The use of 'top-up' experiments to investigate the effect of very small doses per fraction in mouse skin

The partial tolerance type of 'top-up' experiment has been investigated to determine the resolution of this approach for studying the damage to mouse skin from very small doses of X-rays and neutrons. The effect of 20 fractions, each as small as 0.10 Gy of X-rays or of 0.05 Gy of neutrons, can be detected if 3 MeV neutrons are used as the 'top-up' reference radiation. This capability results from the almost linear underlying dose-response curve and highly reproducible dose-effect relationship for the low energy neutrons. The data fit the linear quadratic model of dose fractionation for X-rays down to fractional doses of 0.75 Gy, but at lower doses there is a trend towards an increase in the skin radiosensitivity. Modelling shows that this might be consistent with a sub-population of the cells showing an exceptional radiosensitivity, and a replenishment of this subpopulation occurring in the 8 h between small dose fractions. More experiments are needed at very low doses in order to confirm this hypothesis for skin and for other tissues.     

Spindle assembly checkpoint genes reveal distinct as well as overlapping expression that implicates MDF-2/Mad2 in postembryonic seam cell proliferation in Caenorhabditis elegans

Background

The spindle assembly checkpoint (SAC) delays anaphase onset by inhibiting the activity of the anaphase promoting complex/cyclosome (APC/C) until all of the kinetochores have properly attached to the spindle. The importance of SAC genes for genome stability is well established; however, the roles these genes play, during postembryonic development of a multicellular organism, remain largely unexplored.

Results

We have used GFP fusions of 5' upstream intergenic regulatory sequences to assay spatiotemporal expression patterns of eight conserved genes implicated in the spindle assembly checkpoint function in Caenorhabditis elegans. We have shown that regulatory sequences for all of the SAC genes drive ubiquitous GFP expression during early embryonic development. However, postembryonic spatial analysis revealed distinct, tissue-specific expression of SAC genes with striking co-expression in seam cells, as well as in the gut. Additionally, we show that the absence of MDF-2/Mad2 (one of the checkpoint genes) leads to aberrant number and alignment of seam cell nuclei, defects mainly attributed to abnormal postembryonic cell proliferation. Furthermore, we show that these defects are completely rescued by fzy-1(h1983)/CDC20, suggesting that regulation of the APC/C^CDC20 by the SAC component MDF-2 is important for proper postembryonic cell proliferation.

Conclusion

Our results indicate that SAC genes display different tissue-specific expression patterns during postembryonic development in C. elegans with significant co-expression in hypodermal seam cells and gut cells, suggesting that these genes have distinct as well as overlapping roles in postembryonic development that may or may not be related to their established roles in mitosis. Furthermore, we provide evidence, by monitoring seam cell lineage, that one of the checkpoint genes is required for proper postembryonic cell proliferation. Importantly, our research provides the first evidence that postembryonic cell division is more sensitive to SAC loss, in particular MDF-2 loss, than embryonic cell division.     

Context-dependent memory: colour versus odour

An olfactory stimulus and a visual stimulus were employed in a context- dependent memory study using a prose passage as the to-be-remembered item. Ninety-five university students (aged 17-35 years) learned the passage of prose in the presence of one of the stimuli and were then asked to recall the passage with the original context either reinstated or not reinstated. The results revealed a significant context-dependent memory effect for the olfactory cue but not for the visual cue. They demonstrate support for the effectiveness of odours as context cues and it is suggested that context-dependent memory processes may underlie the formation and retrieval of odour-evoked autobiographical memories.      

Protein osmotic pressure and the state of water in frog myoplasm

We measured the osmotic pressure of diffusible myoplasmic proteins in frog (Rana temporaria) skeletal muscle fibers by using single Sephadex beads as osmometers and dialysis membranes as protein filters. The state of the myoplasmic water was probed by determining the osmotic coefficient of parvalbumin, a small, abundant diffusible protein distributed throughout the fluid myoplasm. Tiny sections of membrane (3.5- and 12-14-kDa cutoffs) were juxtaposed between the Sephadex beads and skinned semitendinosus muscle fibers under oil. After equilibration, the beads were removed and calibrated by comparing the diameter of each bead to its diameter measured in solutions containing 3-12% Dextran T500 (a long-chain polymer). The method was validated using 4% agarose cylinders loaded with bovine serum albumin (BSA) or parvalbumin. The measured osmotic pressures for 1.5 and 3.0 mM BSA were similar to those calculated by others. The mean osmotic pressure produced by the myoplasmic proteins was 9.7 mOsm (4 degrees C). The osmotic pressure attributable to parvalbumin was estimated to be 3.4 mOsm. The osmotic coefficient of the parvalbumin in fibers is approximately 3.7 mOsm mM(-1), i.e., roughly the same as obtained from parvalbumin-loaded agarose cylinders under comparable conditions, suggesting that the fluid interior of muscle resembles a simple salt solution as in a 4% agarose gel.     

Inhibition of InhA activity,but not KasA activity,induces formation of a KasA-containing complex in mycobacteria

Isoniazid (INH) remains one of the key drugs used to control tuberculosis, with the enoyl-AcpM reductase InhA being the primary target. However, based on the observation that INH-treated Mycobacterium tuberculosis overproduces KasA, an enzyme involved in the biosynthesis of mycolic acids, and induces the formation of a covalent complex consisting of AcpM, KasA, and INH, it has been proposed that KasA represents the primary target of INH. However, the relevance of this complex to INH action remains obscure. This study was aimed at clarifying the role of InhA and KasA in relation to INH activity. By using anti-KasA antibodies we detected the KasA-containing complex in INH-treated Mycobacterium smegmatis. In addition, INH-treated cells also produced constant levels of KasA that were not sequestered in the complex and presumably were sufficient to ensure mycolic acid biosynthesis. Interestingly, a furA-lacking strain induced the complex at lower concentrations of INH compared with the control strain, whereas higher INH concentrations were necessary to induce the complex in a strain that lacks katG, suggesting that INH needs to be activated by KatG to induce the KasA-containing complex. The InhA inhibitors ethionamide and diazaborine also induced the complex; thus, its formation was not specifically relevant to INH action but was because of InhA inhibition. In addition, in vitro assays using purified InhA and KasA demonstrated that KatG-activated INH, triclosan, and diazaborine inhibited InhA but not KasA activity. Moreover, several thermosensitive InhA mutant strains of M. smegmatis constitutively expressed the KasA-containing complex. This study provides the biochemical and genetic evidence. 1) Only inhibition of InhA, but not KasA, induces the KasA-containing complex. 2) INH is not part of the complex. 3) INH does not target KasA, consistent with InhA being the primary target of INH.     

A ‘minimum stress inflexion’ in relation to environmental and biotic influences on the dynamics of subtidal encrusting communities?

The sedentary fauna of sublittoral boulders was studied at five sites with very different environmental conditions within Lough Hyne Marine Nature Reserve (51° 30 N, 9° 18 W). The degree of competition was assessed through the construction of competitive hierarchies built up from the results of nearly 3000 interactions between organisms. Communities at all sites were predominantly hierarchically organised with few stand-offs recorded. Density of interactions was correlated with total space occupation. Flow speed at all sites was logged over 24 h and this factor also correlated with space occupation. Degree of disturbance was assessed through the use of marked boulders at each site. Species diversity peaked where all factors were moderate and was lowest at extreme sites. The effects of these environmental variables and biotic factors in determining community composition are discussed, and a working model detailing a Minimum Stress Inflexion is described. This represents an extension of the intermediate disturbance hypothesis in that it is proposed that disturbance in this system may serve to reduce sediment load as opposed to preventing space monopolisation in the classical model.     

Morphology and transverse stiffness of Drosophila myofibrils measured by atomic force microscopy

Atomic force microscopy was used to investigate the surface morphology and transverse stiffness of myofibrils from Drosophila indirect flight muscle exposed to different physiologic solutions. I- and A-bands were clearly observed, and thick filaments were resolved along the periphery of the myofibril. Interfilament spacings correlated well with estimates from previous x-ray diffraction studies. Transverse stiffness was measured by using a blunt tip to indent a small section of the myofibrillar surface in the region of myofilament overlap. At 10 nm indention, the effective transverse stiffness (K( perpendicular)) of myofibrils in rigor solution (ATP-free, pCa 4.5) was 10.3 +/- 5.0 pN nm(-1) (mean +/- SEM, n = 8); in activating solution (pCa 4.5), 5.9 +/- 3.1 pN nm(-1); and in relaxing solution (pCa 8), 4.4 +/- 2.0 pN nm(-1). The apparent transverse Young's modulus (E( perpendicular)) was 94 +/- 41 kPa in the rigor state and 40 +/- 17 kPa in the relaxed state. The value of E( perpendicular) for calcium-activated myofibrils (55 +/- 29 kPa) was approximately a tenth that of Young's modulus in the longitudinal direction, a difference that at least partly reflects the transverse flexibility of the myosin molecule.     

Alphavirus Replicon Particles Expressing TRP-2 Provide Potent Therapeutic Effect on Melanoma through Activation of Humoral and Cellular Immunity

Background

Malignant melanoma is the deadliest form of skin cancer and is refractory to conventional chemotherapy and radiotherapy. Therefore alternative approaches to treat this disease, such as immunotherapy, are needed. Melanoma vaccine design has mainly focused on targeting CD8⁺ T cells. Activation of effector CD8⁺ T cells has been achieved in patients, but provided limited clinical benefit, due to immune-escape mechanisms established by advanced tumors. We have previously shown that alphavirus-based virus-like replicon particles (VRP) simultaneously activate strong cellular and humoral immunity against the weakly immunogenic melanoma differentiation antigen (MDA) tyrosinase. Here we further investigate the antitumor effect and the immune mechanisms of VRP encoding different MDAs.

Methodology/Principal Findings

VRP encoding different MDAs were screened for their ability to prevent the growth of the B16 mouse transplantable melanoma. The immunologic mechanisms of efficacy were investigated for the most effective vaccine identified, focusing on CD8⁺ T cells and humoral responses. To this end, ex vivo immune assays and transgenic mice lacking specific immune effector functions were used. The studies identified a potent therapeutic VRP vaccine, encoding tyrosinase related protein 2 (TRP-2), which provided a durable anti-tumor effect. The efficacy of VRP-TRP2 relies on a novel immune mechanism of action requiring the activation of both IgG and CD8⁺ T cell effector responses, and depends on signaling through activating Fcγ receptors.

Conclusions/Significance

This study identifies a VRP-based vaccine able to elicit humoral immunity against TRP-2, which plays a role in melanoma immunotherapy and synergizes with tumor-specific CD8⁺ T cell responses. These findings will aid in the rational design of future immunotherapy clinical trials.