Dominant-negative p53-overexpression in skeletal muscle induces cell death and fiber atrophy in rats

The tumor suppressor p53 is thought to play a key role in the maintenance of cell size and homeostasis, but relatively little is known about its role in skeletal muscle. Based on its ability to suppress cell growth, we hypothesized that inhibiting the function of wild-type p53 through the overexpression of a dominant-negative p53 mutant (DDp53) could result in muscle fiber hypertrophy. To test this hypothesis, we electroporated adult rat tibialis anterior muscles with DDp53 and collected the tissue three weeks later. We confirmed successful overexpression of DDp53 on a histological and biochemical level and found pronounced changes to muscle architecture, metabolism, and molecular signaling. Muscle mass, fiber cross-sectional area, and fiber diameter significantly decreased with DDp53 overexpression. We found histopathological changes in DDp53 transfected muscle which were accompanied by increased levels of proteins that are associated with membrane damage and repair. In addition, DDp53 decreased oxidative phosphorylation complex I and V protein levels, and despite its negative effects on muscle mass and fiber size, caused an increase in muscle protein synthesis as assessed via the SUnSET technique. Interestingly, the increase in muscle protein synthesis was concomitant with a decrease in phospho-S6K1 (Thr389). Furthermore, the muscle wasting in the DDp53 electroporated leg was accompanied by a decrease in global protein ubiquitination and an increase in proteasome activity. In conclusion, overexpression of a dominant-negative p53 mutant in skeletal muscle results in decreased muscle mass, myofiber size, histological muscle damage, a metabolic phenotype, and perturbed homeostasis between muscle protein synthesis and degradation.Subject terms: Proteasome, Phosphorylation, Contractile proteins     

Chemokine-independent preference for T-helper-1 cells in transendothelial migration

We analyzed differences in the transendothelial migration (TEM) ability of T-helper (Th)-1 and Th2 cells across a murine endothelial cell line (F-2) under static conditions. The TEM abilities of Th1 cells from mice bearing autoimmune diseases and antigen-specific Th1 cell lines were severalfold higher than those of Th2 cells and lines of the same origin. These preferences were observed without exogenous chemoattractant and were insensitive to pertussis toxin, which completely blocks TEM induced by exogenous chemoattractants. Antibodies against LFA-1 and ICAM-1 as well as CD44 markedly blocked the TEM of Th1 cells. TEM ability was also blocked by pharmacological inhibitors of Src family protein-tyrosine kinases (PP2 and herbimycin A), phosphatidylinositol 3-kinase (wortmannin), and phosphatidylinositol-specific phospholipase C (). Cross-linking of CD44 strongly induced highly elongated morphology in Th1 lines, but weakly in Th2 lines. The pharmacological inhibitors that blocked TEM also inhibited this morphological change, whereas pertussis toxin did not. These data indicate that there are signaling pathways for TEM independent of chemokine attraction, but through adhesion molecules including CD44, and that the preferential TEM ability of Th1 over Th2 cells is formed, at least in part, by intrinsic differences in these pathways.     

Changes of markers of oxidative stress during menstrual cycle

The levels of urinary hydrogen peroxide and thiobarbituric acid reactive substances have been compared during the menstrual cycle of 12 regularly menstruating women. Higher level of both indices of oxidative stress (normalized with respect to creatinine content) were found in the luteal phase of the cycle. These results give further evidence for the usefulness of urinary hydrogen peroxide and thiobarbituric acid reactive substances as potential biomarkers of oxidative stress and for the antioxidant action of estrogens.     

Brain regeneration from pluripotent stem cells in planarian

How can planarians regenerate their brain? Recently we have identified many genes critical for this process. Brain regeneration can be divided into five steps: (1) anterior blastema formation, (2) brain rudiment formation, (3) pattern formation, (4) neural network formation, and (5) functional recovery. Here we will describe the structure and process of regeneration of the planarian brain in the first part, and then introduce genes involved in brain regeneration in the second part. Especially, we will speculate about molecular events during the early steps of brain regeneration in this review. The finding providing the greatest insight thus far is the discovery of the nou-darake (ndk; ‘brains everywhere’ in Japanese) gene, since brain neurons are formed throughout the entire body as a result of loss of function of the ndk gene. This finding provides a clue for elucidating the molecular and cellular mechanisms underlying brain regeneration. Here we describe the molecular action of the nou-darake gene and propose a new model to explain brain regeneration and restriction in the head region of the planarians.     

Time and dose dependence of pluronic bioactivity in hyperthermia-induced tumor cell death

Pluronic block copolymers have been shown to sensitize cancer cells resulting in an increased activity of antineoplastic agents. In the current study we examined a new application of Pluronic bioactivity in potentiating hyperthermia-induced cancer cell injury. DHD/K12/TRb rat adenocarcinoma cells were exposed to low-grade hyperthermia at 43 degrees C with or without Pluronic P85 or Pluronic L61. A range of Pluronic doses, pre-exposure and heat exposure durations were investigated, and the test conditions were optimized. Treatment efficacy was assessed by measurement of intracellular ATP and mitochondrial dehydrogenase activity. Both P85 and L61 in synergy with heat reduced cell viability appreciably compared to either heat or Pluronic alone. Under optimal conditions, P85 (10 mg/ml, 240 mins) combined with 15 mins heat reduced intracellular ATP to 60.1 +/- 3.5% of control, while heat alone and P85 without heat caused a negligible decrease in ATP of 1.2% and 3.8%, respectively. Similarly, cells receiving 120 mins pre-exposure of L61 (0.3 mg/ml) showed reduction in intracellular ATP to 14.1 +/- 2.1% of control. Again, heat or L61 pre-exposure alone caused a minor decrease in levels of intracellular ATP (1.5% and 4.4%, respectively). Comparable results were observed when viability was assessed by mitochondrial enzyme activity. Survival studies confirmed that the loss of viability translates to a long-term reduction in proliferative activity, particularly for L61 treated cells. Based on these results, we conclude that Pluronic is effective in improving hyperthermic cancer treatment in vitro by potentiating heat-induced cytotoxicity in a concentration and time dependent manner.     

Modulation of Alpha-Synuclein Aggregation by Dopamine Analogs

The action of dopamine on the aggregation of the unstructured alpha-synuclein (α-syn) protein may be linked to the pathogenesis of Parkinson''s disease. Dopamine and its oxidation derivatives may inhibit α-syn aggregation by non-covalent binding. Exploiting this fact, we applied an integrated computational and experimental approach to find alternative ligands that might modulate the fibrillization of α-syn. Ligands structurally and electrostatically similar to dopamine were screened from an established library. Five analogs were selected for in vitro experimentation from the similarity ranked list of analogs. Molecular dynamics simulations showed they were, like dopamine, binding non-covalently to α-syn and, although much weaker than dopamine, they shared some of its binding properties. In vitro fibrillization assays were performed on these five dopamine analogs. Consistent with our predictions, analyses by atomic force and transmission electron microscopy revealed that all of the selected ligands affected the aggregation process, albeit to a varying and lesser extent than dopamine, used as the control ligand. The in silico/in vitro approach presented here emerges as a possible strategy for identifying ligands interfering with such a complex process as the fibrillization of an unstructured protein.     

Population demography of the endangered large blue butterfly <Emphasis Type="Italic">Maculinea arion</Emphasis> in Europe

Demographic parameters such as survival, sex ratio and abundance can profoundly affect the viability of populations and thus are of primary importance in species of conservation concern. Although numerous studies have been published on certain aspects of the ecology and evolution of the endangered Large Blue butterfly Maculinea arion, there is still a lack of detailed knowledge on its populations’ demography. Moreover, M. arion populates a variety of xerothermic habitats throughout its European range using various food plants and host ants, which leads to complications in its conservation. Our aim was to estimate demographic parameters of M. arion populations in different parts of its European range. Detailed mark-recapture sampling was conducted on populations in four different countries. We often found that daily apparent survival probability declined with increasing age of individuals, but there was no difference between male and female survival. In smaller populations, the sex ratio was rather female-biased. Our most interesting result was the lack of protandry in some populations that might be a consequence of selection against reproductive asynchrony in small populations or a polyandrous mating system. The perfect coincidence of male and female phenology can positively affect the effective population size, because the lack of reproductive asynchrony increases the chance of male–female encounters. Abundance of the studied populations ranged between 100 and 1,600 individuals, smaller populations were on the verge of extinction. Habitat of the threatened small populations was either overgrazed or abandoned, while habitat of larger, stable populations was lightly grazed.     

Cytotoxicity of PP(Arg)(2)- and Hp(Arg)(2)-mediated photodynamic therapy and early stage of apoptosis induction in prostate carcinoma in vitro

Porphyrin photosensitizers tend to localize in mitochondria. The depolarization of mitochondrial membrane is one of the early stages of apoptosis and Laser Scanning Fluorescence Microscopy allows to determine changes in transmembrane mitochondrial potential under influence of PDT depending on the kind of photosensitizer (PP(Arg)(2), Hp(Arg)(2)), the energy dose (5, 10, 30 and 50 J/cm(2)) and time periods (24 and 48 hours after irradiation) in the LNCaP (lymphonodal metastasis of prostate carcinoma, the androgen dependent cell line). Cyototoxicity induced by PP(Arg)(2)- and Hp(Arg)(2)-based PDT depending on energy dose and time after irradiation in prostate carcinoma is determined with MTT. Generally, it was shown that lower energy doses induce greater changes in transmembrane mitochondrial potential. Hp(Arg)(2)-based PDT was more effective causing greater mitochondrial membrane depolarization and cell viability decrease in comparison to PP(Arg)(2)-mediated PDT (in the case of maximal nontoxic photosensitizer doses used).     

α1-Antichymotrypsin inactivates staphylococcal cysteine protease in cross-class inhibition

Staphylococcal cysteine proteases are implicated as virulence factors in human and avian infections. Human strains of Staphylococcus aureus secrete two cysteine proteases (staphopains A and B), whereas avian strains express staphopain C (ScpA2), which is distinct from both human homologues. Here, we describe probable reasons why the horizontal transfer of a plasmid encoding staphopain C between avian and human strains has never been observed. The human plasma serine protease inhibitor α₁-antichymotrypsin (ACHT) inhibits ScpA2. Together with the lack of ScpA2 inhibition by chicken plasma, these data may explain the exclusively avian occurrence of ScpA2. We also clarify the mechanistic details of this unusual cross-class inhibition. Analysis of mutated ACHT variants revealed that the cleavage of the Leu383-Ser384 peptide bond results in ScpA2 inhibition, whereas hydrolysis of the preceding peptide bond leads to ACHT inactivation. This evidence is consistent with the suicide-substrate-like mechanism of inhibition.