Overexpression of the CT GalNAc transferase in skeletal muscle alters myofiber growth, neuromuscular structure, and laminin expression.

Carbohydrates have been shown to mediate or modulate a number of important events in the development of the nervous system; however, there is little evidence that they participate directly in the development of synapses. One carbohydrate structure that is likely to be important in synaptic development of the neuromuscular junction is the CT carbohydrate antigen [GalNAcbeta1,4[NeuAcalpha2,3]Galbeta1(-3GalNAc or -4GlcNAc)]. The synaptic localization of the CT antigen is due to the presence of the terminal beta1,4 GalNAc linkage, and such linkages are localized to the neuromuscular junction in many species. Here we show that an enzyme that can create the synaptic CT structure, the CT GalNAc transferase, is also confined to the neuromuscular junction in mice. Using transgenic mice, we show that overexpression of the CT GalNAc transferase in extrasynaptic regions in skeletal myofibers caused as much as a 60% reduction in the diameter of adult myofibers and an order of magnitude increase in satellite cells. Neuromuscular junctions of transgenic mice had severely reduced numbers of secondary folds, Schwann cell processes were present in the synaptic cleft, and secondary folds were often misaligned with active zones. In addition, multiple presynaptic specializations occurred on individual myofibers. In addition, some normally synaptic proteins, including laminin alpha4, laminin alpha5, utrophin, and NCAM, were expressed along extrasynaptic regions of myofibers. One of the muscle proteins that displayed increased glycosylation with the CT antigen in the transgenic mice was alpha-dystroglycan. These experiments provide the first in vivo evidence that a synaptic carbohydrate antigen has important roles in the development of the neuromuscular synapse and suggest that the CT antigen is involved in controlling the expression of synaptic molecules.     

Cancer research by means of tissue engineering – is there a rationale?

Tissue engineering (TE) has evoked new hopes for the cure of organ failure and tissue loss by creating functional substitutes in the laboratory. Besides various innovations in the context of Regenerative Medicine (RM), TE also provided new technology platforms to study mechanisms of angiogenesis and tumour cell growth as well as potentially tumour cell spreading in cancer research. Recent advances in stem cell technology – including embryonic and adult stem cells and induced pluripotent stem cells – clearly show the need to better understand all relevant mechanisms to control cell growth when such techniques will be administered to patients. Such TE‐Cancer research models allow us to investigate the interactions that occur when replicating physiological and pathological conditions during the initial phases of replication, morphogenesis, differentiation and growth under variable given conditions. Tissue microenvironment has been extensively studied in many areas of TE and it plays a crucial role in cell signalling and regulation of normal and malignant cell functions. This article is intended to give an overview on some of the most recent developments and possible applications of TE and RM methods with regard to the improvement of cancer research with TE platforms. The synthesis of TE with innovative methods of molecular biology and stem‐cell technology may help investigate and potentially modulate principal phenomena of tumour growth and spreading, as well as tumour‐related angiogenesis. In the future, these models have the potential to investigate the optimal materials, culture conditions and material structure to propagate tumour growth.     

Dual or triple activation of TLR7, TLR8, and/or TLR9 by single-stranded oligoribonucleotides

The toll-like receptors (TLRs) 7, 8, and 9 stimulate innate immune responses upon recognizing pathogen nucleic acids. Certain GU- or AU-rich RNA sequences were described to differentiate between human TLR7- and TLR8-mediated immune effects. Those single-stranded RNA molecules require endosomal delivery for stabilization against ribonucleases. We have discovered RNA sequences that preferentially activate TLR7, form higher ordered structures, and do not require specific cellular delivery. In addition, a dual activation of TLR8 and TLR9 without affecting TLR7 can be achieved by chimeric molecules consisting of GU-rich RNA and Cytosin (C) phosphordiester or phosphorthioat (p) guanine (CpG) motif DNA sequences. Such chimeras stimulate TLR9-mediated type I interferon (IFN) and TLR8-depending proinflammatory cytokine and chemokine production upon primary human cell activation. However, an RNA-dependent TLR7 IFN-α cytokine release is suppressed by the phosphorothioate DNA sequence contained in the chimeric molecule. To convert the immune response of a single-stranded RNA from TLR7/8 to TLR9, a simple chemical modification at the 5' end proves to be sufficient. Such 8-oxo-2'-deoxy-guanosine or 8-bromo-2'-deoxy-guanosine modifications of the first guanosine in GU-rich single-stranded RNAs convert the immune response to include TLR9 activation and demonstrate strong additive effects for type I IFN immune responses in human primary cells.     

DeltaC and DeltaD interact as Notch ligands in the zebrafish segmentation clock

We describe the production and characterisation of two monoclonal antibodies, zdc2 and zdd2, directed against the zebrafish Notch ligands DeltaC and DeltaD, respectively. We use our antibodies to show that these Delta proteins can bind to one another homo- and heterophilically, and to study the localisation of DeltaC and DeltaD in the zebrafish nervous system and presomitic mesoderm (PSM). Our findings in the nervous system largely confirm expectations from previous studies, but in the PSM we see an unexpected pattern in which the localisation of DeltaD varies according to the level of expression of DeltaC: in the anterior PSM, where DeltaC is plentiful, the two proteins are colocalised in intracellular puncta, but in the posterior PSM, where DeltaC is at a lower level, DeltaD is seen mainly on the cell surface. Forced overexpression of DeltaC reduces the amount of DeltaD on the cell surface in the posterior PSM; conversely, loss-of-function mutation of DeltaC increases the amount of DeltaD on the cell surface in the anterior PSM. These findings suggest an explanation for a long-standing puzzle regarding the functions of the two Delta proteins in the somite segmentation clock--an explanation that is based on the proposition that they associate heterophilically to activate Notch.     

Anaerobic sulfide oxidation with nitrate by a freshwater Beggiatoa enrichment culture

A lithotrophic freshwater Beggiatoa strain was enriched in O2-H2S gradient tubes to investigate its ability to oxidize sulfide with NO3- as an alternative electron acceptor. The gradient tubes contained different NO3- concentrations, and the chemotactic response of the Beggiatoa mats was observed. The effects of the Beggiatoa sp. on vertical gradients of O2, H2S, pH, and NO3- were determined with microsensors. The more NO3- that was added to the agar, the deeper the Beggiatoa filaments glided into anoxic agar layers, suggesting that the Beggiatoa sp. used NO3- to oxidize sulfide at depths below the depth that O2 penetrated. In the presence of NO3- Beggiatoa formed thick mats (>8 mm), compared to the thin mats (ca. 0.4 mm) that were formed when no NO3- was added. These thick mats spatially separated O2 and sulfide but not NO3- and sulfide, and therefore NO3- must have served as the electron acceptor for sulfide oxidation. This interpretation is consistent with a fourfold-lower O2 flux and a twofold-higher sulfide flux into the NO3- -exposed mats compared to the fluxes for controls without NO3-. Additionally, a pronounced pH maximum was observed within the Beggiatoa mat; such a pH maximum is known to occur when sulfide is oxidized to S0 with NO3- as the electron acceptor.     

Ultrastructural analysis of ESCRT proteins suggests a role for endosome-associated tubular-vesicular membranes in ESCRT function

The endosomal sorting complex required for transport (ESCRT) is thought to support the formation of intralumenal vesicles of multivesicular bodies (MVBs). The ESCRT is also required for the budding of HIV and has been proposed to be recruited to the HIV-budding site, the plasma membrane of T cells and MVBs in macrophages. Despite increasing data on the function of ESCRT, the ultrastructural localization of its components has not been determined. We therefore localized four proteins of the ESCRT machinery in human T cells and macrophages by quantitative electron microscopy. All the proteins were found throughout the endocytic pathway, including the plasma membrane, with only around 10 and 3% of the total labeling in the cytoplasm and on the MVBs, respectively. The majority of the labeling (45%) was unexpectedly found on tubular-vesicular endosomal membranes rather than on endosomes themselves. The ESCRT labeling was twice as concentrated on early and late endosomes/lysosomes in macrophages compared with that in T cells, where it was twice more abundant at the plasma membrane. The ESCRT proteins were not redistributed on HIV infection, suggesting that the amount of ESCRT proteins located at the budding site suffices for HIV release. These results represent the first systematic ultrastructural localization of ESCRT and provide insights into its role in uninfected and HIV-infected cells.     

Association of HLA class I antigen abnormalities with disease progression and early recurrence in prostate cancer

Defects in HLA class I antigen processing machinery (APM) component expression often have a negative impact on the clinical course of tumors and on the response to T cell-based immunotherapy. Since only scant information is available about the frequency and clinical significance of HLA class I APM component abnormalities in prostate cancer, the APM component expression pattern was analyzed in 59 primary prostate carcinoma, adjacent normal tissues, as well as in prostate carcinoma cell lines. The IFN-γ inducible proteasome subunits LMP2 and LMP7, TAP1, TAP2, calnexin, calreticulin, ERp57, and tapasin are strongly expressed in the cytoplasm of normal prostate cells, whereas HLA class I heavy chain (HC) and β₂-microglobulin are expressed on the cell surface. Most of the APM components were downregulated in a substantial number of prostate cancers. With the exception of HLA class I HC, TAP2 and ERp57 not detectable in about 0.5% of tumor lesions, all other APM components were not detected in at least 21% of lesions analyzed. These APM component defects were associated with a higher Gleason grade of tumors and an early disease recurrence. Prostate carcinoma cell lines also exhibit a heterogeneous, but reduced constitutive APM component expression pattern associated with lack or reduced HLA class I surface antigens, which could be upregulated by IFN-γ. Our results suggest that HLA class I APM component abnormalities are mainly due to regulatory mechanisms, play a role in the clinical course of prostate cancer and on the outcome of T cell-based immunotherapies.     

Fumarate dissimilation and differential reductant flow by Clostridium formicoaceticum and Clostridium aceticum

Methanol and the O-methyl group of vanillate did not support the growth of Clostridium formicoaceticum in defined medium under CO₂-limited conditions; however, they were growth supportive when fumarate was provided concomitantly. Fumarate alone was not growth supportive under these conditions. Fumarate reduction (dissimilation) to succinate was the predominant electron-accepting, energy-conserving process for methanol-derived reductant under CO₂-limited conditions. However, when both reductant sinks, i.e., fumarate and CO₂, were available, reductant was redirected towards CO₂ in defined medium. In contrast, in undefined medium with both reductant sinks available, C. formicoaceticum simultaneously engaged fumarate dismutation and the concomitant usage of CO₂ and fumarate as reductant sinks. With Clostridium aceticum, fumarate also substituted for CO₂, and H₂ became growth supportive under CO₂-limited conditions. Fumarate dissimilation was the predominant electron-accepting process under CO₂-limited conditions; however, when both reductant sinks were available, H₂-derived reductant was routed towards CO₂, indicating that acetogenesis was the preferred electron-accepting process when reductant flow originated from H₂. Collectively, these findings indicate that fumarate dissimilation, not dismutation, is selectively used under certain conditions and that such usage of fumarate is subject to complex regulation.