75 PLACENTA PROTEIN PROFILE CHARACTERIZATION BY PLACENTOME SIZE AND GESTATIONAL AGE IN CATTLE

2017 ◽  
Vol 29 (1) ◽  
pp. 145 ◽  
Author(s):  
M. M. Ramirez ◽  
J. F. Martins ◽  
P. R. Villamil ◽  
M. Bertolini ◽  
L. R. Bertolini ◽  
...  
Keyword(s):  
In Silico ◽  
Protein Identification ◽  
Protein Extraction ◽  
Tissue Sample ◽  
Protein Profile ◽  
Biological Significance ◽  
Cell Protein ◽  
Protein Pool ◽  
Freeze Dried ◽  
Conceptus Development

The placenta plays a key role in normal conceptus development and in mediating effects of the maternal system on the fetus. Changes in the environment or in placental function may affect fetal developmental and lead to what is known as “developmental origins of health and disease.” Thus, the aim of this study was to analyse the protein profile of bovine placentomes of different sizes at distinct gestation ages to determine spatial and temporal differences or similarities in the protein pool of biological significance to the conceptus and the newborn. Samples of placentomes (n = 36) representing small, medium, and large sizes among placentomes at 60, 90, 120, and 160 days of gestation were collected at a local slaughterhouse and freeze-dried for protein analysis. For that, 5 mg of each tissue sample was used for protein extraction with 1% Triton X and sonication for 10 min at 4°C, with protein concentration per sample determined by the Bradford method. A total of 300 μg of protein was subjected to 2D-SDS PAGE electrophoresis (GE Healthcare®), and gels were stained with Coomassie Blue G-250. Spots were analysed using the PDQuest, with data compared by ANOVA or the Kruskall-Wallis test (SAS). In silico protein identification was performed using ExPASy. On average, 74 proteins were detected in the gels, regardless of placentome sizes and gestational ages. No interactions were detected between placentome sizes and gestational periods. However, differences were observed in the mean number of proteins between small (n = 69), medium (n = 90), and large (n = 64) placentomes (P = 0.002), and between Days 60 (n = 113), 90 (n = 65), 120 (n = 44), and 160 (n = 74) of gestation (P = 0.001). One specific spot (32.45 kDa, 4.6 pI) represented 1.0, 10.8, 12.1, and 2.1% of the intensity of all valid spots for Day 60, 90, 120, and 160 placentomes, respectively, indicating a bell-like expression. For small placentomes, all the spots had similar intensities, whereas for medium placentomes, the 32.45 kDa (4.6 pI) spot represented 3.0% of the intensity of all valid spots. For large placentomes, the 32.45 kDa (4.6 pI) spot and a 46.19 kDa (5.4 pI) spot represented 5.8 and 5.0% of the total intensity for all spots, respectively. Six and 10 spots were differentially identified between placentome sizes and between gestational periods, respectively, from which male-enhanced antigen-1 proteins and angio-associated migratory cell protein were identified by in silico analysis, with the former only present on Day 90 placentomes, and the latter for 120 days of gestation. Further analyzes on the identities of such placental proteins and their profiles is underway, which will be crucial to the comprehension of placentome function and growth during pregnancy, and for the understanding of physiological mechanisms and processes associated with normal conceptus development and life ex utero.

scholarly journals Two-dimensional gel electrophoretic protein profile analysis during seed development of Ocotea catharinensis: a recalcitrant seed species

2010 ◽  
Vol 22 (1) ◽  
pp. 23-33 ◽  
Author(s):  
Leonardo L.C. Dias ◽  
Tiago S. Balbuena ◽  
Vanildo Silveira ◽  
Claudete Santa-Catarina ◽  
Andrej Schevchenko ◽  
...  
Keyword(s):  
Seed Development ◽  
Protein Identification ◽  
Protein Extraction ◽  
Profile Analysis ◽  
Protein Profile ◽  
Two Dimensional ◽  
Two Dimensional Gel Electrophoresis ◽  
Recalcitrant Seed ◽  
And Storage ◽  
Cotyledonary Stage

The aim of the present work was to characterize changes in the protein profile throughout seed development in O. catharinensis, a recalcitrant species, by two-dimensional gel electrophoresis. Protein extraction was undertaken by using a thiourea/urea buffer, followed by a precipitation step with 10% TCA. Comparative analysis during seed development showed that a large number of proteins were exclusively detected in each developmental stage. The cotyledonary stage, which represents the transition phase between embryogenesis and the beginning of metabolism related to maturation, presents the highest number of stage-specific spots. Protein identification, through MS/MS analysis, resulted in the identification of proteins mainly related to oxidative metabolism and storage synthesis. These findings contribute to a better understanding of protein metabolism during seed development in recalcitrant seeds, besides providing information on established markers that could be useful in defining and improving somatic embryogenesis protocols, besides monitoring the development of somatic embryos in this species.

scholarly journals Biochemical aspects of seeds from Cannabis sativa L. plants grown in a mountain environment

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Chiara Cattaneo ◽  
Annalisa Givonetti ◽  
Valeria Leoni ◽  
Nicoletta Guerrieri ◽  
Marcello Manfredi ◽  
...  
Keyword(s):  
Fatty Acid ◽  
Protein Identification ◽  
Cannabis Sativa ◽  
Protein Extraction ◽  
Protein Profile ◽  
Climatic Conditions ◽  
Mountain Area ◽  
Mountain Environments ◽  
Mountain Environment ◽  
Analytical Approaches

AbstractCannabis sativa L. (hemp) is a versatile plant which can adapt to various environmental conditions. Hempseeds provide high quality lipids, mainly represented by polyunsaturated acids, and highly digestible proteins rich of essential aminoacids. Hempseed composition can vary according to plant genotype, but other factors such as agronomic and climatic conditions can affect the presence of nutraceutic compounds. In this research, seeds from two cultivars of C. sativa (Futura 75 and Finola) grown in a mountain environment of the Italian Alps were analyzed. The main purpose of this study was to investigate changes in the protein profile of seeds obtained from such environments, using two methods (sequential and total proteins) for protein extraction and two analytical approaches SDS-PAGE and 2D-gel electrophoresis, followed by protein identification by mass spectrometry. The fatty acids profile and carotenoids content were also analysed. Mountain environments mainly affected fatty acid and protein profiles of Finola seeds. These changes were not predictable by the sole comparison of certified seeds from Futura 75 and Finola cultivars. The fatty acid profile confirmed a high PUFA content in both cultivars from mountain area, while protein analysis revealed a decrease in the protein content of Finola seeds from the experimental fields.

scholarly journals Effects of the Varietal Diversity and the Thermal Treatment on the Protein Profile of Peanuts and Hazelnuts

2018 ◽  
Vol 2018 ◽  
pp. 1-10 ◽  
Author(s):  
Elisabetta De Angelis ◽  
Simona Lucia Bavaro ◽  
Linda Monaci ◽  
Rosa Pilolli
Keyword(s):  
Thermal Treatment ◽  
Protein Modification ◽  
Protein Identification ◽  
Protein Extraction ◽  
Protein Profile ◽  
Protein Solubility ◽  
Differential Resistance ◽  
Extraction Yield ◽  
Significant Difference ◽  
Before And After

Several buffer compositions were compared for their efficiency in protein extraction from both raw and roasted peanut and hazelnut samples, the final goal being to understand the modification of protein solubility upon roasting and maximize the extraction yield. Denaturant conditions provided by urea-TBS buffer resulted in satisfactory extraction yields for both peanut and hazelnut samples, before and after the thermal treatment. In addition, different varieties of peanuts and hazelnuts were characterized to highlight the extent of variability in the protein profile accounted by the varietal factor and eventual differential resistance among cultivars to protein modification induced by the thermal processing. The protein profile was characterized by gel electrophoresis, and specific bands were analyzed by micro-HPLC-MS/MS coupled to software-based protein identification. No significant difference was observed for the investigated hazelnut cultivars, namely, Campana, Romana, and Georgia, whereas interesting features were presented for the peanut varieties Virginia, Zambia, and China. In particular, Zambia variety lacked two bands of approximately 36 and 24 kDa that were visible in Virginia and China varieties, which could suggest a lower allergenic potential of this particular variety which deserves to be further investigated before drawing final conclusions.

scholarly journals Functional properties of protein isolates from camelina (Camelina sativa (L.) Crantz) and flixweed (sophia, Descurainis sophia L.) seed meals

2021 ◽  
Vol 3 (1) ◽  
Author(s):  
Na Thi Ty Ngo ◽  
Fereidoon Shahidi
Keyword(s):  
Functional Properties ◽  
Protein Extraction ◽  
Protein Profile ◽  
Protein Solubility ◽  
Protein Isolate ◽  
Absorption Capacity ◽  
Foaming Properties ◽  
Oil Absorption ◽  
Protein Isolates ◽  
Water Holding

AbstractCamelina and flixweed (sophia) seed protein isolates were prepared using both the conventional extraction and ultrasonic-assisted extraction methods at 40 kHz for 20 min, and their functional properties investigated. SDS-PAGE showed that both ultrasound-assisted and conventional extractions resulted in a similar protein profile of the extract. The application of ultrasound significantly improved protein extraction/content and functional properties (water holding capacity, oil absorption capacity, emulsifying foaming properties, and protein solubility) of camelina protein isolate and sophia protein isolate. The water-holding and oil absorption capacities of sophia protein isolate were markedly higher than those of camelina protein isolate. These results suggest that camelina protein isolate and sophia protein isolate may serve as natural functional ingredients in the food industry. Graphical Abstract

Preliminary screening for toxin genes amongst stock cultures of Clostridium perfringens strains isolated from dogs and calves

2014 ◽  
Vol 4 (3) ◽  
pp. 127-132
Author(s):  
Egwari L. O. ◽  
Oghogho E. S. ◽  
Okwumabua O. E. ◽  
Oniha M. I.
Keyword(s):  
Clostridium Perfringens ◽  
Protein Profile ◽  
Animal Origin ◽  
Cell Protein ◽  
Toxin Gene ◽  
Whole Cell ◽  
University Of Wisconsin ◽  
Gene Type ◽  
Whole Cell Protein ◽  
Gene 4

The spectrum of Clostridium perfringens infections ranges from food toxinosis to myonecrosis. In the current study, whole cell protein and toxin gene types were profiled in 12 randomly selected C. perfringens veterinary stock cultures from the University of Wisconsin, Madison to determine epidemio-logical similarity, or diversity amongst strains of animal origin. Whole cell protein analysis was done by SDS-PAGE while toxin gene typing was achieved by extracting DNA by boiling, DNA concentration and purity was determined by spectrophotometer and nanodrop while separation was carried out by checking it on gel electrophoresis. Multiplex PCR was used to identify the toxigenic gene-type. C. perfringens B and C. perfringens EE with established profiles were used as control strains. Isolates typed included strains cp 296, 309, 12872 (from dogs) and 304, 305, 306, 341, 342, 10754, 12218-2, 12218-3, 12473 (from calves). All 12 strains possess the cpa gene, 4 strains have cpb2, 3 strains etx, 2 strains positive for cpe and 1 for cpb. None of the strains carries the itx gene. Two strains have only cpa gene however no strains has more than two toxin gene types, with cpa-cpb2 combination be-ing more frequent. C. perfringens 305 (etx and cpa) and 342 (cpe and cpa) shared the same protein profile but belong to different toxinotype. It is evi-dent that the cpa gene is a marker for all C. perfringens strains, and similarity in protein profile is not sine qua non for toxin gene type.

scholarly journals Effects of preservation on protein extraction in four seaweed species

2020 ◽  
Vol 32 (5) ◽  
pp. 3401-3409
Author(s):  
Tom Wijers ◽  
Alwin Hylkema ◽  
Tsjippie Visser ◽  
Klaas Timmermans
Keyword(s):  
Protein Content ◽  
Protein Extraction ◽  
Saccharina Latissima ◽  
Chondrus Crispus ◽  
Seaweed Species ◽  
Preservation Method ◽  
Freeze Dried ◽  
The Right ◽  
Kjeldahl Nitrogen ◽  
Protein Extractability

Abstract Using either freshly pulped or preserved seaweed biomass for the extraction of protein can have a great effect on the amount of protein that can be extracted. In this study, the effect of four preservation techniques (frozen, freeze-dried, and air-dried at 40 and 70 °C) on the protein extractability, measured as Kjeldahl nitrogen, of four seaweed species, Chondrus crispus (Rhodophyceae), Ascophyllum nodosum, Saccharina latissima (both Phaeophyceae) and Ulva lactuca (Chlorophyceae), was tested and compared with extracting freshly pulped biomass. The effect of preservation is species dependent: in all four seaweed species, a different treatment resulted in the highest protein extractability. The pellet (i.e., the non-dissolved biomass after extraction) was also analyzed as in most cases the largest part of the initial protein ended up in the pellet and not in the supernatant. Of the four species tested, freeze-dried A. nodosum yielded the highest overall protein extractability of 59.6% with a significantly increased protein content compared with the sample before extraction. For C. crispus extracting biomass air-dried at 40 °C gave the best results with a protein extractability of 50.4%. Preservation had little effect on the protein extraction for S. latissima; only air-drying at 70 °C decreased the yield significantly. Over 70% of the initial protein ended up in the pellet for all U. lactuca extractions while increasing the protein content significantly. Extracting freshly pulped U. lactuca resulted in a 78% increase in protein content in the pellet while still containing 84.5% of the total initial total protein. These results show the importance of the right choice when selecting a preservation method and seaweed species for protein extraction. Besides the extracted protein fraction, the remaining pellet also has the potential as a source with an increased protein content.

A mass spectrometry-based approach to host cell protein identification and its application in a comparability exercise

2014 ◽  
Vol 463 ◽  
pp. 1-6 ◽  
Author(s):  
Veronika Reisinger ◽  
Hansjoerg Toll ◽  
Robert Ernst Mayer ◽  
Jan Visser ◽  
Florian Wolschin
Keyword(s):  
Mass Spectrometry ◽  
Host Cell ◽  
Protein Identification ◽  
Cell Protein ◽  
Host Cell Protein ◽  
Comparability Exercise

Developmental Changes in the PH of Enamel Fluid and Its Effects On Matrix-Resident Proteinases

1996 ◽  
Vol 10 (2) ◽  
pp. 159-169 ◽  
Author(s):  
C.E. Smith ◽  
M. Issid ◽  
H.C. Margolis ◽  
E.C. Moreno
Keyword(s):  
Acetic Acid ◽  
Protein Extraction ◽  
Maturation Stage ◽  
Activity Levels ◽  
Freeze Dried ◽  
Mineral Components ◽  
Acidic Conditions ◽  
Pre Treatment

The objectives of this study were to measure pH in developing enamel at progressively older (more mature) stages of amelogenesis in vivo, and then to formulate synthetic enamel fluid mixtures that approximated these pH values for in vitro studies. The ultimate goal was to characterize the molecular weights of proteinases visualized by enzymograms incubated in synthetic enamel fluid using gelatin and casein as substrates. For most experiments. the proteinases were extracted en masse from small freeze-dried enamel strips directly into a non-reducing sample preparation buffer. In some experiments, we pre-treated the enamel strips with acetic acid to determine if this common method for demineralization and protein extraction caused any changes in the activity levels of the enamel proteinases. In other experiments, we first soaked enamel strips in synthetic enamel fluid to determine solubility of the proteinases within an aqueous phase. The results indicated that the pH of developing enamel remained fairly constant near pH 7.23 across the secretory stage, but it was generally more acidic (6.93) and fluctuated in focal areas between mildly acidic (6.2-6.8) and near-neutral (7.2) conditions across the maturation stage. The pH then slowly rose to near 7.35 when the enamel was almost mature (hard). The acidic conditions were generally inhibitory to most enamel proteinases, but there were some caseinase activities in mid-maturation-stage enamel near 23-30 kDa which appeared to be activated by weakly acidic conditions (pH 6.28). Pre-treatment of enamel samples with 0.5 M acetic acid markedly altered the overall profile of enamel proteinases, causing activation of some latent proteinase activities and permanent inhibition of other activities. Most proteinases in whole homogenates were insoluble in synthetic enamel fluid. This suggests that they may be tightly bound, directly or indirectly, to matrix proteins or mineral components in situ.

scholarly journals Pseudomonas aeruginosa Displays Multiple Phenotypes during Development as a Biofilm

2002 ◽  
Vol 184 (4) ◽  
pp. 1140-1154 ◽  
Author(s):  
Karin Sauer ◽  
Anne K. Camper ◽  
Garth D. Ehrlich ◽  
J. William Costerton ◽  
David G. Davies
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Quorum Sensing ◽  
Protein Identification ◽  
Electrophoretic Analysis ◽  
Cell Protein ◽  
Biofilm Growth ◽  
Alginate Production ◽  
Multiple Phenotypes ◽  
Reporter Gene Analysis ◽  
Biofilm Development

ABSTRACT Complementary approaches were employed to characterize transitional episodes in Pseudomonas aeruginosa biofilm development using direct observation and whole-cell protein analysis. Microscopy and in situ reporter gene analysis were used to directly observe changes in biofilm physiology and to act as signposts to standardize protein collection for two-dimensional electrophoretic analysis and protein identification in chemostat and continuous-culture biofilm-grown populations. Using these approaches, we characterized five stages of biofilm development: (i) reversible attachment, (ii) irreversible attachment, (iii) maturation-1, (iv) maturation-2, and (v) dispersion. Biofilm cells were shown to change regulation of motility, alginate production, and quorum sensing during the process of development. The average difference in detectable protein regulation between each of the five stages of development was 35% (approximately 525 proteins). When planktonic cells were compared with maturation-2 stage biofilm cells, more than 800 proteins were shown to have a sixfold or greater change in expression level (over 50% of the proteome). This difference was higher than when planktonic P. aeruginosa were compared with planktonic cultures of Pseudomonas putida. Las quorum sensing was shown to play no role in early biofilm development but was important in later stages. Biofilm cells in the dispersion stage were more similar to planktonic bacteria than to maturation-2 stage bacteria. These results demonstrate that P. aeruginosa displays multiple phenotypes during biofilm development and that knowledge of stage-specific physiology may be important in detecting and controlling biofilm growth.

Whole-cell protein and ITS rDNA profiles as diagnostic tools to discriminate Fusarium avenaceum intraspecific variability and associated virulence

2009 ◽  
Vol 55 (2) ◽  
pp. 117-125 ◽  
Author(s):  
V. Vujanovic ◽  
S. Vidovic ◽  
M. R. Fernandez ◽  
P. Daida ◽  
D. Korber
Keyword(s):  
Protein Profile ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Intraspecific Variability ◽  
Fusarium Avenaceum ◽  
Cell Protein ◽  
Diagnostic Tools ◽  
Group Method ◽  
Head Blight ◽  
Sds Page

A total of 91 isolates of Fusarium avenaceum were regrouped into 15 phenotypes and 10 vegetative compatibility groups showing specific one-dimensional sodium dodecyl sulphate polyacrylamide gel electrophoresis (1-D SDS–PAGE) protein profiles and less-specific internal transcribed spacer rDNA profiles. Each isolate possessed reproducible signature protein bands. Indeed, the unweighted pair group method with arithmetic averages clustering revealed that the protein profile of each group of isolates correlated with fungus virulence. The use of SDS–PAGE offers a simple and sensitive technique for routine differentiation between pathogenic and nonpathogenic isolates within unknown F. avenaceum populations. The discovery has significant implications for risk assessment of cereal yield to ensure food and feed safety. This low-cost approach has the potential to be optimized and extended to a broad spectrum of Fusarium head blight pathogens.

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