Harmless Protein Research Articles

Impact of different classification schemes on discrimination of proteins with noise-contaminated spectra using laboratory-measured fluorescence data

Biological agents are important to detect and identify with respect to environmental contamination and public health. Noise contamination in fluorescent spectra is one of the contributors to the uncertainties of identification. In order to investigate the noise-tolerant capability provided by laboratory-measured excitation-emission matrix (EEM) fluorescence spectra that are used as a database, fluorescence properties of four proteinaceous biotoxin samples and ten harmless protein samples were characterized by EEM fluorescence spectra, and the predicting performance of models trained by laboratory-measured fluorescence data was tested and verified from validation data with noise-contaminated spectra. By means of peak signal of noise (PSNR) as an indicator of noise levels, the potential impact of noise contaminations on the characterization and discrimination of these samples was evaluated quantitatively. Different classification schemes utilizing multivariate analysis techniques of Principal Component Analysis (PCA), Random Forest (RF), and Multi-layer Perceptron (MPL) coupled with feature descriptors of differential transform (DT), Fourier transform (FT) and wavelet transform (WT) were conducted under different PSNR values. We systematically analyzed the performance of classification schemes by the case study at 20 PSNR and by statistical analysis from 1-100 PSNR. The results show that the spectral features with EEM-WT decreased the demanding number of input variables while retaining high performances in sample classification. The spectral features with EEM-FT presented the worst performance although having the largest number of features. The distributions of feature importance and contribution were found sensitive to noise contaminations. The classification scheme of PCA prior to MPL with EEM-WT as input presented an improvement in lower PSNR. These results indicate that robust features extracted by corresponding techniques are critical to enhancing the spectral differentiation capabilities among these samples and play an important role in eliminating the noise effect. The study of classification schemes for discriminating protein samples with noise-contaminated spectra presents tremendous potential for future developments in the rapid detection and identification of proteinaceous biotoxins based on three-dimensional fluorescence spectrometry.

Toxic Prion: Translation of a Genome's gene with a toxic change

Toxic prion is an abnormal form of a normally harmless protein found in the brain that is responsible for a variety of fatal neurodegenerative diseases of animals including humans. These neurodegenerative diseases are called transmissible spongiform encephalopathies. The key objective of this study is to give a spectacular proof of Nature’s Controlled Experiment for the fact that Genome’s designed/coded directives are effected by its transcripts [7] & proteins serving as implementing engineers in synthesizing its individuals in each species of all genomic-things whereas its usable chemical energy-containing nutritive substances including minerals are used as raw materials in its aerobic or anaerobic compatible environment. The micrographs, Figures, videos were designed be the targets of the study and impartive results in aimful approach of the investigator; and, that was why it had been entitled as Methodology & Results. Genome is not only the true automatic molecular machine capable of synthesizing genomic-things, it is also a supersensitive molecule where symptoms, syndromes, epigenetic modifications, immunological defensive reactions, and the capability of toxic prion proteins (PrP 27-30) to induce conversion of normal prion proteins (PrP 33-35) into toxic prion proteins to cause fatal neurodegenerative diseases are evidences for the fact that the Genome is an unbelievably supersensitive automatic molecular machine!!!! Epigenetics is actually the study of supersensitivity of the Genome by attaching epigenetic groups or chemicals to Genome that are not structural parts of the Genome. When toxicity of prion is caused by change, modification, or mutation in one’s own Genome, it is called autotoxic prion and when it is transmitted from other individuals, it is referred to as acquired toxic prion.

Widely Targeted Lipidomics and Transcriptomics Analysis Revealed Changes of Lipid Metabolism in Spleen Dendritic Cells in Shrimp Allergy.

Shrimp allergy (SA) is pathological type 2 inflammatory immune responses against harmless shrimp protein allergen, which is caused by complex interactions between dendritic cells (DCs) and other immune cells. Lipid metabolism in different DCs states are significantly changed. However, the lipid metabolism of spleen DCs in SA remain ambiguous. In this study, we established a BALB/c mouse shrimp protein extract-induced allergy model to determine the lipid profile of spleen DCs in SA, and the molecular mechanism between lipid metabolism and immune inflammation was preliminarily studied. Spleen DCs were sorted by fluorescence-activated cell sorting, and then widely targeted lipidomics and transcriptomics analysis were performed. Principal component analysis presented the lipidome alterations in SA. The transcriptomic data showed that Prkcg was involved in lipid metabolism, immune system, and inflammatory signaling pathway. In the correlation analysis, the results suggested that Prkcg was positively correlated with triacylglycerol (Pearson correlation coefficient = 0.917, p = 0.01). The lipidomics and transcriptomics integrated pathway analysis indicated the activated metabolic conversion from triacylglycerol to 1,2-diacyl-sn-glycerol and the transmission of lipid metabolism to immune inflammation (from triacylglycerol and ceramide to Prkcg) in SA spleen DCs, and cellular experiments in vitro showed that glyceryl trioleate and C16 ceramide treatment induced immune function alteration in DCs.

Massive expression of cysteine-containing proteins causes abnormal elongation of yeast cells by perturbing the proteasome.

The enhanced green fluorescent protein (EGFP) is considered to be a harmless protein because the critical expression level that causes growth defects is higher than that of other proteins. Here, we found that overexpression of EGFP, but not a glycolytic protein Gpm1, triggered the cell elongation phenotype in the budding yeast Saccharomyces cerevisiae. By the morphological analysis of the cell overexpressing fluorescent protein and glycolytic enzyme variants, we revealed that cysteine content was associated with the cell elongation phenotype. The abnormal cell morphology triggered by overexpression of EGFP was also observed in the fission yeast Schizosaccharomyces pombe. Overexpression of cysteine-containing protein was toxic, especially at high-temperature, while the toxicity could be modulated by additional protein characteristics. Investigation of protein aggregate formation, morphological abnormalities in mutants, and transcriptomic changes that occur upon overexpression of EGFP variants suggested that perturbation of the proteasome by the exposed cysteine of the overexpressed protein causes cell elongation. Overexpression of proteins with relatively low folding properties, such as EGFP, was also found to promote the formation of SHOTA (Seventy kDa Heat shock protein-containing, Overexpression-Triggered Aggregates), an intracellular aggregate that incorporates Hsp70/Ssa1, which induces a heat shock response, while it was unrelated to cell elongation. Evolutionary analysis of duplicated genes showed that cysteine toxicity may be an evolutionary bias to exclude cysteine from highly expressed proteins. The overexpression of cysteine-less moxGFP, the least toxic protein revealed in this study, would be a good model system to understand the physiological state of protein burden triggered by ultimate overexpression of harmless proteins.

Proteomics of diphtheria toxoid vaccines reveals multiple proteins that are immunogenic and may contribute to protection of humans against Corynebacterium diphtheriae

Introduced for mass immunization in the 1920s, vaccines against diphtheria are among the oldest and safest vaccines known. The basic principle of their production is the inactivation of purified diphtheria toxin by formaldehyde cross-linking, which converts the potentially fatal toxin in a completely harmless protein aggregate, which is still immunogenic. Since in addition to diphtheria toxin also other proteins may be secreted by Corynebacterium diphtheriae during cultivation, we assumed that diphtheria toxoid might not be the only component present in the vaccine. To address this question, we established a protocol to reverse formaldehyde cross-linking and carried out mass spectrometric analyses. Different secreted, membrane-associated and cytoplasmic proteins of C. diphtheriae were detected in several vaccine preparations from across the world. Based on these results, bioinformatics and Western blot analyses were applied to characterize if these proteins are immunogenic and may therefore support protection against C. diphtheriae. In frame of this study, we could show that the C. diphtheriae toxoid vaccines induce antibodies against different C. diphtheriae proteins and against diphtheria toxin secreted by Corynebacterium ulcerans, an emerging pathogen which is outnumbering C. diphtheriae as cause of diphtheria-like illness in Western Europe.

The Streptococcus pneumoniae yefM-yoeB and relBE Toxin-Antitoxin Operons Participate in Oxidative Stress and Biofilm Formation

Type II (proteic) toxin-antitoxin systems (TAs) are widely distributed among bacteria and archaea. They are generally organized as operons integrated by two genes, the first encoding the antitoxin that binds to its cognate toxin to generate a harmless protein–protein complex. Under stress conditions, the unstable antitoxin is degraded by host proteases, releasing the toxin to achieve its toxic effect. In the Gram-positive pathogen Streptococcus pneumoniae we have characterized four TAs: pezAT, relBE, yefM-yoeB, and phD-doc, although the latter is missing in strain R6. We have assessed the role of the two yefM-yoeB and relBE systems encoded by S. pneumoniae R6 by construction of isogenic strains lacking one or two of the operons, and by complementation assays. We have analyzed the phenotypes of the wild type and mutants in terms of cell growth, response to environmental stress, and ability to generate biofilms. Compared to the wild-type, the mutants exhibited lower resistance to oxidative stress. Further, strains deleted in yefM-yoeB and the double mutant lacking yefM-yoeB and relBE exhibited a significant reduction in their ability for biofilm formation. Complementation assays showed that defective phenotypes were restored to wild type levels. We conclude that these two loci may play a relevant role in these aspects of the S. pneumoniae lifestyle and contribute to the bacterial colonization of new niches.

Secreted gelsolin desensitizes and induces apoptosis of infiltrated lymphocytes in prostate cancer.

Loss of immunosurveillance is a major cause of cancer progression. Here, we demonstrate that gelsolin, a constituent of ejaculate, induces apoptosis of activated lymphocytes in prostate cancer. Gelsolin was highly expressed in prostate cancer cells, and was associated with tumor progression, recurrence, metastasis, and poor prognosis. In vitro, secreted gelsolin inactivated CD4+ T cells by binding to CD37, and induced apoptosis of activated CD8+ T lymphocytes by binding to Fas ligand during cell contact dependent on major histocompatibility complex I. Moreover, secreted gelsolin bound to sortilin, which in turn bound to Wiskott-Aldrich syndrome protein family member 3, thereby enhancing the endocytosis and intracellular transport of essential lipids needed to facilitate tumor growth and expansion. Under normal conditions, gelsolin is a seemingly harmless protein that prevents immune responses in female recipients. In disease states, however, this protein can inhibit immunosurveillance and promote cancer progression.

Allergen-Associated Immunomodulators: Modifying Allergy Outcome.

The prevalence of allergies is increasing since mid twentieth century; however the underlying causes of this increase are not fully clear. Understanding the mechanism by which a harmless protein becomes an allergen provides us with the basis to prevent and treat these diseases. Although most studies on allergen immunogenicity have traditionally focused on structural properties of the proteins, it is increasingly clear that allergenicity cannot be determined only based on structural features of the allergenic proteins. In fact, allergens do not encounter humanfacings as isolated molecules but contained in complex mixtures of proteins, carbohydrates and lipids, such as pollen grains or foods. As a result, attention has lately been directed to examine whether allergen-associated molecules exhibit immune-regulatory properties. The present review aims to illustrate some examples of how non-protein molecules accompanying the allergen can modulate allergic responses.

A yeast toxic mutant of HET-s amyloid disrupts membrane integrity

Many studies have pointed out the interaction between amyloids and membranes, and their potential involvement in amyloid toxicity. Previously, we generated a yeast toxic amyloid mutant (M8) from the harmless amyloid protein by changing a few residues of the Prion Forming Domain of HET-s (PFD HET-s218–289) and clearly demonstrated the complete different behaviors of the non-toxic Wild Type (WT) and toxic amyloid (called M8) in terms of fiber morphology, aggregation kinetics and secondary structure. In this study, we compared the interaction of both proteins (WT and M8) with membrane models, as liposomes or supported bilayers. We first demonstrated that the toxic protein (M8) induces a significant leakage of liposomes formed with negatively charged lipids and promotes the formation of microdomains inside the lipid bilayer (as potential “amyloid raft”), whereas the non-toxic amyloid (WT) only binds to the membrane without further perturbations. The secondary structure of both amyloids interacting with membrane is preserved, but the anti-symmetric PO2− vibration is strongly shifted in the presence of M8. Secondly, we established that the presence of membrane models catalyzes the amyloidogenesis of both proteins. Cryo-TEM (cryo‐transmission electron microscopy) images show the formation of long HET-s fibers attached to liposomes, whereas a large aggregation of the toxic M8 seems to promote a membrane disruption. This study allows us to conclude that the toxicity of the M8 mutant could be due to its high propensity to interact and disrupt lipid membranes.

Proximate Analysis Of Castor Seeds And Cake

Castor seed obtained from Ilorin was evaluated analytically using its un-decorticated and decorticated full-fat and cake forms for chemical composition and nutritional constituents. Proximate analyses of un-decorticated and decorticated seeds and cake revealed that both the oil seed and cake contain valuable nutrients namely soluble carbohydrate: seed-7.96, cake-24.69, protein: seed-20.78, cake-31.06, fat: seed-51.20, cake-19.40, mineral matter: seed-7.75, cake-11.10 for undecorticated castor seeds and cake respectively. Thedecorticated seed cake gave higher values of these nutrients than the un-decorticated, for instance carbohydrate: seed-8.86, cake-24.88, nitrogen: seed-21.87, cake-35.43, ether extract: seed-55.50, cake-25.10, total ash: seed- 9.40, cake-7.14. Amino acid profile analysis of un-decorticated and decorticated seed and cake compared with soybeans as standard plant protein indicated that the cake is deficient in some indispensable amino acids like lysine (4.11), iso-leucine (3.09), tryptophan (0) compared with 6.30, 7.90 and 1.30 lysine, iso-leucine and tryptopha in soybeans respectively.Based on results of the analyses, it is observed that the seed and cake containvaluable nutrients especially when decorticated and may serve useful alternatives to food/feedstuffs if the castor seed is detoxified of the phytotoxins, ricin (a toxic protein), ricinine (a relatively harmless protein), hydrocyanides and allergens, the extremely potent toxins.

355 Immune Tolerance Induced By Peptide Immunotherapy in An HLA Dq2-Dependent Mouse Model of Gluten Immunity

Patients with Coeliac disease (CD) mount inflammatory T-cell responses to deamidated gluten peptides. Encounter of harmless protein via mucosal surfaces generally induces mucosal tolerance. Using animal models we have shown that oral tolerance to the harmless protein ovalbumin (OVA) depends on differentiation of regulatory T-cells (Treg) in the mucosa draining lymphoid tissue. How this translates to the uptake of gluten is difficult to predict as little is known with respect to localization and presentation of gluten after uptake via the gastrointestinal tract. The aim of this study was to identify the phenotype and localization of the gliadin specific T-cell response In Vivo. Mice transgenically expressing disease predisposing human HLA-DQ2 and human gliadin-specific TCR (CD-TCR) on an MHC-II-/background were used. CD-TCR+CD4+ T-cells from double transgenic mice were isolated and labelled with CFSE. After labelling, the cells were i.v. injected into HLA-DQ2tg x MHC-II-/mice. The acceptor mice received non-deamidated or deamidated chymotrypsin digested gliadin by gavage or deamidated gliadin in the thigh muscle (i.m.). After 72h the response of the gliadin-specific T-cells was analyzed in the gut-draining mesenteric lymph node (MLN), spleen and peripheral lymph nodes (PLN) and compared to the known tolerogenic T-cell response to OVA. At 72h after gavage T-cell response to non-deamidated gliadin was very low suggesting that during homeostasis gliadin is not deamidated by murine tissue transglutaminase. Conversely, a vigorous T-cell response was detected in MLN after gavage of deamidated gliadin. Unlike the response to harmless OVA, deamidated gliadin induced more proliferation in spleen than in MLN implying that the adaptive response to gliadin is not confined to the mucosa draining lymphoid tissue. Proliferating gliadin-specific T-cells in MLN contained minimal numbers of Foxp3+ Treg. By contrast, the cells phenotypically resembled activated T-cells and were comparable to differentiating effector T-cells in the PLN of mice that received deamidated gliadin i.m.. Culture of naive CD4+CD-TCR+ T-cells with HLADQ2+ APC in the presence of deamidated gliadin yielded phenotypically identical effector T-cells. Upon addition of factors that mediate mucosal Treg differentiation, Foxp3+CD4+CD-TCR+ T-cells did differentiate, indicating that the cells do have the intrinsic capacity to develop into adaptive mucosal Treg. Dissecting gliadin-specific T-cell responses in this novel murine system provides important opportunities to study the pathogenesis of CD, investigate efficacy of compounds and treatment strategies but also safety of novel dietary products.

353 Localization and Phenotype of the Gliadin-Specific T-Cell Response After Oral Gliadin Intake in Mice

Patients with Coeliac disease (CD) mount inflammatory T-cell responses to deamidated gluten peptides. Encounter of harmless protein via mucosal surfaces generally induces mucosal tolerance. Using animal models we have shown that oral tolerance to the harmless protein ovalbumin (OVA) depends on differentiation of regulatory T-cells (Treg) in the mucosa draining lymphoid tissue. How this translates to the uptake of gluten is difficult to predict as little is known with respect to localization and presentation of gluten after uptake via the gastrointestinal tract. The aim of this study was to identify the phenotype and localization of the gliadin specific T-cell response In Vivo. Mice transgenically expressing disease predisposing human HLA-DQ2 and human gliadin-specific TCR (CD-TCR) on an MHC-II-/background were used. CD-TCR+CD4+ T-cells from double transgenic mice were isolated and labelled with CFSE. After labelling, the cells were i.v. injected into HLA-DQ2tg x MHC-II-/mice. The acceptor mice received non-deamidated or deamidated chymotrypsin digested gliadin by gavage or deamidated gliadin in the thigh muscle (i.m.). After 72h the response of the gliadin-specific T-cells was analyzed in the gut-draining mesenteric lymph node (MLN), spleen and peripheral lymph nodes (PLN) and compared to the known tolerogenic T-cell response to OVA. At 72h after gavage T-cell response to non-deamidated gliadin was very low suggesting that during homeostasis gliadin is not deamidated by murine tissue transglutaminase. Conversely, a vigorous T-cell response was detected in MLN after gavage of deamidated gliadin. Unlike the response to harmless OVA, deamidated gliadin induced more proliferation in spleen than in MLN implying that the adaptive response to gliadin is not confined to the mucosa draining lymphoid tissue. Proliferating gliadin-specific T-cells in MLN contained minimal numbers of Foxp3+ Treg. By contrast, the cells phenotypically resembled activated T-cells and were comparable to differentiating effector T-cells in the PLN of mice that received deamidated gliadin i.m.. Culture of naive CD4+CD-TCR+ T-cells with HLADQ2+ APC in the presence of deamidated gliadin yielded phenotypically identical effector T-cells. Upon addition of factors that mediate mucosal Treg differentiation, Foxp3+CD4+CD-TCR+ T-cells did differentiate, indicating that the cells do have the intrinsic capacity to develop into adaptive mucosal Treg. Dissecting gliadin-specific T-cell responses in this novel murine system provides important opportunities to study the pathogenesis of CD, investigate efficacy of compounds and treatment strategies but also safety of novel dietary products.

Soluble oligomers from a non‐disease related protein mimic Aβ‐induced tau hyperphosphorylation and neurodegeneration

Protein aggregation and amyloid accumulation in different tissues are associated with cellular dysfunction and toxicity in important human pathologies, including Alzheimer's disease and various forms of systemic amyloidosis. Soluble oligomers formed at the early stages of protein aggregation have been increasingly recognized as the main toxic species in amyloid diseases. To gain insight into the mechanisms of toxicity instigated by soluble protein oligomers, we have investigated the aggregation of hen egg white lysozyme (HEWL), a normally harmless protein. HEWL initially aggregates into beta-sheet rich, roughly spherical oligomers which appear to convert with time into protofibrils and mature amyloid fibrils. HEWL oligomers are potently neurotoxic to rat cortical neurons in culture, while mature amyloid fibrils are little or non-toxic. Interestingly, when added to cortical neuronal cultures HEWL oligomers induce tau hyperphosphorylation at epitopes that are characteristically phosphorylated in neurons exposed to soluble oligomers of the amyloid-beta peptide. Furthermore, injection of HEWL oligomers in the cerebral cortices of adult rats induces extensive neurodegeneration in different brain areas. These results show that soluble oligomers from a non-disease related protein can mimic specific neuronal pathologies thought to be induced by soluble amyloid-beta peptide oligomers in Alzheimer's disease and support the notion that amyloid oligomers from different proteins may share common structural determinants that would explain their generic cytotoxicities.

Human epidermal Langerhans cells lack functional mannose receptors and a fully developed endosomal/lysosomal compartment for loading of HLA class II molecules.

Langerhans cells (LC) represent the dendritic cell (DC) lineage in the epidermis. They capture and process antigens in the skin and subsequently migrate to the draining lymph nodes to activate naive T cells. Efficient uptake and processing of protein antigens by LC would, therefore, seem a prerequisite. We have now compared the capacity of human epidermal LC, blood-derived DC and peripheral blood mononuclear cells to endocytose and present (mannosylated) antigens to antigen-specific T cells. Moreover, we have determined the expression of mannose receptors, and the composition of the intracellular endosomal/lysosomal MHC class II-positive compartment. The results indicate that LC have poor endocytic capacity and do not exploit mannose receptor-mediated endocytosis pathways. Furthermore, the composition of the class II compartment in LC is distinct from that in other antigen-presenting cells and is characterized by the presence of relatively low levels of lysosomal markers. These results underscore the unique properties of LC and indicate that LC are relatively inefficient in antigen uptake, processing and presentation. This may serve to avoid hyper-responsiveness to harmless protein antigens that are likely to be frequently encountered in the skin due to (mechanical) skin damage.

Evidence that secretory IgA antibody is associated with daily mood.

In this study, we examined the secretory immune system, the body's first line of defense against invading organisms, and its relation to daily fluctuations of mood. Specifically, secretory immunoglobulin A (sIgA) was studied. Unlike other psychoimmunity studies that examined all sIgA protein regardless of specificity to invading organisms, ours examined an antigen-specific sIgA response to the oral administration of a harmless protein (rabbit albumin) and monitored the antibody produced in response to the protein. Dental students recorded their daily mood thrice weekly for 8 1/3 weeks, and parotid saliva was obtained from subjects during these contacts. Using a within-subjects analyses strategy, we found that antibody response was lower on days with high negative mood relative to days with lower negative mood, and conversely, sIgA antibody response was higher on days with high positive mood relative to days with lower positive mood. Results from total sIgA protein were in the opposite direction, although not significantly so. These results extend our knowledge of immunological changes and mood, and they suggest that minor life events' role in health may be mediated by the secretory immune system.

Gut Immunity in the Pig - Hypersensitivity Responses to Dietary Antigens

The Intestinal Immune System is obliged to recognise not only antigens associated with pathogenic organisms but also harmless dietary protein. Since chronic immune responses to these ubiquitous antigens present in food are likely to be harmful to normal intestinal function, regulatory mechanisms have evolved which reduce immune responses to such antigens to levels acceptable to the individual. It is essential to the effective working of these mechanisms that the introduction to new antigens should be gradual and not be associated with the sudden presentation of large amounts of a new dietary material. Modern intensive weaning methods pay scant attention to the needs of these immunoregulatory systems, and the resultant immune response may be the cause of the villous atrophy and crypt hyperplasia seen immediately after weaning and which are thought to precipitate post weaning E. coli diarrhoea.

Croonian Lecture.—The biological significance of anaphylaxis

Anaphylaxis was regarded by Richet, who first clearly recognised the phenomenon, as the opposite of immunity or “phylaxis.” At an interval of some weeks, after a first dose of any one of a group of poisonous proteins, the animal was found to be apparently much more susceptible to the action of the poison in question. Further investigation has shown that this suscepti­bility is not connected with the naturally poisonous properties of the substance used, but can be developed in relation to perfectly harmless protein substances, provided they are obtained from a different species and introduced into the system without hydrolytic cleavage. The sensitiveness is highly specific. It discriminates between corresponding substances from different species, between materials from different organs from the same species, and between individual proteins from the same organ. It can be transferred to a normal animal by blood or serum from an anaphylactic animal. In the nature of the substances producing it, in the limits of its specificity, and in the possi­bility of its transfer by serum from a treated animal, it shows a very suggestive correspondence with the type of immunity associated with “pre­cipitin” formation. A highly precipitating serum from an immunised animal confers anaphylaxis on a normal animal more readily, i. e ., in smaller dose, than serum from an animal itself anaphylactic. Nevertheless, the serum from an anaphylactic animal forms no visible precipitate with the antigen, and an animal whose serum has this obvious precipitating quality is not anaphylactic, but immune. Anaphylaxis is not so much the direct opposite of immunity as an anomalous concomitant of a certain phase in its develop­ment. An animal rendered anaphylactic to a naturally poisonous protein is immune to the natural poisonous action, but has acquired a new sensitiveness to it as a protein.