A redescription of Uroproctepisthmium bursicola (Creplin, 1837) n. comb. (Digenea: Echinostomatidae), and re-evaluations of the genera Episthmium Lühe, 1909 and Uroproctepisthmium Fischthal & Kuntz, 1976

Re-examination of the type and newly collected material of Episthmium bursicola (Creplin, 1837) revealed the presence of a uroproct. The species is redescribed and transferred to the genus Uroproctepisthmium as U. bursicola n. comb. E. proximum Travassos, 1922 is also transferred to Uroproctepisthmium as U. proximum n. comb. The generic diagnosis of Uroproctepisthmium is redefined and Episthmium is tentatively retained as a synonym of Echinochasmus, following Odhner (1910), until a thorough revision of its constituent species is made.     

Evidence for the Concerted Evolution between Short Linear Protein Motifs and Their Flanking Regions

Background

Linear motifs are short modules of protein sequences that play a crucial role in mediating and regulating many protein–protein interactions. The function of linear motifs strongly depends on the context, e.g. functional instances mainly occur inside flexible regions that are accessible for interaction. Sometimes linear motifs appear as isolated islands of conservation in multiple sequence alignments. However, they also occur in larger blocks of sequence conservation, suggesting an active role for the neighbouring amino acids.

Results

The evolution of regions flanking 116 functional linear motif instances was studied. The conservation of the amino acid sequence and order/disorder tendency of those regions was related to presence/absence of the instance. For the majority of the analysed instances, the pairs of sequences conserving the linear motif were also observed to maintain a similar local structural tendency and/or to have higher local sequence conservation when compared to pairs of sequences where one is missing the linear motif. Furthermore, those instances have a higher chance to co–evolve with the neighbouring residues in comparison to the distant ones. Those findings are supported by examples where the regulation of the linear motif–mediated interaction has been shown to depend on the modifications (e.g. phosphorylation) at neighbouring positions or is thought to benefit from the binding versatility of disordered regions.

Conclusion

The results suggest that flanking regions are relevant for linear motif–mediated interactions, both at the structural and sequence level. More interestingly, they indicate that the prediction of linear motif instances can be enriched with contextual information by performing a sequence analysis similar to the one presented here. This can facilitate the understanding of the role of these predicted instances in determining the protein function inside the broader context of the cellular network where they arise.     

Proteomic Analysis of Neorickettsia sennetsu Surface-Exposed Proteins and Porin Activity of the Major Surface Protein P51

Neorickettsia sennetsu is an obligate intracellular bacterium of monocytes and macrophages and is the etiologic agent of human Sennetsu neorickettsiosis. Neorickettsia proteins expressed in mammalian host cells, including the surface proteins of Neorickettsia spp., have not been defined. In this paper, we isolated surface-exposed proteins from N. sennetsu by biotin surface labeling followed by streptavidin-affinity chromatography. Forty-two of the total of 936 (4.5%) N. sennetsu open reading frames (ORFs) were detected by liquid chromatography-tandem mass spectrometry (LC/MS/MS), including six hypothetical proteins. Among the major proteins identified were the two major β-barrel proteins: the 51-kDa antigen (P51) and Neorickettsia surface protein 3 (Nsp3). Immunofluorescence labeling not only confirmed surface exposure of these proteins but also showed rosary-like circumferential labeling with anti-P51 for the majority of bacteria and polar to diffuse punctate labeling with anti-Nsp3 for a minority of bacteria. We found that the isolated outer membrane of N. sennetsu had porin activity, as measured by a proteoliposome swelling assay. This activity allowed the diffusion of l-glutamine, the monosaccharides arabinose and glucose, and the tetrasaccharide stachyose, which could be inhibited with anti-P51 antibody. We purified native P51 and Nsp3 under nondenaturing conditions. When reconstituted into proteoliposomes, purified P51, but not Nsp3, exhibited prominent porin activity. This the first proteomic study of a Neorickettsia sp. showing new sets of proteins evolved as major surface proteins for Neorickettsia and the first identification of a porin for the genus Neorickettsia.Neorickettsia spp. are unique environmental, Gram-negative, obligate intracellular bacteria maintained in nature through vertical transmission in trematodes (16, 17, 39, 42). Neorickettsia spp. are polymorphic cocci belonging to the family Anaplasmataceae within the order Rickettsiales in the class Alphaproteobacteria (7). Neorickettsia sennetsu (formerly called Rickettsia sennetsu or Ehrlichia sennetsu) is the first human pathogen in the family Anaplasmataceae to have been isolated and cultured (11, 34). N. sennetsu infects human monocytes and macrophages and causes the disease Sennetsu neorickettsiosis (10, 34, 41). Epidemiologic studies of Sennetsu neorickettsiosis show a strong link between the human ingestion of metacercaria-infested gray mullet fish and acquisition of the disease (11). Symptoms are similar to those of infectious mononucleosis and include swelling of the lymph nodes, pyrexia, inappetence, lethargy, sleeplessness, and overall malaise (10, 34, 41). Geographically, N. sennetsu infections have been reported mainly in western and southern Japan, although antibodies to N. sennetsu have also been found in humans in Malaysia, and one strain of N. sennetsu has been isolated from Malaysia (10, 19, 44). Recently, N. sennetsu infection was found in Laos (35). Treatment of Sennetsu neorickettsiosis involves tetracycline therapy and is normally highly successful at resolving the symptoms (10).Gram-negative bacteria generally have porins spanning their outer membranes. These proteins enable the transport of hydrophilic molecules, such as amino acids, sugars, and other nutrients (36). As seen in other members of the Anaplasmataceae, N. sennetsu is limited in its ability to synthesize necessary compounds, including amino acids and enzymes for intermediary metabolism and glycolysis (20). Therefore, porins are an absolute necessity for the survival of this bacterium. To date, the only porins defined for the order Rickettsiales are major outer membrane proteins of Anaplasma phagocytophilum named P44s (22) and OMP-1F and P28 in Ehrlichia chaffeensis (26). These porins contain 16 (P44s) or 12 (OMP-1F and P28) transmembrane passes, and some are large enough to allow the slow diffusion of tetrasaccharides. P44/Msp2 and OMP-1/P28/P30 proteins belong to the family pfam01617, and the N. sennetsu genome was reported to encode only one hypothetical protein from this family (GenBank accession no. NSE_0875) (20), later named Neorickettsia surface protein 3 (Nsp3) (29).In the present study, surface-exposed proteins of N. sennetsu were isolated from cell culture and identified by proteomics. We first isolated the outer membrane fraction from host cell-free N. sennetsu and examined porin activity by using an in vitro proteoliposome swelling assay. Second, we used antibodies against the dominant protein P51 to examine neutralization of the porin activity. Third, we purified native P51 and Nsp3 from the isolated outer membrane fraction by high-pressure liquid chromatography (HPLC) and tested whether these proteins have porin activity. Identification of surface-exposed proteins and the protein with major porin activity will help in understanding N. sennetsu and the disease that it causes.     

Variation in alcohol dehydrogenase activity in vitro in flies from natural populations ofDrosophila melanogaster

Alcohol dehydrogenase (ADH) activity variation in male flies taken directly from seven natural populations ofDrosophila melanogaster is largely accounted for by segregation of alleles at theAdh structural gene locus. There was little overlap in the ADH activities ofAdh ^F andAdh ^s homozygotes. Body weights varied only slightly betweenAdh genotypes and contributed little to ADH variation. Between and within population variation in ADH activity and ADH protein in flies in the wild is mainly due to the relative frequencies ofAdh ^F andAdh ^s.     

The porcine gonadotropin-releasing hormone receptor gene (GNRHR): genomic organization, polymorphisms, and association with the number of corpora lutea.

The interaction of gonadotropin-releasing hormone (GNRH) and its receptor (GNRHR) is critical in the endocrine regulation of reproduction. The gene (GNRHR) encoding the receptor has been mapped to porcine chromosome 8. There is evidence for three quantitative trait loci (QTL) influencing ovulation rate on this chromosome. We obtained an almost complete sequence (3993 bp, excluding intron 1) of the porcine GNRHR gene using PCR-based comparative genomic walking and inverse genomic walking approaches. Twelve polymorphisms were detected by sequencing of pooled DNA of Chinese Taihu and European Large White pigs, including 7 base substitutions and 5 insertions-deletions (indels). A F2 population of Meishan x European Large White pigs was genotyped for a TG indel in the promoter region, and a C/G substitution in the 3' UTR (untranslated region). A significant association of the C/G substitution with number of corpora lutea at first parity was observed.     

Reovirus-induced apoptosis of MDCK cells is not linked to viral yield and is blocked by Bcl-2.

In this study, we investigated the relationship between reovirus-induced apoptosis and viral growth. Madin-Darby canine kidney (MDCK) epithelial cells infected with prototype reovirus strains type 1 Lang (T1L) or type 3 Dearing (T3D) were found to undergo apoptosis, and T3D induced apoptosis of MDCK cells to a substantially greater extent than T1L. By using T1L x T3D reassortant viruses, we found that differences in the capacities of these strains to induce apoptosis are determined by the viral S1 and M2 gene segments. These genes encode viral outer-capsid proteins that play important roles in viral entry into cells. T1L grew significantly better in MDCK cells than T3D, and these differences in growth segregated with the viral L1 and M1 gene segments. The L1 and M1 genes encode viral core proteins involved in viral RNA synthesis. Bcl-2 overexpression in MDCK cells inhibited reovirus-induced apoptosis but did not substantially affect reovirus growth. These findings indicate that differences in the capacities of reovirus strains to induce apoptosis and grow in MDCK cells are determined by different viral genes and that premature cell death by apoptosis does not limit reovirus growth in MDCK cells.     

Quaternary conformational changes in human hemoglobin studied by laser photolysis of carboxyhemoglobin.

These experiments indicate that absorbance changes observed at the 425 nm isosbestic point of the Hb and HbCO following laser photolysis of HbCO provide a direct measure of the rates of quaternary conformational changes between rapidly reacting Hb (the immediate product of full photolysis) and slowly reacting normal deoxyhemoglobin. Hb, first observed by Gibson (Gibson, Q.H. (1959) Biochem. J. 71, 293-303), Has been interpreted as deoxyhemoglobin remaining in the liganded quaternary conformation following rapid removal of ligand by a light pulse. In borate buffers between pH 8.4 and 9.6 particularly simple pH-independent results were obtained which allowed the use of a Monod. Wyman, and Changeux model (Monod, J., Wyman, J., and Changeux, J (1965) J. Mol. Biol. 12, 88-118) to fit the data. In this case Hb is taken to be R state deoxyhemoglobin. Partial photolysis experiments at 425 nm show that the rate of the R - T conformational change at 20 degrees decreases by about a factor of 2 for each additional bound ligand. The rate of the ligand-free conformational change is found to be 920 +/- 60s(-1), 6400 +/- 600s(-1), and 15,700 +/- 700(-1) respectively at 3 degrees, 20 degrees, and 30 degrees. The previously uninterpreted effects of flash length and partial photolysis on the CO recombination kinetics can be explained in terms of the present model. Kinetic results obtained below pH 8 are found to be inconsistent with a two-state model. It appears that binding of inositol hexaphosphate produces a new rapidly reacting quaternary conformation of HbCO.     

Biosynthesis of sialylated lipooligosaccharides in Haemophilus ducreyi is dependent on exogenous sialic acid and not mannosamine. Incorporation studies using N-acylmannosamine analogues, N-glycolylneuraminic acid, and 13C-labeled N-acetylneuraminic acid

Haemophilus ducreyi is a Gram-negative bacterium that causes chancroid, a sexually transmitted disease. Cell surface lipooligosaccharides (LOS) of H. ducreyi are thought to play important biological roles in host infection. The vast majority of H. ducreyi strains contain high levels of sialic acid (N-acetylneuraminic acid, NeuAc) in their LOS. Here we investigate the biosynthetic origin of H. ducreyi sialosides by metabolic incorporation studies using a panel of N-acylmannosamine and sialic acid analogues. Incorporation of sialosides into LOS was assessed by matrix-assisted laser desorption and electrospray ionization mass spectrometry. A Fourier transform ion cyclotron resonance mass spectrometer provided accurate mass measurements, and a quadrupole time-of-flight instrument was used to obtain characteristic fragment ions and partial carbohydrate sequences. Exogenously supplied N-acetylmannosamine analogues were not converted to LOS-associated sialosides at a detectable level. In contrast, exogenous (13)C-labeled N-acetylneuraminic acid ([(13)C]NeuAc) and N-glycolylneuraminic acid (NeuGc) were efficiently incorporated into LOS in a dose-dependent fashion. Moreover, approximately 1.3 microM total exogenous sialic acid was sufficient to obtain about 50% of the maximum production of sialic acid-containing glycoforms observed under in vitro growth conditions. Together, these data suggest that the expressed levels of sialylated LOS glycoforms observed in H. ducreyi are in large part controlled by the exogenous concentrations of sialic acid and at levels one might expect in vivo. Moreover, these studies show that to properly exploit the sialic acid biosynthetic pathway for metabolic oligosaccharide engineering in H. ducreyi and possibly other prokaryotes that share similar pathways, precursors based on sialic acid and not mannosamine must be used.