Degradation of the tumor antigen epitope gp100(280-288) by fibroblast-associated enzymes abolishes specific immunorecognition

Degradation of the tumor antigen epitope gp100(280-288) (YLEPGPVTA) was investigated in the presence of cultured human fibroblasts, and acellular supernatants obtained from these cells; the possible effect of substrate degradation on in vitro immunorecognition was also addressed. In the presence of fibroblasts, gp100(280-288) was degraded to free amino acids with a half-life of less than 4 min; hydrolysis data support the hypothesis that substrate degradation was mainly caused by the activity of cell-expressed amino- and carboxypeptidases. Gp100(280-288) was also degraded in the presence of acellular supernatants: under these conditions, the hydrolysis pattern was similar to that observed in the presence of whole cells, but degradation kinetics was slower. As a result of these phenomena, immunorecognition of gp100(280-288)-specific cytotoxic T lymphocyte (CTL) clones was severely hampered, and was totally suppressed after 90 min. In conclusion, the high activity of fibroblast-expressed proteases, and the presence of wide-scope soluble enzymes, may explain, at least in part, the low activity of peptide-based antineoplastic vaccines, as well as the transient effectiveness of subcutaneously administered peptides in general.     

Molecular localization of a redox-modulated process regulating plant mitochondrial electron transport

Using in organellar assays, we found that significant tobacco alternative oxidase (AOX) activity is dependent on both reduction of a putative regulatory disulfide bond and the presence of pyruvate, which may interact with a Cys sulfhydryl. This redox modulation and pyruvate activation thus may be important in determining the partitioning of electrons to AOX in vivo. To investigate these regulatory mechanisms, we generated tobacco plants expressing mutated AOX proteins. Mutation of the most N-terminal Cys residue (Cys-126) to an Ala residue produced an AOX that could not be converted to the disulfide-linked form, thus identifying this Cys residue as being responsible for redox modulation. Although this mutation might be expected to produce an AOX with constitutive high activity in the presence of pyruvate, we found it to have minimal in organellar activity in the presence of pyruvate. Nonetheless, the Cys-126 mutation did not appear to have compromised the catalytic function of AOX, given that cells expressing the protein displayed high rates of cyanide-resistant respiration in vivo. The striking difference between in vivo and in organellar results suggests that an additional mechanism(s), as yet unidentified by in organellar assays, may promote activity in vivo. Mutation of the Cys residue nearest the presumptive active site (Cys-176) to an Ala residue did not prevent disulfide bond formation or affect the ability of AOX to be stimulated by pyruvate, indicating that this Cys residue is involved in neither redox modulation nor pyruvate activation.     

Stimulation via CD3-Ti but not CD2 induces rapid tyrosine phosphorylation of a 68-kDa protein in the human Jurkat T cell line.

Tyrosine phosphorylation is an early biochemical event associated with surface receptor triggering in many cellular systems. In T lymphocytes, Ag receptor (CD3-Ti) stimulation results in tyrosine phosphorylation of the CD3 zeta subunit. The tyrosine kinase responsible for this modification after CD3-Ti triggering has not been identified. Here we reported that a 68-kDa T cell membrane-associated protein (pp68) in human Jurkat T cells is phosphorylated on tyrosine residues within 1 min after anti-CD3 mAb addition. This induced tyrosine phosphorylation is detected either by in vivo [32P]orthophosphate labeling of the Jurkat T cells or by in vitro [32P]ATP labeling after immunoprecipitation by antiphosphotyrosine antibody. In contrast, mAb stimulation via CD2 and CD4 structures does not induce phosphorylation of pp68. These data are among the first to provide evidence that CD3-Ti and CD2 activation pathways are distinct. Furthermore, they imply that pp68 is itself a tyrosine kinase and/or is a rapidly phosphorylated substrate of a tyrosine kinase.     

Diel movement of smallmouth yellowfish Labeobarbus aeneus in the Vaal River,South Africa

Diel movements of Orange–Vaal smallmouth yellowfish Labeobarbus aeneus (Burchell, 1822) in the Vaal River, South Africa, were determined by externally attaching radio transmitters to 11 adult fish and manually tracking them between March and May 2012. Twenty-four radio telemetry monitoring surveys produced 2 304 diel tracks. At night, yellowfish displayed a preference for slow shallow (<0.3?m s?1, <0.5?m) and fast shallow habitats (>0.3?m s?1, <0.3?m), whereas by day they avoided these habitats, preferring fast deep areas (>0.3?m s?1, >0.3?m). The average total distance of 272?m moved per 24-hour period was three times greater than the diel range, and the average maximum displacement per minute was significantly higher in daytime (4?m) than at night (1.5?m). These findings suggest that L. aeneus is active primarily during the day in fast-flowing, deeper waters, and relatively inactive at night, when it occupies shallower habitats. This behaviour should be further explored to identify causal mechanisms underlying the diel habitat shifts in this species such as water temperature, foraging tactics and/or predator avoidance.     

A finite element inverse analysis to assess functional improvement during the fracture healing process

Assessment of the restoration of load-bearing function is the central goal in the study of fracture healing process. During the fracture healing, two critical aspects affect its analysis: (1) material properties of the callus components, and (2) the spatio-temporal architecture of the callus with respect to cartilage and new bone formation. In this study, an inverse problem methodology is used which takes into account both features and yields material property estimates that can analyze the healing changes. Six stabilized fractured mouse tibias are obtained at two time points during the most active phase of the healing process, respectively 10 days (n=3), and 14 days (n=3) after fracture. Under the same displacement conditions, the inverse procedure estimations of the callus material properties are generated and compared to other fracture healing metrics. The FEA estimated property is the only metric shown to be statistically significant (p=0.0194) in detecting the changes in the stiffness that occur during the healing time points. In addition, simulation studies regarding sensitivity to initial guess and noise are presented; as well as the influence of callus architecture on the FEA estimated material property metric. The finite element model inverse analysis developed can be used to determine the effects of genetics or therapeutic manipulations on fracture healing in rodents.     

An adaptive finite element model for steerable needles

Biomechanics and Modeling in Mechanobiology - Penetration of a flexible and steerable needle into a soft target material is a complex problem to be modelled, involving several mechanical...     

Ryanodine receptor activation by Ca v 1.2 is involved in dendritic cell major histocompatibility complex class II surface expression

Dendritic cells express the skeletal muscle ryanodine receptor (RyR1), yet little is known concerning its physiological role and activation mechanism. Here we show that dendritic cells also express the Ca(v)1.2 subunit of the L-type Ca(2+) channel and that release of intracellular Ca(2+) via RyR1 depends on the presence of extracellular Ca(2+) and is sensitive to ryanodine and nifedipine. Interestingly, RyR1 activation causes a very rapid increase in expression of major histocompatibility complex II molecules on the surface of dendritic cells, an effect that is also observed upon incubation of mouse BM12 dendritic cells with transgenic T cells whose T cell receptor is specific for the I-A(bm12) protein. Based on the present results, we suggest that activation of the RyR1 signaling cascade may be important in the early stages of infection, providing the immune system with a rapid mechanism to initiate an early response, facilitating the presentation of antigens to T cells by dendritic cells before their full maturation.     

Application of molecular dynamics DL_POLY codes to interfaces of inorganic materials

Three recent applications of the DL_POLY molecular dynamics code are described, which demonstrate the flexibility and viability of the code for extending our understanding of the structure, stability and reactivity of ceramics and minerals at the atomic level. The first is an investigation into differences in oxygen atom mobility in bulk and at the most stable {111} surface of ceria. The results show enhanced surface transport but that it is via subsurface oxygen. Secondly, we investigate how polychloro-dibenzo-pdioxins (PCDDs) molecules might adsorb on clay surfaces. The resulting adsorption energies show a clear relationship with chlorine content of the molecule. Finally, we apply DL_POLY to comparing the aggregation of magnesium oxide and calcium carbonate nanoparticles. We find that very small calcium carbonate nanoparticles are amorphous and their aggregation shows no preferred orientation in contrast to magnesium oxide, which remain highly crystalline and combine in a highly structural specific way.     

Gastrointestinal and immunological responses of senior dogs to chicory and mannan-oligosaccharides

Thirty-four senior dogs (pointers 8 - 11 years, beagles 9 - 11 years) were used to evaluate the effects of oligosaccharides on nutritional and immunological characteristics. Dogs were randomly allotted to treatments [1% chicory (CH), 1% mannan-oligosaccharide (MOS), 1% chicory + 1% MOS (CM), or no supplementation (control, CON)] in a parallel design with a 4 week baseline period followed by a 4 week treatment period. Dietary supplementation with MOS or CM tended (P = 0.07) to increase food intake due, in part, to an increase in fermentable fibre and a decrease in energy content of the diet. Although wet faecal output increased (P < 0.05) for dogs supplemented with MOS or CM, when corrected for food intake, no differences were noted. The CM treatment increased (P < 0.05) faecal score (1 = hard and dry, 5 = watery liquid), although these scores remained in a desirable range (3 to 3.5). Chicory supplementation increased (P = 0.07) fat digestibility. Chicory or MOS increased (P  0.05) faecal bifidobacteria concentrations 0.4 and 0.5 log₁₀ cfu/g DM, respectively, compared to the CON, while MOS decreased (P < 0.05) faecal E. coli concentrations. Oligosaccharides did not affect white blood cell (WBC) concentrations, but CH and CM tended to increase (P = 0.10) neutrophil concentrations compared to control dogs. Peripheral lymphocyte concentrations were decreased in dogs supplemented with MOS (P = 0.06) and CM (P < 0.05). Chicory and MOS alter faecal microbial populations and certain indices of the immune system of senior dogs.