Methylotrophic extremophilic yeast Trichosporon sp.: a soil-derived isolate with potential applications in environmental biotechnology

A yeast isolate revealing unique enzymatic activities and substrate-dependent polymorphism was obtained from autochthonous microflora of soil heavily polluted with oily slurries. By means of standard yeast identification procedures the strain was identified as Trichosporon cutaneum. Further molecular PCR product analyses of ribosomal DNA confirmed the identity of the isolate with the genus Trichosporon. As it grew on methanol as a sole carbon source, the strain appeared to be methylotrophic. Furthermore, it was also able to utilize formaldehyde. A multi-substrate growth potential was shown with several other carbon sources: glucose, glycerol, ethanol as well as petroleum derivatives and phenol. Optimum growth temperature was determined at 25 degrees C, and strong inhibition of growth at 37 degrees C together with the original soil habitat indicated lack of pathogenicity in warm-blooded animals and humans. The unusually high tolerance to xenobiotics such as diesel oil (>30 g/l), methanol (50 g/l), phenol (2 g/l) and formaldehyde (7.5 g/l) proved that the isolate was an extremophilic organism. With high-density cultures, formaldehyde was totally removed at initial concentrations up to 7.5 g/l within 24 h, which is the highest biodegradation capability ever reported. Partial biodegradation of methanol (13 g/l) and diesel fuel (20 g/l) was also observed. Enzymatic studies revealed atypical methylotrophic pathway reactions, lacking alcohol oxidase, as compared with the conventional methylotroph Hansenula polymorpha. However, the activities of glutathione-dependent formaldehyde dehydrogenase, formaldehyde reductase, formate dehydrogenase and unspecific aldehyde dehydrogenase(s) were present. An additional glutathione-dependent aldehyde dehydrogenase activity was also detected. Metabolic and biochemical characteristics of the isolated yeast open up new possibilities for environmental biotechnology. Some potential applications in soil bioremediation and wastewater decontamination are discussed.     

Curcumin affects components of the chromosomal passenger complex and induces mitotic catastrophe in apoptosis-resistant Bcr-Abl-expressing cells

The Bcr-Abl oncoprotein plays a major role in the development and progression of chronic myeloid leukemia and is a determinant of chemotherapy resistance occurring during the blast crisis phase of the disease. The aim of this article was to investigate the possibility of combating the resistance to apoptosis caused by Bcr-Abl by inducing an alternative cell death process. As a model of chronic myeloid leukemia, we employed Bcr-Abl-transfected mouse progenitor 32D cells with low and high Bcr-Abl expression levels corresponding to drug-sensitive and drug-resistant cells, respectively. The drug curcumin (diferuloylmethane), a known potent inducer of cell death in many cancer cells, was investigated for efficacy with Bcr-Abl-expressing cells. Curcumin strongly inhibited cell proliferation and affected cell viability by inducing apoptotic symptoms in all tested cells; however, apoptosis was a relatively late event. G(2)-M cell cycle arrest, together with increased mitotic index and cellular and nuclear morphology resembling those described for mitotic catastrophe, was observed and preceded caspase-3 activation and DNA fragmentation. Mitosis-arrested cells displayed abnormal chromatin organization, multipolar chromosome segregation, aberrant cytokinesis, and multinucleated cells-morphologic changes typical of mitotic catastrophe. We found that the mitotic cell death symptoms correlated with attenuated expression of survivin, a member of the chromosomal passenger complex, and mislocalization of Aurora B, the partner of survivin in the chromosomal passenger complex. Inhibition of survivin expression with small interfering RNA exhibited similar mitotic disturbances, thus implicating survivin as a major, albeit not the only, target for curcumin action. This study shows that curcumin can overcome the broad resistance to cell death caused by expression of Bcr-Abl and suggests that curcumin may be a promising agent for new combination regimens for drug-resistant chronic myeloid leukemia.     

Cations and hydration in catalytic RNA: molecular dynamics of the hepatitis delta virus ribozyme

The hepatitis delta virus (HDV) ribozyme is an RNA enzyme from the human pathogenic HDV. Cations play a crucial role in self-cleavage of the HDV ribozyme, by promoting both folding and chemistry. Experimental studies have revealed limited but intriguing details on the location and structural and catalytic functions of metal ions. Here, we analyze a total of approximately 200 ns of explicit-solvent molecular dynamics simulations to provide a complementary atomistic view of the binding of monovalent and divalent cations as well as water molecules to reaction precursor and product forms of the HDV ribozyme. Our simulations find that an Mg2+ cation binds stably, by both inner- and outer-sphere contacts, to the electronegative catalytic pocket of the reaction precursor, in a position to potentially support chemistry. In contrast, protonation of the catalytically involved C75 in the precursor or artificial placement of this Mg2+ into the product structure result in its swift expulsion from the active site. These findings are consistent with a concerted reaction mechanism in which C75 and hydrated Mg2+ act as general base and acid, respectively. Monovalent cations bind to the active site and elsewhere assisted by structurally bridging long-residency water molecules, but are generally delocalized.     

Physiotherapy as an immunoactive therapy? A pilot study

OBJECTIVES: The aim of this study was to confirm the immunoregulatory and anti-inflammatory changes in the immunologic profile after two months of the facilitation physiotherapy in patients with multiple sclerosis; and to determine whether the changes in the immunologic profile correlate with the changes in dehydroepiandrosterone, the brain microstructure and clinical functions. Design & Setting: A group of 12 patients with multiple sclerosis was examined twice: at the beginning and 2 months later after the patients had undergone the facilitation therapy. Standardized tests evaluating chosen clinical functions (balance, righting, equilibrium and protective reactions, tremor, dysdiadochokinesis, dysmetry, fine hand function and walking), immune parameters (parameters of the humoral and cellular immunity), dehydroepiandrosterone and diffusion tensor imaging (the fractional anisotropy, mean diffusivity) were measured. The patients underwent the facilitation physiotherapy in two sessions lasting two hours each week for two months. Results: All clinical and diffusion tensor imaging parameters significantly improved following the therapy. Without the correction for multiple comparisons, there were significant changes in the IgG, IgG1 subclasses, in the numbers of Neutrophils and Lymphocytes, the T cells (CD3+) absolute number, the T cytotoxic subpopulation (CD3+CD8+) absolute number, B cells (CD19+) and the Natural killer cells. In addition, there was a significant correlation between the changes in the clinical functions and the changes in IgG1 (r=0.67), and between the changes in the mean diffusivity and the changes in CD3+CD8+ absolute (r=-0.61). The changes in the immune parameters and the mentioned correlations were not significant in view of the number of comparisons and thus necessitate further validation. No changes in the dehydroepiandrosterone concentration after the therapy were confirmed. Conclusion: The study suggests new possibilities of physiotherapy to influence the psycho-neuro-endocrine-immune response in patients with multiple sclerosis.     

Characterization of callase (β-1,3-d-glucanase) activity during microsporogenesis in the sterile anthers of Allium sativum L. and the fertile anthers of A. atropurpureum

We examined callase activity in anthers of sterile Allium sativum (garlic) and fertile Allium atropurpureum. In A. sativum, a species that produces sterile pollen and propagates only vegetatively, callase was extracted from the thick walls of A. sativum microspore tetrads exhibited maximum activity at pH 4.8, and the corresponding in vivo values ranged from 4.5 to 5.0. Once microspores were released, in vitro callase activity peaked at three distinct pH values, reflecting the presence of three callase isoforms. One isoform, which was previously identified in the tetrad stage, displayed maximum activity at pH 4.8, and the remaining two isoforms, which were novel, were most active at pH 6.0 and 7.3. The corresponding in vivo values ranged from pH 4.75 to 6.0. In contrast, in A. atropurpureum, a sexually propagating species, three callase isoforms, active at pH 4.8-5.2, 6.1, and 7.3, were identified in samples of microsporangia that had released their microspores. The corresponding in vivo value for this plant was 5.9. The callose wall persists around A. sativum meiotic cells, whereas only one callase isoform, with an optimum activity of pH 4.8, is active in the acidic environment of the microsporangium. However, this isoform is degraded when the pH rises to 6.0 and two other callase isoforms, maximally active at pH 6.0 and 7.3, appear. Thus, factors that alter the pH of the microsporangium may indirectly affect the male gametophyte development by modulating the activity of callase and thereby regulating the degradation of the callose wall.     

Immunosuppressant FK506: Focusing on neuroprotective effects following brain and spinal cord injury

The secondary damage that follows central nervous system (CNS) injury is a target for neuroprotective agents aimed at tissue and function sparing. FK506, a clinically used immunosuppressant, acts neuroprotectively in rat models of brain and spinal cord injury and ischemia. Evidence of in vivo experimental studies highlights the neuroprotective role of FK506 by its direct impact on various cell populations within the CNS. The participation of FK506 in modulation of post-traumatic inflammatory processes is a further potential aspect involved in CNS neuroprotection. In this review we provide an overview of the current laboratory research focusing on the multiple effects of FK506 on neuroprotection following CNS injury.     

Mass spectrometry and animal science: protein identification strategies and particularities of farm animal species

Proteomic approaches are gaining increasing importance in the context of all fields of animal and veterinary sciences, including physiology, productive characterization, and disease/parasite tolerance, among others. Proteomic studies mainly aim the proteome characterization of a certain organ, tissue, cell type or organism, either in a specific condition or comparing protein differential expression within two or more selected situations. Due to the high complexity of samples, usually total protein extracts, proteomics relies heavily on separation procedures, being 2D-electrophoresis and HPLC the most common, as well as on protein identification using mass spectrometry (MS) based methodologies. Despite the increasing importance of MS in the context of animal and veterinary science studies, the usefulness of such tools is still poorly perceived by the animal science community. This is primarily due to the limited knowledge on mass spectrometry by animal scientists. Additionally, confidence and success in protein identification is hindered by the lack of information in public databases for most of farm animal species and their pathogens, with the exception of cattle (Bos taurus), pig (Sus scrofa) and chicken (Gallus gallus). In this article, we will briefly summarize the main methodologies available for protein identification using mass spectrometry providing a case study of specific applications in the field of animal science. We will also address the difficulties inherent to protein identification using MS, with particular reference to experiments using animal species poorly described in public databases. Additionally, we will suggest strategies to increase the rate of successful identifications when working with farm animal species.     

Regional differences of proliferation activity in the spinal cord ependyma of adult rats

Increased proliferation activity in the central canal ependyma of adult rodent spinal cord was described after injury and is thought to participate in recovery processes. Proliferation activity is scarce under physiological conditions, but still could be of importance, as in vitro studies showed that the spinal cord ependyma is an internal source of neural stem cells. Data from these studies indicate that there are regional differences in the distribution of proliferation activity along the rostro-caudal axis. We analyzed the proliferation activities in the ependyma within the entire extent of intact adult rat spinal cord. To identify proliferating cells we performed immunohistochemistry either for cell cycle S-phase marker BrdU or for the nuclear protein Ki-67. BrdU and Ki-67 positive cells were counted on sections selected from four spinal cord regions — cervical, thoracic, lumbar and sacral/coccygeal. Analysis showed that the number of BrdU positive cells within the ependyma was very low in all subdivisions of the spinal cord. Both BrdU and Ki-67 labeling revealed a significantly higher number of proliferating cells in the ependyma of sacrococcygeal part in comparison to all other spinal cord regions, suggesting that the caudal spinal cord might have potentially higher regeneration capacity compared to more rostral parts.