Hydration of enzyme in nonaqueous media is consistent with solvent dependence of its activity

Water plays an important role in enzyme structure and function in aqueous media. That role becomes even more important when one focuses on enzymes in low water media. Here we present results from molecular dynamics simulations of surfactant-solubilized subtilisin BPN' in three organic solvents (octane, tetrahydrofuran, and acetonitrile) and in pure water. Trajectories from simulations are analyzed with a focus on enzyme structure, flexibility, and the details of enzyme hydration. The overall enzyme and backbone structures, as well as individual residue flexibility, do not show significant differences between water and the three organic solvents over a timescale of several nanoseconds currently accessible to large-scale molecular dynamics simulations. The key factor that distinguishes molecular-level details in different media is the partitioning of hydration water between the enzyme and the bulk solvent. The enzyme surface and the active site region are well hydrated in aqueous medium, whereas with increasing polarity of the organic solvent (octane --> tetrahydrofuran --> acetonitrile) the hydration water is stripped from the enzyme surface. Water stripping is accompanied by the penetration of tetrahydrofuran and acetonitrile molecules into crevices on the enzyme surface and especially into the active site. More polar organic solvents (tetrahydrofuran and acetonitrile) replace mobile and weakly bound water molecules in the active site and leave primarily the tightly bound water in that region. In contrast, the lack of water stripping in octane allows efficient hydration of the active site uniformly by mobile and weakly bound water and some structural water similar to that in aqueous solution. These differences in the active site hydration are consistent with the inverse dependence of enzymatic activity on organic solvent polarity and indicate that the behavior of hydration water on the enzyme surface and in the active site is an important determinant of biological function especially in low water media.     

Indigenous Knowledge of Rock Kangaroo Ecology in Western Arnhem Land, Australia

Indigenous peoples of western Arnhem Land, central northern Australia, have detailed knowledge of the rock kangaroos of the region, species that are little known to science. Information about the ecology of the species is required for their conservation and management. Ethnoecological studies can assist senior indigenous people with transfer of knowledge and can give respect and meaningful employment to those involved. We used semidirected interviews in the regional vernacular, Bininj Kunwok, to record indigenous knowledge of the ecology of the four rock kangaroo species (Petrogale brachyotis, P. concinna, Macropus bernardus and M. robustus). Discussions focussed on habitat preferences, diet, activity patterns, reproduction, predation, and hunting practices. The ethnoecological knowledge of the rock kangaroo species was extensive, and both complemented and extended that reported in the scientific literature. In contrast to scientific understanding of taxonomy and ecology, consultants recognized the rock kangaroos as a natural group. They also described subtle differences in the species’ comparative ecology. The methodology used proved highly successful and we recommend recording indigenous knowledge of the ecology of fauna species in the local vernacular wherever possible. This study is one of the most comprehensive ethnozoological studies of a group of species undertaken in Australia.

Contribution of the 37-kDa laminin receptor precursor in the anti-metastatic PSP94-derived peptide PCK3145 cell surface binding

PURPOSE: PCK3145 is an anti-metastatic synthetic peptide with promising therapeutic efficacy against hormone-refractory prostate cancer. The characterization of the PCK3145 peptide cell surface binding/internalization mechanisms and of the receptors involved remained to be explored. RESULTS: [(14)C]PCK3145 cell surface binding assays showed rapid and transient kinetic profile, that was inhibited by RGD peptides, laminin, hyaluronan, and type-I collagen. RGD peptides were however unable to inhibit PCK3145 intracellular uptake. Far-Western ligand binding studies enabled the identification of the 37-kDa laminin receptor precursor (37LRP) as a potential ligand for PCK3145. Overexpression of the recombinant 37LRP indeed led to an increase in PCK3145 binding but unexpectedly not to its uptake. CONCLUSIONS: Our data support the implication of laminin receptors in cell surface binding and in transducing PCK3145 anti-metastatic effects, and provide a rational for targeting cancers that express high levels of such laminin receptors.     

Système sémantiquement interopérable de sélection semi-automatique des patients éligibles aux essais thérapeutiques en cancérologie

Automatic Selection of clinical Trial based on Eligibility Criteria (ASTEC) project is to automate, so as to make it systematic, the search of cancer clinical trials, by reusing the patient data contained into an oncologic electronic health record. ASTEC project tackles two major scientific challenges for medical informatics: 1) the syntactic and semantic interoperability between information systems. The oncologic electronic medical records and the recruitment decision system must be interoperable. The ASTEC project proposes a framework of syntactico-semantic interoperability based on international standards. Generic methods of mediation and reasoning based on ontologies are developed to match data from the electronic medical records to the inclusion/exclusion criteria of clinical trials; 2) a decision support system for recruitment. We have developed inference methods on the electronic medical records adapted to the data structure as well as the eligibility criteria. this paper, we present and justify our choices, concerning the medical process in oncology and the scientific and technical aspects. Furthermore the system will be evaluated in real time. The aim is to demonstrate a significative improvement of the prescreening rate of patient.     

Muscle damage and its relationship with muscle fatigue during a half-iron triathlon

Background

To investigate the cause/s of muscle fatigue experienced during a half-iron distance triathlon.

Methodology/Principal Findings

We recruited 25 trained triathletes (36±7 yr; 75.1±9.8 kg) for the study. Before and just after the race, jump height and leg muscle power output were measured during a countermovement jump on a force platform to determine leg muscle fatigue. Body weight, handgrip maximal force and blood and urine samples were also obtained before and after the race. Blood myoglobin and creatine kinase concentrations were determined as markers of muscle damage.

Results

Jump height (from 30.3±5.0 to 23.4±6.4 cm; P<0.05) and leg power output (from 25.6±2.9 to 20.7±4.6 W · kg⁻¹; P<0.05) were significantly reduced after the race. However, handgrip maximal force was unaffected by the race (430±59 to 430±62 N). Mean dehydration after the race was 2.3±1.2% with high inter-individual variability in the responses. Blood myoglobin and creatine kinase concentration increased to 516±248 µg · L⁻¹ and 442±204 U · L⁻¹, respectively (P<0.05) after the race. Pre- to post-race jump change did not correlate with dehydration (r = 0.16; P>0.05) but significantly correlated with myoglobin concentration (r = 0.65; P<0.001) and creatine kinase concentration (r = 0.54; P<0.001).

Conclusions/significance

During a half-iron distance triathlon, the capacity of leg muscles to produce force was notably diminished while arm muscle force output remained unaffected. Leg muscle fatigue was correlated with blood markers of muscle damage suggesting that muscle breakdown is one of the most relevant sources of muscle fatigue during a triathlon.     

Glatiramer Acetate Increases Phagocytic Activity of Human Monocytes In Vitro and in Multiple Sclerosis Patients

Beside its effects on T cells, a direct influence on cells of the myelo-monocytic lineage by GA becomes evident. Recently, we demonstrated that GA drives microglia to adopt properties of type II antigen presenting cells (APC) and increases their phagocytic activity. In the present work, we focused on human blood monocytes in order to examine whether GA may increase phagocytic activity in vivo and to evaluate the molecular mechanisms explaining this new discovered mode of action. Peripheral blood mononuclear cells (PBMC) were obtained using a Biocoll-Isopaque gradient and monocytes were subsequently isolated by using CD14 MicroBeads. Phagocytic activity was determined by flow cytometric measurement of the ingestion of fluorescent beads. Flow cytometry was also used to assess monocytic differentiation and expression of phagocytic receptors. Monocytes of GA treated MS patients exhibited a significantly higher phagocytic activity than those of healthy controls or non-treated MS patients. In vitro, a significant phagocytic response was already detectable after 1 h of GA treatment at the concentrations of 62.5 and 125 µg/ml. A significant increase at all concentrations of GA was observed after 3 h and 24 h, respectively. Only monocytes co-expressing CD16, particularly CD14⁺⁺CD16⁺ cells, were observed to phagocytose. Treatment of monocytes with IL-10 and supernatants from GA-treated monocytes did not alter phagocytosis. We observed a decrease in CD11c expression by GA while no changes were found in the expression of CD11b, CD36, CD51/61, CD91, TIM-3, and CD206. In our blocking assays, treatment with anti-CD14, anti-CD16, anti-TIM3, anti-CD210, and particularly anti-CD36 antibodies led to a decrease in phagocytosis. Our results demonstrate a new mechanism of action of GA treatment that augments phagocytic activity of human monocytes in vivo and in vitro. This activity seems to arise from the CD14⁺⁺CD16⁺ monocyte subset.     

Correlation of the content of hepatotoxin nodularin and glycosidic carotenoids, 4-ketomyxol-2′-fucoside and novel 1′-O-methyl-4-ketomyxol-2′-fucoside,in 20 strains of the cyanobacterium Nodularia spumigena

The carotenoids of 19 different strains of Nodularia spumigena and one Nodularia sphaerocarpa from different global locations were investigated. The molecular structure of the diagnostic pigment in N. spumigena of the Baltic Sea, tentatively named ‘4-keto-myxoxanthophyll-like pigment’ in Schlüter, L., Garde, K., Kaas, H., [2004. A 4-keto-myxoxanthophyll-like pigment is a diagnostic pigment for the toxic cyanobacteria Nodularia spumigena in the Baltic Sea. Mar. Ecol. Prog. Ser. 275, 69–78.] was determined to be a 4-ketomyxol-2′-fucoside. In most of the strains an additional carotenoid was found, identified as the novel 1′-O-methyl-4-ketomyxol-2′-fucoside by 2D NMR. This glycosidic carotenoid methyl ether was found to be a more important diagnostic pigment than the 4-ketomyxol-2′-fucoside for the toxic N. spumigena in the Baltic Sea. Out of the 20 strains 15 were found to produce the hepatotoxin nodularin. The content of carotenoids and nodularin was found to increase relative to chlorophyll a at increasing light intensity and at stationary growth, and nodularin was significantly correlated to both 4-ketomyxol-2′-fucoside and 1′-O-methyl-4-ketomyxol-2′-fucoside, and particular to the sum of these two pigments.     

PCR Amplification of the Gene acmA Differentiates Lactococcus lactis subsp. lactis and L. lactis subsp. cremoris

The occurrence of the acmA gene, encoding the lactococcal N-acetylmuramidase in new lactococcal isolates from raw milk cheeses, has been determined. Isolates were genotypically identified to the subspecies level with a PCR technique. On the basis of PCR amplification of the acmA gene, the presence or absence of an additional amplicon of approximately 700 bp correlated with Lactococcus lactis subspecies. L. lactis subsp. lactis exhibits both the expected 1,131-bp product and the additional amplicon, whereas L. lactis subsp. cremoris exhibits a single 1,131-bp fragment.