Embryonic shell formation in the snail Biomphalaria glabrata: a comparison between scanning electron microscopy (SEM) and synchrotron radiation micro computer tomography (SR{micro}CT)

Embryos of different developmental stages and newly hatchedjuveniles of the freshwater snail Biomphalaria glabrata wereinvestigated by synchrotron radiation micro computer tomography(SRµCT). Because this method is sensitive for objectswith a high X-ray density, it is ideally suited to study mineralizedtissues without the need for dissection of the sample, i.e.removal of the soft tissue. This is a clear advantage over scanningelectron microscopy (SEM). However, the resolution is inferiorto SEM (about 1–2 µm compared to a few nm).After the measurement, computer-processed handling (virtualturning, cutting and measuring) of the object is possible. Thedevelopment of the calcified shell in embryos before hatching(age 60, 72, 96 and 120 h after oviposition) was investigatedand both methods were compared. While it was not possible tofind a calcified shell in 60 h old embryos, the shell in72 h old embryos was almost fully mineralized. By SRµCT,the weight of the calcified shell was estimated to 0.64, 9.59and 30.3 µg for embryos of 72, 96 and 120 h.All juvenile snails, of 5 days and 4 weeks after hatching, containedconcretions in the stomach, mostly consisting of calcium phosphate. (Received 10 May 2007; accepted 20 September 2007)     

Structural determinants of the anti-HIV activity of a CCR5 antagonist derived from Toxoplasma gondii

The protozoan parasite Toxoplasma gondii possesses a protein, cyclophilin-18 (C-18), which binds to the chemokine receptor CCR5, induces interleukin-12 production from murine dendritic cells, and inhibits fusion and infectivity of human immunodeficiency virus 1 (HIV-1) R5 viruses by co-receptor antagonism. Site-directed mutagenesis was employed to identify the domains in C-18 responsible for its CCR5 binding and antiviral functions. To do so we focused on amino acid differences with Plasmodium falciparum cyclophilin, which, although 53% identical with C-18, has minimal binding activity for CCR5, and we generated 22 mutants with substitutions in the regions of non-homology located on the putative surface of the molecule. Two mutations situated on the face of C-18, predicted to be involved in its interaction with the ligand cyclosporin A, were shown to be critical for CCR5-binding and the inhibition of HIV-1 fusion and infectivity. In contrast, four mutations in C-18 specifically designed to abolish the peptidyl-prolyl cis-trans-isomerase activity of the protein failed to inactivate its CCR5 binding and HIV inhibitory activities. Interleukin-12 induction by C-18, on the other hand, was abrogated by mutations effecting either the CCR5 binding or enzymatic function of the molecule. These findings shed light on the structural basis of the molecular mimicry of the chemokine function by a pathogen-derived protein and provide a basis for further modification of C-18 into an antiviral agent.     

Scientific and technological aspects of aqueous glasses

The physical nature of a glass, as related to stable liquid and crystalline solid phases was defined by Kauzmann in 1948. Since then, glass research has been almost exclusively confined to inorganic materials. This review aims to demonstrate that many substances, not falling into the category of classical 'materials', can be rendered into amorphous states. In particular, water itself, but also water soluble and water sensitive organic molecules, some of them biomolecules, can be rendered into supersaturated and solid solutions. New ways of studying and applying amorphisation processes have led to major advances in food and pharmaceutical processing aimed mainly at the stabilisation of labile materials. Because of their molecular similarities to water, polyhydroxy compounds are attracting particular interest as potential matrix elements in the preparation of glassy products.     

De novo insertion of an<Emphasis Type="Italic"> Alu</Emphasis> sequence in the coding region of the<Emphasis Type="Italic"> CLCN5</Emphasis> gene results in Dent's disease

Dent's disease is an X-linked renal tubular disorder characterized by low-molecular-weight proteinuria, hypercalciuria, nephrocalcinosis, nephrolithiasis, and eventual renal failure. Various types of mutations in the renal chloride channel gene, CLCN5, have been identified in patients with this disease. We studied a Spanish patient with Dent's disease and found, by polymerase chain reaction amplification of the CLCN5 exons, an abnormally large exon 11. Sequencing analysis revealed that this was attributable to the insertion in codon 650 of an Alu element of the "young" Ya5 subfamily. The Alu element was inserted with the same orientation as the CLCN5 gene and arose de novo on the maternal chromosome. Polymorphism analysis indicated that the insertion occurred in the germline of the maternal grandfather. The presence of a long poly(A) tract and evidence for a 16-bp target-site duplication implied that the Alu element was integrated by retrotransposition. This mutation predicts a truncated ClC-5 protein that lacks part of the carboxy-terminus and is likely to result in loss of function of the chloride channel. Insertions of Alu sequences, which are rarely found in coding regions, have occasionally been reported to cause other genetic diseases. However, this is the first report of a retrotransposon insertion in the CLCN5 gene associated with Dent's disease.Electronic Supplementary Material Supplementary material is available in the online version of this article at     

Catabolism of volatile sulfur compounds precursors by Brevibacterium linens and Geotrichum candidum, two microorganisms of the cheese ecosystem

Two Brevibacterium linens strains and the cheese-ripening yeast Geotrichum candidum were compared with regard to their ability to produce volatile sulfur compounds (VSCs) from three different precursors namely L-methionine, 4-methylthio-2-oxobutyric acid (KMBA) and 4-methylthio-2-hydroxybutyric acid (HMBA). All microorganisms were able to convert these precursors to VSCs. However, although all were able to produce VSCs from L-methionine, only G. candidum accumulated KMBA when cultivated on this amino acid, contrary to B. linens suggesting that the transamination pathway is not active in this microorganism. Conversely, a L-methionine gamma-lyase activity--which catalyses the one step L-methionine to methanethiol (MTL) degradation route--was only found in B. linens strains. Several other enzymatic activities involved in the catabolism of the precursors tested were investigated. KMBA transiently accumulated in G. candidum cultures, and was then reduced to HMBA by a KMBA dehydrogenase (KDH) activity. This activity was not detected in B. linens. Despite no HMBA dehydrogenase (HDH) was found in G. candidum, a strong HMBA oxidase (HOX) activity was measured in this microorganism. This latter activity was weakly active in B. linens. KMBA and HMBA demethiolating activities were found in all the microorganisms. Our results illustrate the metabolic diversity between cheese-ripening microorganisms of the cheese ecosystem.     

Substitution of a single residue in Stichodactyla helianthus peptide, ShK-Dap22, reveals a novel pharmacological profile

ShK, a peptide isolated from Stichodactyla helianthus venom, blocks the voltage-gated potassium channels, K(v)1.1 and K(v)1.3, with similar high affinity. ShK-Dap(22), a synthetic derivative in which a diaminopropionic acid residue has been substituted at position Lys(22), has been reported to be a selective K(v)1.3 inhibitor and to block this channel with equivalent potency as ShK [Kalman et al. (1998) J. Biol. Chem. 273, 32697-32707]. In this study, a large body of evidence is presented which indicates that the potencies of wild-type ShK peptide for both K(v)1.3 and K(v)1.1 channels have been previously underestimated. Therefore, the affinity of ShK-Dap(22) for both channels appears to be ca. 10(2)-10(4)-fold weaker than ShK. ShK-Dap(22) does display ca. 20-fold selectivity for human K(v)1.3 vs K(v)1.1 when measured by the whole-cell voltage clamp method but not in equilibrium binding assays. ShK-Dap(22) has low affinity for K(v)1.2 channels, but heteromultimeric K(v)1.1-K(v)1.2 channels form a receptor with ca. 200-fold higher affinity for ShK-Dap(22) than K(v)1.1 homomultimers. In fact, K(v)1.1-K(v)1.2 channels bind ShK-Dap(22) with only ca. 10-fold less potency than ShK and reveal a novel pharmacology not predicted from the homomultimers of K(v)1.1 or K(v)1.2. The concentrations of ShK-Dap(22) needed to inhibit human T cell activation were ca. 10(3)-fold higher than those of ShK, in good correlation with the relative affinities of these peptides for inhibiting K(v)1.3 channels. All of these data, taken together, suggest that ShK-Dap(22) will not have the same in vivo immunosuppressant efficacy of other K(v)1.3 blockers, such as margatoxin or ShK. Moreover, ShK-Dap(22) may have undesired side effects due to its interaction with heteromultimeric K(v)1.1-K(v)1.2 channels, such as those present in brain and/or peripheral tissues.     

Artificial chaperone-assisted refolding of bovine carbonic anhydrase using molecular assemblies of stimuli-responsive polymers

An artificial chaperone, which can decrease the protein aggregation and increase the reactivation yield of denatured protein in a fashion similar to natural chaperone, was newly developed using stimuli-responsive polymers. It has previously been reported that the addition of poly(propylene oxide)-phenyl-poly(ethylene glycol) (PPOn-Ph-PEG) with the unit number of PPO (n) 33 could enhance the refolding of bovine carbonic anhydrase (Kuboi et al. J. Chromatogr. B 2000, 243, 213). PPO-Ph-PEG with a large PPO chain (n = 50) was synthesized and the surface properties were characterized by both the relative fluorescence intensity of 1-anilino-8-naphthalene sulfonate (ANS) and the fluidity determined by diphenylhexatriene (DPH). The variation of ANS intensity and DPH fluidity is shown in a diagram as functions of temperature and polymer concentration. The high values of ANS intensity and fluidity of PPO50-Ph-PEG were obtained in a relatively wide conditional range (more than 0.08 mM and more than 15 degrees C) although the conditions showing the high values of PPO33-Ph-PEG were restricted (more than 0.1 mM and more than 40 degrees C). It was also found that molecular assemblies of PPOn-Ph-PEG with diameters of 7-18 nm were formed in the above conditions. On the basis of the surface properties of their polymer self-assemblies, the possibility of using them as an artificial chaperone was investigated. The effect of the addition of PPOn-Ph-PEG on the reactivation yield of a model protein, carbonic anhydrase from bovine (CAB), and the optical density of the solution was examined at various temperatures and concentrations. The reactivation yield of CAB was strongly enhanced and the aggregate formation (the optical density) was suppressed by adding PPOn-Ph-PEG in the above conditions, which show high ANS intensity and DPH fluidity. Especially in the presence of 0.1 mM PPO50-Ph-PEG, the reactivation yield of CAB reached approximately 100% at 40-55 degrees C. It was thus found that self-assemblies of the present polymer could be utilized as an artificial chaperone by selecting suitable stimuli conditions.     

Random peptide libraries displayed on adeno-associated virus to select for targeted gene therapy vectors

Characterizing the molecular diversity of the cell surface is critical for targeting gene therapy. Cell type-specific binding ligands can be used to target gene therapy vectors. However, targeting systems in which optimum eukaryotic vectors can be selected on the cells of interest are not available. Here, we introduce and validate a random adeno-associated virus (AAV) peptide library in which each virus particle displays a random peptide at the capsid surface. This library was generated in a three-step system that ensures encoding of displayed peptides by the packaged DNA. As proof-of-concept, we screened AAV-libraries on human coronary artery endothelial cells. We observed selection of particular peptide motifs. The selected peptides enhanced transduction in coronary endothelial cells but not in control nonendothelial cells. This vector targeting strategy has advantages over other combinatorial approaches such as phage display because selection occurs within the context of the capsid and may have a broad range of applications in biotechnology and medicine.