cellular proteins. The cell surface epitopes recognized by monoclonal antibodies were detected specifically in the genusPhytomonas.These epitopes, which are detected in culture, plant and insect forms, may be useful as targets forPhytomonasidentifica

ISSN:1066-5234    

DOI:10.1111/j.1550-7408.1995.tb01571.x

出版商:Blackwell Publishing Ltd    

年代:1995

数据来源: WILEY

摘要:

ABSTRACT.The lysosomal enzyme N‐acetyl‐β‐hexosaminidase (βhex) has been purified fromAcanthamoeba castellaniigrowth medium by a three step procedure. The enzyme was precipitated with ammonium sulfate, partially purified on a DE52 column and purified to homogeneity on an affinity column. The purified βhex appeared to be a monomer with a molecular mass of 58 kDa and a pI of approximately 5.8. The enzyme activity in growth medium at RT was stable for several months. The purified βhex was enzymatically deglycosylated and injected, into two rabbits to make polyclonal antibodies. One antiserum was specific for βhex, but the other stained many bands on immunoblots of whole cell preparations. Using fluorescently labelled secondary antibodies we have determined that both antisera stain digestive vacuoles in theAcanthamoebacytoplasm, and do not stain the contractile vacuole. The multi‐specific antiserum had high avidity for βhex, but also stained the carbohydrate portion of other molecules. These other molecules may be lysosomal enzymes as well, since the activity of several other lysosomal enzymes was partially immunoprecipitable with the antiserum. We plan to use these antibodies to study traffic patterns among the variety of vacuolar structures inAcantham

ISSN:1066-5234    

DOI:10.1111/j.1550-7408.1995.tb01572.x

出版商:Blackwell Publishing Ltd    

年代:1995

数据来源: WILEY