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1.
Uninucleate and multinucleate stages of a protozoan parasite are described from cultured abalone Haliotis iris Martyn, 1784 in New Zealand. The parasite is identified as a haplosporidian by the occurrence of multinucleate plasmodia, mitochondria with tubular cristae, lipid droplets, anastomosing endoplasmic reticulum (aER), multivesicular bodies (MVBs), haplosporogenesis by the production of haplosporosome-like bodies from nuclear membrane-bound Golgi, and their maturation to haplosporosomes. Coated pits occurred in the plasma membrane and coated vesicles were scattered in the cytoplasm, particularly in association with the Golgi face away from the nucleus, and aER. It is concluded that the outward face of the Golgi may be the trans face, and that aER is the trans-Golgi network. Coated pits and bristle-coated vesicles are reported from a haplosporidian for the first time. The vesicles in the MVBs resembled the cores and inner membranes of haplosporosomes, without the outer layer. The possible inter-relationships of these features are discussed. The abalone parasite differs from previously described haplosporidians in the apparent absence of a persistent mitotic spindle, and the presence of intracytoplasmic coccoid to rod-shaped bacteria resembling Rickettsiales-like prokaryotes. Phylogenetic analysis of the 16S rRNA gene sequence of the Rickettsiales-like prokaryotes indicated that these organisms belong to the Rickettsia cluster. The prokaryotes have a high (7%) sequence divergence from known Rickettsieae, with Rickettsia sp. and R. massiliae being the closest relatives. The lack of non-molecular evidence prevents us from proposing a new rickettsial genus at this time.  相似文献   
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The single most difficult problem in phylogenetic analysis is decidingwhether a shared taxonomic character is due to common ancestry or one thatappeared independently due to convergence, parallelism, or reversion to anancestral state. Mammalian L1 retrotransposons undergo periodicamplifications in which multiple copies of the elements are interspersed inthe genome. Because these elements apparently are transmitted only byinheritance and are retained in the genome, a shared L1 amplification eventcan only be an inherited ancestral character. We propose that L1amplification events can be an excellent tool for analyzing mammalianevolution and demonstrate here how we addressed several refractory problemsin rodent systematics using L1 DNA as a taxonomic character.  相似文献   
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Discovering the function of an unknown protein, particularly one with neither structural nor functional correlates, is a daunting task. Interaction analyses determine binding partners, whereas DNA transfection, either transient or stable, leads to intracellular expression, though not necessarily at physiologically relevant levels. In theory, direct intracellular protein delivery (protein transduction) provides a conceptually simpler alternative, but in practice the approach is problematic. Domains such as HIV TAT protein are valuable, but their effectiveness is protein specific. Similarly, the delivery of intact proteins via endocytic pathways (e.g. using liposomes) is problematic for functional analysis because of the potential for protein degradation in the endosomes/lysosomes. Consequently, recent reports that microspheres can deliver bio-cargoes into cells via a non-endocytic, energy-independent pathway offer an exciting and promising alternative for in vitro delivery of functional protein. In order for such promise to be fully exploited, microspheres are required that (i) are stably linked to proteins, (ii) can deliver those proteins with good efficiency, (iii) release functional protein once inside the cells, and (iv) permit concomitant tracking. Herein, we report the application of microspheres to successfully address all of these criteria simultaneously, for the first time. After cellular uptake, protein release was autocatalyzed by the reducing cytoplasmic environment. Outside of cells, the covalent microsphere–protein linkage was stable for ≥90 h at 37 °C. Using conservative methods of estimation, 74.3% ± 5.6% of cells were shown to take up these microspheres after 24 h of incubation, with the whole process of delivery and intracellular protein release occurring within 36 h. Intended for in vitro functional protein research, this approach will enable study of the consequences of protein delivery at physiologically relevant levels, without recourse to nucleic acids, and offers a useful alternative to commercial protein transfection reagents such as Chariot™. We also provide clear immunostaining evidence to resolve residual controversy surrounding FACS-based assessment of microsphere uptake.Many proteomic techniques can be used to build a picture of a protein with unknown function, but eventually the individual protein''s activity must be studied. Traditional transfection of encoding DNA permits intracellular expression, but often at uncontrolled, nonphysiological levels. Moreover, DNA transfection can neither deliver protein–inhibitor complexes nor readily deliver multiple proteins in a single experiment and thus exploit knowledge from proteomic protein–protein interaction analyses. In contrast, a truly generic protein transduction reagent could theoretically address all possibilities. We believe that polymeric microspheres could fulfill this role, and we have recently synthesized and characterized dual-functionalized, bio-compatible microspheres that permit intracellular tracking (1). Herein, we now report the development of those microspheres into a protein transduction reagent that can carry protein stably, deliver it efficiently to cells, release the protein in the cytoplasm, and concurrently permit fluorescent imaging of transduced cells.Phagocytosis of microspheres was first observed over 30 years ago (2). Perhaps more unexpectedly, uptake of polystyrene microspheres has recently been reported in many other, nonphagocytic cell types, some of which are traditionally considered to be resistant to DNA transfection and/or protein transduction. For example, microspheres are taken up readily by primary immune cells (3), embryonic stem cells (4), human neural stem cells (5), differentiating mouse neural stem cells (5), and several nonphagocytic cell lines (3, 6, 7). In all instances, the reported efficiency of cellular uptake is high, with “beadfection” of up to 90% of cells being typical (4, 5, 8). No additional reagents aside from the microspheres themselves are required in order to promote cellular uptake, and critically, no toxicity has been observed in any of the cell types beadfected, including HEK293T and L929 cells 2 days after beadfection (8), E14g2a embryonic stem cells 3 days after beadfection (4), and mouse and human neural stem cells 30 days after beadfection (5). In the latter case, the microspheres did not have any deleterious effect on the differentiation of human neural stem cells 30 days after beadfection (5).The mechanism of microsphere entry is also nontoxic, and compelling evidence has been published recently that polystyrene-based microspheres (from 0.2 μm to as large as 2 μm) enter cells via a non-endocytosis, energy-independent mechanism (8). Although unusual, such a mechanism is consistent with claims for the commercial reagent Chariot™ (9). Interestingly, a non-endocytic, energy-independent mechanism has also been reported for the entry of rhenium cluster/polymer hybrid particles into HeLa cells (10). Failure of the microspheres to be endocytosed, at least via a clathrin-dependent mechanism, is perhaps to be predicted, as their diameter considerably exceeds that of clathrin-coated vesicles (typically 100 nm). Bradley and co-workers (8) propose that the entry mechanism for polystyrene-based microspheres is one of passive diffusion in which the microsphere interacts with the membrane, anchors, and, after membrane reorganization, enters the cell, resulting in direct cytoplasmic localization.For functional analysis following transduction, the avoidance of endocytosis or phagocytosis is particularly relevant, as endocytosed particles are destined for endosomes and then, normally, for the lysosomes. The lowered pH of the endosome and, more seriously, the acidic and hydrolytic environment of the lysosome risk disruption of the protein structure and/or function. In contrast, for vaccine delivery (where liposomes can be employed), such exposure is advantageous because protein breakdown forms an essential part of antigen presentation. The potential for protein breakdown in endosomes is also irrelevant for the delivery of protein/peptide drugs such as insulin (for which microencapsulation has proven effective for long-term controlled drug release (11, 12)), as these drugs typically function in the extracellular environment, often exerting their effects by binding to membrane-bound receptors. Thus, although vehicles such as liposomes and nanoparticles are employed both extensively and successfully as drug and vaccine delivery vectors in vivo (1316), they are far from ideal for studying the biological effect of a delivered protein in vitro. Colloidal particles are also endocytosed (17), and therefore these delivery vehicles may present similar disadvantages.Traditionally, protein transduction domains such as HIV TAT (1820) or other cell-penetrating peptides (2123) are used to deliver proteins to cells. Whereas positively charged peptides such as TAT are thought to enter the cells via macropinocytosis (reviewed in Ref. 24), a recent publication suggests that at least some cell-penetrating peptide/bio-cargo complexes (siRNA) are endocytosed (25). Here, although the cargoes avoid the lysosomes, acidification of the endosome is required for endosomal escape of the delivered cargo, and indeed, acidification appears to be a recurring requirement for endosomal escape of biomolecular cargoes using cell-penetrating peptides (reviewed in Ref. 24). Consequently, cell-penetrating peptides are unlikely to become generic tools for functional protein delivery.In contrast, the recent demonstrations that polystyrene microspheres can carry a variety of molecular cargoes with them into the cytoplasm (4, 5, 7, 26, 27) make them particularly exciting as potential vectors for delivering functional proteins and/or protein complexes. β-Galactosidase retains its activity when delivered via this route (7), confirming the potential of microspheres to act as generic protein-delivery vehicles. However, delivered proteins have to date remained tethered to the microspheres, and thus existing studies are limited to proteins that are active in the cytoplasm and, critically, retain their activity when immobilized on polystyrene. For the broad-based study of protein function, the subsequent release of the delivered protein within the cell is desirable.An ideal technology would deliver any protein to any cell type and release that protein in the cell, where it could undertake its normal activity. Here we report the first example of such a microsphere-based approach. Protein is delivered on microspheres and then released in the cell by the reducing cytoplasmic environment. This release is mediated by a linker that attaches the protein stably and covalently to the microspheres in vitro but intracellularly is cleaved over a period of hours. It has already been shown that microspheres are taken up with high efficiency by a range of cell types and can carry a variety of cargoes. Because the chemistry of the linker described herein is amenable to linkage with any molecule containing a free amine moiety, the technology provides a new generic platform for in vitro, cell-based delivery of individual proteins, protein complexes, protein mixtures, or other amino-functionalized molecules.  相似文献   
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The blood granulocyte composition of 10 species of dogfish is given, together with ultrastructural observations made on Etmopterus baxteri Leydig organ and blood, and on spleens of Oxynotus bruniensis, Deania calcea, Scymnodon plunketi and blood of Centroscymnus crepidator . Neutrophilic granulocytes, which were common, had spherical granules that developed a dense core, which then lost contents to become lucent. Eosinophilic granulocytes had ovoid or elongated granules with a fibrillar content that became aligned longitudinally, and rarely formed an axial rod. Eosinophils had large spherical granules that were electron-dense but in early stages had a disorganised fibrillar content. These cells correspond to the neutrophils, heterophils and eosinophils, respectively, of other elasmobranchs.
Dogfish granulocytes are compared with those of other elasmobranchs, and their lack of similarity to those of higher vertebrates is noted.  相似文献   
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Four intracellular prokaryotes are reported from the scallops Pecten novaezelandiae Reeve, 1853 and Chlamys delicatula Hutton, 1873. Elongated (1025 x 110 nm), irregular (390 x 200 nm), or toroidal (410 x 200 nm) mollicute-like organisms (M-LOs) occurred free in the cytoplasm in the digestive diverticular epithelial cells of both scallop species. Those in P. novaezelandiae bore osmiophilic blebs that sometimes connected the organisms together, and some had a rod-like protrusion, both of which resemble the blebs and tip structures of pathogenic mycoplasmas. The M-LOs in C. delicatula had a slightly denser core than periphery. Round M-LOs, 335 x 170 nm, occurred free in the cytoplasm of agranular haemocytes in P. novaezelandiae, without apparent harm to the host cell. In P. novaezelandiae, 2 types of highly prevalent (95 to 100%) basophilic inclusions in the branchial epithelium contained Rickettsia-like organisms (R-LOs). Type 1 inclusions occurred in moderately hypertrophied, intensely basophilic cells, 8 to 10 microm in diameter, containing elongate intracellular R-LOs, 2000 x 500 nm. Type 2 inclusions were elongated and moderately basophilic in markedly hypertrophic branchial epithelial cells, 50 x 20 microm in diameter, containing intracellular organisms 500 x 200 nm in diameter. The possible roles of these organisms in pathogenesis is discussed.  相似文献   
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