Isoelectric Focusing Of Soluble Proteins Research Articles

Quantification of Leptosphaeria maculans Growth in Cotyledons of Brassica napus Using ELISA

AbstractThe result of the Leptosphaeria maculans/Brassica napus interaction is usually assessed by symptom scoring following a cotyledon‐inoculation test. However, an early evaluation of the interaction, and reliable quantitaive data of fungal growth inside plant tissues are needed to supplement the visual assessment of the symptoms. For this purpose, we developed a quantitatve double antibody sandwich enzyme‐linked immunosorbent assay (DAS‐ELISA) using rabbit polyclonal antisera directed against soluble mycelial proteins. The specificity of the serum was first assessed by immunoblotting following isoelectric focusing of soluble proteins (Western blot) and by DAS‐ELISA. Except for Alternaria brassicae, no cross‐reactions were observed with my celial extracts of saprophytes or pathogens of B. napus following DAS‐ELISA. Although Tox+ and Tox0 isolates of L. maculans were unequivocally discriminated by Western blot, they were quantitatively indistinguishable following ELISA, thus enabling us to analyse a wide range of L. maculans isolates in planta. The detection limit of the assay was less than 10 ng of fungal proteins per ml of plant extract. For a given isolate, time‐course studies showed that fungal growth in cotyledons was correlated with symptom scoring. In the case of hypersensitive response, only 34% of the plants were ELISA‐positive, and these plants never contained more than 10 ng of fungal protein per cotyledon. In contrast, in the cases, of susceptibility, 100% of the plants were ELISA‐positive and fungal protein content was higher than 10 μg per cotyledon. Moreover, significant differences in ability to colonize the tissues were observed among Tox+ isolates. Finally, using the ELISA quantification, intermediate symptoms could be differentiated as lateresistance responses or susceptibility.

IDENTIFICATION OF SIX COMMERCIALLY CULTURED CRAYFISH SPECIES USING TOTAL PROTEIN SEPARATION BY ISOELECTRIC FOCUSING

Total soluble proteins extracted from the tail muscles of 6 commercially cultured species of crayfishes were separated using isoelectric focusing (IEF). The banding patterns produced could be used to successfully identify 5 of the 6 species. Unique protein bands at pI 5.75 and 6.02 were identified for Pacifasticus leniusculus. A unique group of 3 protein bands were identified between pI 5.46 and 5.6 in Cherax quadricarinatus. Two species of Orconectes, 0. virilis and 0. rusticus, were identified by a pattern shift in a pair of protein bands between pI 5.1 and 5.3. The only 2 species that could not be distinguished from each other based on their protein banding patterns were Procambarus clarkii and P. zonangulus. However, these 2 members of the genus Procambarus could be differentiated from the other species examined by the presence of a protein band at pI 5.1 not found in the other species. Species-specific protein banding patterns were observed in juveniles, females, and Form I and Form II males, suggesting that the patterns used for species identification do not appear to be dependent on age or sex. Furthermore, these protein banding patterns were not dependent on the molt stage. The analysis of IEF of soluble proteins, coupled with the analysis of traditional morphological characteristics, may be a valuable tool for species identification in crayfish.

Protein profiles of Brucella suis and Brucella abortus in isoelectric focusing and sodium dodecyl sulphate — polyacrylamide gel electrophoresis

The protein profiles of five field isolates of Brucella suis and four field isolates of B. abortus were examined. Isoelectric focusing of soluble proteins from cell sonicates produced 28 bands common to both species, with a further 10 bands being unique to a species. No intraspecies variation was detected. Sodium dodecyl sulfate extracted proteins were resolved by polyacrylamide gel electrophoresis. The proteins were transferred to nitrocellulose and then stained. These techniques allowed the demonstration of 35 bands which were common to both species, with a further 15 bands which were unique to one of the two species. Again, no intraspecies variation was detected.

Banding Patterns of Fasciola hepatica and Fasciola gigantica (Trematoda) by Isoelectric Focusing

Liver flukes Fasciola hepatica and Fasciola gigantica are polymorphic and vary morphologically depending upon the host being parasitized. It is known also that mixed infection occurs where both species are present. A technique involving protein separation was used to distinguish the 2 species. Isoelectric focusing of soluble proteins was performed on polyacrylamide gels using whole-body proteins from adult flukes. Although many bands appeared common to both species and some were shared with host tissues, the banding patterns could be used to distinguish 1 species from the other. Soluble protein isoelectric focusing is simple, reproducible, and has very good resolution. It seems well suited to the differentiation of the 2 fluke species.

Morphological variability of potato cyst nematodes (Globodera spp.) in the Canary Islands

Thirty populations of potato cyst nematode (Globodera spp.) from the Island of Tenerife and two populations from the UK were assessed for several morphometric and non-morphometric characters thought to discriminate between the species G. rostochiensis and G. pallida. Also 200 cysts from each population were analysed by isoelectric focusing of soluble proteins. Correlation analysis, analysis of variance and principal component analysis were used to investigate relationships between the morphometric characters, how the relationships varied between species and between populations, and which characters were most useful for discriminating between species. The two species differed significantly for each of the four morphometric characters: stylet length, fenestra length, anus-fenestra distance and the number of ridges. The stylet length and fenestra length also showed differences between populations of G. rostochiensis while stylet length and number of ridges showed differences between populations of C. pallida. In general, populations of G. pallida showed greater variation than populations of C. rostochiensis. Principal component analysis of the population means indicated that over 73% of the variation in the characters could be explained by the contrast of stylet and fenestra lengths against the anus-fenestra distance and number of ridges. A plot of the first two principal components separated the two species. Stepwise discriminant analysis provided a linear combination of these four variables which discriminated between the species. Stylet length was found to be the most useful characteristic for distinguishing the species whilst anus-fenestra distance was the least useful.

Isoelectric focusing of soluble proteins from Fasciola hepatica L, 1758 and Fascioloides magna B, 1875

Summary Isoelectric focusing was performed on the soluble proteins of whole-body and excretory-secretory products (esp) of Fasciola hepatica and Fascioloides magna. Adult F hepatica flukes were recovered from experimentally infected sheep and esp obtained from the flukes; portions of liver were cut and frozen at - 70 C. Fascioloides magna adults were collected from naturally infected white-tailed deer and esp obtained; portions of liver were collected from noninfected white-tailed deer. Adult flukes and their host tissues were homogenized and centrifuged; protein concentrations with their esp were determined and adjusted to < 2.50 mg/ml. Seven esp samples from F hepatica and 1 from Fascioloides magna were subjected to isoelectric focusing with the 2 species of fluke and their respective host liver homogenates. After separation, gels were stained with silver and scanned on a laser densitometer. Protein banding patterns of the 2 species of flukes were dissimilar. In the pH range of 3.5 to 9.6, the body protein had approximately 30 peaks and esp about 23 peaks in both species. Overall banding patterns of the body protein and esp of both species were distinct from those of respective host tissues. Of the peaks reported as dominant, 3 of the body protein and 2 of esp were shared between the 2 species. Fascioloides magna had more dominant peaks than F hepatica. This technique of soluble protein isoelectric focusing is simple and reproducible, and the 2 fluke species can easily be differentiated by this technique, as well as by morphologic characteristics.

Micro-isoelectric focusing of proteins from single stigmas of Populus

Isoelectric focusing of soluble proteins from single stigmas in Populus has been developed using ultrathin polyacrylamide gels. After silver nitrate staining, 50–60 protein bands are shown, thus establishing stigmatic protein fingerprints. The application of this technique to the biochemical identification of poplar clones is described in this paper, and the limits of the technique are discussed.

An investigation of host influence on soluble protein banding profiles of Hymenolepis spp. (Cestoidea), using isoelectric focusing

Protein separation techniques have been utilized in recent years as alternative means of differentiating species and strains of tapeworms. As with the traditional morphological approach, however, there is the potential for host influence. For such separative techniques to be useful taxonomically it is important that the banding profiles differentiating parasites be consistent, regardless of host species. To test for host influence, two Hymenolepis species were maintained both in preferred and alternative definitive host species. The soluble proteins of tapeworms of a given species, from the different host species, were then subjected to isoelectric focusing, and the resulting banding patterns were compared. There were no noticeable differences in the banding profiles of these worms, indicating a lack of host influence. If these findings hold for other tapeworm genera, isoelectric focusing of soluble proteins could have practical value in parasite surveys.

Development of secretory activity in the long hyaline gland of the male migratory grasshopper, Melanoplus sanguinipes (Fabr.) (Orthoptera : Acrididae)

Changes in the ultrastructure of epithelial cells from long hyaline glands of male Melanoplus sanguinipes (Fabr.) (Orthoptera : Acrididae) have been examined during sexual maturation and after allatectomy. In newly emerged males, the long hyaline gland epithelium is composed of 1–3 cell layers. The cells contain almost no rough endoplasmic reticulum, inconspicuous Golgi complexes, and large numbers of free ribosomes and polysomes. Within 24 hr, the cells undergo considerable reorganization to form a 1-cell-thick layer. Changes in cytostructure include proliferation of the rough endoplasmic reticulum and the development of several elaborate Golgi complexes. The developing lumen contains a coarse fibrous material. By 3 days postemergence, columnar epithelial cells are clearly capable of considerable synthesis and export of secretory protein. Rough endoplasmic reticulum, and large, elaborate Golgi complexes are the major structural features of the cytoplasm. From day 3 to sexual maturity (day 7), no major ultrastructural changes occur, although massive accumulation of secretion in the lumen causes the epithelium to become cuboidal or flattened. Isoelectric focusing of soluble proteins from long hyaline gland secretions shows that maturing glands contain increasing numbers and quantities of secretory proteins. Allatectomy has minor effects on long hyaline gland ultrastructure. A reduction in the density of rough endoplasmic reticulum and ribosomes suggests that glands from operated males are metabolically less active. This is confirmed by qualitative and quantitative changes in the amount of secretion as revealed by isoelectric focusing. The observations are discussed in terms of the juvenile hormone control of long hyaline gland maturation.

Isoelectric focusing of soluble proteins in the characterization of three species of Hymenolepis (Cestoda)

A technique involving protein separation was used as an alternative to a morphological approach in the differentiation of the tapeworms Hymenolepis diminuta, H. citelli, and H. microstoma. Isoelectric focusing of soluble proteins was performed on Polyacrylamide gels using extracts from whole, adult worms. Each species of Hymenolepis was found to have a unique protein banding pattern, although some bands appeared to be common to two or all three species. Very little difference was found in the protein banding patterns of worms of a given species, whether they were from a single host individual or two different host individuals of the same species. There was also little difference between gels in the banding patterns of a given species. This technique of soluble protein isoelectric focusing is simple and reproducible, has very good resolution, and seems well suited to taxonomic studies involving tapeworms.

Biochemical characterisation of Australian strains of Echinococcus granulosus by isoelectric focusing of soluble proteins

The existence of three distinct strains of E. granulosus in Australia has been previously demonstrated on the basis of several criteria. In the present study, numerous isolates of E. granulosus from domestic and wild animal populations in different geographical areas of Australia were subjected to detailed biochemical analysis using isoelectric focusing of soluble proteins. Three different populations were recognised which corresponded to the three strains described previously, thus confirming their genetic distinction. One strain is common to all domestic intermediate hosts on the Australian mainland. Evidence is presented that humans and macropod marsupials are also susceptible to infection with this strain and that it is similar to E. granulosus occurring in sheep in New Zealand and the United Kingdom. The other two strains are confined to macropod marsupials on the Australian mainland and sheep in Tasmania respectively.

Field collections of Aphis fabae scopoli S. lat. (Homoptera: Aphididae) studied by starch gel electrophoresis andisoelectric focusing

1. 1. Electrophoresis separations of two enzyme systems and isoelectric focusing of soluble proteins of black bean aphids ( Aphis fabae Scop.) have been examined. 2. 2. Results from the two systems in general supported each other and were reproducible within each single parent stock. 3. 3. Correlations were found between separation patterns and host plants and between patterns and aphids collected from different localitities. 4. 4. Single parent colonies which survived the winter oviparously did not show pattern differences the following year.