The occurrence of polyenoic fatty acids with greater than 22 carbon atoms in mammalian spermatozoa.

Fatty acids with carbon chain lengths greater than 22 (VLCFA) have been detected in boar, ram, bull and human spermatozoa. Saturated and mono-unsaturated fatty acids were present in all spermatozoa but, except for human spermatozoa, polyenoic fatty acids were quantitatively the most important components. Marked differences in polyenoic fatty acid composition were observed. Whereas human spermatozoa contain predominantly di-, tri- and tetraenoic fatty acids with up to 32 carbon atoms, boar, ram and bull spermatozoa also contain pentaenoic and/or hexaenoic acids with up to 34 carbon atoms. Human and boar spermatozoa differ markedly from those of the ram and bull in that only n-6 series acids are present.     

Monitoring of biofilms grown on differentially structured metallic surfaces using confocal laser scanning microscopy

Imaging of biofilms on opaque surfaces is a challenge presented to researchers especially considering pathogenic bacteria, as those typically grow on living tissue, such as mucosa and bone. However, they can also grow on surfaces used in industrial applications such as food production, acting as a hindrance to the process. Thus, it is important to understand bacteria better in the environment they actually have relevance in. Stainless steel and titanium substrata were line structured and dotted surface topographies for titanium substrata were prepared to analyze their effects on biofilm formation of a constitutively green fluorescent protein (GFP)‐expressing Escherichia coli strain. The strain was batch cultivated in a custom built flow cell initially for 18 h, followed by continuous cultivation for 6 h. Confocal laser scanning microscopy (CLSM) was used to determine the biofilm topography. Biofilm growth of E. coli GFPmut2 was not affected by the type of metal substrate used; rather, attachment and growth were influenced by variable shapes of the microstructured titanium surfaces. In this work, biofilm cultivation in flow cells was coupled with the most widely used biofilm analytical technique (CLSM) to study the time course of growth of a GFP‐expressing biofilm on metallic surfaces without intermittent sampling or disturbing the natural development of the biofilm.     

Serine residues 286, 288, and 293 within the CIITA: a mechanism for down-regulating CIITA activity through phosphorylation

CIITA is the primary factor activating the expression of the class II MHC genes necessary for the exogenous pathway of Ag processing and presentation. Strict control of CIITA is necessary to regulate MHC class II gene expression and induction of an immune response. We show in this study that the nuclear localized form of CIITA is a predominantly phosphorylated form of the protein, whereas cytoplasmic CIITA is predominantly unphosphorylated. Novel phosphorylation sites were determined to be located within a region that contains serine residues 286, 288, and 293. Double mutations of these residues increased nuclear CIITA, indicating that these sites are not required for nuclear import. CIITA-bearing mutations of these serine residues significantly increased endogenous MHC class II expression, but did not significantly enhance trans-activation from a MHC class II promoter, indicating that these phosphorylation sites may be important for gene activation from intact chromatin rather than artificial plasmid-based promoters. These data suggest a model for CIITA function in which phosphorylation of these specific sites in CIITA in the nucleus serves to down-regulate CIITA activity.     

Transplantation of mesenchymal stromal cells on mineralized collagen leads to ectopic matrix synthesis in vivo independently from prior in vitro differentiation

BACKGROUND: Tissue engineering using mesenchymal stromal cells (MSC) represents a promising approach for bone regeneration. Nevertheless, the optimal constructs have yet to be determined. It still remains unclear if there is a benefit of in vitro differentiation of MSC prior to transplantation or if undifferentiated MSC hold the optimal potential concerning new tissue formation. METHODS: After isolation and in vitro expansion, MSC were seeded on mineralized collagen sponges and transplanted in a heterotopic SCID mice model (n=12). While group A contained undifferentiated MSC, in group B cells were cultivated for 14 days in vitro under osteogenic conditions prior to implantation. Results were compared with non-loaded scaffolds (group C). Animals were killed for investigation at 4 and at 8 weeks. RESULTS: In situ hybridization demonstrated integration of MSC for up to 8 weeks in groups A and B. Histology revealed significantly more extracellular matrix synthesis in MSC-seeded scaffolds containing calcium phosphate and collagen type I at 4 and 8 weeks after transplantation compared with unloaded controls. At a biochemical level, higher levels of specific alkaline phosphatase expression were detected in MSC-loaded scaffolds (P<0.05). Scaffolds containing undifferentiated and differentiated MSC did not appear to differ in terms of matrix synthesis and protein expression, while the number of avital cells was significant higher in those probes loaded with differentiated MSC (P<0.01). DISCUSSION: The integration of transplanted cells and MSC-associated matrix synthesis encourages the use of MSC-loaded mineralized collagen for tissue engineering of bone. Furthermore, our data suggest that in vitro differentiation of MSC does not have a positive influence in terms of improved matrix synthesis.     

The plastid genome-encoded Ycf4 protein functions as a nonessential assembly factor for photosystem I in higher plants

Photosystem biogenesis in the thylakoid membrane is a highly complicated process that requires the coordinated assembly of nucleus-encoded and chloroplast-encoded protein subunits as well as the insertion of hundreds of cofactors, such as chromophores (chlorophylls, carotenoids) and iron-sulfur clusters. The molecular details of the assembly process and the identity and functions of the auxiliary factors involved in it are only poorly understood. In this work, we have characterized the chloroplast genome-encoded ycf4 (for hypothetical chloroplast reading frame no. 4) gene, previously shown to encode a protein involved in photosystem I (PSI) biogenesis in the unicellular green alga Chlamydomonas reinhardtii. Using stable transformation of the chloroplast genome, we have generated ycf4 knockout plants in the higher plant tobacco (Nicotiana tabacum). Although these mutants are severely affected in their photosynthetic performance, they are capable of photoautotrophic growth, demonstrating that, different from Chlamydomonas, the ycf4 gene product is not essential for photosynthesis. We further show that ycf4 knockout plants are specifically deficient in PSI accumulation. Unaltered expression of plastid-encoded PSI genes and biochemical analyses suggest a posttranslational action of the Ycf4 protein in the PSI assembly process. With increasing leaf age, the contents of Ycf4 and Y3IP1, another auxiliary factor involved in PSI assembly, decrease strongly, whereas PSI contents remain constant, suggesting that PSI is highly stable and that its biogenesis is restricted to young leaves.     

Water-soluble chlorophyll protein (WSCP) of Arabidopsis is expressed in the gynoecium and developing silique

Water-soluble chlorophyll protein (WSCP) has been found in many Brassicaceae, most often in leaves. In many cases, its expression is stress-induced, therefore, it is thought to be involved in some stress response. In this work, recombinant WSCP from Arabidopsis thaliana (AtWSCP) is found to form chlorophyll-protein complexes in vitro that share many properties with recombinant or native WSCP from Brassica oleracea, BoWSCP, including an unusual heat resistance up to 100°C in aqueous solution. A polyclonal antibody raised against the recombinant apoprotein is used to identify plant tissues expressing AtWSCP. The only plant organs containing significant amounts of AtWSCP are the gynoecium in open flowers and the septum of developing siliques, specifically the transmission tract. In fully grown but still green siliques, the protein has almost disappeared. Possible implications for AtWSCP functions are discussed.