Search for substrate-based inhibitors fitting the S2' space of malarial aspartic protease plasmepsin II.

Plasmepsin (Plm) has been identified as an important target for the development of new antimalarial drugs, since its inhibition leads to the starvation of Plasmodium falciparum. A series of substrate-based dipeptide-type Plm II inhibitors containing the hydroxymethylcarbonyl isostere as a transition-state mimic were synthesized. The general design principle was provision of a conformationally restrained hydroxyl group (corresponding to the set residue at the P2' position in native substrates) and a bulky unit to fit the S2' pocket.     

Identification and characterization of allophenylnorstatine-based inhibitors of plasmepsin II, an antimalarial target

Plasmepsin II is a key enzyme in the life cycle of the Plasmodium parasites responsible for malaria, a disease that afflicts more than 300 million individuals annually. Since plasmepsin II inhibition leads to starvation of the parasite, it has been acknowledged as an important target for the development of new antimalarials. In this paper, we identify and characterize high-affinity inhibitors of plasmepsin II based upon the allophenylnorstatine scaffold. The best compound, KNI-727, inhibits plasmepsin II with a K(i) of 70 nM and a 22-fold selectivity with respect to the highly homologous human enzyme cathepsin D. KNI-727 binds to plasmepsin II in a process favored both enthalpically and entropically. At 25 degrees C, the binding enthalpy (DeltaH) is -4.4 kcal/mol and the entropic contribution (-TDeltaS) to the Gibbs energy is -5.56 kcal/mol. Structural stability measurements of plasmepsin II were also utilized to characterize inhibitor binding. High-sensitivity differential scanning calorimetry experiments performed in the absence of inhibitors indicate that, at pH 4.0, plasmepsin II undergoes thermal denaturation at 63.3 degrees C. The structural stability of the enzyme increases with inhibitor concentration in a manner for which the binding energetics of the inhibitor can quantitatively account. The effectiveness of the best compounds in killing the malaria parasite was validated by performing cytotoxicity assays in red blood cells infected with Plasmodium falciparum. EC50s ranging between 6 and 10 microM (3-6 microg/mL) were obtained. These experiments demonstrate the viability of the allophenylnorstatine scaffold in the design of powerful and selective plasmepsin inhibitors.     

Transient state kinetics of enzyme IICBGlc, a glucose transporter of the phosphoenolpyruvate phosphotransferase system of Escherichia coli: equilibrium and second order rate constants for the glucose binding and phosphotransfer reactions

During translocation across the cytoplasmic membrane of Escherichia coli, glucose is phosphorylated by phospho-IIA(Glc) and Enzyme IICB(Glc), the last two proteins in the phosphotransfer sequence of the phosphoenolpyruvate:glucose phosphotransferase system. Transient state (rapid quench) methods were used to determine the second order rate constants that describe the phosphotransfer reactions (phospho-IIA(Glc) to IICB(Glc) to Glc) and also the second order rate constants for the transfer from phospho-IIA(Glc) to molecularly cloned IIB(Glc), the soluble, cytoplasmic domain of IICB(Glc). The rate constants for the forward and reverse phosphotransfer reactions between IIA(Glc) and IICB(Glc) were 3.9 x 10(6) and 0.31 x 10(6) m(-1) s(-1), respectively, and the rate constant for the physiologically irreversible reaction between [P]IICB(Glc) and Glc was 3.2 x 10(6) m(-1) s(-1). From the rate constants, the equilibrium constants for the transfer of the phospho-group from His90 of [P]IIA(Glc) to the phosphorylation site Cys of IIB(Glc) or IICB(Glc) were found to be 3.5 and 12, respectively. These equilibrium constants signify that the thiophospho-group in these proteins has a high phosphotransfer potential, similar to that of the phosphohistidinyl phosphotransferase system proteins. In these studies, preparations of IICB(Glc) were invariably found to contain endogenous, firmly bound Glc (estimated K'(D) approximately 10(-7) m). The bound Glc was kinetically competent and was rapidly phosphorylated, indicating that IICB(Glc) has a random order, Bi Bi, substituted enzyme mechanism. The equilibrium constant for the binding of Glc was deduced from differences in the statistical goodness of fit of the phosphotransfer data to the kinetic model.     

Intercropping patterns and different farming systems affect the yield and yield components of safflower and bitter vetch

This factorial field experiment was done based on a randomized complete block design in Urmia University, Iran, in 2013 and was repeated in 2014. Treatments included two farming systems (high input and organic) and different intercropping patterns that alternated bitter vetch (Vicia ervilia L.) and safflower (Carthamus tinctorius L.) with row ratios of 2:2, 3:2, 4:2 and 5:2. Sole cropping of bitter vetch and safflower was used as the control. In both years, the 2:2 intercropping pattern had biomass yield advantages compared to sole cropping and the other intercropping ratios, based on greater land equivalent ratio values. Safflower had higher relative crowding coef?cients, competitive ratio (CR) and aggressivity (A) values than bitter vetch. High-input farming was more effective than the organic system in both years. Safflower was the superior competitor when grown with bitter vetch, and its productivity dominated the total biomass yields. Thus, intercropping of safflower with bitter vetch has the potential to improve performance with high land-use efficiency.     

Jaw and long bone marrow derived osteoclasts differ in shape and their response to bone and dentin

Increasing evidence suggests the existence of osteoclast diversity. Here we investigated whether precursors obtained from marrow of the mandibula or long bone could give rise to phenotypically different osteoclasts. Formation of multinucleated cells was assessed after culturing mouse marrow cells of the two bone types with macrophage colony stimulating factor (M-CSF) and receptor activator of NFκB ligand (RANKL) for up to 10 days on plastic, bone or dentin. Two times more osteoclasts formed from long bone marrow cells on bone compared to dentin, whereas higher numbers of jaw osteoclasts formed on dentin. Resorption of dentin or bone was similar for osteoclasts formed from both types of precursors. In contrast to jaw marrow derived osteoclasts, long bone osteoclasts predominantly had a multi-compartmented shape, with at least two nuclei containing compartments per cell. Osteoclasts on bone contained two times more actin rings than osteoclasts on dentin, regardless of their precursor origin. However, the area per osteoclast covered by actin rings was similar (20%) for both substrates. This study suggests that marrow cells obtained from different bones give rise to different osteoclasts. The substrate on which the osteoclasts are generated plays a role in steering their formation rather than their resorption.     

Petroleum Degradation,Biosurfactant and Laccase Production by Fusarium neocosmosporiellum RH-10: A Microcosm Study

The aim of this study was to evaluate the potential of crude oil removal by fungal strains isolated from Arak refinery. The results showed that the RH10 strain is a potent strain as a surfactant producer and degrader of petrochemical hydrocarbons. The strain was identified as a Fusarium neocosmosporiellum and could degrade 58% of hydrocarbons in the minimal medium and reduce the surface tension from 45 to 26.5 mN m^?1. Moreover, residual crude oil analysis with Fourier transform infrared spectrophotometry showed that this strain was able to degrade 50% of aliphatic compounds. To investigate the mechanism of degradation, oxidase enzymes were assayed and it was found that F. neocosmosporiellum can produce 1.94 U L^?1 of laccase in 10 g L^?1 crude oil. Carbon, nitrogen, phosphorus and soil pattern optimization in a microcosm study showed that this strain removed 44% and 27% of the crude oil from contaminated soil in 1% and 5% crude oil concentrations, respectively. Under optimum condition, 9.66 g kg^?1 crude oil was removed by F. neocosmosporiellum when the initial oil concentration was 50 g kg^?1, at the end of 150 days microcosm experiment. The results demonstrated the promising potential of fungi strain for cleaning of contaminated soil.     

Patterns of Chemokine Receptor Fusion Cofactor Utilization by Human Immunodeficiency Virus Type 1 Variants from the Lungs and Blood

Human immunodeficiency virus type 1 (HIV-1) infection is highly compartmentalized, with distinct viral genotypes being found in the lungs, brain, and other organs compared with blood. CCR5 and CXCR4 are the principal HIV-1 coreceptors, and a number of other molecules support entry in vitro but their roles in vivo are uncertain. To address the relationship between tissue compartmentalization and the selective use of entry coreceptors, we generated functional env clones from primary isolates derived from the lungs and blood of three infected individuals and analyzed their use of the principal, secondary, orphan, and virus-encoded coreceptors for fusion. All Env proteins from lung viruses used CCR5 but not CXCR4, while those from blood viruses used CCR5 or CXCR4 or both. The orphan receptor APJ was widely used for fusion by Env proteins from both blood and lung viruses, but none used the cytomegalovirus-encoded receptor US28. Fusion mediated by the secondary coreceptors CCR2b, CCR3, CCR8, and CX3CR1 and orphan receptors GPR1, GPR15, and STRL33 was variable and heterogeneous, with relatively broad utilization by env clones from isolates of one subject but limited use by env clones from the other two subjects. However, there was no clear distinction between blood and lung viruses in secondary or orphan coreceptor fusion patterns. In contrast to fusion, none of the secondary or orphan receptors enabled efficient productive infection. These results confirm, at the level of cofactor utilization, previous observations that HIV-1 populations in the lungs and blood are biologically distinct and demonstrate diversity within lung-derived as well as blood-derived quasispecies. However, the heterogeneity in coreceptor utilization among clones from each isolate and the lack of clear distinction between lung- and blood-derived Env proteins argue against selective coreceptor utilization as a major determinant of compartmentalization.     

Thrombospondins are astrocyte-secreted proteins that promote CNS synaptogenesis

The establishment of neural circuitry requires vast numbers of synapses to be generated during a specific window of brain development, but it is not known why the developing mammalian brain has a much greater capacity to generate new synapses than the adult brain. Here we report that immature but not mature astrocytes express thrombospondins (TSPs)-1 and -2 and that these TSPs promote CNS synaptogenesis in vitro and in vivo. TSPs induce ultrastructurally normal synapses that are presynaptically active but postsynaptically silent and work in concert with other, as yet unidentified, astrocyte-derived signals to produce functional synapses. These studies identify TSPs as CNS synaptogenic proteins, provide evidence that astrocytes are important contributors to synaptogenesis within the developing CNS, and suggest that TSP-1 and -2 act as a permissive switch that times CNS synaptogenesis by enabling neuronal molecules to assemble into synapses within a specific window of CNS development.