Synthesis,SAR and intramolecular hydrogen bonding pattern of 1,3,5-trisubstituted 4,5-dihydropyrazoles as potent cannabinoid CB1 receptor antagonists

The synthesis, structure–activity relationship (SAR) studies and intramolecular hydrogen bonding pattern of 1,3,5-trisubstituted 4,5-dihydropyrazoles are described. The target compounds 6–18 represent a novel class of potent and selective CB₁ receptor antagonists. Based on X-ray diffraction data, the orally active 17 is shown to elicit a different intramolecular H-bonding mode as compared to ibipinabant (3) and SLV330 (4).     

Two γ-tubulin isoforms are differentially expressed during development in Helianthus annuus

The cytoskeleton is involved in major developmental events in plant cell growth and differentiation. Nucleation events play a key role in the dynamic and organization of the microtubule (Mt) cytoskeleton. Among many proteins involved in Mt nucleation, γ -tubulin has been identified as an essential component of the Mt organizing centers (MTOC). In protoplasts, somatic embryogenesis induction has been correlated with remodeling of Mt cytoskeleton. We have investigated the specific developmental expression of γ -tubulin in Helianthus annuus . Two γ -tubulin isoforms have been detected by immunoblotting, with bands at 52 and 58 kDa. The larger γ -tubulin (58 kDa) is present in all the sunflower tissues tested and is associated with the nucleus. The smaller γ -tubulin (52 kDa), differing from the former at the carboxy-terminal end, is only present in meristematic and dedifferentiated cells and is not bound to the nucleus. This first demonstration of the presence of two γ -tubulins in plant cells is discussed in terms of distinct roles in the nucleation and organization of Mts.     

Comparison of multilocus sequence typing (MLST), pulsed-field gel electrophoresis (PFGE), and amplified fragment length polymorphism (AFLP) for genetic typing of Staphylococcus aureus

We compared multilocus sequence typing (MLST), pulsed-field gel electrophoresis (PFGE), and amplified fragment length polymorphism (AFLP) for typing of Staphylococcus aureus and show that the methods yield similar results, although with differences in resolving power and reproducibility. Epidemiological conditions should determine which is the optimal typing method to be used.     

Tissue factor coagulant function is enhanced by protein-disulfide isomerase independent of oxidoreductase activity

Protein-disulfide isomerase (PDI) switches tissue factor (TF) from coagulation to signaling by targeting the allosteric Cys186-Cys209 disulfide. Here, we further characterize the interaction of purified PDI with TF. We find that PDI enhances factor VIIa-dependent substrate factor X activation 5-10-fold in the presence of wild-type, oxidized soluble TF but not TF mutants that contain an unpaired Cys186 or Cys209. PDI-accelerated factor Xa generation was blocked by bacitracin but not influenced by inhibition of vicinal thiols, reduction of PDI, changes in redox gradients, or covalent thiol modification of reduced PDI by N-ethylmaleimide or methyl-methanethiosulfonate, which abolished PDI oxidoreductase but not chaperone activity. PDI had no effect on fully active TF on either negatively charged phospholipids or in activating detergent, indicating that PDI selectively acts upon cryptic TF to facilitate ternary complex formation and macromolecular substrate turnover. PDI activation was reduced upon mutation of TF residues in proximity to the macromolecular substrate binding site, consistent with a primary interaction of PDI with TF. PDI enhanced TF coagulant activity on microvesicles shed from cells, suggesting that PDI plays a role as an activating chaperone for circulating cryptic TF.     

Protein kinase CK2 phosphorylation of EB2 regulates its function in the production of Epstein-Barr virus infectious viral particles

The Epstein-Barr Virus (EBV) early protein EB2 (also called BMLF1, Mta, or SM) promotes the nuclear export of a subset of early and late viral mRNAs and is essential for the production of infectious virions. We show here that in vitro, protein kinase CK2alpha and -beta subunits bind both individually and, more efficiently, as a complex to the EB2 N terminus and that the CK2beta regulatory subunit also interacts with the EB2 C terminus. Immunoprecipitated EB2 has CK2 activity that phosphorylates several sites within the 80 N-terminal amino acids of EB2, including Ser-55, -56, and -57, which are localized next to the nuclear export signal. EB2S3E, the phosphorylation-mimicking mutant of EB2 at these three serines, but not the phosphorylation ablation mutant EB2S3A, efficiently rescued the production of infectious EBV particles by HEK293(BMLF1-KO) cells harboring an EB2-defective EBV genome. The defect of EB2S3A in transcomplementing 293(BMLF1-KO) cells was not due to impaired nucleocytoplasmic shuttling of the mutated protein but was associated with a decrease in the cytoplasmic accumulation of several late viral mRNAs. Thus, EB2-mediated production of infectious EBV virions is regulated by CK2 phosphorylation at one or more of the serine residues Ser-55, -56, and -57.     

The gradual onset brain death model: a relevant model to study organ donation and its consequences on the outcome after transplantation

Organs used for transplantation are usually derived from heart-beating brain dead donors. However, brain death is known to have negative effects on donor organ quality, previously studied using a difficult to control sudden onset experimental model. We have now developed a reproducible gradual onset brain death model in rats without requiring inotropic support. Fisher inbred rats weighing 260-300 g were used. Brain death was induced by a gradual inflation of a subdurally placed balloon catheter. During induction and the period following brain death, the animals were mechanically ventilated and blood pressure was continuously monitored. The blood pressure registration showed a characteristic pattern during brain death induction, in which a decrease in blood pressure, a hypotensive period in which the Cushing response occurred, and a sharp peak were consistent findings. After brain death was induced, blood pressure was maintained at normotensive levels up to 4 h. After the experiments, neuropathological evaluation of the brain located haemorrhagic cerebral parenchyma, and immunocytochemistry of liver tissue revealed a significant influx of polymorph nuclear cells, as was previously observed as well. This improved model allows the study of brain death on donor organ quality without the use of inotropic support.     

The protein that binds to DNA base J in trypanosomatids has features of a thymidine hydroxylase

Trypanosomatids contain an unusual DNA base J (beta-d-glucosylhydroxymethyluracil), which replaces a fraction of thymine in telomeric and other DNA repeats. To determine the function of base J, we have searched for enzymes that catalyze J biosynthesis. We present evidence that a protein that binds to J in DNA, the J-binding protein 1 (JBP1), may also catalyze the first step in J biosynthesis, the conversion of thymine in DNA into hydroxymethyluracil. We show that JBP1 belongs to the family of Fe(2+) and 2-oxoglutarate-dependent dioxygenases and that replacement of conserved residues putatively involved in Fe(2+) and 2-oxoglutarate-binding inactivates the ability of JBP1 to contribute to J synthesis without affecting its ability to bind to J-DNA. We propose that JBP1 is a thymidine hydroxylase responsible for the local amplification of J inserted by JBP2, another putative thymidine hydroxylase.     

Does prey capture induce area-restricted search? A fine-scale study using GPS in a marine predator, the wandering albatross

In a patchy environment, predators are expected to increase turning rate and start an area-restricted search (ARS) when prey have been encountered, but few empirical data exist for large predators. By using GPS loggers with devices measuring prey capture, we studied how a marine predator adjusts foraging movements at various scales in relation to prey capture. Wandering albatrosses use two tactics, sit and wait and foraging in flight, the former tactic being three times less efficient than the latter. During flight foraging, birds caught large isolated prey and used ARS at scales varying from 5 to 90 km, with large-scale ARS being used only by young animals. Birds did not show strong responses to prey capture at a large scale, few ARS events occurred after prey capture, and birds did not have high rates of prey capture in ARS. Only at small scales did birds increase sinuosity after prey captures for a limited time period, and this occurred only after they had caught a large prey item within an ARS zone. When this species searches over a large scale, the most effective search rule was to follow a nearly straight path. ARS may be used to restrict search to a particular environment where prey capture is more predictable and profitable.     

Assay of the activity of malonyl-coenzyme A decarboxylase by gas chromatography-mass spectrometry

We developed a gas chromatography-mass spectrometry (GC-MS) assay to measure the activity of malonyl-coenzyme A (CoA) decarboxylase (MCD) in crude tissue homogenates. Liver extracts are incubated with [U-(13)C(3)]malonyl-CoA to form [U-(13)C(2)]acetyl-CoA by the action of MCD. The reaction mixture contains 2 mM ADP to prevent the hydrolysis of [1,2-(13)C(2)]acetyl-CoA by acetyl-CoA hydrolase present in the extracts. Newly formed [U-(13)C(2)]acetyl-CoA and internal standard of [(2)H(3),1-(13)C]acetyl-CoA are analyzed as thiophenol derivatives by GC-MS. This assay was applied to a study of the kinetics of MCD in rat liver. Using the Lineweaver-Burke plot of MCD kinetics, K(m) of 202microM and V(max) of 3.3micromol min(-1) (g liver)(-1) were calculated. The liver MCD activities (micromol min(-1) g(-1)+/-SD) in three groups of rats with different nutritional statuses-fed, 1-day fasted, and 2-day fasted-were 1.80+/-0.41, 2.59+/-0.37 (P<0.05), and 3.07+/-0.70 (P<0.05), respectively. We report a practical, nonradioactive, sensitive assay of MCD in crude tissue extract.