The Immobilization Antigens of Tetrahymena thermophila Are Glycoproteins

ABSTRACT The four immobilization antigens controlled by the SerH locus in Tetrahymena thermophila have been isolated and partially characterized (Doerder, F. P. & Berkowitz, M. S. 1986. Purification and partial characterization of the H immobilization antigens of Tetrahymena thermophila. J. Protozool. , 33 :204–208). We show here, using immunoprecipitation and electrophoresis after labeling with ³⁵S-methionine, ¹⁴C-mannose, ¹⁴C-glucosamine, and N-Acetyl- d -[1-³H]glucosamine, that these proteins are glycosylated. We suggest the immobilization antigens in Tetrahymena may be anchored to the surface membrane by phosphatidylinositol glycans.     

Glycoproteins That Exhibit Extensive Size Polymorphisms in Dictyostelium Discoideum

Electrophoretic variants which arise from amino acid substitutions, leading to charge differences between proteins are ubiquitous and have been used extensively for genetic analysis. Less well documented are polymorphisms in the size of proteins. Here we report that a group of glycoproteins, which share a common carbohydrate epitope, vary in size in different isolates of the cellular slime mould, Dictyostelium discoideum. One of these proteins, PsA, a developmentally regulated prespore-specific surface glycoprotein, has previously been shown to exist in three size forms due to allelic variation at the pspA locus on linkage group I. In this report, a second glycoprotein, PsB, which is also prespore specific but found inside prespore cells, is studied. PsB maps to linkage group II and exhibits at least four different sizes in the isolates examined. We propose that the size polymorphisms are the product of allelic variation at the pspB locus, due to differences in the number of repeat units.     

Imputing Amino Acid Polymorphisms in Human Leukocyte Antigens

DNA sequence variation within human leukocyte antigen (HLA) genes mediate susceptibility to a wide range of human diseases. The complex genetic structure of the major histocompatibility complex (MHC) makes it difficult, however, to collect genotyping data in large cohorts. Long-range linkage disequilibrium between HLA loci and SNP markers across the major histocompatibility complex (MHC) region offers an alternative approach through imputation to interrogate HLA variation in existing GWAS data sets. Here we describe a computational strategy, SNP2HLA, to impute classical alleles and amino acid polymorphisms at class I (HLA-A, -B, -C) and class II (-DPA1, -DPB1, -DQA1, -DQB1, and -DRB1) loci. To characterize performance of SNP2HLA, we constructed two European ancestry reference panels, one based on data collected in HapMap-CEPH pedigrees (90 individuals) and another based on data collected by the Type 1 Diabetes Genetics Consortium (T1DGC, 5,225 individuals). We imputed HLA alleles in an independent data set from the British 1958 Birth Cohort (N = 918) with gold standard four-digit HLA types and SNPs genotyped using the Affymetrix GeneChip 500 K and Illumina Immunochip microarrays. We demonstrate that the sample size of the reference panel, rather than SNP density of the genotyping platform, is critical to achieve high imputation accuracy. Using the larger T1DGC reference panel, the average accuracy at four-digit resolution is 94.7% using the low-density Affymetrix GeneChip 500 K, and 96.7% using the high-density Illumina Immunochip. For amino acid polymorphisms within HLA genes, we achieve 98.6% and 99.3% accuracy using the Affymetrix GeneChip 500 K and Illumina Immunochip, respectively. Finally, we demonstrate how imputation and association testing at amino acid resolution can facilitate fine-mapping of primary MHC association signals, giving a specific example from type 1 diabetes.     

Chimeric Lyssavirus Glycoproteins with Increased Immunological Potential

The rabies virus glycoprotein molecule (G) can be divided into two parts separated by a flexible hinge: the NH₂ half (site II part) containing antigenic site II up to the linear region (amino acids [aa] 253 to 275 encompassing epitope VI [aa 264]) and the COOH half (site III part) containing antigenic site III and the transmembrane and cytoplasmic domains. The structural and immunological roles of each part were investigated by cell transfection and mouse DNA-based immunization with homogeneous and chimeric G genes formed by fusion of the site II part of one genotype (GT) with the site III part of the same or another GT. Various site II-site III combinations between G genes of PV (Pasteur virus strain) rabies (GT1), Mokola (GT3), and EBL1 (European bat lyssavirus 1 [GT5]) viruses were tested. Plasmids pGPV-PV, pGMok-Mok, pGMok-PV, and pGEBL1-PV induced transient expression of correctly transported and folded antigens in neuroblastoma cells and virus-neutralizing antibodies against parental viruses in mice, whereas, pG-PVIII (site III part only) and pGPV-Mok did not. The site III part of PV (GT1) was a strong inducer of T helper cells and was very effective at presenting the site II part of various GTs. Both parts are required for correct folding and transport of chimeric G proteins which have a strong potential value for immunological studies and development of multivalent vaccines. Chimeric plasmid pGEBL1-PV broadens the spectrum of protection against European lyssavirus genotypes (GT1, GT5, and GT6).     

Platelet Glycoproteins and Fibrinogen in Recovery from Idiopathic Sudden Hearing Loss

Background

The pathomechanism and location of idiopathic sudden sensorineural hearing loss (ISSHL) is unclear. In a previous case-control study, we found elevated fibrinogen concentrations and a higher prevalence of T allele carriers of the glycoprotein (Gp) Ia C807T polymorphism in ISSHL patients.

Methodology

127 patients with ISSHL (mean age 53.3 years, 48.8% females), who underwent a standard therapy with high dose steroids, pentoxifyllin and sterofundine over 8 days were included. We examined the influence of GpIa genotype and fibrinogen (BclI-, A312-, HaeIII-) genotype and fibrinogen plasma levels on hearing recovery after 8 weeks (change from baseline: 0 dB  =  no recovery, >0 to 10 dB = moderate recovery, >10 dB = good recovery). In a subsample of 59 patients with ISSHL, we further studied the association of platelet glycoprotein GpIa, Ib and IIIa densities on hearing recovery as well as the possible effect-modification of platelet glycoproteins on hearing recovery by plasma fibrinogen.

Results

In univariate analysis, neither the GpIa genotype nor fibrinogen genotype (all p>0.1) but lower fibrinogen levels (p = 0.029), less vertigo (p = 0.002) and lower GpIIIa receptor density (p = 0.037, n = 59) were associated with hearing recovery. In multivariate analysis, fibrinogen significantly modified the effect of GPIa receptor density on good hearing recovery (effect-modification on multiplicative scale OR = 0.45 (95% confidence interval (0.21–0.94)), p = 0.03). GPIb receptor density below the mean was associated with a 2-fold increase in good hearing recovery both in patients with fibrinogen levels above (p = 0.04) as well as in patients with fibrinogen levels below the mean (p = 0.06). There was no indication for an effect-modification (p = 0.97).

Conclusions

The findings suggest a vascular/rheological origin of ISSHL with unique features of thrombosis in the inner ear artery that may include complex interrelationships among platelet glycoproteins and plasma fibrinogen.     

Ecotilling,一种检测基因多样性的新方法

定向诱导基因组局部突变(TILLING:TargetingInducedLocalLesionsInGenomes)检测技术是将随机化学诱变与PCR方法结合,对目的基因区域进行鉴定筛选的一种低投入、高通量的反向遗传学方法。“Ecotilling”是利用该技术检测自然群体中存在的基因多样性的新方法。目前,TILLING及Ecotilling已被应用于多个物种的基因多样性的研究。本文系统介绍了TILLING及Ecotilling的定义、技术流程与特点、应用概况、常用工具,并展望了该技术的应用前景。     

Immunoproteomic to Identify Antigens in the Intestinal Mucosa of Crohn's Disease Patients

Incidences of Crohn disease (CD) have increased significantly in the last decade. Immunoproteomics are a promising method to identify biomarkers of different diseases. In the present study, we used immunoproteomics to study proteins of intestinal mucosal lesions and neighboring normal intestinal mucosa of 8 CD patients. Reactive proteins were validated by Western blotting. Approximately 50 protein spots localized in the 4 to 7 pI range were detected on two-dimensional electrophoresis gels, and 6 differentially expressed protein spots between 10 and 100 kDa were identified. Reactive proteins were identified as prohibitin, calreticulin, apolipoprotein A-I, intelectin-1, protein disulfide isomerase, and glutathione s-transferase Pi. Western blotting was conducted on the intestinal mucosa of another 4 CD patients to validate the reactive proteins. We found that intestinal mucosal lesions had high levels of prohibitin expression. Glutathione s-transferase expression was detected in 100% of the intestinal mucosa examined. Thus, we report 6 autoantigens of CD, including 3 new and 3 previously reported autoantigens. Intelectin-1, protein disulfide isomerase, and glutathione-s-transferases may be used as biomarkers for CD pathogenesis.     

T Antigens of Bovine and Canine Adenoviruses Demonstrated by Immunofluorescence

The intracellular development in acutely infected cells of bovine and canine adenovirus T antigens was followed by immunofluorescent staining. With both species of adenovirus, antigen was first detected as intranuclear pin-points at 18 hr postinfection and coalescence into large lobular masses was noticed by 24 hr. Cross-reactions between bovine 1 (nononcogenic) and bovine 3 (oncogenic) T antigens were not observed by the direct technique although the more sensitive indirect procedure did reveal cross-reactivity. Extensive cross-reactions were observed between the T antigens of the oncogenic canine hepatitis virus and the "nononcogenic" Toronto strain of canine adenovirus. The magnitude of these reactions places the two canine strains in the same T antigen subgroup. The canine and bovine T antigens were not stained by tumor antisera against any of the known human or simian T antigen subgroups. Antigen synthesis was not prevented by inhibitors of deoxyribonucleic acid synthesis although the appearance was altered markedly.     

Case-Control Approach to Identify Plasmodium falciparum Polymorphisms Associated with Severe Malaria

Background

Studies to identify phenotypically-associated polymorphisms in the Plasmodium falciparum 23 Mb genome will require a dense array of marker loci. It was considered promising to undertake initial allelic association studies to prospect for virulence polymorphisms in Thailand, as the low endemicity would allow higher levels of linkage disequilibrium (LD) than would exist in more highly endemic areas.

Methodology/Principal Findings

Assessment of LD was first made with 11 microsatellite loci widely dispersed in the parasite genome, and 16 microsatellite loci covering a ∼140 kb region of chromosome 2 (an arbitrarily representative non-telomeric part of the genome), in a sample of 100 P. falciparum isolates. The dispersed loci showed minimal LD (Index of Association, I^S_A = 0.013, P = 0.10), while those on chromosome 2 showed significant LD values mostly between loci <5 kb apart. A disease association study was then performed comparing parasites in 113 severe malaria cases and 245 mild malaria controls. Genotyping was performed on almost all polymorphisms in the binding domains of three erythrocyte binding antigens (eba175, eba140 and eba181), and repeat sequence polymorphisms ∼2 kb apart in each of three reticulocyte binding homologues (Rh1, Rh2a/b, and Rh4). Differences between cases and controls were seen for (i) codons 388-90 in eba175, and (ii) a repeat sequence centred on Rh1 codon 667.

Conclusions/Significance

Allelic association studies on P. falciparum require dense genotypic markers, even in a population of only moderate endemicity that has more extensive LD than highly endemic populations. Disease-associated polymorphisms in the eba175 and Rh1 genes encode differences in the middle of previously characterised erythrocyte binding domains, marking these for further investigation.     

The Potential Biomarkers to Identify the Development of Steatosis in Hyperuricemia

Hyperuricemia (HU) often progresses to combine with non-alcoholic fatty liver disease (NAFLD) in the clinical scenario, which further exacerbates metabolic disorders; early detection of biomarkers, if obtained during the HU progression, may be beneficial for preventing its combination with NAFLD. This study aimed to decipher the biomarkers and mechanisms of the development of steatosis in HU. Four groups of subjects undergoing health screening, including healthy subjects, subjects with HU, subjects with HU combined with NAFLD (HU+NAFLD) and subjects with HU initially and then with HU+NAFLD one year later (HU→HU+NAFLD), were recruited in this study. The metabolic profiles of all subjects’ serum were analyzed by liquid chromatography quadruple time-of-flight mass spectrometry. The metabolomic data from subjects with HU and HU+NAFLD were compared, and the biomarkers for the progression from HU to HU+NAFLD were predicted. The metabolomic data from HU→HU+NAFLD subjects were collected for further verification. The results showed that the progression was associated with disturbances of phospholipase metabolism, purine nucleotide degradation and Liver X receptor/retinoic X receptor activation as characterized by up-regulated phosphatidic acid, cholesterol ester (18:0) and down-regulated inosine. These metabolic alterations may be at least partially responsible for the development of steatosis in HU. This study provides a new paradigm for better understanding and further prevention of disease progression.     

A Mutant Library Approach to Identify Improved Meningococcal Factor H Binding Protein Vaccine Antigens

Factor H binding protein (FHbp) is a virulence factor used by meningococci to evade the host complement system. FHbp elicits bactericidal antibodies in humans and is part of two recently licensed vaccines. Using human complement Factor H (FH) transgenic mice, we previously showed that binding of FH decreased the protective antibody responses to FHbp vaccination. Therefore, in the present study we devised a library-based method to identify mutant FHbp antigens with very low binding of FH. Using an FHbp sequence variant in one of the two licensed vaccines, we displayed an error-prone PCR mutant FHbp library on the surface of Escherichia coli. We used fluorescence-activated cell sorting to isolate FHbp mutants with very low binding of human FH and preserved binding of control anti-FHbp monoclonal antibodies. We sequenced the gene encoding FHbp from selected clones and introduced the mutations into a soluble FHbp construct. Using this approach, we identified several new mutant FHbp vaccine antigens that had very low binding of FH as measured by ELISA and surface plasmon resonance. The new mutant FHbp antigens elicited protective antibody responses in human FH transgenic mice that were up to 20-fold higher than those elicited by the wild-type FHbp antigen. This approach offers the potential to discover mutant antigens that might not be predictable even with protein structural information and potentially can be applied to other microbial vaccine antigens that bind host proteins.     

Glycoproteins in prokaryotes

Rather recently it has become clear that prokaryotes (Archaea and Bacteria) are able to glycosylate proteins. A literature survey revealed the different types of glycoproteins. They include mainly surface layer (S-layer) proteins, flagellins, and polysaccharide-degrading enzymes. Only in a few cases is structural information available. Many different structures have been observed that display much more variation than that observed in eukaryotes. A few studies have given evidence for the function of the prokaryotic glycoprotein glycans. Also from the biosynthetic point of view, information is rather scarce. Due to their different cell structure, prokaryotes have to use mechanisms different from those found in eukaryotes to glycosylate proteins. However, from the fragmented data available for the prokaryotic glycoproteins, similarities with the eukaryotic system can be noticed. Received: 24 February 1997 / Accepted: 13 May 1997     

Glycosphingolipids as Potential Diagnostic Markers and/or Antigens in Neurological Disorders

Glycosphingolipids are most abundant in the nervous system within which there are developmental, regional, structural and cellular differences regarding their composition. They are shedded to the cerebrospinal fluid and thus potential markers for pathogenic alterations in the brain, such as developmental abnormalities, demyelination, gliosis, neuronal cell destruction. The glycosphingolipids have also been found to be antigens in autoimmune processes involving the nervous system, in particular in peripheral neuropathies like Guillain Barré syndrome, multifocal motor neuropathy etc. The immune response might have been triggered by infectious agents with an antigen epitope which mimic the glycosphingolipid or by a primary nerve tissue damage leading to release of glycosphingolipids. There is a series of support for a clinical significance of cerebrospinal fluid glycosphingolipid determinations and the presence of anti-glycosphingolipid antibodies but this has to be further explored. This paper is a mini review of the state of the art and discuss methodological aspects and improvements that might help to explore the relevance of glycosphingolipids in neurological disorders.     

Glycoproteins Released by Leishmania donovani: Immunologic Relationships with Host and Bacterial Antigens and Preliminary Biochemical Analysis*

SYNOPSIS. The antigenically active glycoproteins (AAGP) released by Leishmania donovani strain 3S promastigotes into growth media and by amastigotes of this strain into the tissues, e.g. blood, of infected hamsters was found to consist of 6 to 7 antigenically distinct components. The antigenic activity of these glycoproteins was resistant to freeze-thawing, protease treatment, and purification by column chromatography using Sephadex G-100. This activity, however, was destroyed by Na periodate and altered by boiling; AAGP adhered firmly to Amicon filter (UM2). The antigenically active substances absorbed UV at 230, 260, 280 nm and gave positive Folin phenol, phenol sulfuric acid, and orcinol reactions. By gel diffusion, the component glycoproteins were found to form lines with concanavalin A and to give reactions of identity and partial identity with human red cells and Mycobacterium butyricum. The possible involvement of the antigenically active glycoproteins in pathogensis of kala azar is discussed.     

Panicle Water Potential, a Physiological Trait to Identify Drought Tolerance in Rice

Two upland rice varieties (IRAT109, IAPAR9) and one lowland rice variety (Zhenshan 97B) were planted in summer and treated with both normal (full water) and drought stress in the reproductive stage. Panicle water potential (PWP) and leaf water potential (LWP) were measured every 1.0-1.5 h over 24 h on sunny days. Both PWP and LWP of upland varieties started to decrease later, maintained a higher level and recovered more quickly than that of the lowland variety. The results show that PWP can be used as an indicator of plant water status based on the parallel daily changes, and the high correlation between PWP and LWP. Similar correlations were also observed between PWP, LWP and eight traits related to plant growth and grain yield formation. PWP seemed to be more effective for distinguishing the upland rice varieties with different drought-tolerant ability. Differences in PWP and LWP between upland and lowland rice varieties were also observed at noon even under normal water conditions, implying the incorporation of the drought-tolerant mechanism to improve the photosynthesis and yield of traditional paddy rice.     

利用离体叶片鉴定杨树耐盐潜力

以受体杨树107和转基因杨树18-1的一年生枝条为材料,采用Hoagland营养液水培方法,添加不同浓度的NaCl,检测二者植株生长以及叶中Na＋和K＋含量的变化。结果表明,在0.6%NaCl处理下18-1生根率明显高于107,生物量较大,叶片中Na＋积累量是107的1.45倍左右。以107和18-1离体叶片为材料,NaCl处理下测定其生理活性指标,发现18-1叶盘的失绿速度和相对电导率都显著低于107叶盘。把离体叶片接种在改良MS培养基上,1.2%NaCl处理可使107叶盘几乎停止伸长,细胞大小不再增加;而18-1叶盘培养7天后伸长了近50%。以上结果表明利用离体叶片可以鉴定出不同基因型杨树的耐盐潜力。     

Omics Approaches to Identify Potential Biomarkers of Inflammatory Diseases in the Focal Adhesion Complex

Inflammatory diseases such as inflammatory bowel disease(IBD) require recurrent invasive tests, including blood tests, radiology, and endoscopic evaluation both to diagnose and assess disease activity, and to determine optimal therapeutic strategies. Simple ‘bedside' biomarkers could be used in all phases of patient management to avoid unnecessary investigation and guide further management. The focal adhesion complex(FAC) has been implicated in the pathogenesis of multiple inflammatory diseases, including IBD, rheumatoid arthritis, and multiple sclerosis. Utilizing omics technologies has proven to be an efficient approach to identify biomarkers from within the FAC in the field of cancer medicine. Predictive biomarkers are paving the way for the success of precision medicine for cancer patients, but inflammatory diseases have lagged behind in this respect. This review explores the current status of biomarker prediction for inflammatory diseases from within the FAC using omics technologies and highlights the benefits of future potential biomarker identification approaches.     

TaqMan Real-Time PCR Assays for Single-Nucleotide Polymorphisms Which Identify Francisella tularensis and Its Subspecies and Subpopulations

Francisella tularensis, the etiologic agent of tularemia and a Class A Select Agent, is divided into three subspecies and multiple subpopulations that differ in virulence and geographic distribution. Given these differences, there is a need to rapidly and accurately determine if a strain is F. tularensis and, if it is, assign it to subspecies and subpopulation. We designed TaqMan real-time PCR genotyping assays using eleven single nucleotide polymorphisms (SNPs) that were potentially specific to closely related groups within the genus Francisella, including numerous subpopulations within F. tularensis species. We performed extensive validation studies to test the specificity of these SNPs to particular populations by screening the assays across a set of 565 genetically and geographically diverse F. tularensis isolates and an additional 21 genetic near-neighbor (outgroup) isolates. All eleven assays correctly determined the genetic groups of all 565 F. tularensis isolates. One assay differentiates F. tularensis, F. novicida, and F. hispaniensis from the more genetically distant F. philomiragia and Francisella-like endosymbionts. Another assay differentiates F. tularensis isolates from near neighbors. The remaining nine assays classify F. tularensis-confirmed isolates into F. tularensis subspecies and subpopulations. The genotyping accuracy of these nine assays diminished when tested on outgroup isolates (i.e. non F. tularensis), therefore a hierarchical approach of assay usage is recommended wherein the F. tularensis-specific assay is used before the nine downstream assays. Among F. tularensis isolates, all eleven assays were highly sensitive, consistently amplifying very low concentrations of DNA. Altogether, these eleven TaqMan real-time PCR assays represent a highly accurate, rapid, and sensitive means of identifying the species, subspecies, and subpopulation of any F. tularensis isolate if used in a step-wise hierarchical scheme. These assays would be very useful in clinical, epidemiological, and/or forensic investigations involving F. tularensis.