Integrin-linked kinase localizes to the centrosome and regulates mitotic spindle organization

Integrin-linked kinase (ILK) is a serine-threonine kinase and scaffold protein with well defined roles in focal adhesions in integrin-mediated cell adhesion, spreading, migration, and signaling. Using mass spectrometry-based proteomic approaches, we identify centrosomal and mitotic spindle proteins as interactors of ILK. alpha- and beta-tubulin, ch-TOG (XMAP215), and RUVBL1 associate with ILK and colocalize with it to mitotic centrosomes. Inhibition of ILK activity or expression induces profound apoptosis-independent defects in the organization of the mitotic spindle and DNA segregation. ILK fails to localize to the centrosomes of abnormal spindles in RUVBL1-depleted cells. Additionally, depletion of ILK expression or inhibition of its activity inhibits Aurora A-TACC3/ch-TOG interactions, which are essential for spindle pole organization and mitosis. These data demonstrate a critical and unexpected function for ILK in the organization of centrosomal protein complexes during mitotic spindle assembly and DNA segregation.     

Rapid detection of CYP1A1, CYP2D6, and NAT variants by multiplex polymerase chain reaction and allele-specific oligonucleotide assay.

Drugs and carcinogens are excreted from the body after metabolic conversion involving enzymes mediating oxidative metabolism and conjugation. Many of the corresponding genes exhibit functional polymorphisms that contribute to individual cancer susceptibility. To increase the efficiency and to facilitate genotyping, we developed a combined approach (PCR-ASO) which includes multiplex PCR and allele-specific oligonucleotide (ASO) hybridization. PCR primer pairs were used to amplify the following alleles/variants: CYP1A1*1, *2A, *2B; CYP2D6*3, *4; NAT1*4, *3, *10, *11, *14, *15; and NAT2*4, *5A, *5B, *5C, *6A, *7B. The products were dot-blotted and polymorphisms were detected by hybridization with ASO probes for both wild-type and variant sites in parallel. This approach was validated by genotyping DNA samples from a French-Canadian population that was previously analyzed by PCR-RFLP. The variants frequencies were compared with the data on other populations available in the literature. The PCR-ASO assay appears to be simple, efficient, and cost-effective, particularly if a large number of samples are to be screened for several DNA variants. This approach has potential for automation with microplates and robotic workstations for high throughput.     

New polymeric thiocyanatoiron(II) complex with N,N′-diethylnicotinamide - Synthesis, structure, magnetic and spectral properties

The iron(II) compound of formula [Fe(NCS)₂(dena)₂]_n (dena = N,N′-diethylnicotinamide) has been prepared by the reaction between iron(III) thiocyanate and dena in ethanol solution. The complex was characterized by elemental analysis, spectral and magnetic measurements. Single-crystal X-ray diffraction methods show that the complex, crystallizing in the triclinic space group, undergoes a phase transition between 220 K and 230 K, connected with the doubling of cell volume. Crystal structures at 230 K (1a; HT phase) and 150 K (1b; LT phase) are described and a transition mechanism is discussed. In both phases the compound has an extended chain structure, in which the neutral molecule of N,N′-diethylnicotinamide acts as a bridging ligand binding through pyridine N atom to one centre and through amide O atom to the neighbouring Fe centre. The Fe²⁺ ion has a slightly distorted trans-octahedral environment with FeO₂N₄ chromophore, and all Fe-O and Fe-N bonds in the typical for high-spin iron(II) compounds range. Variable-temperature magnetic susceptibility data in the temperature range 1.8-300 K show that iron(II) is high-spin S = 2(⁵T_2g) and as a result effects due to zero-field splitting are anticipated at low temperatures. The IR spectrum suggested the coordination of N,N′-diethylnicotinamide to the central atom of iron(II) as a bridging ligand and NCS group as a monodentate ligand.     

Bradykinin Postconditioning Protects Pyramidal CA1 Neurons Against Delayed Neuronal Death in Rat Hippocampus

Aims The present study was undertaken to evaluate possible neuroprotective effect of bradykinin against delayed neuronal death in hippocampal CA1 neurons if applied two days after transient forebrain ischemia in the rat. Methods Transient forebrain ischemia was induced in male Wistar rats by four-vessel occlusion for 8 min. To assess efficacy of bradykinin as a new stressor for delayed postconditioning we used two experimental groups of animals: ischemia 8 min and 3 days of survival, and ischemia 8 min and 3 days of survival with i.p. injection of bradykinin (150 μg/kg) applied 48 h after ischemia. Results We found extensive neuronal degeneration in the CA1 region at day 3 after ischemia/reperfusion. The postischemic neurodegeneration was preceded by increased activity of mitochondrial enzyme MnSOD in cytoplasm, indicating release of MnSOD from mitochondria in the process of delayed neuronal death. Increased cytosolic cytochrome c and subsequently caspase-3 activation are additional signs of neuronal death via the mitochondrial pathway. Bradykinin administration significantly attenuated ischemia-induced neuronal death, and also suppressed the release of MnSOD, and cytochrome c, and prevented caspase-3 activation. Conclusions Bradykinin can be used as an effective stressor able to prevent mitochondrial failure leading to apoptosis-like delayed neuronal death in postischemic rat hippocampus.     

Amidated pectin derivatives with n-propyl-, 3-aminopropyl-, 3-propanol- or 7-aminoheptyl-substituents

Highly methylated (HM) pectin (DM = 90%) was amidated with four amines under anhydrous conditions. The goal was to prepare modified pectins that could be useful components of composites with new properties. The yields and degree of amidation (DA) of the products were, respectively, 67–92% and 67.3–72.0%. Maximal values were obtained for pectin propylamide (1) and minimal for pectin 7-aminoheptylamide (4). While products 1 and pectin 3-propanolamide (2) showed 95 g/100 g solubility in water, pectin 3-aminopropylamide (3) was 46 g/100 g and 4 only 3 g/100 g water-soluble. The TG/DTA data indicate that the thermal properties were not dramatically altered under inert environment and improved as far as thermooxidation resistance. From the overall prospective product 3 seems to be the most valuable component for further composite preparation.     

Kinetics of the biodegradation of phenol in wastewaters from the chemical industry by covalently immobilized <Emphasis Type="Italic">Trichosporon cutaneum</Emphasis> cells

A simple method for the preparation of the biocatalyst with whole cells is presented, and the applicability of the technique for biodegradation of phenol in wastewater from the chemical industries using the basidomycetes yeast Trichosporon cutaneum is explored. Kinetic studies of the influence of other compounds contained in wastewater as naphthalene, benzene, toluene and pyridine indicate that apart from oil fraction, which is removed, the phenol concentration is the only major factor limiting the growth of immobilized cells. Mathematical models are applied to describe the kinetic behavior of immobilized yeast cells. From the analysis of the experimental curves was shown that the obtained values for the apparent rate parameters vary depending on the substrate concentration (μ_maxapp from 0.35 to 0.09 h⁻¹ and K _sapp from 0.037 to 0.4 g dm⁻³). The inhibitory effect of the phenol on the obtained yield coefficients was investigated too. It has been shown that covalent immobilization of T. cutaneum whole cells to plastic carrier beads is possible, and that cell viability and phenol degrading activity are maintained after the chemical modification of cell walls during the binding procedure. The results obtained indicate a possible future application of immobilized T. cutaneum for destroying phenol in industrial wastewaters.     

Genetic heterogeneity in regional populations of Quebec—Parental lineages in the Gaspe Peninsula

Stable colonization of the Gaspe Peninsula by Europeans started in the middle of the 18th century at the time of the British conquest of New France. The earliest settlers were Acadians, escaping British deportation policies, followed by Loyalists from the US, who preferred to remain under British rule after the Declaration of Independence. In the 19th century, the developing fishing industry attracted French Canadians from the St. Lawrence Valley and newcomers from Europe including Channel Islanders from Jersey and Guernsey. We analyzed parental lineages of the self‐declared descendants of these four groups of settlers by mtDNA D‐loop sequencing and Y‐chromosome genotyping and compared them with French, British, and Irish samples. Their representation in terms of haplotype frequency classes reveals different signatures of founder effects, such as a loss of rare haplotypes, modification of intermediate frequency haplotypes, reduction in genetic diversity (seen in Acadians), but also enrichment by admixture. Parental lineages correlate with group identity. Descendants of early settlers, Acadians and Loyalists, preserved their identity more than those of French Canadian and Channel Islander “latecomers.” Although overall genetic diversity among Gaspesians is comparable with their European source populations, F_ST analysis indicated their greater differentiation. Distinct settlement history, a limited number of founders and relative genetic isolation contributed to the regionalization of the Quebec gene pool that appears less homogenous than usually anticipated. Am J Phys Anthropol, 2009. © 2009 Wiley‐Liss, Inc.