Determination of the structural ensemble of the molten globule state of a protein by computer simulations

We have used computer simulations to investigate the structural nature of the molten globule (MG) state of canine milk lysozyme. To sample the conformational space efficiently, we performed replica-exchange umbrella sampling simulations with the radius of gyration as a reaction coordinate. We applied the Weighted Histogram Analysis Method to the trajectory of the simulations to obtain the potential of mean force, from which we identified representative structures corresponding to local minima in the free energy surface. The representative structures obtained in this way are in accord with the characteristics of the MG state reported previously by experimental studies. We conjecture that the MG state comprises a series of partially structured states undergoing relatively fast conformational interchange.     

Peptides inhibit selectin-mediated cell adhesion in vitro, and neutrophil influx into inflammatory sites in vivo

The selectins are cell adhesion molecules whose carbohydrate-bindingdomain (C-type lectin) is thought to be involved in leukocyteadhesion to activated vascular endothelium in the inflammatoryprocess. A series of peptides, based on a conserved region (⁴⁸YYWIGIRK⁵⁵-NH₂)of the lectin domain of E-, L- and P-selectins, were analysedfor their ability to block selectin-mediated cell adhesion invitro, and neutrophil infiltration into sites of inflammationin vivo. The peptides inhibited the adhesion of myeloid cellsto recombinant forms of E- and P-selectin. The adhesion of myeloidcells to human endothelial cells, stimulated to express E-selectin,was also inhibited by the peptides. Finally, the peptides blockedthe adhesion of lymphocytes, expressing L-selectin, to highendothelial venules in lymph nodes which contain the ligandfor L-selectin. A clear structure-activity relationship wasestablished when peptides of different amino acid chain lengthswere tested in these assays. Peptides lacking tyrosine residues(e.g. WIGIR-NH₂) at their amino terminus were poor inhibitorsof selectin-mediated cell adhesion in vitro. The peptides thatwere found to be inhibitors of cell adhesion in vitro were alsofound to inhibit (up to 70%) neutrophil infiltration into sitesof inflammation in a thioglycollate-induced peritonitis mousemodel system. They also significantly reduced (>50%) themigration of neutrophils into cytokine-treated skin. These resultsstrongly suggest that compounds based on these tyrosine-containing,selectin-derived peptides could be used as anti-inflammatorytherapeutic agents. inflammation neutrophils peptides selectins     

Successive spontaneous abortions including one with whole-arm translocation between chromosomes 2

Summary A family with five induced and seven spontaneous abortions and no live births is described. Four of the seven spontaneous abortuses were available for cytogenetic examination and three were successfully karyotyped. Their karyotypes were 46,XX; 46,XX/46,XX,t(2;2)(2p2p;2q2q); and 46,XY. The karyotypes of the parents were normal. The origin of the 2p/2p and 2p/2p translocation in one of the abortuses was assigned to an interhomologous whole-arm translocation in an early mitotic division in a conceptus with a 46,XX karyotype.     

Tumor-related expression of alpha1,2fucosylated antigens on colorectal carcinoma cells and its suppression by cell-mediated priming using sugar acceptors for alpha1,2fucosyltransferase

The accumulation of alpha1,2fucosylated antigens, such as Y (Fucalpha1,2Galbeta1,4 [Fucalpha1,3]GlcNAcbeta), Le(b) (Fucalpha1,2Galbeta1,3-[Fucalpha1,4]GlcNAcbeta), and H type 2 (Fucalpha1,2 Galbeta1,4GlcNAcbeta) occurs specifically within human colorectal tumor tissues and can be detected by an antifucosylated antigen antibody, such as the YB-2 antibody. In the present investigation, we found that the expression of these antigens bearing an alpha1,2-linked fucose correlated with the resistance of the tumor cells to anticancer treatments. Addition of an exogenous sugar acceptor for alpha1,2fucosyltransferase to the cell medium resulted in suppression of alpha1,2fucosylated antigen expression on the tumor cells and increased susceptibility to anticancer treatment. The increased susceptibility may be attributed to cancer cell-mediated priming by sugar acceptors for alpha1,2fucosyltransferase added to the medium.     

Ligh- and electron-microscopic immunocytochemistry of somatotropes in the anterior pituitary gland of European ferret,Mustela putorius furo

Light-microscopic immunocytochemistry of ferret anterior pituitary revealed the localization of somatotropes in the pars distalis, but no immunoreactive cells were detected in the pars tuberalis. Ultrastructural studies by superimposition immunocytochemistry and immuno-electron microscopy, clucidated the morphological heterogeneity of these somatotropic cells. They were classified into 2 subtypes on the basis of size of the secretory granules. Type-I cells with small granules (mean diameter, 192 nm), were considered to be the immature somatotrop, while Type-II cells, with comparatively larger secretory granules (mean diameter, 257 nm), were considered to be the matured form of Type-I cells and the typical somatotropic cell-type, and were much more predominant than the Type-I cells. The fact that Type-II cells had a distinct Golgi zone and many mitochondria, while in Type-I cells the intracellular organelles were generally less developed, supports this suggestion. In addition to these two extreme subtypes, several intermediate forms were also encountered that may represent different transitional phases during the conversion of Type I to Type II. Protein A-gold immuno-electron microscopy illustrated the specific localization of growth hormone over the granules, with no labelling over any other cytoplasmic organelles of the 2 somatotrope subtypes.     

Establishment of a mouse model for post‐inflammatory hyperpigmentation

Post‐inflammatory hyperpigmentation (PIH) is a common cutaneous condition that can cause a disfigured appearance. However, the pathophysiology of PIH remains poorly understood, at least in part, because an appropriate animal model for research has not been established. In order to analyze the pathomechanism of PIH, we successfully induced PIH in a hairless version of transgenic mice (hk14‐SCF Tg/HRM) that have a human‐type epidermis containing melanin by repeated hapten application of 2,4‐dinitrofluorobenzene. Histopathologic observation showed epidermal hyperplasia, predominant infiltrations of inflammatory cells, and melanin‐containing cells in the dermis just after elicitation of the atopic dermatitis‐like condition. At week 2, the findings were similar to the characteristics of PIH, that is, an increase of melanin without spongiosis or liquid degeneration in the epidermis and an increase in dermal melanophages. Dynamic analysis of melanin showed that the melanin in the dermis remained for a longer duration than in the epidermis. Furthermore, immunohistochemical staining revealed that the majority of cells containing melanin were positive for the anti‐CD68 antibody, but negative for the anti‐F4/80 antibody. These data suggest that novel treatments of PIH should be targeted against macrophages and should eventually lead to the development of new treatment modalities.     

Production of Hydroxyl Radicals and α-dicarbonyl Compounds Associated with Amadori Compound-Cu 2+ Complex Degradation

The chemical properties of Amadori compounds in the presence of transition metal ions were studied, using the analogs 1-deoxy-1- n -butylamino- d -fructose (DBF) and N &#102 -formyl-fructoselysine (fFL). The following characteristics were revealed: (a) DBF combined easily with Cu 2+ (but no other transition metal ions) to form a DBF-Cu 2+ complex in phosphate buffer, pH 7.4; (b) the complex was unstable, and degraded with the release of Cu + during incubation at 37°C; (c) degradation of the complex was associated with the production of hydroxyl radicals by the Fenton reaction and &#102 -dicarbonyl compounds by non-autoxidative degradation; and (d) properties of DBF were similar to those of fFL. The above properties were additionally observed in glycated poly-Lys (GPL). Our findings indicate a novel mechanism for the generation of hydroxyl radicals and &#102 -dicarbonyl compounds from Amadori adducts in the presence of Cu 2+ .     

Structure of the Atg12–Atg5 conjugate reveals a platform for stimulating Atg8–PE conjugation

Atg12 is conjugated to Atg5 through enzymatic reactions similar to ubiquitination. The Atg12–Atg5 conjugate functions as an E3‐like enzyme to promote lipidation of Atg8, whereas lipidated Atg8 has essential roles in both autophagosome formation and selective cargo recognition during autophagy. However, the molecular role of Atg12 modification in these processes has remained elusive. Here, we report the crystal structure of the Atg12–Atg5 conjugate. In addition to the isopeptide linkage, Atg12 forms hydrophobic and hydrophilic interactions with Atg5, thereby fixing its position on Atg5. Structural comparison with unmodified Atg5 and mutational analyses showed that Atg12 modification neither induces a conformational change in Atg5 nor creates a functionally important architecture. Rather, Atg12 functions as a binding module for Atg3, the E2 enzyme for Atg8, thus endowing Atg5 with the ability to interact with Atg3 to facilitate Atg8 lipidation.