scholarly journals Differentiation and numerical analysis of oral yeasts based on SDS-Page profiles. Influence of the culture media on the whole-cell protein extracts

2001 ◽  
Vol 61 (3) ◽  
pp. 507-516 ◽  
Author(s):  
J. F. HÖFLING ◽  
E. A. R. ROSA ◽  
C. V. PEREIRA ◽  
M. F. G. BORIOLLO ◽  
J. A. O. RODRIGUES

The application of gel electrophoresis and numerical analysis of yeast soluble proteins analysis to the investigation of 12 oral yeast strains belonging to five species is described. It involves one-dimensional electrophoresis of SDS-solubilized whole-cell proteins using different culture media for the cultivation of the cells, integration densitometries in the areas of the gels and percentages of the proteins extraction. These extracts were prepared from four isolates of Candida albicans, two of C. tropicalis, C. guilliermondii, C. parapsilosis and C. krusei. The extracts from whole-cells proteins using different culture media for the cultivation of the cells were fractionated by slab electrophoresis using a discontinuous buffer system. The corresponding patterns showed at least 36 polypeptides in the range of 14.4-200 kDa. Different isolates of each species were clearly different in each of the five species. The data obtained suggest that different nutritional compositions led to the expression of different proteins derived from alternatives metabolic pathways expressed by the electrophoretic profiles. The construction of a database of protein fingerprints and numerical analysis based on such data, may have some implications in the classification and identification of such species with epidemiological, ecological and taxonomic purposes. A well defined or synthetic culture media seems to be much properly.

2003 ◽  
Vol 45 (5) ◽  
pp. 249-257 ◽  
Author(s):  
Marcelo Fabiano Gomes Boriollo ◽  
Edvaldo Antonio Ribeiro Rosa ◽  
Wagner Luis de Carvalho Bernardo ◽  
Reginaldo Bruno Gonçalves ◽  
José Francisco Höfling

The aim of this research was to evaluate the protein polymorphism degree among seventy-five C. albicans strains from healthy children oral cavities of five socioeconomic categories from eight schools (private and public) in Piracicaba city, São Paulo State, in order to identify C. albicans subspecies and their similarities in infantile population groups and to establish their possible dissemination route. Cell cultures were grown in YEPD medium, collected by centrifugation, and washed with cold saline solution. The whole-cell proteins were extracted by cell disruption, using glass beads and submitted to SDS-PAGE technique. After electrophoresis, the protein bands were stained with Coomassie-blue and analyzed by statistics package NTSYS-pc version 1.70 software. Similarity matrix and dendrogram were generated by using the Dice similarity coefficient and UPGMA algorithm, respectively, which made it possible to evaluate the similarity or intra-specific polymorphism degrees, based on whole-cell protein fingerprinting of C. albicans oral isolates. A total of 13 major phenons (clusters) were analyzed, according to their homogeneous (socioeconomic category and/or same school) and heterogeneous (distinct socioeconomic categories and/or schools) characteristics. Regarding to the social epidemiological aspect, the cluster composition showed higher similarities (0.788 < S D < 1.0) among C. albicans strains isolated from healthy children independent of their socioeconomic bases (high, medium, or low). Isolates of high similarity were not found in oral cavities from healthy children of social stratum A and D, B and D, or C and E. This may be explained by an absence of a dissemination route among these children. Geographically, some healthy children among identical and different schools (private and public) also are carriers of similar strains but such similarity was not found among other isolates from children from certain schools. These data may reflect a restricted dissemination route of these microorganisms in some groups of healthy scholars, which may be dependent of either socioeconomic categories or geographic site of each child. In contrast to the higher similarity, the lower similarity or higher polymorphism degree (0.499 < S D < 0.788) of protein profiles was shown in 23 (30.6%) C. albicans oral isolates. Considering the social epidemiological aspect, 42.1%, 41.7%, 26.6%, 23.5%, and 16.7% were isolates from children concerning to socioeconomic categories A, D, C, B, and E, respectively, and geographically, 63.6%, 50%, 33.3%, 33.3%, 30%, 25%, and 14.3% were isolates from children from schools LAE (Liceu Colégio Albert Einstein), MA (E.E.P.S.G. "Prof. Elias de Melo Ayres"), CS (E.E.P.G. "Prof. Carlos Sodero"), AV (Alphaville), HF (E.E.P.S.G. "Honorato Faustino), FMC (E.E.P.G. "Prof. Francisco Mariano da Costa"), and MEP (E.E.P.S.G. "Prof. Manasses Ephraim Pereira), respectively. Such results suggest a higher protein polymorphism degree among some strains isolated from healthy children independent of their socioeconomic strata or geographic sites. Complementary studies, involving healthy students and their families, teachers, servants, hygiene and nutritional habits must be done in order to establish the sources of such colonization patterns in population groups of healthy children. The whole-cell protein profile obtained by SDS-PAGE associated with computer-assisted numerical analysis may provide additional criteria for the taxonomic and epidemiological studies of C. albicans.


2019 ◽  
Vol 13 (4) ◽  
pp. 317 ◽  
Author(s):  
Achmad Suhermanto ◽  
Sukenda Sukenda ◽  
Muhammad Zairin Jr. ◽  
Angela Mariana Lusiastuti ◽  
Sri Nuryati

Bakteri Streptococcus agalactiae tipe β-hemolitik dan non-hemolitik menjadi agen penyebab infeksi streptococcosis yang mengakibatkan kematian dan kerugian besar pada budidaya ikan nila. Penelitian ini bertujuan untuk membandingkan toksisitas sel utuh dan extracellular product (ECP) bakteri b-hemolitik dan non-hemolitik yang diinjeksikan pada ikan nila. Karakterisasi S. agalactiae berdasarkan SNI dan API 20 STREP, serta pemisahan protein dengan metode SDS-PAGE. Pengujian toksisitas dilakukan dengan cara menginjeksikan sel utuh dan ECP S. agalactiae secara intraperitoneal (IP) dengan dosis 0,1 mL ekor-1. Hasil uji biokimia, dan konfirmasi dengan API 20 STREP menunjukkan bahwa semua isolat positif S. agalactiae. Fraksinasi protein pada sel utuh bakteri diperoleh pita protein masing-masing sebanyak sembilan dan tujuh pita pada tipe β-hemolitik dan non-hemolitik. Fraksinasi ECP teridentifikasi pada β-hemolitik sebanyak tujuh pita dan non-hemolitik empat pita protein. Konsentrasi protein sel utuh dan ECP b-hemolitik lebih besar dibandingkan bakteri non-hemolitik. Gejala abnormalitas lebih cepat terjadi pada ikan nila yang diinjeksi ECP bakteri b-hemolitik dan berbanding lurus dengan kematian sebanyak 91%-100% pada jam ke-13 pascainjeksi. Hasil ini menunjukkan bahwa ECP bakteri S. agalactiae β-hemolitik lebih virulen dibandingkan tipe non-hemolitik. Hingga akhir pemeliharaan tidak ada kematian pada ikan yang diinjeksi sel utuh bakteri S. agalactiae b-hemolitik dan non-hemolitik. Studi histopatologi ikan yang diinjeksi ECP S. agalactiae pada organ hati, limpa, otak, dan ginjal menunjukkan adanya kongesti, hemoragi, dan nekrosis.The β-hemolytic and non-hemolytic biotype of Streptococcus agalactiae are the agents that cause streptococcosis infection which resulted in high mortality and major losses in tilapia culture. This study aimed to compare the toxicity of whole cell and extracellular product (ECP) b-hemolytic and non-hemolytic bacteria from injected tilapia. Characterization of S. agalactiae was based on SNI and API 20 STREP and protein separation by SDS-PAGE method. Toxicity test was carried out by injecting whole cells and ECP S. agalactiae intraperitoneally with a dose of 0.1 mL fish-1. The results of biochemical tests, with confirmation by API 20 STREP showed that all isolates were positive for S. agalactiae. Protein fractionation of whole bacterial cells obtained as many as nine and seven bands of protein in b-hemolytic and non hemolytic biotype, respectively. ECP fractionation was identified in β-hemolytic biotype as many as seven bands and four protein bands in non-hemolytic. The whole cell protein concentration and ECP β-hemolytic were higher than non-hemolytic bacteria. Symptoms of abnormalities occurred faster in tilapia which was injected with ECP b-hemolytic bacteria and had positive correlation with 91%-100% mortalities at the 13th hours post-injection. This results indicated that ECP of S. agalactiae β-hemolytic are more virulent than non-hemolytic. Until the end of the trial, there were no deaths in fish injected with whole cells of b-hemolytic and non-hemolytic S. agalactiae. Histopathological studies of ECP-injected fish S. agalactiae in the liver, spleen, brain, and kidneys showed congestion, hemorrhage, and necrosis. 


2004 ◽  
Vol 54 (4) ◽  
pp. 1235-1237 ◽  
Author(s):  
Tom Coenye ◽  
Elke Vanlaere ◽  
Enevold Falsen ◽  
Peter Vandamme

Type and reference strains of Stenotrophomonas maltophilia and Stenotrophomonas africana were compared with each other and with the type strains of other Stenotrophomonas species, using SDS-PAGE of whole-cell proteins, DNA–DNA hybridization and extensive biochemical characterization. S. maltophilia LMG 958T and S. africana LMG 22072T had very similar whole-cell-protein patterns and were also biochemically very similar. A DNA–DNA binding level of 70 % between both type strains confirmed that S. africana and S. maltophilia represent the same taxon. It is concluded that S. africana is a later synonym of S. maltophilia.


2010 ◽  
Vol 55 (No. 6) ◽  
pp. 259-263 ◽  
Author(s):  
A. Aksakal

This study was carried out to determine the whole cell protein profiles of Salmonella serovars from chicken, turkey and sheep faeces by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). A total of 34 Salmonella strains were included in the study, 14 of them were isolated from chicken, 14 from turkey and six from sheep. SDS-PAGE was carried out using 12% (w/v) separating and 4% (w/v) stacking gels. The results showed more than 30 protein bands ranging in size from 97 kDa (kilodaltons) to below 14.4 kDa as determined by visual assessment of their approximate molecular masses. Protein bands of 78.1, 51.2, 41.5, 37.3, 35.1, 33.9, 30.7, 27.6, 25.4, and 24 kDa were detected in all Salmonella serovars. Salmonella strains used in this study were closely related and could not be differentiated depending on the whole cell protein profiles using SDS-PAGE.


1996 ◽  
Vol 33 (3) ◽  
pp. 169-171 ◽  
Author(s):  
I.A. Khan ◽  
A. Rattan ◽  
T. Fatima ◽  
F.G. Khan ◽  
A. Kalia

1977 ◽  
Author(s):  
H. Horák ◽  
P.G. Barton ◽  
C.M. Gibbs

Horse platelet membranes isolated by the glycerol lysis technique and subjected to SDS-PAGE showed large amounts of actin and variable amounts of myosin relative to other membrane proteins and glycoproteins. [14C]-2-dinitrothioadenosine diphosphate, when briefly incubated with whole cells, rapidly labeled the membrane actin component. Retention of myosin by the membranes during their isolation was optimized by lysing the cells and resuspending the membranes in Tris-HC1, pH 7.35, with 0.13 M KCl, 0.01 M NaCl, 2 mM MgCl2 and 0.01 mM CaCl2. Subsequently, significant amounts of actin and myosin could be eluted from the membranes with 10-3 M ADP but not with CDP, GDP or UDP. Actin was also eluted effectively from membranes prepared in Tris-NaCl, pH 7.35, by washing with 0.1 mM EDTA (in presence or absence of ADP). Despite repeated washings with either elution system, more than 50% of the actin remained associated with the membranes. When membrane vesicles with right side out (RO) and inside out (IO) orientation, separated by chromatography on Con A-Sepharose, were similarly washed identical results were obtained. Two dimensional electrophoresis of the membrane protein of IO vesicles separated two major actin components, one of which was differentially removed by prior treatment of the vesicles with 10-3 M ADP. It is concluded that (1) platelet myosin and two forms of platelet actin are associated with the cell membrane and that myosin and one form of actin can be displaced by ADP or EDTA while the second form of actin is more firmly attached, and (2) some actin is present on both membrane surfaces.


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