Interaction of ethanol with opiate receptors

Addition of ethanol to rat brain homogenate containing opiate receptors inhibits at a concentration of 50 mM the stereospecific binding of 3H-naloxone at 37 degrees C but not at 0 degree C, with the ID50 being 462 mM under these conditions. The temperature-dependent inhibition of the ligand binding suggests that ethanol does not compete with naloxone for specific binding sites of opiate receptors and changes the structure of lipids in biological membranes. Scatchard's analysis has demonstrated that apart from a decrease in the number of highly affinity binding sites of 3H-naloxone, the total amount of the binding sites remains unchanged both in the presence and absence of ethanol and constitutes 453 and 549 fmol/mg protein. It is assumed that ethanol might interconvert highly and low-affinity binding sites. Analysis of the effect of ethanol on 3H-naloxone binding with opiate receptors contained by synaptic membranes obtained from animals with varying predisposition to voluntary alcoholization has shown that ethanol inhibits to a greater degree ligand binding with membranes obtained from rats predisposed to alcoholization. The possibility of the involvement of receptors in the biochemical mechanisms by which the initial alcoholic motivation is effected is under discussion.     

Measurement of 7-dehydrocholesterol and cholesterol in hair can be used in the diagnosis of Smith-Lemli-Opitz syndrome

7-dehydrocholesterol (7-DHC) and cholesterol (CHOL) are biomarkers of Smith-Lemli-Opitz Syndrome (SLOS), a congenital autosomal recessive disorder characterized by elevated 7-DHC level in patients. Hair samples have been shown to have great diagnostic and research value, which has long been neglected in the SLOS field. In this study, we sought to investigate the feasibility of using hair for SLOS diagnosis. In the presence of antioxidants (2,6-ditert-butyl-4-methylphenol and triphenylphosphine), hair samples were completely pulverized and extracted by micro-pulverized extraction in alkaline solution or in n-hexane. After microwave-assisted derivatization with N,O-Bis(trimethylsilyl)trifluoroacetamide, the analytes were measured by GC-MS. We found that the limits of determination for 7-DHC and CHOL were 10 ng/mg and 8 ng/mg, respectively. In addition, good linearity was obtained in the range of 50–4000 ng/mg and 30–6000 ng/mg for 7-DHC and CHOL, respectively, which fully meets the requirement for SLOS diagnosis and related research. Finally, by applying the proposed method to real hair samples collected from 14 healthy infants and two suspected SLOS patients, we confirmed the feasibility of hair analysis as a diagnostic tool for SLOS. In conclusion, we present an optimized and validated analytical method for the simultaneous determination of two SLOS biomarkers using human hair.     

Structural Insight of a Trimodular Halophilic Cellulase with a Family 46 Carbohydrate-Binding Module

Cellulases are the key enzymes used in the biofuel industry. A typical cellulase contains a catalytic domain connected to a carbohydrate-binding module (CBM) through a flexible linker. Here we report the structure of an atypical trimodular cellulase which harbors a catalytic domain, a CBM46 domain and a rigid CBM_X domain between them. The catalytic domain shows the features of GH5 family, while the CBM46 domain has a sandwich-like structure. The catalytic domain and the CBM46 domain form an extended substrate binding cleft, within which several tryptophan residues are well exposed. Mutagenesis assays indicate that these residues are essential for the enzymatic activities. Gel affinity electrophoresis shows that these tryptophan residues are involved in the polysaccharide substrate binding. Also, electrostatic potential analysis indicates that almost the entire solvent accessible surface of CelB is negatively charged, which is consistent with the halophilic nature of this enzyme.     

Solving the N-Queens problem with a binary Hopfield-type network

The application of a discrete Hopfield-type neural network to solving the NP-Hard optimization problem — the N-Queens Problem (NQP) — is presented. The applied network is binary, and at every moment each neuron potential is equal to either 0 or 1. The network can be implemented in the asynchronous mode as well as in the synchronous one with n parallel running processors. In both cases the convergence rate is up to 100%, and the experimental estimate of the average computational complexity is polynomial. Based on the computer simulation results and the theoretical analysis, the proper network parameters are established. The behaviour of the network is explained.     

Modeling the Role of Negative Cooperativity in Metabolic Regulation and Homeostasis

A significant proportion of enzymes display cooperativity in binding ligand molecules, and such effects have an important impact on metabolic regulation. This is easiest to understand in the case of positive cooperativity. Sharp responses to changes in metabolite concentrations can allow organisms to better respond to environmental changes and maintain metabolic homeostasis. However, despite the fact that negative cooperativity is almost as common as positive, it has been harder to imagine what advantages it provides. Here we use computational models to explore the utility of negative cooperativity in one particular context: that of an inhibitor binding to an enzyme. We identify several factors which may contribute, and show that acting together they can make negative cooperativity advantageous.     

Phosphorylation and ubiquitination of dynamin-related proteins (AtDRP3A/3B) synergically regulate mitochondrial proliferation during mitosis

The balance between mitochondrial fission and fusion is disrupted during mitosis, but the mechanism governing this phenomenon in plant cells remains enigmatic. Here, we used mitochondrial matrix‐localized Kaede protein (mt‐Kaede) to analyze the dynamics of mitochondrial fission in BY‐2 suspension cells. Analysis of the photoactivatable fluorescence of mt‐Kaede suggested that the fission process is dominant during mitosis. This finding was confirmed by an electron microscopic analysis of the size distribution of mitochondria in BY‐2 suspension cells at various stages. Cellular proteins interacting with Myc‐tagged dynamin‐related protein 3A/3B (AtDRP3A and AtDRP3B) were immunoprecipitated with anti‐Myc antibody‐conjugated beads and subsequently identified by microcapillary liquid chromatography–quadrupole time‐of‐flight mass spectrometry (CapLC Q‐TOF) MS/MS. The identified proteins were broadly associated with cytoskeletal (microtubular), phosphorylation, or ubiquitination functions. Mitotic phosphorylation of AtDRP3A/AtDRP3B and mitochondrial fission at metaphase were inhibited by treatment of the cells with a CdkB/cyclin B inhibitor or a serine/threonine protein kinase inhibitor. The fate of AtDRP3A/3B during the cell cycle was followed by time‐lapse imaging of the fluorescence of Dendra2‐tagged AtDRP3A/3B after green‐to‐red photoconversion; this experiment showed that AtDRP3A/3B is partially degraded during interphase. Additionally, we found that microtubules are involved in mitochondrial fission during mitosis, and that mitochondria movement to daughter cell was limited as early as metaphase. Taken together, these findings suggest that mitotic phosphorylation of AtDRP3A/3B promotes mitochondrial fission during plant cell mitosis, and that AtDRP3A/3B is partially degraded at interphase, providing mechanistic insight into the mitochondrial morphological changes associated with cell‐cycle transitions in BY‐2 suspension cells.     

Modulatory effect of Lactobacillus acidophilus KLDS 1.0738 on intestinal short‐chain fatty acids metabolism and GPR41/43 expression in β‐lactoglobulin–sensitized mice

We investigated the correlation between the beneficial effect of Lactobacillus acidophilus on gut microbiota composition, metabolic activities, and reducing cow's milk protein allergy. Mice sensitized with β‐lactoglobulin (β‐Lg) were treated with different doses of L. acidophilus KLDS 1.0738 for 4 weeks, starting 1 week before allergen induction. The results showed that intake of L. acidophilus significantly suppressed the hypersensitivity responses, together with increased fecal microbiota diversity and short‐chain fatty acids (SCFAs) concentration (including propionate, butyrate, isobutyrate, and isovalerate) when compared with the allergic group. Moreover, treatment with L. acidophilus induced the expression of SCFAs receptors, G‐protein–coupled receptors 41 (GPR41) and 43 (GPR43), in the spleen and colon of the allergic mice. Further analysis revealed that the GPR41 and GPR43 messenger RNA expression both positively correlated with the serum concentrations of transforming growth factor‐β and IFN‐γ (p < .05), but negatively with the serum concentrations of IL‐17, IL‐4, and IL‐6 in the L. acidophilus–treated group compared with the allergic group (p < .05). These results suggested that L. acidophilus protected against the development of allergic inflammation by improving the intestinal flora, as well as upregulating SCFAs and their receptors GPR41/43.