The yeast Tor signaling pathway is involved in G2/M transition via polo-kinase

The target of rapamycin (Tor) protein plays central roles in cell growth. Rapamycin inhibits cell growth and promotes cell cycle arrest at G1 (G0). However, little is known about whether Tor is involved in other stages of the cell division cycle. Here we report that the rapamycin-sensitive Tor complex 1 (TORC1) is involved in G2/M transition in S. cerevisiae. Strains carrying a temperature-sensitive allele of KOG1 (kog1-105) encoding an essential component of TORC1, as well as yeast cell treated with rapamycin show mitotic delay with prolonged G2. Overexpression of Cdc5, the yeast polo-like kinase, rescues the growth defect of kog1-105, and in turn, Cdc5 activity is attenuated in kog1-105 cells. The TORC1-Type2A phosphatase pathway mediates nucleocytoplasmic transport of Cdc5, which is prerequisite for its proper localization and function. The C-terminal polo-box domain of Cdc5 has an inhibitory role in nuclear translocation. Taken together, our results indicate a novel function of Tor in the regulation of cell cycle and proliferation.     

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.     

Hyaluronic acid in rabbit pericardial fluid and its production by pericardium

High-Mr (greater than 2 X 10(6)) hyaluronic acid (about 82 micrograms/ml) was found for the first time in rabbit pericardial fluid. Biosynthetic experiments with minced pericardium from rabbit showed that the high-Mr hyaluronic acid in the pericardial fluid was actively synthesized by the pericardium from [3H]glucosamine.     

Induction of rosmarinic acid biosynthesis in Lithospermum erythrorhizon cell suspension cultures by yeast extract

Summary A transient increase in rosmarinic acid (RA) content in cultured cells of Lithospermum erythrorhizon was observed after addition of yeast extract (YE) to the suspension cultures, reaching a maximum at 24 hr. The highest increase of the RA content (2.5-fold) was obtained when 6-day-old cells in the exponential growth phase were treated with YE. Preceding the induced RA accumulation, phenylalanine ammonia-lyase (PAL) activity increased rapidly, whereas tyrosine aminotransferase (TAT) activity was largely unaffected by the treatment. The incorporation of both ¹⁴C-phenylalanine and ¹⁴C-tyrosine into RA was enhanced in the YE-treated cells, consistent with increased synthesis of the ester.Abbreviations 2,4-D 2,4 dichlorophenoxyacetic acid - PAL phenylalanine ammonia-lyase - TAT tyrosine aminotransferase - RA rosmarinic acid - YE yeast extract     

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     

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.     

Purification and identification of intermediate catabolic products in the in vivo degradation of pig liver phosphofructokinase

Phosphofructokinase (ATP:D-fructose-6-phosphate 1-phosphotransferase, EC 2.7.1.11) was purified to homogeneity from pig livers. Polyclonal antibody against the enzyme was induced in a rabbit, and the IgG fraction was obtained by chromatography on a Protein A-Sepharose CL-4B column. The specific antibody was purified further by immunoaffinity chromatography on a phosphofructokinase-conjugated affinity column. Intermediate catabolic products of phosphofructokinase were extracted from fresh pig livers under conditions of inhibition of proteinases, concentrated by chromatography on an anti-phosphofructokinase IgG-conjugated affinity column, and purified by two-dimensional polyacrylamide gel electrophoresis. Their cross-reactivities to the purified phosphofructokinase were assessed by an immunoelectrotransfer blot method. The intact form of phosphofructokinase in pig liver was demonstrated as the major spot of 84 kDa on the blot. Polypeptides of 68, 64, 56, and 51 kDa showed apparent cross-reactivities to phosphofructokinase. The structural homology among them was confirmed by proteinase V8 digestion followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The possibility of artifacts in preparation was ruled out by an internal tracer method. Thus, it is concluded that the predominant isozyme of phosphofructokinase in pig liver (84 kDa) is in vivo degraded via intermediate catabolic products of 68, 64, 56, and 51 kDa.     

Designed and real working situations in machine systems operation.

New work situations designed at the stage when new machine systems are introduced are realized on the assumption that the new systems can maintain their designed functions consistently, generally eliminating previous work habits and without sufficient knowledge about real working processes and skills. This may produce differences between designed and real working situations. Some examples are presented from observations on influence of modern design of cargo ships on their crews. It was difficult for crews to maintain stable working conditions, especially when machine systems deviated from their designed functions. Often the crew had to work in off-duty hours giving up private freetime activities. Among various factors contributing to the discrepancies between designed and real work, lack of availability of the new systems is the most important factor. Also important is lack of back-up systems which would function either when the machine systems are out of order or when previous working skills and habits must be applied. A need for developing methods of evaluation of these two factors from ergonomic points of view is pointed out.     

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.