Molecular diversity of bacteria in Yunnan yellow cattle (Bos taurs) from Nujiang region, China

The rumen content of four Yunnan Yellow Cattle (Bos taurs) were collected to determine the bacteria diversity by using 16S rRNA gene sequence analysis. A total of 129 sequences were examined and the sequences were referred as 107 OTU (Operational Taxonomy Unit) according to the similarity level of 97% in gene sequence. Similarity analysis revealed that Yunnan Yellow Cattle had 12 sequences (10 OTU) shared 97% or greater similarity with cultured rumen bacteria Butyrivibrio fibrisolvens, Succiniclasticum ruminis, Ruminococcus bromii, Clostridium proteoclasticum, Ruminococcus flavefaciens, Pseudobutyrivibrio ruminis, Jeotgalicoccus psychrophilus, and Prevotella ruminicola, which accounting for 9.3% of the total clones (9.2% of the total OTU). The further 12 sequences (9 OTU) shared 90–97% similarity with cultured bacteria Clostridium aminobutyricum, butyrate-producing bacterium, Schwartzia succinivorans, Prevotella ruminicola, Eubacterium ruminantium, Ruminococcus albus, and Clostridium termitidis, also accounting for 9.3% of the total sequences (8.3% of the total OTU). The remaining 105 sequences (90 OTU) shared less than 90% similarity with cultured bacteria, accounting for 81.4% of the total sequences (82.5% of the total OTU). According to the phylogenetic analysis, all sequences were phylogenetically placed within phyla of low G+C subdivision (accounting for 72.1 and 72.5% of the total clones and OTU, respectively) and CFB subdivision (Cytophaga-Flexibacter-Bacteroides; accounting for 27.9 and 27.5% of the total clones and OTU, respectively). Among the examined clones, rare bacteria Jeotgalicoccus psychrophilus was detected in the rumen of cattle.     

The chlamydial OTU domain‐containing protein ChlaOTU is an early type III secretion effector targeting ubiquitin and NDP52

Chlamydia are obligate intracellular pathogens. Upon contact with the host, they use type III secretion to deliver proteins into the cell, thereby triggering actin‐dependent entry and establishing the infection. We observed that Chlamydia caviae elicited a local and transient accumulation of ubiquitinated proteins at the entry sites, which disappeared within 20 min. We investigated the mechanism for the rapid clearance of ubiquitin. We showed that the OTU‐like domain containing protein CCA00261, predicted to have deubiquitinase activity, was detected in infectious particles and was a type III secretion effector. This protein is present in several Chlamydia strains, including the human pathogen Chlamydia pneumoniae, and we further designate it as ChlaOTU. We demonstrated that ChlaOTU bound ubiquitin and NDP52, and we mapped these interactions to distinct domains. NDP52 was recruited to Chlamydia entry sites and was dispensable for infection and for bacterial growth. ChlaOTU functioned as a deubiquitinase in vitro. Heterologousexpression of ChlaOTU reduced ubiquitin accumulation at the entry sites, while a catalytic mutant of the deubiquitinase activity had the opposite effect. Altogether, we have identified a novel secreted protein of chlamydiae. ChlaOTU targets both ubiquitin and NDP52 and likely participates in the clearance of ubiquitin at the invasion sites.     

Effect of exogenous circulating anti-bPL antibodies on bovine placental lactogen measurements in foetal samples

Background  

The involvement of placental lactogen (PL) in the regulation of foetal growth has been investigated in different species by in vivo immunomodulation techniques. However, when circulating antibodies are present together with the hormone, the procedure for hormonal measurement becomes considerably complex. The aim of this study was the immunoneutralization of bovine placental lactogen (bPL) concentrations in bovine foetal circulation by direct infusion of rabbit anti-bPL purified immunoglobulins (IgG) via a foetal catheter (in vivo study). The ability of a RIA based on guinea pig anti-bPL antiserum, for the measurement of bPL concentrations in samples containing exogenous rabbit anti-bPL immunoglobulins, was also analyzed in in vitro and in vivo conditions.     

黔中地区马尾松林下杜鹃根部内生真菌群落组成及其生态功能

探索黔中马尾松(Pinus massoniana)林下杜鹃(Rhododendron simsii)根内生真菌物种多样性和生态功能。采集黔中乌当(WD)、孟关(MG)、龙里(LL) 3个地区马尾松林下杜鹃的发根,提取真菌DNA进行高通量测序。分析真菌Alpha多样性,借助FUNGuild注释平台解析真菌的生态功能类别,探索真菌群落中的核心微生物组,并结合网络图展示菌群之间的关联性。结果,3个地区的杜鹃根部内生真菌多样性非常丰富,WD地区杜鹃内生真菌多样性和丰富度最高。试验共获得有效序列425799条,817个可操作分类单元(OTU),分属于6门19纲52目103科154属,主要隶属于子囊菌门(81.2%)、接合菌门(5.8%)和担子菌门(4.7%);优势纲、目、科分别为:散囊菌纲(43.0%)、散囊菌目(39.2%)、发菌科(39.2%);在属的水平上,青霉属(38.60%)占比最高,其次是木霉属(7.20%)、拟盘多毛孢属(6.10%)。根部的真菌拥有多种生态功能群,如未定义腐生菌(194 OTU)、植物病原菌(20 OTU)、土壤腐生菌(18 OTU)、外生菌(14 OTU)、地衣共生真菌(10 OTU)、杜鹃类菌根真菌(5 OTU)、木腐生菌(5 OTU)、丛枝菌根(4 OTU)、内生菌(2 OTU)、动物病原菌(8 OTU),以及多种混合营养型类群21类,102个Undefined种类在FUNGuild数据库中没有参考信息。根部真菌可以形成生态位共享模式,而且不同功能群之间存在耦合性,核心基因组与关键物种以真菌组形成的生态功能团表现。     

Microbial fingerprinting detects unique bacterial communities in the faecal microbiota of rats with experimentally-induced colitis

An abnormal composition of the gut microbiota is believed to be associated with the pathogenesis of inflammatory bowel disease (IBD). We utilized terminal restriction fragment length polymorphism (T-RFLP) analysis to quantify faecal bacterial communities from rats with experimental colitis. Male Sprague Dawley rats (n=10/group) ingested 2% dextran sulfate sodium (DSS) or water for up to 7 days. Rats were killed and colonic tissues collected for histological analysis. Damage severity score in the distal colon was significantly greater (P<0.001) following DSS consumption compared to controls. T-RFLP faecal bacterial profiles generated with either MspI or CfoI revealed a significant difference (P<0.001) in community composition between healthy and colitic rats, with bacterial composition in healthy rats more variable than in rats with colitis. Operational taxonomic units (OTU: taxonomically related groups of bacteria) associated with either the healthy or colitic state were identified. OTU (116, 226, 360, and 948; CfoI) and (118 and 188; MspI) were strongly associated with untreated healthy rats, while OTU (94, 98, 174, and 384; CfoI) and (94 and 914; MspI) were predominantly associated with DSS-treated colitic rats. Phylogenetic OTU assignment suggested that Bacteroidales and Lactobacillus sp. were predominantly associated with the colitic and healthy rats, respectively. These results show that faecal bacterial profiling is a rapid, sensitive and non-invasive tool for detecting and identifying changes in gut microbiota associated with colitis. Restoring microbial homeostasis by targeting colitis-associated OTU through specific microbiological interventions could form the basis of novel therapeutic strategies for IBD.     

An In Vitro Procedure for Estimation of Lead Relative Bioavailability: With Validation

The relative bioaccessibility leaching procedure (RBALP) is a simple, reproducible, and rapid in vitro procedure for estimating the in vivo (juvenile swine) relative bioavailability (RBA) of lead in solid media. Control of pH, temperature, and agitation are the most critical parameters of this in vitro procedure. The performance of the method was evaluated by triplicate analyses of each of 19 different test substances by the author and three independent laboratories, and comparison of the results to relative bioavailability (RBA) values measured in vivo. The results indicate that the RBALP measurements are strongly correlated with the in vivo RBA values (r² = 0.924, p < 0.0001), with an average absolute error of 10% and an average predictive error of 20%. Comparison of results within and between laboratories indicates that the procedure is highly reproducible, with inter-and intra-laboratory coefficients of variation of 4% and 6%, respectively, and within-sample precision of approximately 7%. Based on the results reported here, the RBALP can be effective in providing reliable estimates of lead RBA as predicted by the immature swine model. The method may also be valuable in evaluating site-specific differences in bioaccessibility, assessing remedial technologies intended to reduce lead RBA, providing a screening mechanism for futurein vivo studies, and providing insight into the chemical and physical factors that control lead bioavailability.     

Effect of long-term nitrogen fertilization on mycorrhizal fungi associated with a dominant grass in a semiarid grassland

We studied the diversity of arbuscular mycorrhizal fungi (AMF) in semiarid grassland and the effect of long-term nitrogen (N) fertilization on this fungal community. Root samples of Bouteloua gracilis were collected at the Sevilleta National Wildlife Refuge (New Mexico, USA) from control and N-amended plots that have been fertilized since 1995. Small subunit rDNA was amplified using AMF specific primers NS31 and AM1. The diversity of AMF was low in comparison with other ecosystems, only seven operational taxonomic units (OTU) were found in B. gracilis and all belong to the genus Glomus. The dominant OTU was closely related to the ubiquitous G. intraradices/G. fasciculatum group. N-amended plots showed a reduction in the abundance of the dominant OTU and an increase in AMF diversity. The greater AMF diversity in roots from N-amended plots may have been the result of displacement of the dominant OTU, which facilitated detection of uncommon AMF. The long-term implications of AMF responses to N enrichment for plant carbon allocation and plant community structure remain unclear.     

Genetic variability,community structure,and horizontal transfer of endosymbionts among three Asia II‐Bemisia tabaci mitotypes in Pakistan

Endosymbionts associated with the whitefly Bemisia tabaci cryptic species are known to contribute to host fitness and environmental adaptation. The genetic diversity and population complexity were investigated for endosymbiont communities of B. tabaci occupying different micro‐environments in Pakistan. Mitotypes of B. tabaci were identified by comparative sequence analysis of the mitochondria cytochrome oxidase I (mtCOI) gene sequence. Whitefly mitotypes belonged to the Asia II‐1, ‐5, and ‐7 mitotypes of the Asia II major clade. The whitefly–endosymbiont communities were characterized based on 16S ribosomal RNA operational taxonomic unit (OTU) assignments, resulting in 43 OTUs. Most of the OTUs occurred in the Asia II‐1 and II‐7 mitotypes (r² = .9, p < .005), while the Asia II‐5 microbiome was less complex. The microbiome OTU groups were mitotype‐specific, clustering with a basis in phylogeographical distribution and the corresponding ecological niche of their whitefly host, suggesting mitotype‐microbiome co‐adaptation. The primary endosymbiont Portiera was represented by a single, highly homologous OTU (0%–0.67% divergence). Two of six Arsenophonus OTUs were uniquely associated with Asia II‐5 and ‐7, and one occurred exclusively in Asia II‐1, two only in Asia II‐5, and one in both Asia II‐1 and ‐7. Four other secondary endosymbionts, Cardinium, Hemipteriphilus, Rickettsia, and Wolbachia OTUs, were found at ≤29% frequencies. The most prevalent Arsenophonus OTU was found in all three Asia II mitotypes (55% frequency), whereas the same strain of Cardinium and Wolbachia was found in both Asia II‐1 and ‐5, and a single Hemipteriphilus OTU occurred in Asia II‐1 and ‐7. This pattern is indicative of horizontal transfer, suggestive of a proximity between mitotypes sufficient for gene flow at overlapping mitotype ecological niches.     

Exploring Symbiodinium diversity and host specificity in Acropora corals from geographical extremes of Western Australia with 454 amplicon pyrosequencing

Scleractinian corals have demonstrated the ability to shuffle their endosymbiotic dinoflagellate communities (genus Symbiodinium) during periods of acute environmental stress. This has been proposed as a mechanism of acclimation, which would be increased by a diverse and flexible association with Symbiodinium. Conventional molecular techniques used to evaluate Symbiodinium diversity are unable to identify genetic lineages present at background levels below 10%. Next generation sequencing (NGS) offers a solution to this problem and can resolve microorganism diversity at much finer scales. Here we apply NGS to evaluate Symbiodinium diversity and host specificity in Acropora corals from contrasting regions of Western Australia. The application of 454 pyrosequencing allowed for detection of Symbiodinium operational taxonomic units (OTUs) occurring at frequencies as low as 0.001%, offering a 10 000‐fold increase in sensitivity compared to traditional methods. All coral species from both regions were overwhelmingly dominated by a single clade C OTU (accounting for 98% of all recovered sequences). Only 8.5% of colonies associated with multiple clades (clades C and D, or C and G), suggesting a high level of symbiont specificity in Acropora assemblages in Western Australia. While only 40% of the OTUs were shared between regions, the dominance of a single OTU resulted in no significant difference in Symbiodinium community structure, demonstrating that the coral‐algal symbiosis can remain stable across more than 15° of latitude and a range of sea surface temperature profiles. This study validates the use of NGS platforms as tools for providing fine‐scale estimates of Symbiodinium diversity and can offer critical insight into the flexibility of the coral‐algal symbiosis.     

Improved Strategy for Comparing Microbial Assemblage Fingerprints

Microbial fingerprinting techniques permit the rapid visualization of entire assemblages in single assays, allowing direct comparison of communities in different samples, where the null hypothesis of such analyses is that all samples are the same. The comparison of fingerprints relies upon the precise estimation of all amplified DNA fragment lengths, which correspond to operational taxonomic units (OTU; analogous, but not equal to, a taxon in macroorganism studies). However, computer interpolation of size standards (and consequently OTU size calling) can be imprecise between gel runs, which can lead to imprecise calculation of similarity indices between multiple assemblages. To account for OTU size calling imprecision, all fragments within a range of sizes (a window) can be combined (i.e., “binned”) where the window is as wide as the imprecision of OTU size calling. However, artifacts may occur upon binning samples that may cause samples to appear less similar to each other, caused by splitting of OTU between adjacent bin windows. In this work we present an improved binning technique that accounts for OTU size calling imprecision in the comparison of multiple fingerprints. This technique comprises binning all pairwise comparisons in multiple bin window frames, where the starting size of the window (i.e., frame) is shifted by +1 bp for a total of x frames, where x bp is the width of the maximum bin window size in any binning scheme. Pairwise similarity indices between different community fingerprints are calculated for each of the x frames. To best address the null hypothesis of the community comparison, the maximum similarity value of all x frames is then used in downstream analyses to compare the communities. We believe this binning technique provides the most accurate and least biased comparison between different microbial fingerprints.     

Characterization of the cyanobacteria and associated bacterial community from an ephemeral wetland in New Zealand

New Zealand ephemeral wetlands are ecologically important, containing up to 12% of threatened native plant species and frequently exhibiting conspicuous cyanobacterial growth. In such environments, cyanobacteria and associated heterotrophs can influence primary production and nutrient cycling. Wetland communities, including bacteria, can be altered by increased nitrate and phosphate due to agricultural practices. We have characterized cyanobacteria from the Wairepo Kettleholes Conservation Area and their associated bacteria. Use of 16S rRNA amplicon sequencing identified several operational taxonomic units (OTUs) representing filamentous heterocystous and non‐heterocystous cyanobacterial taxa. One Nostoc OTU that formed macroscopic colonies dominated the cyanobacterial community. A diverse bacterial community was associated with the Nostoc colonies, including a core microbiome of 39 OTUs. Identity of the core microbiome associated with macroscopic Nostoc colonies was not changed by the addition of nutrients. One OTU was highly represented in all Nostoc colonies (27.6%–42.6% of reads) and phylogenetic analyses identified this OTU as belonging to the genus Sphingomonas. Scanning electron microscopy showed the absence of heterotrophic bacteria within the Nostoc colony but revealed a diverse community associated with the colonies on the external surface.     

Performance and microbial community composition dynamics of aerobic granular sludge from sequencing batch bubble column reactors operated at 20 °C, 30 °C, and 35 °C

Two bubble column sequencing batch reactors fed with an artificial wastewater were operated at 20 °C, 30 °C, and 35 °C. In a first stage, stable granules were obtained at 20 °C, whereas fluffy structures were observed at 30 °C. Molecular analysis revealed high abundance of the operational taxonomic unit 208 (OTU 208) affiliating with filamentous bacteria Leptothrix spp. at 30 °C, an OTU much less abundant at 20 °C. The granular sludge obtained at 20 °C was used for the second stage during which one reactor was maintained at 20 °C and the second operated at 30 °C and 35 °C after prior gradual increase of temperature. Aerobic granular sludge with similar physical properties developed in both reactors but it had different nutrient elimination performances and microbial communities. At 20 °C, acetate was consumed during anaerobic feeding, and biological phosphorous removal was observed when Rhodocyclaceae-affiliating OTU 214 was present. At 30 °C and 35 °C, acetate was mainly consumed during aeration and phosphorous removal was insignificant. OTU 214 was almost absent but the Gammaproteobacteria-affiliating OTU 239 was more abundant than at 20 °C. Aerobic granular sludge at all temperatures contained abundantly the OTUs 224 and 289 affiliating with Sphingomonadaceae indicating that this bacterial family played an important role in maintaining stable granular structures.     

Studies of antigen-RNA and immunity

Summary This review is about the involvement of antigen, and more specifically that of antigen-RNA complexes, in the inductive phase of an immune response. Early studies are reviewed that were begun by the senior author more than twenty years ago to probe the question of the role of antigen in antibody production in rabbits. It was shown that the quantity of antigen declined with time after injection but persisted at significant levels for very long intervals of time after injection, suggesting that all the antigen may never be lost. Since the liver yielded more detectable antigen than any other tissue it was the main source of in vivo antigen for biological and chemical characterization. As more extensive experiments were undertaken these showed stability of the radioactive hapten labels used to detect antigen and also the dynamic involvement of the liver in handling antigen. A body of evidence was thus accumulating that implied, at least indirectly, an immunological role for the liver-retained antigen. As for the antigen findings, one of the earliest was that antigen persisted, not in the original form of the injected protein, but as antigen fragments associated with ribonucleic acid, from whence originated the term antigen-RNA complexes. In later studies that utilized deproteinizing techniques, aimed at obtaining pure ribonucleic acid, antigen peptide was found to persist as a moiety of the isolated nucleic acid, thus providing convincing evidence for the in vivo origin of these complexes. The antigen-RNA complexes were immunologically active by both in vivo and in vitro tests. Recent experiments, using rats injected with aniline-azo-BSA have extended the earlier findings and it is these studies upon which attention will be focused in the experimental section. Liver polysomes and isolates of pure hepatocytes have been found to contain antigen material associated with RNA, and studies are in progress using these sources of material for elucidation of antigen-RNA function. It seems timely to suggest that an equally important application may be the use of these products of in vivo metabolism as probes to study the many inductive phenomena in an immune response.Abbreviations Ag antigen - BGG bovine gamma globulin - BSA bovine serum albumin - cFA complete Freund's adjuvant - ³H-BGG ³H-aniline-azo-BGG - ³H-BSA ³H-aniline-azo-BSA - ³H-RSA ³H-aniline-azo-rat serum albumin - ip intraperitoneal - iv intravenous - KLH keyhole limpet hemocyanin - np nucleopeptide - RSA rat serum albumin - [³⁵S]-BSA [³⁵S]-sulfanilate-azo-bovine serum albumin - S-BSA sulfanilate-azo-bovine serum albumin     

THE BINDING OF BOTULINUM TOXIN TO MEMBRANE LIPIDS: PHOSPHOLIPIDS AND PROTEOLIPID

A number of phospholipids known to be constituents of nerve endings were tested for their ability to inactivate botulinum toxin. Substances tested included phosphatidylcholine, phosphatidalcholine, phosphatidylethanolamine, phosphatidalethanolamine, β-acyl lysolecithin, sphingomyelin, phosphatidylserine, phosphatidic acid, phosphatidylinositol and cardiolipin. Proteolipid from bovine white matter was also tested. Neutral phospholipids potentiated the toxicity in vivo of botulinum toxin, but they had no effect on the toxicity in vitro. Some, but not all, acidic phospholipids caused loss of toxicity of botulinum toxin in solutions at low pH both in vivo and in vitro. However, none of these substances when incubated with toxin under physiological conditions of temperature, pH and ionic strength, caused loss of toxin potency. The data suggest that none of these phospholipids is likely to be a toxin receptor.     

A single Thaumarchaeon drives nitrification in deep oligotrophic Lake Constance

Ammonia released during organic matter mineralization is converted during nitrification to nitrate. We followed spatiotemporal dynamics of the nitrifying microbial community in deep oligotrophic Lake Constance. Depth-dependent decrease of total ammonium (0.01–0.84 μM) indicated the hypolimnion as the major place of nitrification with ¹⁵N-isotope dilution measurements indicating a threefold daily turnover of hypolimnetic total ammonium. This was mirrored by a strong increase of ammonia-oxidizing Thaumarchaeota towards the hypolimnion (13%–21% of bacterioplankton) throughout spring to autumn as revealed by amplicon sequencing and quantitative polymerase chain reaction. Ammonia-oxidizing bacteria were typically two orders of magnitude less abundant and completely ammonia-oxidizing (comammox) bacteria were not detected. Both, 16S rRNA gene and amoA (encoding ammonia monooxygenase subunit B) analyses identified only one major species-level operational taxonomic unit (OTU) of Thaumarchaeota (99% of all ammonia oxidizers in the hypolimnion), which was affiliated to Nitrosopumilus spp. The relative abundance distribution of the single Thaumarchaeon strongly correlated to an equally abundant Chloroflexi clade CL500-11 OTU and a Nitrospira OTU that was one order of magnitude less abundant. The latter dominated among recognized nitrite oxidizers. This extremely low diversity of nitrifiers shows how vulnerable the ecosystem process of nitrification may be in Lake Constance as Central Europe's third largest lake.     

Improved protein sequence coverage by on resin deglycosylation and cysteine modification for biomarker discovery

Membrane proteins and secreted factors (soluble proteins or extracellular matrix components) are the targets of most monoclonal antibodies, which are currently in clinical development. These proteins are frequently post‐translationally modified, e.g. by the formation of disulfide bonds or by glycosylation, which complicates their identification using proteomics technologies. Here, we describe a novel methodology for the on resin deglycosylation and cysteine modification of proteins after in vitro, in vivo or ex vivo biotinylation. Biotinylated proteins are captured on streptavidin resin and all subsequent modifications, as well as the proteolytic digestion, which yields peptides for MS analysis, are performed on resin. Using biotinylated bovine fetuin‐A as a test protein, an improvement in sequence coverage from 7.9 to 58.7% could be shown, including the identification of all three glycosylation sites. Furthermore, a complex mixture derived from the ex vivo biotinylation of vascular structures in human kidney with cancer obtained by perfusion after surgical resection revealed almost a doubling of sequence coverage for all checked proteins when analyzed by LC‐MALDI TOF/TOF.     

Peptide aptamers as new tools to modulate clathrin-mediated internalisation — inhibition of MT1-MMP internalisation

Background  

Peptide aptamers are combinatorial protein reagents that bind to targets with a high specificity and a strong affinity thus providing a molecular tool kit for modulating the function of their targets in vivo.     

Distribution of Arsenite-Oxidizing Bacteria and its Correlation with Temperature in Hot Springs of the Tibetan-Yunnan Geothermal Zone in Western China

The distribution of arsenite-oxidizing bacteria in response to temperature is of great importance to an understanding of biogeochemical cycling of arsenic in geothermal features. The abundance and diversity of arsenite-oxidizing bacteria were investigated in the geothermal features of Tengchong County of Yunnan Province, Dachaidan County of Qinghai Province, and Tibet. The abundance of aioA genes, which encode the large subunit of arsenite oxidase, was determined by quantitative polymerase chain reactions. The diversity of aioA genes was studied by PCR-cloning-based phylogenetic analyses. The results showed that the aioA gene abundance increased as temperature decreased, whereas its diversity at the OTU level (97% cutoff) increased with increasing temperature. This suggests that temperature played an important role in affecting aioA gene distribution and thus arsenic speciation. The aioA gene population (at OTU level) differed among the studied regions, indicating geographic isolation may be an important factor controlling aioA gene distribution in hot springs.     

Structural Basis for Ovarian Tumor Domain-containing Protein 1 (OTU1) Binding to p97/Valosin-containing Protein (VCP)

Valosin-containing protein (VCP), also known as p97, is an AAA⁺ ATPase that plays an essential role in a broad array of cellular processes including the endoplasmic reticulum-associated degradation (ERAD) pathway. Recently, ERAD-specific deubiquitinating enzymes have been reported to be physically associated with VCP, although the exact mechanism is not yet clear. Among these enzymes is ovarian tumor domain-containing protein 1 (OTU1). Here, we report the structural basis for interaction between VCP and OTU1. The crystal structure of the ubiquitin regulatory X-like (UBXL) domain of OTU1 (UBXL_OTU1) complexed to the N-terminal domain of VCP (N_VCP) at 1.8-Å resolution reveals that UBXL_OTU1 adopts a ubiquitin-like fold and binds at the interface of two subdomains of N_VCP using the ³⁹GYPP⁴² loop of UBXL_OTU1 with the two prolines in cis- and trans-configurations, respectively. A mutagenesis study shows that this loop is not only critical for the interaction with VCP but also for its role in the ERAD pathway. Negative staining EM shows that one molecule of OTU1 binds to one VCP hexamer, and isothermal titration calorimetry suggests that the two proteins bind with a K_D of 0.71 μm. Analytical size exclusion chromatography and isothermal titration calorimetry demonstrates that OTU1 can bind VCP in both the presence and absence of a heterodimer formed by ubiquitin fusion degradation protein 1 and nuclear localization protein 4.     

In Vitro Binding of Trypanosoma congolense to Erythrocytes*

Synopsis. Trypanosoma congolense Broden, an intravascular parasite, binds to vessel walls and erythrocytes of infected hosts. In an attempt to characterize T. congolense adhesion to host cells, an in vitro assay was devised. It was shown in the in vitro experiments that T. congolense binds to bovine, sheep, and goat erythrocytes, but not always to erythrocytes of rats, mice, rabbits, horses or humans. Only the anterior part of live trypanosomes adheres to erythrocytes, and the attachment site on the trypanosomes is destroyed by trypsin and chymotrypsin. Trypanosomes did not adhere to bovine erythrocytes that had been incubated with neuraminidase, sodium periodate and poly-L-lysine. The foregoing experiments suggest that the surface of T. congolense contains a protein-associated site which binds to sialic acid of some host cells. This surface site is most likely responsible for attachment to blood vessels in vivo.