Human glioma cell culture: two FCS-free media could be recommended for clinical use in immunotherapy

Immunotherapy, particularly active vaccination, may be developed as an effective and safe treatment modality for malignant gliomas, which continue to have a poor prognosis, despite advances in surgical techniques and adjuvant chemotherapy and radiotherapy. Since no glioma-specific tumor-associated antigens (TAAs) have been discovered, autologous tumor cells or well-established glioma cell lines could be used in future vaccination protocols to induce antitumour immunity against unknown TAAs. One obstacle for clinical use of these tumour cell vaccines is related to foetal calf serum (FCS). Efforts are currently being directed toward developing FCS-free media and serum-free alternatives to culture these cell vaccines. In this study, a medium containing human serum and one serum-free medium (UltraCulture), supplemented or not with epidermal growth factor, were tested on morphology, survival, DNA content and TAA expression of human glioma cell lines and glioma biopsy primary cultures. Their effects were compared on FCS-containing medium. Results show that, whatever the medium used, no significant variations in morphology and survival were observed. Furthermore, human serum-containing medium or UltraCulture preserved at early passage cultures the cell population of interest present in the biopsies before culture. In addition, the expression profile of eight TAAs was similar between these media. These data indicate that human serum-containing medium and UltraCulture serum-free medium could be promising candidates to produce tumour-cell vaccines.     

Tri-locus sequence data reject a “Gondwanan origin hypothesis” for the African/South Pacific crab genus Hymenosoma

Crabs of the family Hymenosomatidae are common in coastal and shelf regions throughout much of the southern hemisphere. One of the genera in the family, Hymenosoma, is represented in Africa and the South Pacific (Australia and New Zealand). This distribution can be explained either by vicariance (presence of the genus on the Gondwanan supercontinent and divergence following its break-up) or more recent transoceanic dispersal from one region to the other. We tested these hypotheses by reconstructing phylogenetic relationships among the seven presently-accepted species in the genus, as well as examining their placement among other hymenosomatid crabs, using sequence data from two nuclear markers (Adenine Nucleotide Transporter [ANT] exon 2 and 18S rDNA) and three mitochondrial markers (COI, 12S and 16S rDNA). The five southern African representatives of the genus were recovered as a monophyletic lineage, and another southern African species, Neorhynchoplax bovis, was identified as their sister taxon. The two species of Hymenosoma from the South Pacific neither clustered with their African congeners, nor with each other, and should therefore both be placed into different genera. Molecular dating supports a post-Gondwanan origin of the Hymenosomatidae. While long-distance dispersal cannot be ruled out to explain the presence of the family Hymenosomatidae on the former Gondwanan land-masses and beyond, the evolutionary history of the African species of Hymenosoma indicates that a third means of speciation may be important in this group: gradual along-coast dispersal from tropical towards temperate regions, with range expansions into formerly inhospitable habitat during warm climatic phases, followed by adaptation and speciation during subsequent cooler phases.     

Regulation of convergence and extension movements during vertebrate gastrulation by the Wnt/PCP pathway

Vertebrate gastrulation entails massive cell movements that establish and shape the germ layers. During gastrulation, the individual cell behaviors are strictly coordinated in time and space by various signaling pathways. These pathways instruct the cells about proliferation, shape, fate and migration into proper location. Convergence and extension (C&E) movements during vertebrate gastrulation play a major role in the shaping of the embryonic body. In vertebrates, the Wnt/Planar Cell Polarity (Wnt/PCP) pathway is a key regulator of C&E movements, essential for several polarized cell behaviors, including directed cell migration, and mediolateral and radial cell intercalation. However, the molecular mechanisms underlying the acquisition of Planar Cell Polarity by highly dynamic mesenchymal cells engaged in C&E are still not well understood. Here we review new evidence implicating the Wnt/PCP pathway in specific cell behaviors required for C&E during zebrafish gastrulation, in comparison to other vertebrates. We also discuss findings on the molecular regulation and the interaction of the Wnt/PCP pathway with other signaling pathways during gastrulation movements.     

Mitochondrial F1F0-ATP synthase and organellar internal architecture

The mitochondrial F₁F₀-ATP synthase adopts supramolecular structures. The interaction domains between monomers involve components belonging to the F₀ domains. In Saccharomyces cerevisiae, alteration of these components destabilizes the oligomeric structures, leading concomitantly to the appearance of monomeric species of ATP synthase and anomalous mitochondrial morphologies in the form of onion-like structures. The mitochondrial ultrastructure at the cristae level is thus modified. Electron microscopy on cross-sections of wild type mitochondria display many short cristae with narrowed intra-cristae space, whereas yeast mutants defected in supramolecular ATP synthases assembly present a low number of large lamellar cristae of constant thickness and traversing the whole organelle. The growth of these internal structures leads finally to mitochondria with sphere-like structures with a mean diameter of 1 μm that are easily identified by epifluorescence microscopy. As a result, ATP synthase is an actor of the mitochondrial ultrastructure in yeast. This paper reviews the ATP synthase components whose modifications lead to anomalous mitochondrial morphology and also provides a schema showing the formation of the so-called onion-like structures.     

Mutations in CNNM4 Cause Recessive Cone-Rod Dystrophy with Amelogenesis Imperfecta

Cone-rod dystrophies are inherited dystrophies of the retina characterized by the accumulation of deposits mainly localized to the cone-rich macular region of the eye. Dystrophy can be limited to the retina or be part of a syndrome. Unlike nonsyndromic cone-rod dystrophies, syndromic cone-rod dystrophies are genetically heterogeneous with mutations in genes encoding structural, cell-adhesion, and transporter proteins. Using a genome-wide single-nucleotide polymorphism (SNP) haplotype analysis to fine map the locus and a gene-candidate approach, we identified homozygous mutations in the ancient conserved domain protein 4 gene (CNNM4) that either generate a truncated protein or occur in highly conserved regions of the protein. Given that CNNM4 is implicated in metal ion transport, cone-rod dystrophy and amelogenesis imperfecta may originate from abnormal ion homeostasis.     

Functional studies of membrane-bound and purified human Hedgehog receptor Patched expressed in yeast

The Sonic Hedgehog (Shh) signalling pathway plays an important role both in embryonic development and in adult stem cell function. Inappropriate regulation of this pathway is often due to dysfunction between two membrane receptors Patched (Ptc) and Smoothened (Smo), which lead to birth defects, cancer or neurodegenerative diseases. However, little is known about Ptc, the receptor of the Shh protein, and the way Ptc regulates Smo, the receptor responsible for the transduction of the signal. To develop structure-function studies of these receptors, we expressed human Ptc (hPtc) in the yeast Saccharomyces cerevisiae. We demonstrated that hPtc expressed in a yeast membrane fraction is able to interact with its purified ligand Shh, indicating that hPtc is produced in yeast in its native conformational state. Using Surface Plasmon Resonance technology, we showed that fluorinated surfactants preserve the ability of hPtc to interact with its ligand after purification. This is the first report on the heterologous expression and the purification of a native and stable conformation of the human receptor Ptc. This work will allow the scale-up of hPtc production enabling its biochemical characterization, allowing the development of new therapeutic approaches against diseases induced by Shh signalling dysfunction.     

Classical cadherins control nucleus and centrosome position and cell polarity

Control of cell polarity is crucial during tissue morphogenesis and renewal, and depends on spatial cues provided by the extracellular environment. Using micropatterned substrates to impose reproducible cell–cell interactions, we show that in the absence of other polarizing cues, cell–cell contacts are the main regulator of nucleus and centrosome positioning, and intracellular polarized organization. In a variety of cell types, including astrocytes, epithelial cells, and endothelial cells, calcium-dependent cadherin-mediated cell–cell interactions induce nucleus and centrosome off-centering toward cell–cell contacts, and promote orientation of the nucleus–centrosome axis toward free cell edges. Nucleus and centrosome off-centering is controlled by N-cadherin through the regulation of cell interactions with the extracellular matrix, whereas the orientation of the nucleus–centrosome axis is determined by the geometry of N-cadherin–mediated contacts. Our results demonstrate that in addition to the specific function of E-cadherin in regulating baso-apical epithelial polarity, classical cadherins control cell polarization in otherwise nonpolarized cells.     

X-Ray-Radiation-Induced Cooperative Atomic Movements in Protein

X-rays interact with biological matter and cause damage. Proteins and other macromolecules are damaged primarily by ionizing X-ray photons and secondarily by reactive radiolytic chemical species. In particular, protein molecules are damaged during X-ray diffraction experiments with protein crystals, which is, in many cases, a serious hindrance to structure solution. The local X-ray-induced structural changes of the protein molecule have been studied using a number of model systems. However, it is still not well understood whether these local chemical changes lead to global structural changes in protein and what the mechanism is.We present experimental evidence at atomic resolution indicating the movement of large parts of the protein globule together with bound water molecules in the early stages of radiation damage to the protein crystal. The data were obtained from a crystal cryocooled to ~ 100 K and diffracting to 1 ?. The movement of the protein structural elements occurs simultaneously with the decarboxylation of several glutamate and aspartate residues that mediate contacts between moving protein structural elements and with the rearrangement of the water network. The analysis of the anisotropy of atomic displacement parameters reveals that the observed atomic movements occur at different rates in different unit cells of the crystal. Thus, the examination of the cooperative atomic movement enables us to better understand how radiation-induced local chemical and structural changes of the protein molecule eventually lead to disorder in protein crystals.