Simultaneous LC/ESI‐MS Separation Method for the Enantioseparation of Some New Anticonvulsant Drugs

A sensitive and specific method for the simultaneous determination of the enantiomeric purity of 2,6‐dimethylphenoxyacetyl derivatives as trans or cis racemic and enantiomeric forms with 2‐ or 4‐aminocyclohexanol moiety ( 1 , 2 , 3 , 6 ) and their amine analogs ( 8 , 9 ) was developed. The compounds studied are known for their anticonvulsant activity and the most interesting pharmacological results were those for (±)‐trans‐2‐(2,6‐dimethylphenoxy)‐N‐(2‐hydroxycyclohexyl)acetamide ( 1 ) as well as (±)‐trans‐2‐[(2,6‐dimethylphenoxy)ethyl]aminocyclohexanol ( 8 ). The analytical method for determining the enantiomeric purity of the compounds studied is based on direct separation of the analytes using a chiral stationary phase (Chiralpak AS column). The mass spectrometric analysis was done on a coupled liquid chromatograph–mass spectrometer system with an electrospray ionization source (LC/ESI‐MS). For the compounds 1 , 8 , and 9 , the method allows an excellent separation of enantiomers, with a resolution higher than 3.2, and a tailing factor of less than 1.67 with a final enantiomer purity better than 97.5%. Chirality 26:144–149, 2014. © 2014 Wiley Periodicals, Inc.     

Virulence Structure of the Powdery Mildew (Blumeria graminis) Population Occurring on Triticale (x Triticosecale) in Poland

Powdery mildew, caused by Blumeria graminis is an important disease of cereals in many production regions. Until end of the last century triticale had been regarded as a species characterized by high level of resistance for this disease. However, after several years of intensive production on a big area in Poland, Germany and other European countries it start to be susceptible for many pathogens including B. graminis. Because of this, virulence structure of this pathogen population on triticale in Poland was evaluated across 2008–2010. Leaf samples with symptoms of the powdery mildew disease were collected randomly from nineteen localities. As a total, 1402 B. graminis isolates were collected: 23–25 isolates per locality in each year. Standard differential set of 28 genotypes was used: twenty‐one wheat with known resistance genes and seven triticale. Based on the obtained results it was possible to observe significant differences in virulence structure between years and localities. No virulence's against Pm21 (Yangmai5), and Pm3d + 4b (Kadett) were found in any year. All tested isolates were virulent on Moreno and Lamberto cultivars. In a total, 36% of tested isolates possessed 9, 11 or 12 virulence's per genotypes. Twenty five percent of tested isolates were virulent to 5 triticale cultivars. Correlation between pathotypes frequency and sampling region were not found what suggest that local epidemics play the most important role in triticale growing regions in Poland.     

Copper-induced structural propensities of the amyloidogenic region of human prion protein

Transmissible spongiform encephalopathies are associated with the misfolding of the cellular Prion Protein (PrP^C) to an abnormal protein isoform, called scrapie prion protein (PrP^Sc). The structural rearrangement of the fragment of N-terminal domain of the protein spanning residues 91–127 is critical for the observed structural transition. The amyloidogenic domain of the protein encloses two copper-binding sites corresponding to His-96 and His-111 residues that act as anchors for metal ion binding. Previous studies have shown that Cu(II) sequestration by both sites may modulate the peptide’s tendency to aggregation as it inflicts the hairpin-like structure that stabilizes the transition states leading to β-sheet formation. On the other hand, since both His sites differ in their ability to Cu(II) sequestration, with His-111 as a preferred binding site, we found it interesting to test the role of Cu(II) coordination to this single site on the structural properties of amyloidogenic domain. The obtained results reveal that copper binding to His-111 site imposes precise backbone bending and weakens the natural tendency of apo peptide to β-sheet formation.     

Chondrocyte suspension in fibrin glue

Autologous chondrocyte implantation has been shown to be a promising method for treatment of deep articular cartilage defects. The hyaline cartilage formed by implanted autologous chondrocytes has biomechanical properties similar to those of natural articular cartilage. Between June 2006 and September 2008 we performed Autologous chondrocyte implantation (ACI) in 50 patients and the chondrocytes were supported in fibrin glue. The cartilage biopsy samples were taken from the non-weight bearing area of the patient’s femoral condyle and the samples were transferred to the cell culture laboratory. Chondrocyte were kept in culture about 20 days. Fibrin glue was used as a three dimensional carrier for chondrocyte implantation. A 450 ml of patient’s own blood was collected prior to transplantation to produce autologous fibrinogen. Alternatively the allogenic fibrinogen was prepared from Regional Blood Center voluntary donors. Before surgery the chondrocyte suspension was mixed with fibrin glue and gel—like fibrograft was prepared. The total number of cells and the size of fibrograft depended on the defect size in the knee. Our results suggest that ACI technique with fibrin glue is a promising method for treatment of cartilage defect.     

Acid–base and metal ion binding properties of 2-thiocytidine in aqueous solution

The thionucleoside 2-thiocytidine (C2S) occurs in nature in transfer RNAs; it receives attention in diverse fields like drug research and nanotechnology. By potentiometric pH titrations we measured the acidity constants of H(C2S)(+) and the stability constants of the M(C2S)(2+) and M(C2S-H)(+) complexes (M(2+) = Zn(2+), Cd(2+)), and we compared these results with those obtained previously for its parent nucleoside, cytidine (Cyd). Replacement of the (C2)=O unit by (C2)=S facilitates the release of the proton from (N3)H(+) in H(C2S)(+) (pK (a) = 3.44) somewhat, compared with H(Cyd)(+) (pK (a) = 4.24). This moderate effect of about 0.8 pK units contrasts with the strong acidification of about 4 pK units of the (C4)NH(2) group in C2S (pK (a) = 12.65) compared with Cyd (pK (a) approximately 16.7); the reason for this result is that the amino-thione tautomer, which dominates for the neutral C2S molecule, is transformed upon deprotonation into the imino-thioate form with the negative charge largely located on the sulfur. In the M(C2S)(2+) complexes the (C2)S group is the primary binding site rather than N3 as is the case in the M(Cyd)(2+) complexes, though owing to chelate formation N3 is to some extent still involved in metal ion binding. Similarly, in the Zn(C2S-H)(+) and Cd(C2S-H)(+) complexes the main metal ion binding site is the (C2)S(-) unit (formation degree above 99.99% compared with that of N3). However, again a large degree of chelate formation with N3 must be surmised for the M(C2S-H)(+) species in accord with previous solid-state studies of related ligands. Upon metal ion binding, the deprotonation of the (C4)NH(2) group (pK (a) = 12.65) is dramatically acidified (pK (a) approximately 3), confirming the very high stability of the M(C2S-H)(+) complexes. To conclude, the hydrogen-bonding and metal ion complex forming capabilities of C2S differ strongly from those of its parent Cyd; this must have consequences for the properties of those RNAs which contain this thionucleoside.     

Structure of rough,smooth, stripped and reconstituted rough membranes derived from rat liver as visualized by the freeze fracture technique

The structure of purified fractions of rough, smooth, stripped rough and reconstituted rough membranes have been investigated by the freeze etching technique. Preparations of rough and reconstituted rough membranes, active in protein synthesis, show vesicles whose outer surface is covered with ribosome-like particles. The inner surface of these vesicles contains also numerous particles of the same size. The particles located on the outer surface are largely absent in the stripped rough membrane preparations which, however, retain the particles located on the inner face. Particles were not seen either on the outer nor on the inner face of the smooth membranes. The possibility is considered that the particles located on the inner face are specific to the rough membranes and might play a role in the specific binding of ribosomes to the membranes.