Structure and folding of a designed knotted protein

A very small number of natural proteins have folded configurations in which the polypeptide backbone is knotted. Relatively little is known about the folding energy landscapes of such proteins, or how they have evolved. We explore those questions here by designing a unique knotted protein structure. Biophysical characterization and X-ray crystal structure determination show that the designed protein folds to the intended configuration, tying itself in a knot in the process, and that it folds reversibly. The protein folds to its native, knotted configuration approximately 20 times more slowly than a control protein, which was designed to have a similar tertiary structure but to be unknotted. Preliminary kinetic experiments suggest a complicated folding mechanism, providing opportunities for further characterization. The findings illustrate a situation where a protein is able to successfully traverse a complex folding energy landscape, though the amino acid sequence of the protein has not been subjected to evolutionary pressure for that ability. The success of the design strategy--connecting two monomers of an intertwined homodimer into a single protein chain--supports a model for evolution of knotted structures via gene duplication.     

Application of neural networks for the analysis of intravascular ultrasound and histological aortic wall appearance-an in vitro tissue characterization study

The role of tissue characterization by intravascular ultrasound (IVUS) imaging of the aortic wall has not been well established. The artificial neural networks (ANNs) are a promising tool for image classification. The aim of the study was to assess the texture correlation between matching IVUS and histologic images of the aortic wall. The computer-based discrimination of pathology within the data sets was also evaluated. In vitro IVUS images and histologic sections from 36 aortic segments were compared using texture parameters that produced the best correlation or the highest discriminative value. The images were classified as normal or abnormal with variable degrees of pathology. Tissue characterization was performed by a nearest neighbor classifier, linear discriminant analysis (LDA) and the ANN-based approach. Good agreement was observed between IVUS and the histologic reference with a correlation coefficient of r = 0.89, r = 0.76 and r = 0.71 for the three most successful texture parameters. The ANN-based approach was the most effective in discriminant analysis, with a correct classification rate of 87.5% for histologic images and 79.2% for IVUS data. The study shows that ANNs are a potentially effective tool for assessment of IVUS aortic images.     

Phylogenetic analysis of the genus Plasmodium based on the gene encoding adenylosuccinate lyase.

Phylogenetic studies of the genus Plasmodium have been performed using sequences of the nuclear, mitochondrial and plastid genes. Here we have analyzed the adenylosuccinate lyase (ASL) gene, which encodes an enzyme involved in the salvage of host purines needed by malaria parasites for DNA synthesis. The ASL gene is present in several eukaryotic as well as prokaryotic organisms and does not have repeat regions, which facilitates the accuracy of the alignment. Furthermore, it has been shown that ASL is not subject to positive natural selection. We have sequenced the ASL gene of several different Plasmodium species infecting humans, rodents, monkeys and birds and used the obtained sequences along with the previously known P. falciparum ASL sequence, for structural and phylogenetic analysis of the genus Plasmodium. The genetic divergence of ASL is comparable with that observed in other nuclear genes such as cysteine proteinase, although ASL cannot be considered conserved when compared to aldolase or superoxide dismutase, which exhibit a slower rate of evolution. Nevertheless, a protein like ASL has a rate of evolution that provides enough information for elucidating evolutionary relationships. We modeled 3D structures of the ASL protein based on sequences used in the phylogenetic analysis and obtained a consistent structure for four different species despite the divergence observed. Such models would facilitate alignment in further studies with a greater number of plasmodial species or other Apicomplexa.     

Structure of natural killer receptor 2B4 bound to CD48 reveals basis for heterophilic recognition in signaling lymphocyte activation molecule family

Natural killer (NK) cells eliminate virally infected and tumor cells. Among the receptors regulating NK cell function is 2B4 (CD244), a member of the signaling lymphocyte-activation molecule (SLAM) family that binds CD48. 2B4 is the only heterophilic receptor of the SLAM family, whose other members, e.g., NK-T-B-antigen (NTB-A), are self-ligands. We determined the structure of the complex between the N-terminal domains of mouse 2B4 and CD48, as well as the structures of unbound 2B4 and CD48. The complex displayed an association mode related to, yet distinct from, that of the NTB-A dimer. Binding was accompanied by the rigidification of flexible 2B4 regions containing most of the polymorphic residues across different species and receptor isoforms. We propose a model for 2B4-CD48 interactions that permits the intermixing of SLAM receptors with major histocompatibility complex-specific receptors in the NK cell immune synapse. This analysis revealed the basis for heterophilic recognition within the SLAM family.     

Do G protein‐coupled receptors expressed in human lingual epithelium interact with HPV11?

Human papillomaviruses infect epithelia but little is known about the nature of cell surface receptors interacting with the viral particles. It has been proposed that glycosaminoglycans and integrins may be involved in the attachment process. In the present study, the putative interactions of virus-like particles of human papillomavirus type 11 (HPV11), which present a tropism for nasopharyngeal epithelia, with olfactory and taste receptors expressed in the human lingual epithelium were studied. The L1 protein of HPV11 was produced in insect cells. The presence of L1 virus-like particles was analyzed by ELISA using monoclonal antibodies specific for full-size particles and by electron microscopy. Using immunofluorescence, it was observed that virus-like particles interacted with taste buds from murine tongue, with the tagged human olfactory receptor hJCG5 expressed in HEK-293 but not with the tagged taste receptor hT2R4. This therefore suggests that hJCG5 may be involved in the adsorption process of HPV11 to lingual epithelium serving as a so-called "adsorption-adhesive molecule."     

Perceived intensity and pleasantness of sucrose taste in male alcoholics

AIMS: The aim of the present study was to evaluate a possible relationship between taste responses to sweet solutions and alcoholic status. METHODS: The rated intensity and pleasantness of sucrose taste was compared in male alcoholics (n = 45) and non-alcoholic controls (n = 33). RESULTS: The rated intensity, but not pleasantness, of water taste (0% sucrose) was higher in the alcoholics. The two groups did not differ with respect to the rated intensity or pleasantness of sucrose solutions (1-30%). The proportion of sweet-likers, i.e. subjects rating 30% sucrose as most pleasant, was similar in both groups (the controls: 57.6%, the alcoholics: 62.2%). A subgroup of alcoholics with a paternal history of alcoholism (n = 22) rated the highest sucrose concentration as more pleasant compared to alcoholics without alcoholic fathers. The proportion of sweet-likers among the alcoholics with a paternal history of alcoholism (77.3%) was significantly higher than that found in the alcoholics without a familial history of alcoholism (47.8%). CONCLUSIONS: The present results suggest the following: (i) alcohol dependence is not associated with any major alterations in taste responses to sucrose solutions, (ii) sweet liking is a phenotypic marker of male alcoholics with a paternal history of alcoholism.     

Classification of thyroid gland disease on the basis of selenium concentration in serum

The aim of this study was to estimate the concentration of total selenium in serum women with thyroid gland disease. Selenium was determined by atomic absorption spectrometry using the hydride generation method (HG-AAS). Research was determined in 94 patients with thyroid gland disease and in 28 healthy controls. Selenium concentration of serum was variously in patients than in control group and patients with different thyroid gland diseases. Concentration in control group was 0.06231 microg/ml, in goitre group--0.05612 microg/ml, in hyperthyroidism--0.07149 microg/ml and in hypothyroidism--0.09088 microg/ml.