Relationship Of Proteins Research Articles

Proteomic Landscape of De Novo Pediatric Acute Myeloid Leukemia

Background: Many genetic drivers that are implicated in disease pathology and risk stratification have been identified for pediatric acute myeloid leukemia (pedi-AML). However, only a minority have been exploited for therapeutic interventions and most of the identified genetic events currently lack targeted therapy to address the mutations. The combined consequences of genetic and epigenetic events culminate in a net effect manifested at the protein level and most of the chemotherapies target proteins. Yet little is known about the proteomic landscape of pedi-AML.Methods: Reverse Phase Protein Array (RPPA) was performed with 291 strictly validated antibodies to determine the protein expression levels of bulk leukemic cells from 505 pedi-AML patient samples that were collected prior to therapy. All patients participated on the COG AAML1031 Phase 3 clinical trial, that compared standard therapy (ADE) to ADE plus bortezomib (ADE+B). Proteins were allocated into 31 protein functional groups (PFG) (e.g. cell cycle, apoptosis) to analyze proteins in relation to related proteins. Progeny clustering was performed to identify patients with correlated protein expression patterns within each PFG (protein cluster). Block clustering searched for protein clusters that recurrently co-occurred (protein constellation), and for subgroups of patients that expressed similar combinations of protein constellations (protein signatures). Protein signatures were correlated with known cytogenetics and mutational state.Results: For each PFG, cluster analysis identified an optimal number of protein clusters, resulting in a total of 120. From this we constructed 11 protein constellations (PRCON) and 10 protein signatures (SIG) (Fig. 1A). A training set (n=334) and test set (n=171) showed high reproducibility (Pearson's X2; p < 0.001). SIG were prognostic for event-free survival (EFS) (p = 0.029), with a favorable EFS for SIG 1, 2, 3, 5, 7 & 9, and an unfavorable EFS for SIG 4, 6, 8, 10. Notably, patients that formed SIG 3 had a significant better EFS after receiving ADE+B than patients that received ADE (p = 0.039) (Fig. 1B). This SIG was highly enriched for CEBPA mutated cases; 43% vs. 9% overall (p < 0.001). SIG were associated with cytogenetic aberrations (p < 0.001) and mutational state, as well as with the traditional risk groups (p < 0.001). For example, t(8;21) was overrepresented in SIG 9 (39% vs. 16% overall) and MLL-rearrangement in SIG 6 (61% vs. 19% overall). Multivariate analysis was performed using variables definable at time of diagnosis and known prognostic factors. This resulted in a final model including unfavorable protein SIG together with low risk cytogenetics and NPM1 mutation state as independent prognostic factors, suggesting that proteins add to known prognostic factors.Proteomics could also identify aberrantly expressed proteins within each SIG compared to normal CD34+ cells. This recognized 31 proteins as universally down regulated (e.g. CDKN1A, PPP2R2A) and 13 as universally up regulated (e.g. PIK3CA, NCL). Many other proteins were different between SIG, and thus potentially targetable in particular patient groups: high KIT (SIG 3, 4 & 5), high BCL2 (SIG 3, 4, 5, 6, 9 & 10) and high BRD4 (SIG 6). SIG 1 & 2 both had a very characteristic expression pattern and were most distinct from the others. IGFR1, IGFR1.pY1135, AKT3, SMAD5.pS463-7 and RICTOR.pT1135 were all high, whereas they were low in most other SIG. Conversely, STAT3 was normal in SIG 1&2, but changed in the other SIG. SIG 6, 7 & 8 also had relatively similar expression patterns, but had different outcomes. Compared to SIG 6 & 8, SIG 7 had higher GAB2 and MCL1 and lower RAD50, ERCC1.Conclusions: Pedi-AML is characterized by a finite number (n=10) of recurrent protein signatures. SIG were only partially correlated with cytogenetics and mutation state, indicating that protein expression could add to genetics in the process of risk stratification. We identified SIG that did well vs. SIG that did not, independent of known risk factors, and identified a group of patients that could potentially benefit from ADE+B. Recognition of differentially expressed proteins suggest potential targets for combinational treatment (Hoff et al. ASH HSF1 abstract).Figure 1. A. Identification of 11 PRCON (horizontally) and 10 SIG (vertically). B. SIG were prognostic for EFS. C. SIG 3 significantly benefitted from ADE+B (green) compared to ADE alone (blue). [Display omitted] DisclosuresKolb:Roche- Genentech: Membership on an entity's Board of Directors or advisory committees; Servier: Membership on an entity's Board of Directors or advisory committees.

Specific Proteins in Relation with Iron Overload in Experimental Study

Iron is an essential mineral for organisms and due to environment iron deficiency and pollution it is very important to understand and monitor the iron homeostasis. Thus, the concentration of iron is very important in hematological and biochemical parameters because iron is component of hemoglobin, erythrocytes, and myoglobin -being part of oxygen-binding proteins. The aim of our experiment was to try a natural protection with fresh, organic plants administration � of iron overload in rabbits. Thereby, we administrated ferrous gluconate (hydro-solution, 15g Fe2+/body weight) as intraperitoneal injection for 43 days to German Lop Eared breed rabbits, and in the same time we tried to protect the animals with a diet consisting in black cumin seeds; clover, chives, leek, parsley, coriander, rucola, radish, fenugreek - leaves; cucumber and carrots. At the end of experiment we analyzed the protein electrophoresis (albumin, a1-globulin, a2-globulin, b1-globulin, b2-globulin and g-globulin), total iron binding capacity, and transferrin concentration. The analytical results demonstrated that plant administration was beneficial for iron homeostasis and the tested blood parameters did not record significant variations between control and experimental group, which proves that diet is very important in health and homeostatic mechanisms in living organisms.

Severity of clinical dry eye manifestations influences protein expression in tear fluid of patients with primary Sjögren's syndrome.

Ocular dryness is a characteristic feature of primary Sjögren’s syndrome (pSS). This may result in dry eye disease (DED), leading to damage of the ocular surface. Additional, non-invasive diagnostic techniques are needed when evaluating pSS patients. Hence, screening for disease-specific biomarkers in biological fluid could be promising. We have previously examined the proteome of tear fluid from pSS patients through Liquid chromatography-mass spectrometry (LC-MS), and conducted a thorough ocular evaluation of patients with pSS. In this study we further explored the association between dry eye manifestations and protein expression in tear fluid of pSS patients. Medical history of 27 patients and 32 healthy controls was gathered. Subjective complaints were registered through questionnaires. Objective findings including tear osmolarity, tear film break up time (TFBUT), Schirmer’s test, and ocular and corneal surface staining were also recorded. LC-MS was conducted formerly on tear fluid from all subjects in order to generate proteomic biomarker profiles. Scaffold was employed to analyse the LC-MS data for quantitative differences between patient and control groups, and the mean spectral counts were calculated for the five most upregulated proteins in relation to DED manifestations. Dysregulated cellular processes were identified in pSS patients using FunRichv3 enrichment analysis. The five most upregulated proteins previously identified in pSS patients were DNA (apurinic or apyrimidinic site) lyase (APEX1), thioredoxin-dependent peroxidase reductase (PRDX3), copine (CPNE1), aconitate hydratase (ACO2), and LIM domain only protein 7 (LMO7), in descending order. A significant increase in mean spectral counts for these proteins were observed in pSS patients with pathological DED manifestations compared to healthy controls (p<0.0001). Consequently, dysregulated cellular pathways involving innate and adaptive immunity were also detected. In conclusion, our observations suggest a relationship between presence of dry eye signs and upregulated proteins in tear fluid from patients with pSS. Further studies are needed in order to replicate the concepts explored and analyses performed in a greater cohort of pSS patients, where sensitivity and specificity of the methods conducted can also be verified further.

Age-related Changes in the Expression of Heat Shock Protein 70 and 90 on the Gastric Mucosa During Gastric Ulcer Healing

Heat shock proteins 70 and 90 (HSP-70 and HSP-90) are associated with gastroprotective and ulcer healing potentials. Reports in literatures have shown that age affects gastric ulcer healing, but the role of these heat shock proteins in relation to age has not been fully understood. This study, therefore, investigated changes in the expression of HSP-70 and HSP-90 in the gastric mucosa of 3, 6 and 18-month old rats during healing of Acetic acid-induced gastric ulcers. Male Wister rats (aged 3, 6 and 18 months) were divided into 3 groups according to their ages. Acetic acid ulcer model was used for this study. Ulcer area, oxidative stress, antioxidant markers, HSP-70 and HSP-90 concentration by ELISA and expression by immunohistochemistry was assessed. Results obtained indicate the highest percentage area healed on day 14 in 3 months old rats (100%), while percentage healing for 6 and 18 months old rats was 89.00% and 55.29%, respectively. Malondialdehyde (MDA) concentration was directly proportional to age, while antioxidant enzyme (Superoxide dismutase (SOD) and catalase (CAT)) activities were inversely proportional to age. The concentration and expression of HSP-70 were inversely proportional to age while HSP-90 had directly proportionality to age. The histological architecture also confirmed the faster rate of healing in 3-month old rats recorded in this study.This study indicates that HSP-70 and HSP-90 play different roles in age-related healing of gastric ulcers.

Abstract 425: A Pro-reductive Redox State Protects the Myocardium From Isoproterenol-Induced Pathological Remodeling in Nrf2 Transgenic Mouse

Background: Nuclear factor (erythroid-derived 2)-like 2 (NEFL2/Nrf2) is an inducible transcription factor that is crucial to fight against stress caused by various means (i.e. oxidative or xenobiotic stresses). While the Nrf2 has a negligible role on regulating the basal transcription of antioxidant and cytoprotective genes, its activation in response to stress is indispensable. Here, we hypothesize that augmentation of myocardial Nrf2, through transgene expression, will protect the myocardium from isoproterenol-induced pathological remodeling. Methods: Cardiac specific Nrf2 transgenic (Nrf2-Tg) and non-transgenic (NTg) litter mates in the C57/BL6 background were subjected to isoproterenol (ISO) treatment (50 mg/kg bw/day, for 7 days), and assessed the myocardial structure, function (Echocardiography) and Nrf2-dependent defense mechanisms. Results: Severe infarction and fibrosis along with increased inflammation leading to myocardial dysfunction in NTg mice exposed to ISO, but the Nrf2-TG hearts were found to be resistant to ISO insult. Suppression of infarction and protection of myocardial structure and function in the Nrf2-Tg mice could be attributed to a pro-reductive condition displayed in these hearts. In particular, ISO administration resulted in an excessive generation of reactive oxygen species and oxidation of proteins along with significant downregulation of antioxidant genes (p&lt;0.05)and their proteins lead to myocardial damage in NTg mice. Of note, myocardium of ISO-treated TG mice displayed significantly reduced protein oxidation and comparable levels of antioxidant genes and proteins (p&lt;0.05) in relation to PBS-treated NTg mice. Taken together, it is suggested that a pre-existing pro-reductive redox milieu is likely to combat ISO-induced oxidative stress, thereby protecting the myocardium from pathological remodeling. Conclusion: Thus, we conclude that a modest trans-activation of the global antioxidant defense system might have therapeutic potential than either targeting single antioxidant (i.e. SOD or catalase or GPX) or supplementation of antioxidant combos to protect the myocardium in response to stress.

Abstract 3222: Evaluation of associations between circulating proteins and breast cancer risk using genetic variants

Abstract Breast cancer is the most commonly diagnosed cancer in women in many countries. Several circulating protein biomarkers have been identified in relation to breast cancer risk. However, previous studies either had small sample sizes or employed low-throughput techniques. To search for novel biomarkers, we utilized genetic variants as instruments and evaluated over 1,400 proteins in relation to breast cancer risk using data from the Breast Cancer Association Consortium (BCAC). We extracted beta coefficients from reported protein quantitative trait loci (pQTL) derived from genome-wide association studies of circulating proteins. Summary statistics of these pQTL variants associated with breast cancer risk were obtained from 122,977 cases and 105,974 controls of European descent in the BCAC. Associations of genetically predicted protein levels with breast cancer risk were evaluated using the inverse-variance weighted method. For proteins with a significant association, expression levels of the corresponding gene were predicted using genotyping and transcriptomic data from the Genotype-Tissue Expression project and then evaluated for their associations with breast cancer risk. We identified 56 protein biomarkers showing a significant association with breast cancer risk after accounting for multiple comparisons (false discovery rate &amp;lt; 0.05). Among them, levels of 32 proteins were influenced by variants close to a newly reported breast cancer susceptibility locus (9q34.2, ABO). Inverse associations of breast cancer risk were found with membrane proteins such as insulin receptor, insulin-like growth factor receptor 1, hepatocyte growth factor receptor, neurogenic locus notch homolog protein 1, and vascular endothelial growth factor receptor 2, with odds ratios ranging from 0.82 to 0.97 per unit increase in genetic risk scores (p-values ranging from 6.53x10-4 to 3.28x10-8). Genetically predicted expression of five genes, CPNE1, CTSF, TFPI, SCG3, and QSOX2, was found to be associated with breast cancer risk at p &amp;lt;0.05 in the same direction as the associations observed for the corresponding proteins. Results from our study suggest that multiple membrane proteins related to insulin resistance may be linked to breast cancer risk through genetic variants at 9q34.2. Citation Format: Xiang Shu, Jiandong Bao, Lang Wu, Jirong Long, Xingyi Guo, Kyriaki Michailidou, Manjeet K. Bolla, Qin Wang, Joe Dennis, Jacques Simard, Douglas F. Easton. Evaluation of associations between circulating proteins and breast cancer risk using genetic variants [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3222.

Maize grain soluble sugar and protein contents in response to simulated hail damage

Production of grain of high nutritional quality is essential in maize (Zea mays L.) as a food grain crop. Grain quality may be influenced by environmental and management factors, such as hail, plant density and grain selection. A study was conducted to determine the effect of hail damage on maize grain contents of soluble sugars and total proteins in relation to grain position on the cob and planting density. Maize was grown at three planting densities (65 000 plants ha−1 [high], 46 000 plants ha−1 [moderate] and 28 000 plants ha−1 [low]) at two sites (Baynesfield and Swayimane) in KwaZulu-Natal, South Africa. Approximately 80% hail damage was simulated on the leaves at the seventh leaf (V7) and tasseling (VT) stages of development of three maize cultivars (‘SC701’, ‘Mac Medium Pearl’ and ‘Zama Star’), whereas undamaged plants served as the control. After harvest at maturity, grains from the proximal and distal portions of the cob were divided and analysed for grain nutritional quality, i.e. soluble sugars and proteins. Soluble sugar analysis focused on the predominant sugars, namely glucose, fructose and sucrose. Although not significant, hail damage typically at VT reduced grain soluble sugar and protein contents by at least 3.2% and 3.8%, respectively, and grains produced at Baynesfield were more resilient to damage compared with those produced at Swayimane. Selection of seeds from the distal cob portion, especially for ‘SC701’, and low plant density reduced the detrimental effect of hail damage on grain sugar and protein content. The present results indicate that in areas prone to hail ‘SC701’ is a cultivar of choice, and a low plant density and selection of grains from the distal cob portion can mitigate the detrimental effects of hail on grain nutritional quality.

Cell Migration Related to MDR-Another Impediment to Effective Chemotherapy?

Multidrug resistance, mediated by members of the ATP-binding cassette (ABC) proteins superfamily, has become one of the biggest obstacles in conquering tumour progression. If the chemotherapy outcome is considered successful, when the primary tumour volume is decreased or completely abolished, modulation of ABC proteins activity is one of the best methods to overcome drug resistance. However, if a positive outcome is represented by no metastasis or, at least, elongation of remission-free time, then the positive effect of ABC proteins inhibition should be compared with the several side effects it causes, which may inflict cancer progression and decrease overall patient health. Clinical trials conducted thus far have shown that the tested ABC modulators add limited or no benefits to cancer patients, as some of them are merely toxic and others induce unwanted drug–drug interactions. Moreover, the inhibition of certain ABC members has been recently indicated as potentially responsible for increased fibroblasts migration. A better understanding of the complex role of ABC proteins in relation to cancer progression may offer novel strategies in cancer therapy.

Molecular Evolution and Functional Divergence of the IgLON Family.

IgLON family is a subgroup of cell adhesion molecules which is known to have diverse roles in neuronal development. IgLONs are characterized by possessing 3 Ig-like C2 domains, which play a part in mediating various cellular interactions. Recently, IgLONs have been shown to be expressed at the blood-brain barrier (BBB). However, our understanding of the genetic divergence patterns and evolutionary rates of these proteins in relation to their functions, in general, and at the BBB, in particular, remains inadequate. In this study, 12 species were explored to shed more light on the phylogenetic origins, structure, functional specificity, and divergence of this family. A total of 40 IgLON genes were identified from vertebrates and invertebrates. The absence of IgLON family genes in Hydra vulgaris and Nematostella vectensis but not in Drosophila melanogaster suggests that this family appeared during the time of divergence of Arthropoda 455 Mya. In general, IgLON genes have been subject to strong positive selection in vertebrates. Our study, based on IgLONs’ structural similarity, suggests that they may play a role in the evolutionary changes in the brain anatomy towards complexity including regulating neural growth and BBB permeability. IgLONs’ functions seem to be performed through complex interactions on the level of motifs as well as single residues. We identified several IgLON motifs that could be influencing cellular migration and proliferation as well as BBB integrity through interactions with SH3 or integrin. Our motif analysis also revealed that NEGR1 might be involved in MAPK pathway as a form of a signal transmitting receptor through its motif (KKVRVVVNF). We found several residues that were both positively selected and with highly functional specificity. We also located functional divergent residues that could act as drug targets to regulate BBB permeability. Furthermore, we identified several putative metalloproteinase cleavage sites that support the ectodomain shedding hypothesis of the IgLONs. In conclusion, our results present a bridge between IgLONs’ molecular evolution and their functions.

Single-Particle Tracking of Cell Surface Proteins.

Single-particle tracking has been used extensively to advance our understanding of the plasma membrane and the mechanisms controlling the movement of cell surface proteins. These studies provide fundamental insights into the regulation of membrane receptor activation and the assembly of signaling clusters. Here, we describe a method to label and track B cell receptor (BCR) and other cell surface proteins and how this method can be adapted to simultaneously track two molecular species or examine the movement of membrane proteins in relation to membrane microdomains. We recently used this method to study the role of the actin cytoskeleton in the regulation of B cell receptor dynamics at the cell surface.

Winter ambient training conditions are associated with increased bronchial hyperreactivity and with shifts in serum innate immunity proteins in young competitive speed skaters.

IntroductionRegular training modulates airway inflammation and modifies susceptibility to respiratory infections. The impact of exercise and ambient conditions on airway hyperreactivity and innate immunity has not been well studied. We aimed to assess exercise-related symptoms, lung function, airway hyperresponsiveness and innate immunity proteins in relation to meteorological conditions and exercise load in competitive athletes.Material and methodsThirty-six speed skaters were assessed during winter (WTP) and summer (STP) periods. The control group comprised 22 non-exercising subjects. An allergy questionnaire for athletes (AQUA) and IPAQ (International Physical Activity Questionnaire) were used to assess symptoms and exercise. Meteorological parameters were acquired from World Meteorological Organization resources. Serum innate immunity proteins were measured by ELISA.ResultsExercise-associated respiratory symptoms were reported by 79.4% of skaters. Despite similar exercise load and lung parameters during both periods, positive methacholine challenge was more frequent during winter (p = 0.04). Heat shock protein HSPA1 and IL-1RA were significantly decreased during STP compared to WTP and controls. During WTP, IL-1RA was elevated in skaters reporting exercise-induced symptoms (p = 0.007). sCD14 was elevated in athletes versus controls in both periods (p < 0.05). HSPA1 was significantly higher in WTP compared to STP irrespective of presence of respiratory tract infections (RTIs). IL-1RA in WTP was elevated versus STP (p = 0.004) only in RTI-negative athletes. Serum IL-1RA negatively correlated with most meteorological parameters during WTP.ConclusionsAmbient training conditions, but not training load, influence bronchial hyperreactivity and the innate immune response in competitive athletes assessed during winter. The protective effect of regular exercise against respiratory infections is associated with a shift in serum innate immunity proteins.

Structural and functional changes of fibrinogen due to aging

Different factors affect coagulation process. Since fibrinogen is the main coagulation factor, the influence of aging on fibrinogen structure and function was investigated in this study. Fibrinogen was isolated from plasma obtained from healthy persons in the age range 21–83 and examined. Lectin microarray analysis demonstrated increased glycosylation of fibrinogen due to aging, with predominant increase in high-mannose or hybrid type N-glycans, as well as tri-/tetraantennary complex N-glycans with greater content of galactose and N-acetylglucosamine residues. Spectrofluorimetric analysis indicated that fibrinogen molecules have more densely packed structure, but there are no additional advanced glycation end products with increasing age. According to the results of functional analysis, fibrinogen molecules isolated from older persons exhibited reduced clotting time, with significant positive correlation with age, but there were no differences in clotting speed, maximal optical density of fibrin clot, diameter of fibrin fibres, fibrin porosity or reactivity with the insulin-like growth factor binding protein 1. Glycosylation changes of fibrinogen in healthy aging most likely affect its structure and function, namely clotting time. Structural and functional studies of proteins in relation to healthy aging contribute to deeper understanding of mechanisms responsible for longevity.

Correlative Super-resolution and Electron Microscopy to Resolve Protein Localization in Zebrafish Retina.

We present a method to investigate the subcellular protein localization in the larval zebrafish retina by combining super-resolution light microscopy and scanning electron microscopy. The sub-diffraction limit resolution capabilities of super-resolution light microscopes allow improving the accuracy of the correlated data. Briefly, 110 nanometer thick cryo-sections are transferred to a silicon wafer and, after immunofluorescence staining, are imaged by super-resolution light microscopy. Subsequently, the sections are preserved in methylcellulose and platinum shadowed prior to imaging in a scanning electron microscope (SEM). The images from these two microscopy modalities are easily merged using tissue landmarks with open source software. Here we describe the adapted method for the larval zebrafish retina. However, this method is also applicable to other types of tissues and organisms. We demonstrate that the complementary information obtained by this correlation is able to resolve the expression of mitochondrial proteins in relation with the membranes and cristae of mitochondria as well as to other compartments of the cell.

Correlative Super-resolution and Electron Microscopy to Resolve Protein Localization in Zebrafish Retina

We present a method to investigate the subcellular protein localization in the larval zebrafish retina by combining super-resolution light microscopy and scanning electron microscopy. The sub-diffraction limit resolution capabilities of super-resolution light microscopes allow improving the accuracy of the correlated data. Briefly, 110 nanometer thick cryo-sections are transferred to a silicon wafer and, after immunofluorescence staining, are imaged by super-resolution light microscopy. Subsequently, the sections are preserved in methylcellulose and platinum shadowed prior to imaging in a scanning electron microscope (SEM). The images from these two microscopy modalities are easily merged using tissue landmarks with open source software. Here we describe the adapted method for the larval zebrafish retina. However, this method is also applicable to other types of tissues and organisms. We demonstrate that the complementary information obtained by this correlation is able to resolve the expression of mitochondrial proteins in relation with the membranes and cristae of mitochondria as well as to other compartments of the cell.

Glycosylated Chromogranin A: Potential Role in the Pathogenesis of Heart Failure.

Endocrine and paracrine factors influence the cardiovascular system and the heart by a number of different mechanisms. The chromogranin-secretogranin (granin) proteins seem to represent a new family of proteins that exerts both direct and indirect effects on cardiac and vascular functions. The granin proteins are produced in multiple tissues, including cardiac cells, and circulating granin protein concentrations provide incremental prognostic information to established risk indices in patients with myocardial dysfunction. In this review, we provide recent data for the granin proteins in relation with cardiovascular disease, and with a special focus on chromogranin A and heart failure. Chromogranin A is the most studied member of the granin protein family, and shorter, functionally active peptide fragments of chromogranin A exert protective effects on myocardial cell death, ischemia-reperfusion injury, and cardiomyocyte Ca2+ handling. Granin peptides have also been found to induce angiogenesis and vasculogenesis. Protein glycosylation is an important post-translational regulatory mechanism, and we recently found chromogranin A molecules to be hyperglycosylated in the failing myocardium. Chromogranin A hyperglycosylation impaired processing of full-length chromogranin A molecules into physiologically active chromogranin A peptides, and patients with acute heart failure and low rate of chromogranin A processing had increased mortality compared to other acute heart failure patients. Other studies have also demonstrated that circulating granin protein concentrations increase in parallel with heart failure disease stage. The granin protein family seems to influence heart failure pathophysiology, and chromogranin A hyperglycosylation could directly be implicated in heart failure disease progression.

Proteins in Relation to Vigor and Viability of White Lupin (Lupinus albus L.) Seed Stored for 26 Years.

The aim of the study was to evaluate the vigor and viability as well as to determine and compare the contents of selected protein fractions of white lupin (Lupinus albus L.) seeds stored for 26 years at temperatures of -14°C and +20°C. The seeds stored at -14°C germinated in 86.3%, while the seeds stored at +20°C did not germinate at all. The viability evaluation was confirmed by the measuring electroconductivity of seed exudates. In seeds stored at -14°C the contents of γ, δ, and β conglutin were 14, 4 and 69 mg g-1 fresh mass, respectively, while in seed stored at +20°C they were 15.5, 3, 65 mg g-1 fresh mass, respectively. One-dimensional electrophoresis of γ and δ conglutin fractions indicated the presence of several intense polypeptide bands with molecular weights from 23.0 to 10.3 kDa. Polypeptide bands with a molecular weight of 22.4 and 19.8 kDa exhibited almost two times higher expression in the seeds stored at -14°C compared to the seeds stored at +20°C. Electrophoresis revealed 310 protein spots on the maps generated for seeds stored at -14°C, and 228 spots for seeds stored at +20°C. In seeds stored at +20°C most polypeptide subunits had a pI ranging from 4.5 to 7 and a molecular weight of 10–97 kDa. The greatest differences in the contents of polypeptides between the analyzed variants was observed within the range of 20–45 kDa (-14°C: 175, +20°C: 115 protein spots) and within the range of 65–97 kDa (-14°C: 103, +20°C: 75 protein spots). In seeds stored at +20°C, a clear decline in basic (8–10 pI) polypeptides was observed. The study demonstrated that the polypeptides identified as γ and δ conglutins are probably closely related to vigor and viability of seeds.

Characteristics of six small heat shock protein genes from Bactrocera dorsalis: Diverse expression under conditions of thermal stress and normal growth.

To explore the functions of small heat shock proteins (sHsps) in relation to thermal stress and development in Bactrocera dorsalis (Hendel), one of the most economically important pest species attacking a wide range of fruits and vegetables, six full-length cDNAs of sHsp genes (BdHsp17.7, 18.4, 20.4, 20.6, 21.6 and 23.8) were cloned, and the expression patterns in different developmental stages and tissues, as well as in response to both thermal and 20-hydroxyecdysone (20E) exposures, were examined using real time quantitative PCR. The open reading frames (ORFs) of six sHsps are 453, 489, 537, 543, 567 and 630bp in length, encoding proteins with molecular weights of 17.7, 18.4, 20.4, 20.6, 21.6 and 23.8kDa, respectively. BdHsp18.4 and BdHsp20.4 maintained lower expression levels in both eggs and larvae, whereas remarkably up-regulated after the larval-pupal transformation, suggesting that these two sHsps may be involved in metamorphosis. Significant tissue specificity exists among sHsps: the highest expression of BdHsp20.6 and BdHsp23.8 in the Malpighian tubules and ovary, respectively, versus a peak in the fat body for others. BdHsp20.4 and BdHsp20.6 were significantly up-regulated by thermal stress. In contrast, BdHsp18.4 and BdHsp23.8 reacted only to heat stress. BdHsp17.7 and BdHsp21.6 were insensitive to both heat and cold stresses. The degree of sHsps response depends on intensity of 20E treatment, i.e., dose and time. These results strongly suggest functional differentiation within the sHsp subfamily in B. dorsalis. The physiological function of sHsp members under thermal stress and normal growth remains the subjects of further investigation.

Haemocytes collected from experimentally infected Pacific oysters, Crassostrea gigas: Detection of ostreid herpesvirus 1 DNA, RNA, and proteins in relation with inhibition of apoptosis.

Recent transcriptomic approaches focused on anti-viral immunity in molluscs lead to the assumption that the innate immune system, such as apoptosis, plays a crucial role against ostreid herpesvirus type 1 (OsHV-1), infecting Pacific cupped oyster, Crassostrea gigas. Apoptosis constitutes a major mechanism of anti-viral response by limiting viral spread and eliminating infected cells. In this way, an OsHV-1 challenge was performed and oysters were monitored at three times post injection to investigate viral infection and host response: 2h (early after viral injection in the adductor muscle), 24h (intermediate time), and 48h (just before first oyster mortality record). Virus infection, associated with high cumulative mortality rates (74% and 100%), was demonstrated in haemocytes by combining several detection techniques such as real-time PCR, real-time RT PCR, immunofluorescence assay, and transmission electron microscopy examination. High viral DNA amounts ranged from 5.46×104 to 3.68×105 DNA copies ng-1 of total DNA, were detected in dead oysters and an increase of viral transcripts was observed from 2, 24, and 48hpi for the five targeted OsHV-1 genes encoding three putative membrane proteins (ORFs 25, 41, and 72), a putative dUTPase (ORF 75), and a putative apoptosis inhibitor (ORF 87). Apoptosis was studied at molecular and cellular levels with an early marker (phosphatidyl-serine externalisation measured by flow cytometry and epifluorescence microscopy) and a later parameter (DNA fragmentation by terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling assay (TUNEL)). The down-regulation of genes encoding proteins involved in the activation of the apoptotic pathway (TNF and caspase 3) and the up-regulation of genes encoding anti-apoptotic proteins (IAP-2, and Bcl-2) suggested an important anti-apoptosis phenomenon in haemocytes from OsHV-1 infected oysters at 24 and 48hpi. Additionally, more phosphatidyl-serines were externalized and more cells with DNA fragmentation were observed in haemocytes collected from artificial seawater injected oysters than in haemocytes collected from OsHV-1 infected oysters at 24 and 48hpi, suggesting an inhibition of the apoptotic process in presence of the virus. In conclusion, this study is the first to focus on C. gigas haemocytes, cells involved in the host immune defense, during an OsHV-1 challenge in controlled conditions by combining various and original approaches to investigate apoptosis at molecular and cellular levels.

Involvement of Gaucher Disease Mutations in Parkinson Disease.

Gaucher disease is an autosomal recessive lysosomal storage disorder, caused by mutations in the GBA gene. The frequency of Gaucher disease patients and heterozygote carriers that developed Parkinson disease has been found to be above that of the control population. This fact suggests that mutations in the GBA gene can be involved in Parkison's etiology. Analysis of large cohorts of patients with Parkinson disease has shown that there are significantly more cases bearing GBA mutations than those found among healthy individuals. Functional studies have proven an interaction between α-synuclein and GBA, the levels of which presented an inverse correlation. Mutant GBA proteins cause increases in α-synuclein levels, while an inhibition of GBA by α-synuclein has been also demonstrated. Saposin C, a coactivator of GBA, has been shown to protect GBA from this inhibition. Among the GBA variants associated with Parkinson disease, E326K seems to be one of the most prevalent. Interestingly, it is involved in Gaucher disease only when it forms part of a double-mutant allele, usually with the L444P mutation. Structural analyses have revealed that both residues (E326 and L444) interact with Saposin C and, probably, also with α-synuclein. This could explain the antagonistic role of these two proteins in relation to GBA.

Changes in proteins, physical stability and structure in directly heated UHT milk during storage at different temperatures

Changes occurring in directly heated UHT milk were studied during storage at 5, 22, 30 and 40 °C. Industrially produced UHT milk samples were analysed for changes in enzymatic activity, protein modification, destabilisation of casein micelles and relocation of milk proteins in relation to sedimentation and gel formation. Sedimentation occurred at all temperatures, and the protein composition of the sediments reflected the composition of its liquid phase; however, there was no α-lactalbumin, β-lactoglobulin or κ-casein present in sediments. Tendrils composed of β-lactoglobulin and κ-casein were seen on casein micelles after UHT treatment and grew in length prior to gelation. High degrees of lactosylation of proteins and peptides were clearly correlated with the absence of gelation and long tendrils. Gelled samples showed complete hydrolysis of intact β-casein, and limited lactosylation of β-lactoglobulin and κ-casein.