Robots, pipelines, polyproteins: enabling multiprotein expression in prokaryotic and eukaryotic cells

Multiprotein complexes catalyze vital biological functions in the cell. A paramount objective of the SPINE2 project was to address the structural molecular biology of these multiprotein complexes, by enlisting and developing enabling technologies for their study. An emerging key prerequisite for studying complex biological specimens is their recombinant overproduction. Novel reagents and streamlined protocols for rapidly assembling co-expression constructs for this purpose have been designed and validated. The high-throughput pipeline implemented at IGBMC Strasbourg and the ACEMBL platform at the EMBL Grenoble utilize recombinant overexpression systems for heterologous expression of proteins and their complexes. Extension of the ACEMBL platform technology to include eukaryotic hosts such as insect and mammalian cells has been achieved. Efficient production of large multicomponent protein complexes for structural studies using the baculovirus/insect cell system can be hampered by a stoichiometric imbalance of the subunits produced. A polyprotein strategy has been developed to overcome this bottleneck and has been successfully implemented in our MultiBac baculovirus expression system for producing multiprotein complexes.     

A study to sustain the hypothesis of the multiple genesis of oligoasthenoteratospermia in human idiopathic infertile males

Interdependence between sperm concentration, motility, morphology, and the percentage of aneuploid sperm was explored to test whether oligoasthenoteratospermia (OAT) may have a multiple origin in idiopathic infertile males. A total of 174 men (age, 35.8 +/- 4.3 yr) with idiopathic infertility were studied. Seven patients had nonobstructive azoospermia, 55 had severe OAT, 30 had OAT, 27 had isolated alterations of motility, 45 had alterations of morphology and of motility, and 10 had isolated alterations of morphology. The sperm morphology was assessed with strict criteria. The percentage of aneuploid sperm was assessed with fluorescent in situ hybridization for chromosomes X, Y, 13, 15, 16, 17, 18, 21, and 22. Relationships between sperm features, and the relationship between sperm features and aneuploidies were analyzed with multivariate regression analysis. Statistical analysis did not find any significant relationship between the percentage of typical forms and sperm concentration or between morphology and motility. On the other hand, a positive and significant relationship was found between sperm concentration and motility. The percentage of aneuploid sperm was inversely and significantly related to the percentage of typical forms but not to motility and concentration. Sperm morphology is an independent characteristic with respect to concentration and motility, whereas it showed a significant inverse relationship with respect to the percentage of aneuploid sperm. This means that idiopathic OAT may occur by means of at least two independent pathways, the first affecting concentration and/or motility and the second affecting morphology.     

Large and dissimilar repertoire of Melan-A/MART-1-specific CTL in metastatic lesions and blood of a melanoma patient

It is widely accepted that the repertoire of Melan-A-specific T cells naturally selected in melanoma patients is diverse and mostly nonoverlapping among different individuals. To date, however, no studies have addressed the TCR profile in different tumor sites and the peripheral blood from the same patient. We compared the TCR usage of Melan-A-specific T cells from different compartments of a single melanoma patient to evaluate possible clonotype expansion or preferential homing over a 4-mo follow-up period. Using HLA-A2 peptide tetramers, CD8(+) T cells recognizing the modified Melan-A immunodominant ELAGIGILTV peptide were isolated from four metastatic lesions resected from a single melanoma patient, and their TCR repertoire was studied. A panel of T cell clones was generated by cell cloning of tetramer-positive cells. Analysis of the TCR beta-chain V segment and the complementarity-determining region 3 (CDR3) length and sequence revealed a large diversity in the TCR repertoire, with only some of the clones showing a partial conservation in the CDR3. A similar degree of diversity was found by analyzing a number of T cell clones obtained after sorting a Melan-A-specific population derived from PBLs of the same patient after in vitro culture with the immunodominant epitope. Moreover, clonotypes found at one site were not present in another, suggesting the lack of expansion and circulation of one or more clonotypes. Taken together, these results buttress the notion that the CTLs recognizing the immunodominant Ag of Melan-A comprise a high number of different clonotypic TCR, of which only some exhibit common features in the CDR3.     

Differentiation of repetitive DNA sites and sex chromosome systems reveal closely related group in Parodontidae (Actinopterygii: Characiformes)

Parodon and Apareiodon lack sufficiently consistent morphological traits to be considered a monophyletic group in Parodontidae. Species within this family are either sex-homomorphic or sex-heteromorphic (i.e., lacking a differentiated sex chromosome system, ZZ/ZW or ZZ/ZW(1)W(2)). In this study, a DNA fragment from the heterochromatin segment of the W chromosome of Apareiodon ibitiensis (named WAp) was microdissected and used for in situ mapping of nine Parodontidae species. The species were also characterized using a satellite DNA probe (pPh2004). The species were phylogenetically clustered according to 17 characters, which were examined by both classical and molecular cytogenetic techniques. Given the present results, the single ZZ/ZW sex chromosome system seems to have been derived from a paracentric inversion of a terminal WAp site onto the proximal regions of the short arms of a metacentric chromosome pair, followed by WAp site amplification. We reason that these events restrained recombination and favored differentiation of the W chromosome in some species. Moreover, co-hybridization experiments targeting the WAp and pPh2004 repetitive DNA sites of A. affinis suggest that the ZZ/ZW(1)W(2) sex chromosomes of this species may have arisen from a translocation between the proto-sex chromosome and an autosome. Our phylogenetic analysis corroborates the hypothesis of sex chromosome differentiation and establishes groups of closely related species. The phylogenetic reorganization in response to these new data supports the presence of internal monophyletic groups within Parodontidae.     

P2RY1/ALK3-Expressing Cells within the Adult Human Exocrine Pancreas Are BMP-7 Expandable and Exhibit Progenitor-like Characteristics

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Regulation of RasGRP via a Phorbol Ester-Responsive C1 Domain

As part of a cDNA library screen for clones that induce transformation of NIH 3T3 fibroblasts, we have isolated a cDNA encoding the murine homolog of the guanine nucleotide exchange factor RasGRP. A point mutation predicted to prevent interaction with Ras abolished the ability of murine RasGRP (mRasGRP) to transform fibroblasts and to activate mitogen-activated protein kinases (MAP kinases). MAP kinase activation via mRasGRP was enhanced by coexpression of H-, K-, and N-Ras and was partially suppressed by coexpression of dominant negative forms of H- and K-Ras. The C terminus of mRasGRP contains a pair of EF hands and a C1 domain which is very similar to the phorbol ester- and diacylglycerol-binding C1 domains of protein kinase Cs. The EF hands could be deleted without affecting the ability of mRasGRP to transform NIH 3T3 cells. In contrast, deletion of the C1 domain or an adjacent cluster of basic amino acids eliminated the transforming activity of mRasGRP. Transformation and MAP kinase activation via mRasGRP were restored if the deleted C1 domain was replaced either by a membrane-localizing prenylation signal or by a diacylglycerol- and phorbol ester-binding C1 domain of protein kinase C. The transforming activity of mRasGRP could be regulated by phorbol ester when serum concentrations were low, and this effect of phorbol ester was dependent on the C1 domain of mRasGRP. The C1 domain could also confer phorbol myristate acetate-regulated transforming activity on a prenylation-defective mutant of K-Ras. The C1 domain mediated the translocation of mRasGRP to cell membranes in response to either phorbol ester or serum stimulation. These results suggest that the primary mechanism of activation of mRasGRP in fibroblasts is through its recruitment to diacylglycerol-enriched membranes. mRasGRP is expressed in lymphoid tissues and the brain, as well as in some lymphoid cell lines. In these cells, RasGRP has the potential to serve as a direct link between receptors which stimulate diacylglycerol-generating phospholipase Cs and the activation of Ras.     

Correlative immunocytochemical and electron microscopic studies: Identification of (Entero)glucagon-somatostatin- and pancreatic polypeptide-like-containing cells in the human colon

Summary Correlative immunocytochemical and electron microscopic studies, using the semi thin-thin technic, were performed to identify the (entero) glucagon, somatostatin and pancreatic polypeptide-like immunoreactive cells of the human colonic mucosa. Mean granule diameter for each cell type was estimated according to two methods and histograms showing the granule size distribution were constructed. A total of 139 immunostained cells identified at the ultrastructural level were analyzed. Mean granule diameter for (entero)glucagon-containing cells was 318±11 nm but a reduction of granule size with age was noteworthy: granules were larger in the fetus (mean diameter 350±15) than in adults (mean diameter 310±10 nm). Somatostatin-containing cells, very rare in adults, were present in the fetal distal colon. Their general mean granule diameter was 354±18 nm but many cells had a mean granule diameter of more than 400 nm. A pancreatic polypeptidelike immunoreactivity was found only in (entero)glucagon-containing cells, pointing out the possible occurrence of both peptides (or of similar sequences) in the same cells. Previous ultrastructural studies dealing with a tentative classification of the human colonic endocrine cells were compared with the present data.This work was supported by the Institut National de la Santé et de la Recherche Médicale (INSERM).     

Microbial interactions in alcoholic beverages

International Microbiology - This review examines the different types of interactions between the microorganisms involved in the fermentation processes of alcoholic beverages produced all over the...     

Changes in native and introduced host–parasite networks

Biological Invasions - Introduced species can alter the dynamics and structure of a native community. Network analysis provides a tool to study host–parasite interactions that can help to...