Storage protein dynamics in zygotic and somatic embryos of white fir

Composition and accumulation patterns of storage proteins in female gametophyte and embryos of the white fir (Abies concolor) were investigated during embryogenesis and germination of mature seeds using SDS-PAGE and immunological approach. Altogether 9 major and minor protein components with molecular masses of 14, 16, 22, 24, 27, 30, 35, 38, and 43 kDa were detected in female gametophytes and 9 protein bands in the embryos with the molecular sizes of 14, 16, 22, 24, 25, 27, 34, 38, and 43 kDa. The species seems to deviate in this respect from other representatives of Pinaceae. A conspicuous increase of storage protein synthesis was observed at the stage of fully cellularized female gametophytes and at the cotyledonary stage of embryo development. There exists a high degree of similarity between storage protein profiles of white fir zygotic and somatic embryos. Successive stages of somatic embryogenesis exhibited a high degree of similarity of storage proteins except for cotyledonary stage when a noticeable increase in storage protein synthesis was registered. Conversely, during germination of somatic embryos, an overwhelming majority of storage proteins was depleted.     

Patterns of protein synthesis during the cell cycle of Chinese hamster ovary cells

The protein synthesis patterns at various stages of the cell cycle of Chinese hamster ovary cells were examined by labelling cells with [35S]methionine and then separating the proteins by isoelectric focussing and two-dimensional, nonequilibrium pH gradient gel electrophoresis. We have observed a number of proteins which display quantitative differences in synthesis at specific cell cycle stages and of these the alpha- and beta-tubulins have been identified. A few proteins appear to be uniquely synthesized at specific times during the cell cycle. These include the histones and a modified version of them, which are synthesized only in S phase, and a pair of 21 kilodalton (kDa), pI 5.5 proteins, which appear only in late G2 and mitosis. We have also identified a 58-kDa, pI 7.5 protein which is present at all cell cycle stages except during late G2. This protein appears to have the same temporal properties as a 57-kDa protein called "cyclin" originally described in sea urchin embryos.     

Protein degradation in mammary gland explants evidence for limited heterogeneity of protein degradation rates

Mammary gland explants in organ culture were subjected to hormonal manipulation, and rates of protein degradation during 1 and 2 day periods were measured by a double-isotope method. Isotope ratios for protein subunits in subcellular fractions were measured after resolution by two-dimensional polyacrylamide gel electrophoresis. Frequency distribution analysis shows that the isotope ratios for each fraction are coupled predominantly in exponential distributions corresponding to populations of protein subunits with different mean degradation rates. The result also suggest that heterogeneity of protein degradation rates within each population is limited. There is no consistent correlation of degradation rate with protein isoelectric point or subunit molecular weight either overall or within any population of degradation rates. Therefore, the similarity of protein degradation rates within each population is clearly not related to these molecular properties of the proteins.     

Changes in protein synthetic activity in early Drosophila embryos mutant for the segmentation gene Krüppel

We have identified early embryo proteins related to the segmentation gene Krüppel by [35S]methionine pulse labelling and two-dimensional gel electrophoresis. Protein synthesis differences shared by homozygous embryos of two Krüppel alleles when compared to heterozygous and wild-type embryos are reported. The study was extended to syncytial blastoderm stages by pulse labelling and gel analysis of single embryos, using Krüppel-specific proteins from gastrula stages as molecular markers for identifying homozygous Krüppel embryos. Localized expression of interesting proteins was examined in embryo fragments. The earliest differences detected at nuclear migration stages showed unregulated synthesis in mutant embryos of two proteins that have stage specific synthesis in normal embryos. At the cellular blastoderm stage one protein was not synthesized and two proteins showed apparent shifts in isoelectric point in mutant embryos. Differences observed in older embryos included additional proteins with shifted isoelectric points and a number of qualitative and quantitative changes in protein synthesis. Five of the proteins with altered rates of synthesis in mutant embryos showed localized synthesis in normal embryos. The early effects observed are consistent with the hypothesis that the Krüppel product can be a negative or positive regulator of expression of other loci, while blastoderm and gastrula stage shifts in isoelectric point indicate that a secondary effect of Krüppel function may involve post-translational modification of proteins.     

Two-dimensional gel patterns of protein synthesis before and after fertilization of sea urchin eggs

Eggs and embryos of the sea urchin Lytechinus pictus were labeled with [35S]methionine. Aqueous extracts of protein were prepared and analyzed by a high resolution two-dimensional polyacrylamide gel electrophoresis system described recently by O'Farrell utilizing isoelectric focusing and sodium dodecyl sulfate electrophoresis. Out of about 400 distinctly resolved newly synthesized proteins, all but a few detectable in the zygote were being synthesized in the egg. Thus, the activation of translation of stored maternal messenger RNA following fertilization is due to a quantitative rather than qualitative change in the population of messenger RNA available for translation. The patterns of protein synthesis change only slightly during cleavage, but major differences appear by the beginning of gastrulation.     

Storage protein accumulation during zygotic and somatic embryo development in Picea abies (Norway spruce)

Total protein was extracted from zygotic embryos and from somatic embryos of Picea abies (L.) Karst. (Norway spruce) cultured in vitro at different times during their development. An analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 2-dimensional gel electrophoresis of the protein extracts showed that protein composition and the temporal changes in protein abundance were very similar in the two embryo types. Both zygotic and somatic embryos accumulated storage proteins in abundance during their maturation phase of growth; the somatic embryos when cultured on medium containing 90 m M sucrose and 7.6 μ M ABA. The major storage proteins are composed of polypeptides with molecular masses of about 22, 28, 33 and 42 kDa and they are identical in both embryo types according to their molecular mass and average isoelectric points. These proteins are also the most abundant proteins in the female gametophytic tissue of the mature seed.     

Changes in the pattern of protein synthesis during differentiation of the Ascomycete Sphaerostilbe repens

The vegetative mycelium of Sphaerostilbe repens Berkeley and Broome (strain CBS 275-60) gives rise, within 48 h, to aggregated organs composed of coremia and rhi-zomorphs. Developmental changes in polypeptide patterns were studied by one- and two-dimensional polyacrylamide gel electrophoresis after cells had been induced to undergo synchronized differentiation. One-dimensional gel electrophoresis revealed only minor changes during the morphogenesis. Of the 300 polypeptides resolved by two-dimensional gel electrophoresis, nearly 12% either increased or decreased during coremium and rhizomorph differentiation. Some polypeptides appeared to be unique to one or the other of the cell preparations and represented apparent qualitative differences. During the first 24 h of differentiation, about 20 polypeptide spots appeared, 6 were enhanced, 4 were reduced and 32 disappeared. Over the next 24 h changes in the population of proteins were less marked: 14 new proteins were revealed and 9 increased in intensity while 15 declined and 9 were no longer detectable. Five proteins which were present at a significant level only during the first stages of differentiation, may therefore, putatively be designated as aggregation-specific polypeptides.     

Post-transcriptional regulation of protein synthesis in Ilyanassa embryos and isolated polar lobes

The experimental removal of the polar lobe, an anucleate cytoplasmic protrusion formed in preparation for the first cleavage, from the egg of Ilyanassa obsoleta results in grossly abnormal embryonic development. In experiments reported here normal and delobed embryos, as well as isolated polar lobes, were incubated with [³⁵S]methionine for 4 hr beginning at the completion of the first cleavage or 21 hr later during epiboly. Proteins were extracted and examined by fluorography after resolution by two-dimensional polyacrylamide gel electrophoresis. In normal embryos the synthesis of several proteins begins or ends between the two stages investigated. In isolated polar lobes a subset of these developmental changes in protein synthesis occurs, indicating that the regulation of these events is independent of concomitant nuclear activity and probably involves selective regulation of the translation of mRNA stored in the eggs. The patterns of protein synthesis in normal embryos and delobed embryos are qualitatively extremely similar, though quantitative differences are also observed. No proteins can be detected which are synthesized exclusively in polar lobes.     

Proteomic profiles of embryonic development in the ascidian Ciona intestinalis

We report here proteomics-based protein profiles of three embryonic stages of the ascidian Ciona intestinalis. Two-dimensional gel electrophoresis revealed 416, 539, and 695 protein spots in the unfertilized eggs, 16 cell-stage embryos, and tadpole larvae, respectively. Comparative and quantitative analyses of the spot patterns identified proteins showing an increase or decrease in amount during embryonic development. Protein identification by MALDI-TOF/MS indicated not only the abundance and importance of metabolic enzymes and translation elongation factors but also the functional importance of actin-binding proteins and molecular chaperones during ascidian development. Global changes in spots for vitellogenin-like protein suggested post-translational modification or proteolytic digestion of this protein during embryogenesis. Comparison between mRNA and protein levels among unfertilized eggs, 16 cell-stage embryos and tadpole larvae indicated nonparallel expression patterns of genes and proteins. Ascidians provide an excellent system for studying gene expression and cell differentiation during development, and the present study should shed light on the associated molecular mechanism at the protein level.     

Vimentin and 70K Neurofilament Protein Co-exist in Embryonic Neurones from Spinal Ganglia

Abstract: The mesenchymal intermediate filament protein vimentin and the 70K component of neurofilament were detected by two-dimensional gel electrophoresis in cultures of pure sensory and sympathetic neurones derived from chick embryos. The identities of these neuronal intermediate filament proteins were confirmed by comparison of their molecular weights, isoelectric points, and peptide patterns from limited papain digestions with those of the corresponding proteins from fibroblasts and brain, respectively. A specific antibody to vimentin stained filamenteous structures and colcemid-induced coils in both neurones and associated satellite cells. In contrast, a specific antibody to the 70K neurofilament protein stained these structures solely in neurones. This neurone-specific staining, as well as its molecular weight and isoelectric point, distinguishes the 70K neurofilament protein from the 68K neurofilament as sociated protein described by others, which has been claimed to resemble the tubulin assembly protein.     

Changes in liver nuclear sap proteins during chick embryo development

Summary Nuclear sap proteins from liver of 12-, 15-, 19-day-old embryos and 1-day-old chicks were resolved by one-and two-dimensional gel electrophoresis. Although the protein patterns from various stages of development have remarkable similarities, some qualitative and quantitative differences were found among these patterns. The most pronounced changes were detected in protein with molecular weight of 100 K which was very abundant in nuclei of 12-day-old embryos and disappeared in nuclei of older embryos and in protein with molecular weight of 40 K which rapidly diminished after hatching.     

Changes in ovine conceptus and endometrial function following asynchronous embryo transfer or administration of progesterone

The secretory protein profile from conceptuses collected from naturally mated ewes on Days 10, 12, 14, and 16 was characterized by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and fluorography. The presence of the anti-luteolysin ovine trophoblast protein-1 (oTP-1) in culture medium from Day 10 conceptuses was confirmed by fluorography, Western blotting, and radioimmunoassay (RIA). On each of the days studied, oTP-1 was the dominant secretory protein, and was secreted in increasing quantities as pregnancy progressed. In a second experiment, Day 6 embryos were transferred to either Day 6 (SR) or Day 4 (AR) recipients. Three mated ewes (P) received daily injections of 50 mg progesterone on Days 4-9. Controls consisted of 2 groups of pregnant ewes (D8 and D10). Conceptuses and ipsilateral endometrium were collected 4 days following transfer of conceptuses to SR and AR ewes, on Day 10 in P and D10 ewes, and on Day 8 in D8 ewes. Conceptus volume was estimated upon recovery from the uterus. Tissues were cultured with 35S-methionine, and the medium was analyzed for total and trichloroacetic acid-precipitable radiolabeled proteins. Levels of specific endometrial secretory proteins were determined after protein separation by 2D-PAGE and estimation of the radioactivity associated with discrete radiolabeled proteins on fluorographs. The concentration of oTP-1 in conceptus culture medium was estimated by RIA. Thirty endometrial proteins were investigated. All 30 proteins were present in endometrial cultures from SR, AR, D10, and P ewes, but 13 proteins were absent from D8 ewes. Levels of three proteins were higher in AR compared to D8 (p less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)     

A new analytical procedure for two-dimensional electrophoresis of cellular proteins: comparison of protein compositions of parent strain and a K+-accumulation mutant of E. coli.

An improved two-dimensional analytical electrophoretic technique fractionates according to molecular weight in the presence of dedecyl sulfate in the first dimension, then fractionates according to isoelectric point in a perpendicular dimension. Electrofocusing in the second dimension achieves nearly complete removal of most protein components while providing true estimates of their isoelectric points. Because not all proteins penetrate isoelectric focusing gels, some proteins may go unrecognized using conventional two-dimensional systems where isoelectric focusing precedes electrophoresis. However, such components do enter dodecyl sulfate gels; hence the presence and molecular weight of those components can be established by the new procedure. A concurrent finding was that, in general, penetration of isoelectric focusing gels by discrete protein subunits dissolved in 9 M urea is an all-or-none phenomenon depending on the solubility of the specific subunit. The procedure was applied to comparison of the protein compositions of a parental strain (CBH) of Escherichia coli and a derived mutant strain (RD-2) deficient in ability to accumulate K+. The strains showed similar two-dimensional patterns except for one discrete isoelectric component absent in the parent strain but present in the mutant.     

Predominant identification of RNA-binding proteins in Fas-induced apoptosis by proteome analysis

Proteome analysis of Jurkat T cells was performed in order to identify proteins that are modified during apoptosis. Subtractive analysis of two-dimensional gel patterns of apoptotic and nonapoptotic cells revealed differences in 45 protein spots. 37 protein spots of 21 different proteins were identified by peptide mass fingerprinting using matrix-assisted laser desorption/ionization mass spectrometry. The hnRNPs A0, A2/B1, A3, K, and R; the splicing factors p54(nrb), SRp30c, ASF-2, and KH-type splicing regulatory protein (FUSE-binding protein 2); and alpha NAC, NS1-associated protein 1, and poly(A)-binding protein 4 were hitherto unknown to be involved in apoptosis. The putative cleavage sites of the majority of the proteins could be calculated by the molecular masses and isoelectric points in the two-dimensional electrophoresis gel, the peptide mass fingerprints, and after translation by treatment with recombinant caspase-3. Remarkably, 15 of the 21 identified proteins contained the RNP or KH motif, the best characterized RNA-binding motifs.     

Set of proteins shows abnormal posttranslational modification in embryos homozygous for dominant T-mutations

T and Tc are dominant mutations in the mouse that affect neuroaxial development when heterozygous and cause embryonic death when homozygous. Embryos were analyzed individually by two-dimensional gel electrophoresis at 9 1/2 days gestation, 1 day before homozygotes die in utero. A comparison of the protein patterns of mutant homozygotes with those of their littermates revealed a set of proteins (T-proteins) that showed isoelectric point (pl) polymorphism. All the T-proteins were more basic in mutant homozygotes. These polymorphisms could be detected, although they were less pronounced, in embryos as young as 7 1/2-day presomite stages, when it is impossible to distinguish homozygous mutants grossly. Interestingly, the same proteins show a pl shift from basic to acidic in wild-type embryos during development from 7 1/2 to 9 1/2 days. Thus, it appears that in T and Tc mutants a developmentally specific posttranslational acidic modification of these proteins is disturbed. The likely cause of the abnormality is a defect in some mechanism for phosphorylation, since the T-proteins of wild-type embryos were shifted to higher pls by phosphatase treatment. This disturbance appears to be localized to axial structures (neural tube, somites, and surrounding mesenchyme) since only these structures, and not the rest of the mutant homozygous embryos, contain abnormally basic T-proteins.     

Identification of AGR2 protein, a novel potential cancer marker, using proteomics technologies

A comparative analysis of the proteins in prostate tissues of the patients operated for hyperplasia (n = 7) or cancer (n = 5) was performed aiming to search for protein diagnostic markers. Differences in several minor proteins were detected using two-dimensional electrophoresis according to O’Farrel; among them, an additional protein with a molecular weight of 19 kDa and an isoelectric point of 9.0 was observed in four of the cancer cases. Mass spectrometry allowed this protein to be identified as the androgen-induced secreted protein AGR2. The possibility of using AGR2 as a diagnostic marker of prostate cancer is discussed.     

A rice protein library: a data-file of rice proteins separated by two-dimensional electrophoresis

Proteins extracted from embryos, endosperms and leaves of rice were separated by two-dimensional electrophoresis and relative molecular weights and isoelectric points were determined. The separated proteins were electroblotted onto a polyvinylidene difluoride membrane and 85 electroblotted proteins were analyzed by a gas-phase protein sequencer. The N-terminal amino-acid sequences of 27 out of 85 proteins were determined in this manner. The N-terminal regions of the remaining proteins could not be sequenced and they were inferred to have a blocking group at the N-terminus. Among proteins, 11 could be sequenced after deblocking by in situ treatment with pyroglutamyl peptidase. The internal amino-acid sequences of 23 proteins were determined by sequence analysis of peptides obtained by Cleveland peptide mapping. The amino-acid sequences determined here were compared with those of known plant and animal proteins. The concanavalin A-peroxidase method was used to determine whether the 85 proteins were glycosylated and the diagonal electrophoresis method was used to determine whether they contained disulphide bonding. Finally, we constructed a data-file of rice proteins including information on relative molecular weight, isoelectric point, amino-acid sequence, sequence homology, glycosylation, and the presence of disulphide bonding.     

Changes in plasma membrane protein composition during early development of the frog Rana ridibunda.

Plasma membranes of fully-grown oocytes and early developmental stage embryos of Rana ridibunda were isolated by differential and density gradient centrifugation; they were purified 18-fold as indicated by 5'-nucleotidase. The plasma membrane protein pattern of five different developmental stages was studied by two-dimensional polyacrylamide gel electrophoresis. More than 60 protein species could be detected by silver staining. Most of them are largely conserved during development. The rest either show precise stage specificity or exhibit stronger staining intensity at particular stages. The number of proteins specific for each stage is small, neurula being exempted. The changes observed in the plasma membrane profile during development are mostly prominent in a group of proteins with similar molecular weights (40-45 kDa) but with different pI values. The differences observed in the plasma membrane patterns are discussed in relation to the significance of each stage during development.     

Schistosoma mansoni: protein composition and synthesis during early development; evidence for early synthesis of heat shock proteins

There are alterations in the proteins synthesized during different stages of development of Schistosoma mansoni. The protein profiles at different stages were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. When stained by Coomassie blue, no significant differences were seen in protein profiles derived from cultured schistosomula from Days 0 to 6 and from adults. Newly synthesized proteins were detected by [35S]methionine incorporation. There were only a few differences in the protein profiles of schistosomula from Days 1 to 6 and from adults. Profiles derived from Day 0 schistosomula showed striking differences. Only a few proteins appear to be synthesized on Day 0 under these conditions. Schistosomula on Day 0 synthesized several minor proteins as well as a major protein of approximately 69,000 Da. This protein was immunoprecipitated by rabbit antiserum against bovine uncoating adenosine triphosphatase which recognizes the constitutive and induced 70,000 Da heat shock proteins in a wide variety of eukaryotic cells. More significant differences were observed when the newly synthesized proteins were analyzed by two dimensional gel electrophoresis. The profiles of newly synthesized proteins showed a specific repertoire of expression during the early stages of development in the parasite. A shift in temperature and medium during transformation from aquarium water to isotonic medium may initiate the synthesis of heat shock protein in these parasites.     

The purification and characterization of multiple forms of mouse submaxillary gland renin

Five forms of renin, A₀, A, C, D and E, from mouse submaxillary gland were purified by a two-step procedure including chromatography on the immunoaffinity column and CM-cellulose column. Four renin fractions, A₀, A, C and E were purified to homogeneity by the criteria of polyacrylamide gel electrophoresis, analytical isoelectric focusing and Ouchterlony double immunodiffusion. All these forms of renin have molecular weights of 40 000 as determined by gel filtration on Sephadex G-100 column. No high molecular weight renin could be demonstrated. Individual renin fractions showed similar angiotensin I formation activity, 52–158 ng angiotensin I/ng protein per h. No other protease activity could be detected with hemoglobin or casein as substrate. These purified proteins showed a discrete pattern of migration under polyacrylamide gel electrophoresis. Under denaturing condition in SDS-gel electrophoresis, all but fraction D showed a protein band with a molecular weight of 30 000. Fraction D showed a major component with molecular weight of 33 000. The isoelectric points of these renin forms varied from 5.46 to 5.76. They all reacted with antibody raised against renin A and showed similar pressor response activity with 20 ng quantities of the purified proteins. The closely related characteristics of these five forms of renin were further demonstrated by their similarity in peptide mapping patterns after limited digestion with Staphylococcus aureus V8 protease. The data suggest that these proteins are homologous proteins.