Cutaneous Phaeohyphomycosis due to Alternaria Infectoria

Here we report a case of cutaneous alternariosis in a 74-year-old man treated by corticotherapy for myasthenia, and presenting with papular, crusted lesions on the left elbow and the right knee. Histological examination of the biopsy specimens showed fungal hyphae associated with round-shaped cells which were highly suggestive of alternariosis. Mycological culture allowed the isolation of a dematiaceous fungus which was identified as a member of the Alternaria infectoria species-group. This was confirmed by PCR amplification and sequencing of the internal transcribed spacer domain of the gene encoding nuclear ribosomal DNA and of the mitochondrial small subunit ribosomal DNA domain. The fungus was therefore referred to the Scientific Institute of Public Health where it was identified as Alternaria infectoria, on the basis of its very small 1 or 2-celled conidia often arranged in long chains and presenting with very long secondary conidiophores. Corticotherapy was stopped and a local antifungal treatment with ketoconazole was initiated, allowing the stabilisation of the cutaneous lesions within 2 months.     

A toy model for predicting the rate of amyloid formation from unfolded protein

We develop a toy model for predicting the rate of amyloid formation from an unfolded polypeptide. The model assumes irreversible amyloid growth, employs a collision encounter scheme and uses a Gaussian chain approximation to describe the polypeptide sequence. A principal feature of the model is its dependence on a number of key sequence residues whose correct placement, geometric arrangement and orientation in relation to their interacting partners define the success, or otherwise, of the amyloid formation reaction. Although not realistic at the molecular level, the model captures some essential features of the system and is therefore useful from a heuristic standpoint. For the case of amyloid formation from an unstructured state, the model suggests that the major determinants of the rate of fibril formation are the length of the sequence separating the critical amino acids promoting amyloid formation and the positional placement of the critical residues within the sequence. Our findings suggest also that the sequence distance between the key interacting amino acid residues may play a role in defining the maximum width of a fibril and that the addition of non-interacting segments of long structure-less polypeptide chain to an amyloidogenic peptide may act to inhibit fibril formation. We discuss these findings with reference to the placement of critical sequence residues within the polypeptide chain, the design of polypeptides with lower amyloid formation propensities and the development of aggregation inhibitors as potential therapeutics for protein depositional disorders.     

Cryptochrome 1 contributes to blue-light sensing in pea

Cryptochromes are widespread in higher plants but their physiological roles as blue-light photoreceptors have been examined in relatively few species. Screening in a phyA null mutant background has identified several blue-light response mutants in pea (Pisum sativum), including one that carries a substitution of a highly conserved glycine residue in the N-terminal photolyase-homologous domain of the pea CRY1 gene. Analyses of cry1, phyA, and phyB mutants show that all three photoreceptors contribute to seedling photomorphogenesis under high-irradiance blue light, whereas phyA is the main photoreceptor active under low irradiances. Triple phyA phyB cry1 mutants grown under high-irradiance blue light are indistinguishable from dark-grown wild-type plants in length and leaf expansion but show a small residual response to higher-irradiance white light. Monogenic cry1 mutants have little discernable phenotype at the seedling stage, but later in development are more elongated than wild-type plants. In addition, the loss of cry1 moderates the short-internode phenotype of older phyA mutants, suggesting an antagonism between phyA and cry1 under some conditions. Pea cry1 has a small inhibitory effect on flowering under long and short days. However, the phyA cry1 double mutant retains a clear promotion of flowering in response to blue-light photoperiod extensions, indicating a role for one or more additional blue-light photoreceptors in the control of flowering in pea.     

Application of a two-stage Syrian hamster embryo cell transformation assay to cigarette smoke particulate matter

The induction of transformation in Syrian hamster embryo (SHE) cells is a multifactorial process, in comparison to endpoints induced in in vitro genotoxicity assays such as Ames, mouse lymphoma and cytogenetics [Y. Berwald, L. Sachs, In vitro cell transformation with chemical carcinogens, Nature (London) 200 (1963) 1182-1184]. Furthermore, a number of non-genotoxic carcinogens and promoters such as clofibrate and diethylhexylphthalate, have been positively identified in this assay, while giving false negative results in traditional genotoxicity assays [H. Yamasaki, J. Ashby, M. Bignami, W. Jongen, K. Linnainmaa, R.F. Newbold, G. Nguyen-Ba, S. Parodi, E. Rivedal, D. Schiffmann, J.W.I.M. Simons, P. Vasseur, Nongenotoxic carcinogens: development of detection methods based on mechanisms: a European project, Mutat. Res. 353 (1996) 47-63]. A high concordance between results obtained in this assay when compared with rodent carcinogenesis bioassays has also been noted [R.J. Isfort, G.A. Kerckaert, R.A. LeBoeuf, Comparison of the standard and reduced pH Syrian hamster embryo (SHE) in vitro cell transformation assays to predict the carcinogenic potential of chemicals, Mutat. Res. 356 (1996) 11-63]. Carcinogenesis is known to be a multistage process, with agents potentially acting at each stage. Specifically, mouse skin painting experiments established that tumour induction could be mechanistically divided into two distinct phases, termed initiation and promotion. Initiation, is defined as the stage at which a normal cell is converted to a latent tumour cell, followed by promotion where the latent tumour cell progresses to a tumour [W.F. Friedwald, P. Rous, The initiating and promoting elements in tumour production: analysis of the effects of tar, benzpyrene and methylcholanthrene on rabbit skin, J. Exp. Med. 80 (1944) 101-125]. A protocol for the pH 6.7 SHE transformation assay has been developed which allows separation of cell transformation process into two phases, potentially analogous to initiation and promotion in vivo. This allows chemicals found to be positive in the traditional SHE cell transformation assay to be further classified as initiators or promoters. Following validation with known initiators, benzo(a)pyrene and N-methyl-N'-nitro-N-nitrosoguanidine and promoters, 12-O-tetradecanoyl-phorbol-13-acetate and phenobarbitone, the two-stage model was applied to cigarette smoke particulates which was found to act both at the initiation and promotion stage of cell transformation.     

Coalescence of membrane tethers: experiments, theory, and applications

Tethers are nanocylinders of lipid bilayer membrane, arising in situations ranging from micromanipulation experiments on synthetic vesicles to the formation of dynamic tubular networks in the Golgi apparatus. Relying on the extensive theoretical and experimental works aimed to understand the physics of individual tethers formation, we addressed the problem of the interaction between two nanotubes. By using a combination of micropipette manipulation and optical tweezers, we quantitatively studied the process of coalescence that occurred when the separation distance between both vesicle-tether junctions became smaller than a threshold length. Our experiments, which were supported by an original theoretical analysis, demonstrated that the measurements of the tether force and angle between tethers at coalescence directly yield the bending rigidity, kappa, and the membrane tension, sigma, of the vesicles. Contrary to other methods used to probe the bending rigidity of vesicles, the proposed approach permits a direct measurement of kappa without requiring any control of the membrane tension. Finally, after validation of the method and proposal of possible applications, we experimentally investigated the dynamics of the coalescence process.     

Dielectric control of counterion-induced single-chain folding transition of DNA

In the presence of condensing agents, single chains of giant double-stranded DNA undergo a first-order phase transition between an elongated coil state and a folded compact state. To connect this like-charged attraction phenomenon to counterion condensation, we performed a series of single-chain experiments on aqueous solutions of DNA, where we varied the extent of counterion condensation by varying the relative dielectric constant epsilon(r) from 80 to 170. Single-chain observations of changes in the conformation of giant DNA were performed by transmission electron microscopy and fluorescence microscopy, with tetravalent spermine (SPM(4+)) as a condensing agent. At a fixed dielectric constant, single DNA chains fold into a compact state upon the addition of spermine, whereas at a constant spermine concentration single DNA chains unfold with an increase in epsilon(r). In both cases, the transition is largely discrete at the level of single chains. We found that the critical concentration of spermine necessary to induce the single-chain folding transition increases exponentially as the dielectric constant increases, corresponding to 87-88% of the DNA charge neutralized at the onset of the transition. We also observed that the toroidal morphology of compact DNA partially unfolds when epsilon(r) is increased.     

EPR kinetic studies of the S−1 state in spinach thylakoids

The Y_Z decay kinetics in a formal S₋₁ state, regarded as a reduced state of the oxygen evolving complex, was determined using time-resolved EPR spectroscopy. This S₋₁ state was generated by biochemical treatment of thylakoid membranes with hydrazine. The steady-state oxygen evolution of the sample was used to optimize the biochemical procedure for performing EPR experiments. A high yield of the S₋₁ state was generated as judged by the two-flash delay in the first maximum of oxygen evolution in Joliot flash-type experiments. We have shown that the Y_Z re-reduction rate by the S₋₁ state is much slower than that of any other S-state transition in hydrazine-treated samples. This slow reduction rate in the S₋₁ to S₀ transition, which is in the order of the S₃ to S₀ transition rate, suggests that this transition is accompanied by some structural rearrangements. Possible explanations of this unique, slow reduction rate in the S₋₁ to S₀ transition are considered, in light of earlier observations by others on hydrazine/hydroxylamine reduced PS II samples.     

Identifying interactions between transmembrane helices from the adenosine A2A receptor

The human adenosine A(2A) receptor (A(2A)R) is an integral membrane protein and a member of the G-protein-coupled receptor (GPCR) superfamily, characterized by seven transmembrane (TM) helices. Although helix-helix association in the lipid bilayer is known to be an essential step in the folding of GPCRs, the determinants of their structures, folding, and assembly in the cell membrane are poorly understood. Previous studies in our group showed that while peptides corresponding to all seven TM domains of A(2A)R form stable helical structures in detergent micelles and lipid vesicles, they display significant variability in their helical propensity. This finding suggested to us that some TM domains might need to interact with other domains to properly insert and fold in hydrophobic environments. In this study, we assessed the ability of TM peptides to interact in pairwise combinations. We analyzed peptide interactions in hydrophobic milieus using circular dichroism spectroscopy and F?rster resonance energy transfer. We find that specific interactions between TM helices occur, leading to additional helical content, especially in weakly helical TM domains, suggesting that some TM domains need a partner for proper folding in the membrane. The approach developed in this study will enable complete analysis of the TM domain interactions and the modeling of a folding pathway for A(2A)R.