A Tyrosinase missense mutation causes albinism in the Wistar rat

Tyrosinase serves as a key enzyme in the synthesis of melanin. In humans mutations in the TYR gene are associated with type 1 oculocutaneous albinism (OCA1) that leads to reduced or absent pigmentation of skin, hair and eye. Various mutations causing OCA in man, mouse, rabbit and cattle have been identified throughout the Tyrosinase gene including nonsense, missense, frameshift and splice site alterations. Here we report a missense substitution at codon R299H in exon 2 of the Tyr gene in the albino Wistar rat. As this very exchange has already been described in OCA patients, our findings reinforce the significance of this region for normal catalytic activity of tyrosinase protein.     

Annexin II is required for apical transport in polarized epithelial cells

The sorting of apical proteins comprises an initial recognition step in the trans Golgi network and a final partitioning of the apical pool of proteins into at least two different types of vesicular carriers. One criteria of these carriers is the association or non-association of the protein content with lipid rafts. We have previously characterized a population containing the raft-associated sucrase-isomaltase-carrying vesicles (SAVs) and another one, the non-raft-associated lactase-phlorizin hydrolase-carrrying vesicles (LAVs) that are targeted separately to the apical membrane. Here, we demonstrate biochemically and by employing confocal laser microscopy that the annexin II-S100A10 complex is a component of SAVs and is absent from LAVs. The unequivocal role of annexin II in the apical targeting of SI is clearly demonstrated when down-regulation of this protein by annexin II-specific small interfering RNA drastically decreases the apical delivery of SI in the epithelial cell line Madin-Darby canine kidney. The annexin II-S100A10 complex plays therefore a crucial role in routing SAVs to the apical membrane of epithelial cells.     

Polypyrimidine tract-binding protein promotes insulin secretory granule biogenesis

Pancreatic beta-cells store insulin in secretory granules that undergo exocytosis upon glucose stimulation. Sustained stimulation depletes beta-cells of their granule pool, which must be quickly restored. However, the factors promoting rapid granule biogenesis are unknown. Here we show that beta-cell stimulation induces the nucleocytoplasmic translocation of polypyrimidine tract-binding protein (PTB). Activated cytosolic PTB binds and stabilizes mRNAs encoding proteins of secretory granules, thus increasing their translation, whereas knockdown of PTB expression by RNA interference (RNAi) results in the depletion of secretory granules. These findings may provide insight for the understanding and treatment of diabetes, in which insulin secretion is typically impaired.     

Cancellous bone allograft seeded with human mesenchymal stromal cells: a potential good manufacturing practice-grade tool for the regeneration of bone defects

Background aimsCombining autologous bone precursor cells with cancellous bone allograft (CBA) offers an appealing strategy for skeletal regeneration. In this context, multipotent mesenchymal stromal cells (MSC) provide an excellent cell source because they are readily harvested from donors, expanded and differentiated in vitro. The aim of this study was to evaluate the proliferation, morphology, osteogenic differentiation and stem cell-related gene expression during static long-term ex vivo cultivation using human MSC and CBA under good manufacturing practice (GMP)-conforming conditions.MethodsMSC were isolated from healthy donors (n = 5) and cultivated on peracetic acid-sterilized CBA in the presence of 10% human platelet-rich plasma without osteogenic supplements. Total protein content, cell-specific alkaline phosphatase (ALP) activity and osteogenic marker gene expression levels were assessed. Stem cell-related gene expression was compared with MSC monolayer cultivation using microarray analysis. Furthermore, cellular distribution and morphology within the porous CBA were visualized by histology and scanning electron microscopy.ResultsEffective adhesion, spreading, proliferation and intercellular contact of human MSC within the pores of CBA were observed during the study (≤42 days). Cell-specific ALP activity peaked after 3 weeks of cultivation. Gene expression of early, intermediate and late osteogenic marker genes was detectable during long-term cultivation. Microarray-based annotation and biologic interaction network data analysis indicated that expression levels of genes encoding crucial differentiation-regulating proteins and extracellular matrix components involved in the process of osteogenesis were induced in CBA-cultivated MSC.ConclusionsMSC-vitalized CBA offers an attractive GMP-grade bone-filling material. Further research is warranted to evaluate its bone-healing potential in vivo.     

Assessment of protein spot components applying correspondence analysis for peptide mass fingerprint data

Proteins separated by two-dimensional gel electrophoresis (2-DE) may be distributed over several spots. Otherwise, one spot may contain more than one component. The same protein occurring in several spots supposedly represents differently modified protein species that might be of biological relevance. Identification of spots with peptide mass fingerprinting and database searching leads only to the detection of the major spot components. If a spot also contains additional minor protein components, quantitation of spots with protein staining techniques or antibody detection becomes misleading. In order to find spots containing minor components we applied correspondence analysis, a multivariate data exploration method, to peptide mass fingerprint data. Correspondence analysis using peak lists revealed groups of spots containing the same protein with their characteristic mass-to-charge ratio (m/z) values. In order to detect different protein spot components an interactive threshold setting and removal of m/z values with subsequent recalculation of the correspondence analysis using our software tool CorrAn are performed. The usefulness of this methodical approach was shown by a data set of peptide mass fingerprints of 284 spots of Helicobacter pylori 26695 separated by 2-DE.     

The Relationship between Antigenic Compounds Produced by Sweet Potato in Response to Black Rot Infection and the Magnitude of Disease Resistance

Tissue extracts of healthy, sliced and black-rotted sweet potato roots of several Japanese varieties showed immunochemical precipitation lines with antisera toward sliced and diseased tissue extracts prepared from an American resistant variety, Sunnyside. The immunochemical precipitation patterns of healthy and sliced tissue extracts and those of diseased tissue extract of the Japanese varieties respectively were almost the same as those of sliced and diseased tissue extracts of the American Sunnyside. Antigenic components designated as A and Cs were distributed in all tissue extracts of both Japanese and American varieties. Components B and D were produced in response to the infection in root tissues of Japanese varieties as well as of American ones. The amount of component B produced in several Japanese varieties was correlated with the magnitude of resistance action of root tissues to the fungus infection and the order was as follows: Norm No. 10 (highly resistant) >Norin No. 1 and Okimasari (resistant) >Norin No. 4 and Norin No. 5 (susceptible). Components B and D seemed to be present in healthy root tissue in very small amounts, and showed an increase in response to the simple injury or slicing, though the magnitude of this increase was much less than the response to the pathogenic infection.     

Expression of a luteoviral movement protein in transgenic plants leads to carbohydrate accumulation and reduced photosynthetic capacity in source leaves

Elucidating the role of viral genes in transgenic plants revealed that the movement protein (MP) from tobacco mosaic virus is responsible for altered carbohydrate allocation in tobacco and potato plants. To study whether this is a general feature of viral MPs, the movement protein MP17 of potato leafroll virus (PLRV), a phloem-restricted luteovirus, was constitutively expressed in tobacco plants. Transgenic lines were strongly reduced in height and developed bleached and sometimes even necrotic areas on their source leaves. Levels of soluble sugars and starch were significantly increased in source leaves. Yet, in leaf laminae the hexose—phosphate content was unaltered and ATP reduced to only a small extent, indicating that these leaves were able to maintain homeostatic conditions by compartmentalization of soluble sugars, probably in the vacuole. On the contrary, midribs contained lower levels of soluble sugars, ATP, hexose—phosphates and UDP-glucose supporting the concept of limited uptake and catabolism of sucrose in the phloem. The accumulation of carbohydrates led to a decreased photosynthetic capacity and carboxylation efficiency of ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) probably owing to decreased expression of photosynthetic proteins. In parallel, levels of pathogenesis-related proteins were elevated which may be the reason for the obtained limited resistance against the unrelated potato virus Y (PVY)^N in the transgenic tobacco plants. Ultrathin sections of affected leaves harvested from 2-week-old plants revealed plasmodesmal alterations in the phloem tissue while plasmodesmata between mesophyll cells were indistinguishable from wild-type. These data favour the phloem tissue to be the primary site of PLRV MP17 action in altering carbohydrate metabolism.     

The dual role of annexin II in targeting of brush border proteins and in intestinal cell polarity

Functional intestinal epithelium relies on complete polarization of enterocytes marked by the formation of microvilli and the accurate trafficking of glycoproteins to relevant membrane domains. Numerous transport pathways warrant the unique structural identity and protein/lipid composition of the brush border membrane. Annexin II (Ca(2+)-dependent lipid-binding protein) is an important component of one of the apical protein transport machineries, which involves detergent-resistant membranes and the actin cytoskeleton. Here, we investigate in intestinal Caco-2 cells the contribution of annexin II to the sorting and transport of brush border hydrolases and role in intestinal cell polarity. Downregulation of annexin II in Caco-2-A4 cell line results in a severe reduction of the levels of the brush border membrane resident enzyme sucrase isomaltase (SI) as well as structural components such as ezrin. This reduction is accompanied by a redistribution of these proteins to intracellular compartments and a striking morphological transition of Caco-2 cells to rudimentary epithelial cells that are characterized by an almost flat apical membrane with sparse and short microvilli. Concomitant with this alteration is the redistribution of the intermediate filament protein keratin 19 to the intracellular membranes in Caco-2-A4 cells. Interestingly, keratin 19 interacts with annexin II in wild type Caco-2 cells and this interaction occurs exclusively in lipid rafts. Our findings suggest a role for annexin II and K19 in differentiation and polarization of intestinal cells.