Successful reconstitution of the non-regenerating adult telencephalon by cell transplantation in Xenopus laevis

The South African clawed frog (Xenopus laevis) can regenerate the anterior half of the telencephalon only during larval life, but such regeneration is no longer possible after metamorphosis. In order to gain a better understanding of differences between larvae and adults that are potentially related to regeneration, several experiments were conducted on larvae and froglets after the partial removal of the telencephalon. As a result, it was found that the cells in the brain proliferated actively, even in non-regenerating froglets, just as was observed in regenerating larvae after the partial removal of the telencephalon. Moreover, it was shown that although the structure was usually imperfect, even isolated single cells derived from the frog brain were able to reconstitute the lost portion when the cells were transplanted to the partially truncated telencephalon. It is therefore likely to be critical for massive organ regeneration that ependymal layer cells promptly cover the cerebral lateral ventricles at an initial stage of wound healing, as is the case observed in larvae. However, in froglets, these cells strongly adhere to one another, and they are therefore unable to move to seal off the exposed ventricle, which in turn is likely to render the froglet brain non-regenerative.     

Isolation of immunoreactive beta-endorphin-related and Met-enkephalin-related peptides from the posterior pituitary of the amphibian, Xenopus laevis

Acid extracts of the posterior pituitary of the amphibian, Xenopus laevis, were analyzed with two heterologous region specific β-endorphin RIAs. Following gel filtration chromatography and cation exchange chromatography four peaks of immunoreactivity were detected. All four peaks were detected with a N-acetyl specific β-endorphin RIA. Peak I represented 92% of the total immunoreactivity isolated following cation exchange chromatography. This peak had a net positive charge at pH 2.5 of +1 and an apparent molecular weight of 1.4 Kd. Following reverse phase HPLC, Peak I fractionated into two peaks: Peak Ia and Peak Ib. Both peaks were detected with the N-acetyl specific β-endorphin RIA and a Met-enkephalin RIA, however, neither peak co-migrated with either Met-enkephalin or N-acetyl-β-endorphin(1–16). At present it is not clear whether Peak I is derived from pro-opiomelanocortin or one of the other opioid polyproteins. Peaks II, III, and IV represented 8% of the total immunoreactivity recovered following cation exchange chromatography. These peaks had net positive charges of +3, +4, and +5, respectively, and apparent molecular weights of 2.8, 3.2, and 3.5 Kd, respectively. These apparently N-acetylated β-endorphin-sized forms are minor end products of the pro-opiomelanocortin biosynthetic pathway.     

A comprehensive proteome map of bovine cerebrospinal fluid

Cerebrospinal fluid (CSF) is considered as the most promising body fluid target for the discovery of biomarkers for early diagnosis of neurodegenerative diseases such as Creutzfeldt–Jakob disease in humans and bovine spongiform encephalopathy in cattle. For the recognition of disease‐associated changes in bovine CSF protein patterns, a detailed knowledge of this proteome is a prerequisite. The absence of a high‐resolution CSF proteome map prompted us to determine all bovine CSF protein spots that can be visualised on 2‐D protein gels. Using state‐of‐the‐art 2‐DE technology for proteome mapping of bovine ante mortem CSF combined with sensitive fluorescent protein staining and MALDI‐TOF/TOF MS for protein identification, a highly detailed 2‐DE map of the bovine CSF proteome was established. Besides the proteins mapped by earlier studies, this map contains 66 different proteins, including 58 which were not annotated in bovine 2‐DE CSF maps before.     

Proteome reference map of Drosophila melanogaster head

Drosophila melanogaster has been used as a genetic model organism to understand the fundamental molecular mechanisms in human biology including memory formation that has been reported involving protein synthesis and/or post-translational modification. In this study, we employed a proteomic platform based on fluorescent 2DE and MALDI-TOF MS to build a standard D. melanogaster head proteome map for proteome-proteome comparison. In order to facilitate the comparison, an interactive database has been constructed for systematically integrating and analyzing the proteomes from different conditions and further implicated to study human diseases related to D. melanogaster model. In summary, the fundamental head proteomic database and bioinformatic analysis will be useful for further elucidating the biological mechanisms such as memory formation and neurodegenerative diseases.     

2D DIGE analysis of the bursa of Fabricius reveals characteristic proteome profiles for different stages of chicken B‐cell development

Antibody producing B‐cells are an essential component of the immune system. In contrast to human and mice where B‐cells develop in the bone marrow, chicken B‐cells develop in defined stages in the bursa of Fabricius, a gut associated lymphoid tissue. In order to gain a better understanding of critical biological processes like immigration of B‐cell precursors into the bursa anlage, their differentiation and final emigration from the bursa we analyzed the proteome dynamics of this organ during embryonic and posthatch development. Samples were taken from four representative developmental stages (embryonic day (ED) 10, ED18, day 2, and day 28) and compared in an extensive 2D DIGE approach comprising six biological replicates per time point. Cluster analysis and PCA demonstrated high reliability and reproducibility of the obtained data set and revealed distinctive proteome profiles for the selected time points, which precisely reflect the differentiation processes. One hundred fifty three protein spots with significantly different intensities were identified by MS. We detected alterations in the abundance of several proteins assigned to retinoic acid metabolism (e.g. retinal‐binding protein 5) and the actin‐cytoskeleton (e.g. vinculin and gelsolin). By immunohistochemistry, desmin was identified as stromal cell protein associated with the maturation of B‐cell follicles. Strongest protein expression difference (10.8‐fold) was observed for chloride intracellular channel 2. This protein was thus far not associated with B‐cell biology but our data suggest an important function in bursa B‐cell development.     

Larval-to-adult conversion of a myogenic system in the frog, Xenopus laevis, by larval-type myoblast-specific control of cell division, cell differentiation, and programmed cell death by triiodo-L-thyronine

For the clarification of larval-to-adult muscle conversion, the authors established primary culture methods for adult- and larval-type myoblasts in the frog, Xenopus laevis, and examined the hormonal response in each case. The cell types were enzymatically dissociated from adult frog leg and tadpole tail muscles, respectively. The cells became attached to culture plates, proliferated, and fused with each other to form multinucleated myotubes within one week. Five significant differences between the two cell types were noted. (1) Adult cells showed greater proliferation activity than larval cells, the former increasing 5.5-fold over 6 days while the latter increase only 2.5-fold. (2) Differentiation (fusion) of larval type myoblasts started earlier. Cell fusion began on day 2 or 3 in larval cells and on day 4 in adult cells. (3) The metamorphic hormone, triiodo-L-thyronine (T3) decreased larval cell numbers to 56% of that of control-cultures on day 7 but had no effect on adult cell number. DNA synthetic activity (3H-thymidine incorporation) in larval cells decreased under T3 (10(-8) M) to 45% of the control level on day 7. (4) Differentiation of adult myoblasts into myotubes was promoted by T3, whereas that of larval cells diminished by half. (5) Myotube death was induced by T3 specifically in larval but not in adult cultures. In addition to the myotube death, double staining with TUNEL (in situ DNA nick end labeling) and anti-desmin antibody indicated that T3 induces myoblast (desmin+ cell) death specifically in larval but not in adult cells. It is thus evident that the conversion of a larval-type myogenic system during metamorphosis becomes possible through nearly totally specific control of cell division, cell differentiation, and programmed cell death at a precursor cell level by T3.     

Over 4100 protein identifications from a Xenopus laevis fertilized egg digest using reversed‐phase chromatographic prefractionation followed by capillary zone electrophoresis–electrospray ionization–tandem mass spectrometry analysis

A tryptic digest generated from Xenopus laevis fertilized embryos was fractionated by RPLC. One set of 30 fractions was analyzed by 100‐min CZE‐ESI‐MS/MS separations (50 h total instrument time), and a second set of 15 fractions was analyzed by 3‐h UPLC‐ESI‐MS/MS separations (45 h total instrument time). CZE‐MS/MS produced 70% as many protein IDs (4134 versus 5787) and 60% as many peptide IDs (22 535 versus 36 848) as UPLC‐MS/MS with similar instrument time (50 h versus 45 h) but with 50 times smaller total consumed sample amount (1.5 μg versus 75 μg). Surprisingly, CZE generated peaks that were 25% more intense than UPLC for peptides that were identified by both techniques, despite the 50‐fold lower loading amount; this high sensitivity reflects the efficient ionization produced by the electrokinetically pumped nanospray interface used in CZE. This report is the first comparison of CZE‐MS/MS and UPLC‐MS/MS for large‐scale eukaryotic proteomic analysis. The numbers of protein and peptide identifications produced by CZE‐ESI‐MS/MS approach those produced by UPLC‐MS/MS, but with nearly two orders of magnitude lower sample amounts.     

Xenopus laevis RIC‐3 enhances the functional expression of the C. elegans homomeric nicotinic receptor,ACR‐16, in Xenopus oocytes

RIC‐3 enhances the functional expression of certain nicotinic acetylcholine receptors (nAChRs) in vertebrates and invertebrates and increases the availability of functional receptors in cultured cells and Xenopus laevis oocytes. Maximal activity of RIC‐3 may be cell‐type dependent, so neither mammalian nor invertebrate proteins is optimal in amphibian oocytes. We cloned the X. laevis ric‐3 cDNA and tested the frog protein in oocyte expression studies. X. laevis RIC‐3 shares 52% amino acid identity with human RIC‐3 and only 17% with that of Caenorhabditis elegans. We used the C. elegans nicotinic receptor, ACR‐16, to compare the ability of RIC‐3 from three species to enhance receptor expression. In the absence of RIC‐3, the proportion of oocytes expressing detectable nAChRs was greatly reduced. Varying the ratio of acr‐16 to X. laevis ric‐3 cRNAs injected into oocytes had little impact on the total cell current. When X. laevis, human or C. elegans ric‐3 cRNAs were co‐injected with acr‐16 cRNA (1 : 1 ratio), 100 μM acetylcholine induced larger currents in oocytes expressing X. laevis RIC‐3 compared with its orthologues. This provides further evidence for a species‐specific component of RIC‐3 activity, and suggests that X. laevis RIC‐3 is useful for enhancing the expression of invertebrate nAChRs in X. laevis oocytes.     

Two‐dimensional proteome reference map of Prototheca zopfii revealed reduced metabolism and enhanced signal transduction as adaptation to an infectious life style

Biochemical, serological, and genetic analyses have identified two genotypes of Prototheca zopfii, a unicellular microalga belonging to the family Chlorellaceae. The P. zopfii genotype 1, abundantly present in cow barns and environment, remains nonpathogenic, while P. zopfii genotype 2 has been isolated from cows with bovine mastitis. The present study was carried out to identify the protein expression level difference between the pathogenic and nonpathogenic strains of P. zopfii. A total of 782 protein spots were observed on the 2D fluorescence difference gel electrophoresis (2D DIGE) gels among which 63 and 44 proteins were identified to be overexpressed in genotypes 1 and 2, respectively. The limited number of protein entries specific for Prototheca in public repositories resulted mainly in the identification of proteins described in other algae, microorganisms, or plants. Gene ontology (GO) analysis indicated reduced carbohydrate metabolism in genotype 1, while genotype 2 displayed enhanced DNA binding, kinase activity, and signal transduction. These effects point to metabolic and signaling adaptations in the pathogenic strain and provide insights into the evolution of otherwise highly similar strains. All MS data have been deposited in the ProteomeXchange with identifier PXD000126.     

Plant‐extract‐induced changes in the proteome of the soil‐borne pathogenic fungus Thielaviopsis basicola

Thielaviopsis basicola is a hemibiotroph fungus that causes black root rot disease in diverse plants with significant impact on cotton production in Australia. To elucidate how T. basicola growth and proteome are influenced by interactions with natural sources, this fungus was cultured in the presence of root extracts from non‐host (wheat, hairy vetch) and susceptible host (cotton, lupin) plants. We found that T. basicola growth was significantly favored in the presence of host extracts, while hierarchical clustering analysis of 2‐DE protein profiles of T. basicola showed plant species had a larger effect on the proteome than host/non‐host status. Analysis by LC‐MS/MS of unique and differentially expressed spots and identification using cross‐species similarity searching and de novo sequencing allowed successful identification of 41 spots. These proteins were principally involved in primary metabolism with smaller numbers implicated in other diverse functions. Identification of several “morpho” proteins suggested morphological differences that were further microscopically investigated. Identification of several highly expressed spots suggested that vitamin B₆ is important in the T. basicola response to components present in hairy vetch extract, and finally, three spots, induced in the presence of lupin extract, may correspond to malic enzyme and be involved in lipid accumulation.     

Comprehensive two-dimensional liquid chromatography mass spectrometric profiling of the rat hippocampal proteome

In this study, we performed the first high‐throughput and comprehensive proteomic profiling of the rat hippocampal proteome. Using a combination of 2‐D LC‐MS and data analysis with the Rosetta Elucidator^® system, we identified 1340 unique proteins. Functional classification showed that most of these were associated with synaptic function and comprised a high proportion of phosphorylated proteins and analytically challenging classes of membrane proteins such as ion channel receptor subunits.     

Proteomic studies support the use of multi-product immunoassays to monitor host cell protein impurities

In the biopharmaceutical industry, recombinant protein drugs are commonly produced in Chinese hamster ovary (CHO) cells. During the development process, removal of CHO cell-derived proteins from the biopharmaceutical product is monitored using multi-product immunoassays. Such immunoassays are developed by raising antibodies to a single CHO cell protein preparation. However, these assays are utilized to monitor CHO cell protein impurities during the recovery of products from different CHO cell lines. To address whether underlying differences between CHO cell lines result in sufficient protein expression changes to exclude the suitability of multi-product immunoassays, a comparative proteomics study of three independently generated CHO cell lines was performed. Statistical analysis of over 1000 proteins resolved by 2-D PAGE demonstrated that the protein expression profiles of three different CHO cell lines exhibit very few differences in protein expression. Only 11 qualitative changes in protein expression and 26 quantitative changes greater than two-fold were observed. Identification of protein spots by mass spectrometry revealed that many of the observed changes were due to post-translational modifications rather than expression of novel proteins in each cell line. These results suggest that multi-product immunoassays are suitable for monitoring host cell proteins in biopharmaceuticals produced in different CHO cell lines.     

A novel S3S‐TAP‐tag for the isolation of T‐cell interaction partners of adhesion and degranulation promoting adaptor protein

The identification of modular units of cellular function is a major goal for proteomic research. Protein complexes represent important building blocks defining functionality and deciphering their composition remains a major challenge. Here, we have designed a new tandem affinity purification (TAP) tag (termed S3S‐tag) for the isolation of protein complexes. Specifically, the immune cell protein ADAP that regulates integrin adhesion was fused either C‐ or N‐terminally to the S3S‐tag. After retroviral transduction of a vector containing S3S‐tagged ADAP and internal ribosomal entry site encoded enhanced green fluorescent protein (eGFP), Jurkat T cells were sorted according to eGFP expression and further selected for expression of TAP‐tagged protein close to endogenous levels. The combination of a cleavable S‐tag and a Strep‐tag II allowed for the isolation of ADAP and associated proteins. Subsequently, stable isotope labeling with amino acids in cell culture‐based mass spectrometric analysis was performed to identify potentially specific interaction partners. Co‐purification of the known interaction partner Src kinase‐associated phosphoprotein of 55 kDa indicates the validity of our approach, while the identification of the ENA/VASP family member EVL, the guanine nucleotide exchange factor GEF‐H1 and the adaptor protein DOCK2 corroborates a link between ADAP‐mediated integrin regulation and the cytoskeleton.     

Functional demonstration of the ability of a primary spermatogonium as a stem cell by tracing a single cell destiny in Xenopus laevis

In Xenopus, although primary spermatogonium (PG), the largest cell in the testis, is believed to be spermatogonial stem cell by histological observations, functional evidence has never been obtained. In the present study, we first indicated that culture of juvenile testis in a medium supplemented with follicle stimulating hormone resulted in no proliferation of PG. In this culture system, early secondary spermatogonia could undergo mitotic divisions with a concomitant decrease in their size, so that they became distinguishable in size from PG. Because the subcutaneous environment of juveniles permitted aggregates of the dissociated testicular cells to reconstruct the normal testis structure, we next inserted a genetically marked PG isolated from cultured testes into the aggregate and transplanted it subcutaneously. In this system, 73.9% of the aggregates contained a marked PG. When we observed the aggregates 12 weeks after transplantation, most aggregates (70.0%) contained marked PG that had self-renewed. Among these, fully growing aggregates contained many spermatogenic cells at the later developmental stage. These results suggested that isolated PG from the cultured testes had the ability as stem cells, and that purification of the spermatogenic stem cells became reliable in Xenopus.     

Expanding the mouse embryonic stem cell proteome: Combining three proteomic approaches

The current study used three different proteomic strategies, which differed by their extent of intact protein separation, to examine the proteome of a pluripotent mouse embryonic stem cell line, R1. Proteins from whole‐cell lysates were subjected either to 2‐D‐LC, or 1‐DE, or were unfractionated prior to enzymatic digestion and subsequent analysis by MS. The results yielded 1895 identified non‐redundant proteins and, for 128 of these, the specific isoform could be determined based on detection of an isoform‐specific peptide. When compared with two previously published proteomic studies that used the same cell line, the current study reveals 612 new proteins.     

Comparative two‐dimensional polyacrylamide gel electrophoresis of the salivary proteome of children with autism spectrum disorder

In the last decades, prevalence of autism spectrum disorder (ASD) has been on the rise. However, clear aetiology is still elusive and improvements in early diagnosis are needed. To uncover possible biomarkers present in ASD, we used two‐dimensional polyacrylamide gel electrophoresis and nanoliquid chromatography‐tandem mass spectrometry (nanoLC‐MS/MS), to compare salivary proteome profiling of children with ASD and controls. A total of 889 spots were compared and only those spots with a fold change ≥1.7 and a P‐value <0.05 or a fold change of ≥3.0 between ASD cases and controls were analysed by nanoLC‐MS/MS. Alpha‐amylase, CREB‐binding protein, p532, Transferrin, Zn alpha2 glycoprotein, Zymogen granule protein 16, cystatin D and plasminogen were down‐regulated in ASD. Increased expression of proto‐oncogene Frequently rearranged in advanced T‐cell lymphomas 1 (FRAT1), Kinesin family member 14, Integrin alpha6 subunit, growth hormone regulated TBC protein 1, parotid secretory protein, Prolactin‐inducible protein precursor, Mucin‐16, Ca binding protein migration inhibitory factor‐related protein 14 (MRP14) was observed in individuals with ASD. Many of the identified proteins have previously been linked to ASD or were proposed as risk factors of ASD at the genetic level. Some others are involved in pathological pathways implicated in ASD causality such as oxidative stress, lipid and cholesterol metabolism, immune system disturbances and inflammation. These data could contribute to protein signatures for ASD presence, risk and subtypes, and advance understanding of ASD cause as well as provide novel treatment targets for ASD.     

Mapping the proteome of thylakoid membranes by de novo sequencing of intermembrane peptide domains

The proteome of a membrane compartment has been investigated by de novo sequence analysis after tryptic in gel digestion. Protein complexes and corresponding protein subunits were separated by a 2-D Blue Native (BN)/SDS-PAGE system. The transmembrane proteins of thylakoid membranes from a higher plant (Hordeum vulgare L.) were identified by the primary sequence of hydrophilic intermembrane peptide domains using nano ESI-MS/MS-analysis. Peptide analysis revealed that lysine residues of membrane proteins are primarily situated in the intermembrane domains. We concluded that esterification of lysine residues with fluorescent dyes may open the opportunity to label membrane proteins still localized in native protein complexes within the membrane phase. We demonstrate that covalent labelling of membrane proteins with the fluorescent dye Cy3 allows high sensitive visualization of protein complexes after 2-D BN/SDS-PAGE. We show that pre-electrophoretic labelling of protein subunits supplements detection of proteins by post-electrophoretic staining with silver and CBB and assists in completing the identification of the membrane proteome.     

Analysis of the rat hypothalamus proteome by data‐independent label‐free LC‐MS/MS

Studies of neuronal, endocrine, and metabolic disorders would be facilitated by characterization of the hypothalamus proteome. Protein extracts prepared from 16 whole rat hypothalami were measured by data‐independent label‐free nano LC‐MS/MS. Peptide features were detected, aligned, and searched against a rat Swiss‐Prot database using ProteinLynx Global Server v.2.5. The final combined dataset comprised 21 455 peptides, corresponding to 622 unique proteins, each identified by a minimum of two distinct peptides. The majority of the proteins (69%) were cytosolic, and 16% were membrane proteins. Important proteins involved in neurological and synaptic function were identified including several members of the Ras‐related protein family and proteins involved in glutamate biosynthesis.