Influence of global fluorination on chloramphenicol acetyltransferase activity and stability

Varied levels of fluorinated amino acid have been introduced biosynthetically to test the functional limits of global substitution on enzymatic activity and stability. Replacement of all the leucine (LEU) residues in the enzyme chloramphenicol acetyltransferase (CAT) with the analog, 5',5',5'-trifluoroleucine (TFL), results in the maintenance of enzymatic activity under ambient temperatures as well as an enhancement in secondary structure but loss in stability against heat and denaturants or organic co-solvents. Although catalytic activity of the fully substituted CAT is preserved under standard reaction conditions compared to the wild-type enzyme both in vitro and in vivo, as the incorporation levels increase, a concomitant reduction in thermostability and chemostability is observed. Circular dichroism (CD) studies reveal that although fluorination greatly improves the secondary structure of CAT, a large structural destabilization upon increased levels of TFL incorporation occurs at elevated temperatures. These data suggest that enhanced secondary structure afforded by TFL incorporation does not necessarily lead to an improvement in stability.     

Involvement of calcium mobilization from caffeine-sensitive stores in mechanically induced cell cycle arrest in the dinoflagellate Crypthecodinium cohnii

Mechanical loads can profoundly alter cell growth and cell proliferation. The dinoflagellates are especially sensitive to mechanical stimulation. Many species will be arrested in cell cycle in response to turbulence or shear stress. We demonstrate here that mechanical shaking and caffeine, the ryanodine-receptor agonist, induced an elevation of cytosolic calcium in the dinoflagellate Crypthecodinium cohnii. Dantrolene, a ryanodine-receptor antagonist, dose-dependently inhibited both shaking-induced and caffeine-induced calcium release. Similar to the effect of mechanical shaking, caffeine alone dose-dependently and reversibly induced cell cycle arrest in dinoflagellates. Prolonged shaking substantially abolished the magnitude of caffeine-induced calcium release and vice-versa, suggesting that both agents released calcium from similar stores through ryanodine receptors. Fluorescence-conjugated ryanodine gave positive labeling, which could be blocked by ryanodine, in the cortice of C. cohnii cells. In addition, caffeine or shaking mobilized intracellular chlortetracycline (CTC)-positive membrane-bound calcium, which could be similarly depleted by t-BuBHQ, a SERCA pump inhibitor. Prior treatment with shaking or caffeine also inhibited the ability of the other agent in mobilizing CTC-positive calcium. CTC-positive microsomal fractions could also be induced to release calcium by caffeine and cADPR, the ryanodinee receptor modulator. t-BuBHQ, but not calcium ionophores, induced cell cycle arrest, and the calcium chelator BAPTA-AM was unable to rescue caffeine-induced cell cycle arrest. These data culminate to suggest that mobilization or depletion of caffeine-sensitive calcium stores, but not calcium elevation per se, is involved in the induction of cell cycle arrest by mechanical stimulation. The present study establishes the role of caffeine-sensitive calcium stores in the regulation of cell cycle progression.     

Discovery of bitriazolyl compounds as novel antiviral candidates for combating the tobacco mosaic virus

Bitriazolyl compounds were synthesized and their activity against tobacco mosaic virus was assessed. Two of them showed promising antiviral activity and were more potent than the reference compounds. Moreover, these compounds are predicted not to be carcinogenic or mutagenic based on the prediction systems. Therefore, the bitriazolyl compounds may provide interesting new leads or scaffolds for use in further attempts to screen novel antiviral candidates.     

Phosphatidylserine recognition by phagocytes: a view to a kill

The redistribution of phosphatidylserine (PS) to the external surface of the plasma membrane is a key element of apoptotic cell recognition and is a molecular cue that dying cells should be engulfed. Phagocytes interact with PS on apoptotic cells through either the PS receptor or secreted bridging proteins called opsonins. The study of two secreted PS opsonins, MFG-E8 and Gas6 and their receptors alphavbeta5 (and alphavbeta3) integrin and Mer tyrosine kinase, respectively, have provided insights into the temporal and spatial aspects of Rac1 activation following the recognition and internalization of apoptotic cells. Disruption of PS opsonins and their signaling pathways often manifest conditions of inflammation and autoimmune disease. Here, we review recent studies involving PS opsonins, their receptors and their role in the phagocytosis of apoptotic cells.     

Acid hydrolysis of fibers from dairy manure

Concentrated acid hydrolysis of lignocellulosic materials is a conventional treatment process for the production of mono-sugars. However, this method has been proved ineffective and undesirable for the treatment of dairy manure due to the high nitrogen content of dairy manure and the environmental issues caused by the use of highly concentrated acid solution. In an effort to overcome these barriers, a modified acid hydrolysis process with short reaction time was introduced that involved a nitrogen-removing pretreatment followed by decrystallization with concentrated acid and then hydrolysis using dilute acid. The effects of nitrogen, acid concentration, reaction time, and temperature were investigated. A pretreated manure with a low nitrogen content of 1.3% was used as the substrate. The results indicated that the optimal conditions for fiber decrystallization were 75% acid concentration, 3:5 sample to acid ratio (weight basis), and 30 min of reaction time; while the optimal conditions for acid hydrolysis were 12.5% acid and 10% dry sample at 135 degrees C for 10 min. These conditions produced 26 g/L glucose at a yield of 84% and 11 g/L hemicellulose-sugars at a yield of 80%.     

Synthesis and evaluation of novel 8-oxo-8H-cyclopenta[a]acenaphthylene-7-carbonitriles as long-wavelength fluorescent markers for hypoxic cells in solid tumor

Novel bioreductive and long-wavelength fluorescent markers for hypoxic cells in solid tumor, 9-isocyano-8H-acenaphtho[l,2-b]pyrrol-8-one with the side chain of 2-nitroimidazole, were designed, synthesized, and evaluated in V79 379A Chinese hamster cells in vitro. Compounds A(2) and A(4) showed good hypoxic-oxic fluorescence differential in vitro (V79 cells) by using fluorescence scan ascent.     

Pentacyclic triterpenes. Part 2: Synthesis and biological evaluation of maslinic acid derivatives as glycogen phosphorylase inhibitors

The synthesis of a series of maslinic acid derivatives is described and their effect on rabbit muscle glycogen phosphorylase a evaluated. Within this series of compounds, 15 (IC(50)=7 microM) is the most potent GPa inhibitor. SAR of the maslinic acid derivatives are discussed.     

Molecular dynamics simulations of folding processes of a beta-hairpin in an implicit solvent

Computer simulations of beta-hairpin folding are relatively difficult, especially those based on the explicit water model. This greatly limits the complete analysis and understanding of their folding mechanisms. In this paper, we use the generalized Born/solvent accessible implicit solvent model to simulate the folding processes of a nine-residue beta-hairpin. We find that the beta-hairpin can fold into its native structure very easily, even using the traditional molecular dynamics method. This allows us to extract 21 complete folding events and investigate the folding process sufficiently. Our results show that there exist four most stable states on the free energy landscape of the short peptide, one native state and three intermediates. We find that two of the non-native stable states have almost the same potential energy as the native state but with lower entropy. This suggests that the native state can be stabilized entropically. Furthermore, we find that the folding processes of this peptide have common features: to fold into its native state, the peptide undergoes a continuous collapsing-extending-recollapsing process to adjust the positions of the side chains in order to form the native middle inter-strand hydrogen bonds. The formations of these bonds are the key step of the folding process. Once these bonds are formed, the peptide can fold into the native state quickly.