Are Hox Genes Ancestrally Involved in Axial Patterning? Evidence from the Hydrozoan Clytia hemisphaerica (Cnidaria)

Background: The early evolution and diversification of Hox-related genes in eumetazoans has been the subject of conflicting hypotheses concerning the evolutionary conservation of their role in axial patterning and the pre-bilaterian origin of the Hox and ParaHox clusters. The diversification of Hox/ParaHox genes clearly predates the origin of bilaterians. However, the existence of a "Hox code' predating the cnidarian-bilaterian ancestor and supporting the deep homology of axes is more controversial. This assumption was mainly based on the interpretation of Hox expression data from the sea anemone, but growing evidence from other cnidarian taxa puts into question this hypothesis. Methodology/Principal Findings: Hox, ParaHox and Hox-related genes have been investigated here by phylogenetic analysis and in situ hybridisation in Clytia hemisphaerica, an hydrozoan species with medusa and polyp stages alternating in the life cycle. Our phylogenetic analyses do not support an origin of ParaHox and Hox genes by duplication of an ancestral ProtoHox cluster, and reveal a diversification of the cnidarian HOX9-14 genes into three groups called A, B, C. Among the 7 examined genes, only those belonging to the HOX9-14 and the CDX groups exhibit a restricted expression along the oralaboral axis during development and in the planula larva, while the others are expressed in very specialised areas at the medusa stage. Conclusions/Significance: Cross species comparison reveals a strong variability of gene expression along the oral-aboral axis and during the life cycle among cnidarian lineages. The most parsimonious interpretation is that the Hox code, collinearity and conservative role along the antero-posterior axis are bilaterian innovations

Two-dimensional crystallization of a histidine-tagged protein on monolayers of fluidity-enhanced Ni2+-chelating lipids

Protein two-dimensional (2D) crystallization on lipid monolayers is a powerful method for structure determination. This method has been extended using the specific and strong interaction between histidine residues (of an overexpressed protein) and Ni2+ ions tethered at the headgroup of synthetic lipids. Understanding and then improving the process of adsorption and crystallization of proteins on a lipid monolayer are prerequisites for the production of large and well-ordered crystals of any soluble or membrane His-tagged proteins. These large high-quality arrays are necessary for structural studies at high resolution. We have investigated the steps of adsorption and 2D crystallization of His-HunR using three different lipids: (i) 2-(bis-carboxymethyl-amino)-6-[2-(1,3-di-O-oleyl-glyceroxy)-acetyl-amino] hexanoic acid nickel- (II) (Ni-NTA-DOGA), which has been previously used, and two specifically designed Ni2+-chelating lipids, (ii) Ni-NTA-BB, which has two branched (B) alkyl chains and (iii) Ni-NTA-BF, a nonsymmetrical lipid with one branched (B) and one fluorinated (F) chain. These three lipids, when spread at the air-water interface, exhibit various fluidity properties. The adsorption and crystallization process have been monitored in situ and in real time using a variety of complementary techniques such as ellipsometry, shear rigidity measurements of the monolayer, and Brewster angle microscopy, and we have also developed X-ray reflectivity analysis to investigate the evolution of the electron density profile of the lipid-protein monolayer. Electron microscopy observations of the protein-lipid layers were also performed. We have found that the fluidity of the lipid monolayer has a marked influence on the rates of protein adsorption and crystallization of His-HupR. When Ni-NTA-BB is used to form the monolayer, it accelerates the process of protein adsorption and the protein crystallization is three times faster than when Ni-NTA-DOGA is used

Synchrotron radiation diffraction from two-dimensional protein crystals at the air/water interface.

Protein structure determination by classical x-ray crystallography requires three-dimensional crystals that are difficult to obtain for most proteins and especially for membrane proteins. An alternative is to grow two-dimensional (2D) crystals by adsorbing proteins to ligand-lipid monolayers at the surface of water. This confined geometry requires only small amounts of material and offers numerous advantages: self-assembly and ordering over micrometer scales is easier to obtain in two dimensions; although fully hydrated, the crystals are sufficiently rigid to be investigated by various techniques, such as electron crystallography or micromechanical measurements. Here we report structural studies, using grazing incidence synchrotron x-ray diffraction, of three different 2D protein crystals at the air-water interface, namely streptavidine, annexin V, and the transcription factor HupR. Using a set-up of high angular resolution, we observe narrow Bragg reflections showing long-range crystalline order in two dimensions. In the case of streptavidin the angular range of the observed diffraction corresponds to a resolution of 10 A in plane and 14 A normal to the plane. We show that this approach is complementary to electron crystallography but without the need for transfer of the monolayer onto a grid. Moreover, as the 2D crystals are accessible from the buffer solution, the formation and structure of protein complexes can be investigated in situ

Dual biological functions of an interleukin-1 receptor antagonist–interleukin-10 fusion protein and its suppressive effects on joint inflammation

The aim of this study was to construct and purify a novel interleukin-1 receptor antagonist (IL-1ra)–interleukin-10 (IL-10) fusion protein and determine its biological function and anti-inflammatory effects. The isolated cDNAs of two inhibitory cytokines (IL-1ra, IL-10) were used to construct a cDNA for the IL-1ra–IL-10 fusion protein. The expressed recombinant cytokines and fusion product were purified and their biological properties analysed. The anti-IL-1 effect was evaluated by using a thymocyte-proliferation assay, and the IL-10 effect was investigated by the inhibition of interferon-γ (IFN-γ) production from splenocytes. The clinical response and histological analyses were studied in an adjuvant arthritic rat model. The fusion protein was 38 000 molecular weight in size. Sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) and immunoblotting demonstrated that the purified protein was recognized by both IL-1ra and IL-10 antibodies. The fusion protein significantly inhibited IL-1-mediated thymocyte proliferation and concanavalin A (ConA)-primed IFN-γ production from splenocytes. The fusion protein also suppressed joint swelling (paw circumference reduced from 5·0 ± 0·2 to 4·1 ± 0·1 cm; paw thickness ≈ 2 mm in difference) and synovial inflammation in adjuvant arthritis of rats. Our investigations indicate that this fusion protein effectively suppresses inflammatory arthritis and may initiate a trend for future clinical application to target multiple molecules at the same time