Molecular cloning, cDNA sequence analysis, and chromosomal localization of mouse Pkd2

The gene responsible for the second form of autosomal dominant polycystic kidney disease, PKD2, has recently been identified. We now describe the cloning, genomic localization, cDNA sequence, and expression analysis of its murine homologue, Pkd2. The cloned cDNA sequence is 5134 bp long and is predicted to encode a 966-amino-acid integral membrane protein with six membrane-spanning domains and intracellular NH2 and COOH termini. Pkd2 is highly conserved with 91% identity and 98% similarity to polycystin-2 at the amino acid level. Pkd2 mRNA is widely expressed in mouse tissues. Pkd2 maps to mouse Chromosome 5 and is excluded as a candidate gene for previously mapped mouse mutations resulting in a polycystic kidney phenotype.     

Modelling and measurements of the velocity gradient and local flow direction at the pore scale of a packed bed

In a packed bed with single phase liquid flow, velocity gradient and local flow direction are measured at the pore scale using tri-segmented microelectrodes flush mounted at the surface of a sphere equator area. The experimental measurements are compared to numerical predictions deduced from the solution of a 3D model based on continuity and momentum balance equations.     

Dual roles of proteasome in the metabolism of presenilin 1

Presenilin 1 (PS1) has been identified as a causative gene for most early-onset familial Alzheimer's disease. Biochemical studies revealed that PS1 exists predominantly as two processed fragments in cells and brain tissues. We prepared stably transfected cells expressing the wild-type and familial Alzheimer's disease-associated mutants of PS1 and investigated the enzyme that participates in the metabolism of PS1. After treatment of the cells with proteasome inhibitors, the full-length PS1 was significantly accumulated. The levels of N- and C-terminal fragments were also increased. The accumulation of PS1 with a deletion of exon 10, which is unable to be processed, on treatment of the transfected cells with lactacystin indicated that proteasome can degrade full-length PS1. A synthetic peptide that includes the processing region of PS1 was cleaved by 20S proteasome at the putative processing sites after Met288 and Glu299. Metabolic labeling experiments showed that the appearance of the N-terminal fragment was attenuated by the inhibitor. Finally, 28-kDa N- and 20-kDa C-terminal fragments were generated by purified PS1 in vitro. These data indicated that the proteasome pathway is involved in PS1 processing. These results demonstrate that the proteasome pathway plays dual roles in processing and degradation of PS1.     

CVT-313, a specific and potent inhibitor of CDK2 that prevents neointimal proliferation

The activity of cyclin-dependent kinase 2 (CDK2) is essential for progression of cells from G1 to the S phase of the mammalian cell cycle. CVT-313 is a potent CDK2 inhibitor, which was identified from a purine analog library with an IC50 of 0.5 microM in vitro. Inhibition was competitive with respect to ATP (Ki = 95 nM), and selective CVT-313 had no effect on other, nonrelated ATP-dependent serine/threonine kinases. When added to CDK1 or CDK4, a 8.5- and 430-fold higher concentration of CVT-313 was required for half-maximal inhibition of the enzyme activity. In cells exposed to CVT-313, hyperphosphorylation of the retinoblastoma gene product was inhibited, and progression through the cell cycle was arrested at the G1/S boundary. The growth of mouse, rat, and human cells in culture was also inhibited by CVT-313 with the IC50 for growth arrest ranging from 1.25 to 20 microM. To evaluate the effects of CVT-313 in vivo, we tested this agent in a rat carotid artery model of restenosis. A brief intraluminal exposure of CVT-313 to a denuded rat carotid artery resulted in more than 80% inhibition of neointima formation. These observations suggest that CVT-313 is a promising candidate for evaluation in other disease models related to aberrant cell proliferation.     

A 1D High Performance Thin Layer Chromatography Method Validated to Quantify Phospholipids Including Cardiolipin and Monolysocardiolipin from Biological Samples

The aim of this study is to identify high performance thin layer chromatography (HPTLC) conditions allowing the separation and quantification of mammalian cellular phospholipids (PLs) (sphingomyelin, phosphatidylcholine, phosphatidylserine, phosphatidylinositol, phosphatidylethanolamine, and especially phosphatidic acid, cardiolipin, and monolysocardiolipin, these latter two being specifically located in mitochondria membranes). In order to make this method faster and easier, a 1D HPTLC method is chosen, testing several eluents as well as several staining methods. A pre‐conditioning of HPTLC plates with boric acid and a copper staining reagent followed by carbonization are selected for the quality of PL separation and homogeneity of staining. The selected conditions are discussed and the method validation is performed according to the International Conference on Harmonization guidelines. Linearity is effective between 1 and 8 µg and limit of quantification is between 0.5 and 2.3 µg depending on PL classes. Precision measurements show coefficients of variation <6%, and when amounts are close to the detection limit, <12%. Lipid extracts of tumor cell lines or isolated mitochondria are used to assess PL profiles. This shows that the HPTLC method can be used routinely to follow level variations of PLs. Practical Applications: The changes in PL composition play a crucial role in tumor processes and regulate cellular functions modulating cellular signaling or mitochondrial metabolism. The simple and cost‐effective 1D HPTLC method that is developed is applied to lipid extracts of whole tumor cells or hepatocyte‐isolated mitochondria. It is sensitive as well as precise to detect variations of phosphatidic acid or cardiolipin levels linked to physio‐pathological conditions. It can also be used to investigate the composition changes of other membrane PLs. Moreover, with a simultaneous analysis of 14 samples/standards on the same plate (six plates per day), this method is adapted for large series of samples.     

A concept of reactive compatibilizer‐tracer for studying reactive polymer blending processes

A concept called reactive compatibilizer‐tracer is proposed. The latter bears reactive groups capable of reacting with its counterpart on forming a copolymer for in situ compatibilization, and fluorescent labels allowing determining very small amounts of the in situ formed compatibilizer and formation of micelles when it occurs. Owing to this concept, it is shown that a reactive compatibilizer may be very efficient at the beginning of a reactive blending process and may suddenly become completely inefficient, resulting in an abrupt and drastic increase in size of the dispersed phase domains. © 2015 American Institute of Chemical Engineers AIChE J, 62: 359–366, 2016     

Psychological disorder and mortality in French older adults: do social relations modify the association?

The possible modifying effect of social relations on the association between depression and mortality was examined in a community-based cohort study. A total of 3,777 randomly selected persons 65 years of age and older in southwest France were followed over a 5-year period from 1988 in the Personnes Agees Quid (PAQUID). At study entry, the prevalence of elevated depressive symptomatology was 12.9% for men and 14.7% for women, and the reported relative isolation was 14.1% for men and 26.0% for women. During a total of 16,984 person-years of follow-up, 849 deaths occurred. Among participants with high levels of depressive symptomatology, the age-adjusted mortality rate ratio was 2.10 (95% confidence interval 1.7-2.7) in men and 1.76 (95% confidence interval 1.4-2.3) in women. When compared with individuals with the most connections, men and women with few social network connections were also at increased risk of mortality: age-adjusted rate ratio = 2.69 (95% confidence interval 1.9-3.8) for men and 1.56 (95% confidence interval 1.0-2.4) for women. Satisfaction with social support had a small but nonsignificant effect on mortality risk. For women, the excess risks due to depressive symptoms and few network connections are observed only in the 65- to 74-year age group, after adjusting for health and health behaviors. Social relations did not significantly modify the depression-mortality associations for either men or women, although the depression-mortality effect was reduced by 12.8% in men. The latter findings do not appear to be compatible with the buffering hypothesis, whereby we would expect social relations to decrease the depression-mortality association. Nonetheless, there are independent effects from these two factors, and older men who are depressed and not socially connected are at increased risk of dying earlier.     

Immobilization of acrylamide-modified oligonucleotides by co-polymerization

A flexible chemistry for solid phase attachment of oligonucleotides is described. Oligonucleotides bearing 5'-terminal acrylamide modifications efficiently co-polymerize with acrylamide monomers to form thermally stable DNA-containing polyacrylamide co-polymers. Co-polymerization attachment is specific for the terminal acrylamide group. Stable probe-containing layers are easily fabricated on supports bearing exposed acrylic groups, including plastic microtiter plates and silanized glass. Attachment can be accomplished using standard polyacrylamide gel recipes and polymerization techniques. Supports having a high surface density of hybridizable oligonucleotide (approximately 200 fmol/mm2) can be produced.     

Application of dried blood spot specimens for serologic subtyping of human immunodeficiency virus type 1 in Thailand

Dried blood spot (DBS) specimens were assessed as an alternative to plasma for human immunodeficiency virus type 1 (HIV-1) serotyping by V3 loop peptide enzyme immunoassay. Nested PCR capable of distinguishing HIV-1 subtypes B and E was used as the reference standard. Ninety-two percent of DBS samples were typeable as either HIV-1 subtype B or E. Serotype results with DBS and plasma were identical for 254 of 257 specimens. A simple DBS collection method provides a convenient alternative for conducting HIV-1 serotype surveillance while retaining sensitivity and specificity.     

Behavioral correlates of maternal depressive symptomatology in conduct-disorder children.

We contrasted two predictive models of the impact of maternal depressive symptomatology on child behavior in a study of 51 mothers and their conduct-disorder children. Relations between global measures of maternal distress and child adjustment and observational measures of mother–child interaction were examined. Children of distressed mothers were more maladjusted than children of nondistressed mothers, when maladjustment was measured on the basis of a global rating, but "better" adjusted when measured on the basis of interactional measures. Measures of maternal indiscriminate responding to the child may account for these findings. Results suggest that (a) although conduct-disorder children are generally more maladjusted when their mothers are distressed, they display this maladjustment in a selective fashion, and (b) maternal distress acts as an adverse contextual factor that maintains mother–child interactional difficulties by disrupting the attentional and monitoring skills required for contingent responding. (PsycINFO Database Record (c) 2010 APA, all rights reserved)