Cell surface distribution and intracellular fate of asialoglycoproteins: a morphological and biochemical study of isolated rat hepatocytes and monolayer cultures

A combination of biochemistry and morphology was used to demonstrate that more than 95 percent of the isolated rat hepatocytes prepared by collagenase dissociation of rat livers retained the pathway for receptor-mediated endocytosis of asialoglycoproteins (ASGPs). Maximal specific binding of (125)I-asialoorosomucoid ((125)I-ASOR) to dissociated hepatocytes at 5 degrees C (at which temperature no internalization occurred) averaged 100,000-400,000 molecules per cell. Binding, uptake, and degredation of (125)I- ASOR at 37 degrees C occurred at a rate of 1 x 10(6) molecules per cell over 2 h. Light and electron microscopic autoradiography (LM- and EM-ARG) of (125)I-ASOR were used to visualize the surface binding sites at 5 degrees C and the intracellular pathway at 37 degrees C. In the EM-ARG experiments, ARG grains corresponding to (125)I-ASOR were distributed randomly over the cell surface at 5 degrees C but over time at 37 degrees C were concentrated in the lysosome region. Cytochemical detection of an ASOR-horseradish peroxidase conjugate (ASOR-HRP) at the ultrastructural level revealed that at 5 degrees C this specific ASGP tracer was concentrated in pits at the cell surface as well as diffusely distributed along the rest of the plasma membrane. Such a result indicates that redistribution of ASGP surface receptors had occurred. Because the number of surface binding sites of (125)I-ASOR varied among cell preparations, the effect of collagenase on (125)I-ASOR binding was examined. When collagenase-dissociated hepatocytes were re-exposed to collagenase at 37 degrees C, 10-50 percent of control binding was observed. However, by measuring the extent of (125)I-ASOR binding at 5 degrees C in the same cell population before and after collagenase dissociation, little reduction in the number of ASGP surface receptors was found. Therefore, the possibility that the time and temperature of the cell isolations allowed recovery of cell surface receptors following collagenase exposure was tested. Freshly isolated cells, dissociated cells that were re-exposed to collagenase, and perfused livers exposed to collagenase without a Ca(++)-free pre-perfusion, were found to bind 110-240 percent more(125)I-ASOR after 1 h at 37 degrees C that they did at 0 time. This recovery of surface ASGP binding activity occurred in the absence of significant protein synthesis (i.e., basal medium or 1 mM cycloheximide). Suspensions of isolated, unpolarized hepatocytes were placed in monolayer culture for 24 h and confluent cells were demonstrated to reestablish morphologically distinct plasma membrane regions analogous to bile canalicular, lateral, and sinusoidal surfaces in vivo. More than 95 percent of these cells maintained the capacity to bind, internalize, and degrade (125)I-ASOR at levels comparable to those of the freshly isolated population. ASOR-HRP (at 5 degrees C) was specifically bound to all plasma membrane surfaces of repolarized hepatocytes (cultured for 24 h) except those lining bile canalicular-like spaces. Thus, both isolated, unpolarized hepatocytes and cells cultured under conditions that promote morphological reestablishment of polarity maintain the pathway for receptor- mediated endocytosis of ASGPs.     

Polyamine requirement for streptomycin action on protein synthesis in bacteria

Self-association between various heparan sulphate species and oligosaccharide fragments thereof have been studied by affinity chromatography. Polysaccharides or oligosaccharides were coupled to agarose and free chains were applied at low concentrations (less than or equal to 2 mg/ml) in 0.15 M NaCl to minimize self-association between free chains. The results show that the interaction may be specific. Heparan sulphate chains chiefly bind to gels substituted with cognate chains, i.e. the same kind or closely similar ones. Oligosaccharides of the general structure glucosamine-(iduronate/glucuronate-glucosamine)n--O--C(=CH2)--CHO were prepared by periodate oxidation/alkaline elimination and also coupled to agarose via the --CHO group. Cognate heparan sulphate chains were bound to this affinity matrix with the same affinity as in the case of heparan-sulphate--agarose. Free oligosaccharides were not bound to oligosaccharide-agarose, nor to the corresponding heparan-sulphate--agarose. Oligosaccharides of the same size and containing only iduronate were ineffective as affinity ligands. It is concluded that the segments comprising both iduronate and glucuronate may serve as contact zones in the heparan sulphate/heparan sulphate self-association and that the strength of binding is dependent on cooperative interactions between a number of such zones. The putative contact zones, as ligands on the matrix, showed an emerging lack of specificity as non-associating or unrelated and associating chains were bound to this gel. This is ascribed to a randomization of the contact zones which, in the polymeric chains, are placed in their proper register by the intervening (glucuronate-N-acetylglucosamine)n segments.     

Isolation and characterization of microsatellite loci in the sand fly Phlebotomus papatasi (Diptera: Psychodidae)

Phlebotomus papatasi is a proven vector of Leishmania major which is one of the causative agents of cutaneous leishmaniasis in the Old World. Although it has a wide geographical range, its population structure is not yet well understood. In an effort to better understand the population dynamics of this vector, we developed a panel of di‐ and trinucleotide microsatellite markers, using a magnetic bead hybridization enrichment protocol. These microsatellite loci showed three to seven alleles with an expected heterozygosity range between 0.702 and 0.876. The level of polymorphisms found in this study suggests that these microsatellite loci can be used for population analysis of P. papatasi.     

Morphology and morphogenesis of a new marine hypotrichous ciliate (Protozoa,Ciliophora, Pseudoamphisiellidae), including a report on the small subunit rRNA gene sequence

The morphology and morphogenesis of a new marine hypotrichous ciliate Pseudoamphisiella elongata sp. nov. isolated from mussel‐farming waters near Qingdao, China, are described based on living and protargol‐impregnated specimens. Morphologically, the new species can be distinguished from its known congeners by its elongate body shape, narrow oral field, having fewer dorsal kineties and caudal cirri, more marginal cirri, and differentiated pretransverse cirri. The identification as a new species is firmly supported by the sequences of the small subunit ribosomal rRNA (SSU rRNA) gene, compared with other known Pseudoamphisiella species, and the phylogenetic analysis. The morphogenetic characteristics can be summarized as follows: (1) the parental adoral zone of membranelles and undulating membranes are entirely rebuilt by the oral primordium, which develops de novo in the outermost region of the cortex; (2) the oral primordium in the opisthe and the frontoventral–transverse (FVT) anlagen in both dividers are formed independently on the cell surface; (3) an ‘extra’ marginal anlage originates to the right of the right marginal anlage, and develops into two or three ‘extra’ marginal cirri; (4) the FVT anlagen develop in the primary mode, and the last FVT streak contributes two migratory cirri (frontoterminal cirri), which are probably resorbed; (5) the right marginal anlagen in both dividers occur close together, independent of the old structure. © 2010 The Linnean Society of London, Zoological Journal of the Linnean Society, 2010, 158 , 231–243.     

The genome sequence of the biocontrol fungus Metarhizium anisopliae and comparative genomics of Metarhizium species

Background

Metarhizium anisopliae is an important fungal biocontrol agent of insect pests of agricultural crops. Genomics can aid the successful commercialization of biopesticides by identification of key genes differentiating closely related species, selection of virulent microbial isolates which are amenable to industrial scale production and formulation and through the reduction of phenotypic variability. The genome of Metarhizium isolate ARSEF23 was recently published as a model for M. anisopliae, however phylogenetic analysis has since re-classified this isolate as M. robertsii. We present a new annotated genome sequence of M. anisopliae (isolate Ma69) and whole genome comparison to M. robertsii (ARSEF23) and M. acridum (CQMa 102).

Results

Whole genome analysis of M. anisopliae indicates significant macrosynteny with M. robertsii but with some large genomic inversions. In comparison to M. acridum, the genome of M. anisopliae shares lower sequence homology. While alignments overall are co-linear, the genome of M. acridum is not contiguous enough to conclusively observe macrosynteny. Mating type gene analysis revealed both MAT1-1 and MAT1-2 genes present in M. anisopliae suggesting putative homothallism, despite having no known teleomorph, in contrast with the putatively heterothallic M. acridum isolate CQMa 102 (MAT1-2) and M. robertsii isolate ARSEF23 (altered MAT1-1). Repetitive DNA and RIP analysis revealed M. acridum to have twice the repetitive content of the other two species and M. anisopliae to be five times more RIP affected than M. robertsii. We also present an initial bioinformatic survey of candidate pathogenicity genes in M. anisopliae.

Conclusions

The annotated genome of M. anisopliae is an important resource for the identification of virulence genes specific to M. anisopliae and development of species- and strain- specific assays. New insight into the possibility of homothallism and RIP affectedness has important implications for the development of M. anisopliae as a biopesticide as it may indicate the potential for greater inherent diversity in this species than the other species. This could present opportunities to select isolates with unique combinations of pathogenicity factors, or it may point to instability in the species, a negative attribute in a biopesticide.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-660) contains supplementary material, which is available to authorized users.     

A structured overview of simultaneous component based data integration

Background  

Data integration is currently one of the main challenges in the biomedical sciences. Often different pieces of information are gathered on the same set of entities (e.g., tissues, culture samples, biomolecules) with the different pieces stemming, for example, from different measurement techniques. This implies that more and more data appear that consist of two or more data arrays that have a shared mode. An integrative analysis of such coupled data should be based on a simultaneous analysis of all data arrays. In this respect, the family of simultaneous component methods (e.g., SUM-PCA, unrestricted PCovR, MFA, STATIS, and SCA-P) is a natural choice. Yet, different simultaneous component methods may lead to quite different results.     

Morphology and Phylogeny of Two New Pleurostomatid Ciliates,Epiphyllum shenzhenense n. sp. and Loxophyllum spirellum n. sp. (Protozoa,Ciliophora) from A Mangrove Wetland,South China

ABSTRACT. The morphology, infraciliature, and small subunit (SSU) rRNA gene sequences of two new pleurostomatid ciliates, Epiphyllum shenzhenense n. sp. and Loxophyllum spirellum n. sp., isolated from a mangrove wetland near Shenzhen, South China, were investigated. Epiphyllum shenzhenense n. sp. is morphologically characterized by leaf‐shaped cell about 150 × 35 μm in vivo, usually with four contractile vacuoles, 20–29 right kineties and 10–26 left kineties, ca. four macronuclear nodules, and two types of extrusomes (i.e. short spindle‐shaped and long bar‐shaped). As a new species, L. spirellum n. sp. is distinguished from its congeners by its posterior dorsal margin twisted onto the left side, the distribution of extrusomes (evenly arranged along the oral slit, the posterior end, and clustered to 7–13 warts on dorsal margin), the subterminally positioned contractile vacuole, the number of kineties (8–10 on right side, 4–5 on left side), and its genetic distance from congeners. Phylogenetic trees based on the SSU rRNA gene sequence for both organisms were constructed, which indicate that Epiphyllum is a distinct genus and occupies a basal position in the Pleurostomatida clade; L. spirellum n. sp. falls well into the Loxophyllum clade, which has a close relationship with Litonotus and Spiroloxophyllum.     

Glycoproteins histochemistry of the gills of Odontesthes bonariensis (Teleostei, Atherinopsidae)

The histochemistry of glycoproteins (GP) in the mucous cells of the gills of the silverside Odontesthes bonariensis was identified with: (1) oxidizable vicinal diols; (2) sialic acid and some of their chain variants, carbon 7 ((7) C), carbon 8 ((8) C) or carbon 9 ((9) C); (3) sialic acid residues without O-acyl substitution and with O-acyl substitution at (7) C, (8) C or (9) C; (4) carboxyl groups and (5) sulphate groups. A battery of seven biotinylated lectins allowed GPs sugar residues to be distinguished. Mucous cells showed the presence of neutral, sulphated and sialylated GPs. Dolichos biflorus agglutinin (DBA) and Glycine max agglutinin (SBA) showed strong positive staining; Arachis hypogaea agglutinin (PNA), Ricinus communis agglutinin-I (RCA-I) and Triticum vulgaris agglutinin (WGA) showed moderate staining, while Ulex europaeus agglutinin-I (UEA-I) was completely negative.