The Type II restriction endonuclease MvaI has dual specificity

The MvaI restriction endonuclease cuts 5′-CC↓AGG-3′/5′-CC↑TGG-3′ sites as indicated by the arrows. N4-methylation of the inner cytosines (C^m4CAGG/C^m4CTGG) protects the site against MvaI cleavage. Here, we show that MvaI nicks the G-strand of the related sequence (CCGGG/CCCGG, BcnI site) if the inner cytosines are C5-methylated: C^m5C↓GGG/CC^m5CGG. At M.SssI-methylated SmaI sites, where two oppositely oriented methylated BcnI sites partially overlap, double-nicking leads to double-strand cleavage (CC^m5C↓GGG/CC^m5C↑GGG) generating fragments with blunt ends. The double-strand cleavage rate and the stringency of substrate site recognition is lower at the methylation-dependent site than at the canonical target site. MvaI is the first restriction endonuclease shown to possess, besides the ‘normal’ activity on its unmethylated recognition site, also a methylation-directed activity on a different sequence.     

Carbamoyloximes as novel non-competitive mGlu5 receptor antagonists

Hit-to-lead optimization of a HTS hit led to new carbamoyloxime derivatives. After identification of an advanced hit (8d) the CYP enzyme inhibitory activity of this class of compounds was successfully eliminated. Systematic exploration of different parts of the advanced hit led us to some promising lead compounds with mGluR5 affinities comparable to that of MPEP.     

Hit-to-lead optimization of disubstituted oxadiazoles and tetrazoles as mGluR5 NAMs

Here we report the discovery and early SAR of a series of mGluR5 negative allosteric modulators (NAMs). Starting from a moderately active HTS hit we synthesized 3,5-disubstituted-oxadiazoles and tetrazoles as mGluR5 NAMs. Based on the analysis of ligand efficiency and lipophilic efficiency metrics we identified a promising lead candidate as a starting point for further optimization.     

Development and characterization of a FIA system for selective assay of l-ascorbic acid in food samples

An amperometric detector and an enzymatic reaction were combined for the measurement of l-ascorbic acid. The enzyme cell (containing immobilized ascorbate oxidase) was connected to a flow injection analyzer (FIA) system with a glassy carbon electrode as an amperometric detector. During optimization and measurements two sample injectors were used, one before and one after the enzyme cell, thus eliminating the background interferences. Subtraction of the signal area given in the presence of enzyme from the one given in the absence of enzyme was applied for measuring analyte concentrations and calibration at 400 mV. Analysis capacity of system is 25 samples/hour. The relative standard deviation (RSD) was below 5% (5 times repeated, 400 μmol/L conc.), linearity up to 400 μmol/L, limit of detection (LOD) 5 μmol/L, fitting of calibration curve in 25–400 μmol/L range was R ² = 0.99.     

Large-scale properties of clustered networks: implications for disease dynamics

We consider previously proposed procedures for generating clustered networks and investigate how these procedures lead to differences in network properties other than clustering. We interpret our findings in terms of the effect of the network structure on the disease outbreak threshold and disease dynamics. To generate null-model networks for comparison, we implement an assortativity-conserving rewiring algorithm that alters the level of clustering while causing minimal impact on other properties. We show that many theoretical network models used to generate networks with a particular property often lead to significant changes in network properties other than that of interest. For high levels of clustering, different procedures lead to networks that differ in degree heterogeneity and assortativity, and in broader scale measures such as ?(0) and the distribution of shortest path lengths. Hence, care must be taken when investigating the implications of network properties for disease transmission or other dynamic process that the network supports.     

Diverse subcellular locations of cryptogein-induced reactive oxygen species production in tobacco Bright Yellow-2 cells

Reactive oxygen species (ROS) play a crucial role in many cellular responses and signaling pathways, including the oxidative burst defense response to pathogens. We have examined very early events in cryptogein-induced ROS production in tobacco (Nicotiana tabacum) Bright Yellow-2 suspension cells. Using Amplex Red and Amplex Ultra Red reagents, which report real-time H2O2 accumulation in cell populations, we show that the internal signal for H2O2 develops more rapidly than the external apoplastic signal. Subcellular accumulation of H2O2 was also followed in individual cells using the 2',7'-dichlorofluorescein diacetate fluorescent probe. Major accumulation was detected in endomembrane, cytoplasmic, and nuclear compartments. When cryptogein was added, the signal developed first in the nuclear region and, after a short delay, in the cell periphery. Interestingly, isolated nuclei were capable of producing H2O2 in a calcium-dependent manner, implying that nuclei can serve as a potential active source of ROS production. These results show complex spatial compartmentalization for ROS accumulation and an unexpected temporal sequence of events that occurs after cryptogein application, suggesting novel intricacy in ROS-signaling cascades.     

Ca2+ channel blockers reverse iron overload by a new mechanism via divalent metal transporter-1

Hereditary hemochromatosis and transfusional iron overload are frequent clinical conditions associated with progressive iron accumulation in parenchymal tissues, leading to eventual organ failure. We have discovered a new mechanism to reverse iron overload-pharmacological modulation of the divalent metal transporter-1 (DMT-1). DMT-1 mediates intracellular iron transport during the transferrin cycle and apical iron absorption in the duodenum. Its additional functions in iron handling in the kidney and liver are less well understood. We show that the L-type calcium channel blocker nifedipine increases DMT-1-mediated cellular iron transport 10- to 100-fold at concentrations between 1 and 100 microM. Mechanistically, nifedipine causes this effect by prolonging the iron-transporting activity of DMT-1. We show that nifedipine mobilizes iron from the liver of mice with primary and secondary iron overload and enhances urinary iron excretion. Modulation of DMT-1 function by L-type calcium channel blockers emerges as a new pharmacological therapy for the treatment of iron overload disorders.     

Rapid detection of sex chromosomal aneuploidies by QF-PCR: application in 200 men with severe oligozoospermia or azoospermia

Klinefelter syndrome is the most common genetic cause of severe male factor infertility. Cytogenetic evaluation of metaphase chromosomes generally has a long turnaround time. We describe a reliable molecular genetic method that can be completed in 2 working days to identify the presence of any extra X chromosomes. The quantitative fluorescent (QF) 5-plex PCR includes the amplification of amelogenin, which is present on both sex chromosomes in a biallelic form, a polymorphic short tandem repeat (STR) on the pseudoautosomal region of X and Y (X22), two polymorphic X-specific STRs (DXS6803, DXS6809), and a Y-specific marker (SY134), in a single tube. The presence of an extra X chromosome is recognized either by a supernumerary peak or an increased peak area based on criteria we have developed. The application of the method on 200 patients resulted in the identification of 14 patients (7%) with Klinefelter syndrome or a variant form (2 SRY-positive 46,XX men), as well as an additional patient with 47,XYY karyotype. The QF-PCR method, along with Y chromosome microdeletion testing, can be used as a first-step genetic analysis in azoospermic or severely oligozoospermic patients for the rapid identification of sex chromosome aneuploidies.