Complex formation between endogenous toxin bilirubin and polyamidoamine dendrimers: a spectroscopic study

The effects of 4th and 5th generation cationic, neutral and anionic polyamidoamine (PAMAM) dendrimers on bilirubin absorbance and fluorescence were studied. Cationic and neutral PAMAM dendrimers shifted the bilirubin absorption maximum from 435 to 442-455 nm, increased the peak absorbance 1.5-fold, shifted the bilirubin fluorescence excitation and emission maxima, increased the fluorescence emission several-fold and significantly protected bilirubin against photodestruction. Using double fluorescence titration technique allowed to receive such constant of binding and the number of binding centers at 20 degrees C: for PAMAM G4 dendrimer, (2.4+/-1.4) x 10(6) (mol/l)(-1) and 0.07+/-0.012; for PAMAM G4-OH dendrimer, (3.1+/-1.3) x 10(6) (mol/l)(-1) and 0.08+/-0.014; for PAMAM G5 dendrimer, (7.6+/-3.6) x 10(6) (mol/l)(-1) and 0.09+/-0.02; and for PAMAM G5-OH dendrimer, (8.5+/-3.2) x 10(6) (mol/l)(-1) and 0.09+/-0.02. These effects can be explained by the formation of bilirubin-PAMAM dendrimer complexes and the formation of bilirubin monomers from tetramers. The formation of complexes sharply increased bilirubin solubility. We conclude that cationic and neutral PAMAM dendrimers bind bilirubin effectively and suggest that such dendrimers may serve as detoxication agents for hydrophobic endogenous toxins.     

Cytotoxicity, haematotoxicity and genotoxicity of high molecular mass arborescent polyoxyethylene polymers with polyglycidol-block-containing shells

We have examined the impact on biological systems of some newly synthesised polyoxyethylene polymers using in vitro assays. Toxicity was tested by the 3-[4,5-dimethylthiazole-2-yl]-2,5 diphenyltetrazolium bromide (MTT) assay, haemolysis was assessed, and an ethidium bromide (EB) assay was used to study interactions between the polymers and DNA. All the assay data showed that the polymers are biocompatible. No differences were found between generations (i.e. macromolecule sizes). The results encourage continuing studies on the clinical use of these molecules as drug carriers.     

Transcutaneous measurement of renal function in conscious mice

Determination of glomerular filtration rate (GFR) in conscious mice is cumbersome for the experimenter and stressful for the animals. Here we report on a simple new technique allowing the transcutaneous measurement of GFR in conscious mice. This approach extends our previously developed technique for rats to mice. The technique relies on a miniaturized device equipped with an internal memory that permits the transcutaneous measurement of the elimination kinetics of the fluorescent renal marker FITC-sinistrin. This device is described and validated compared with FITC-sinistrin plasma clearance in healthy, unilaterally nephrectomized and pcy mice. In summary, we describe a technique allowing the measurement of renal function in freely moving mice independent of blood or urine sampling as well as of laboratory assays.     

Discriminant models for high-throughput proteomics mass spectrometer data

We use several different multivariate analysis methods to discriminate between diseased and healthy patients using protein mass spectrometer data provided by Duke University. Two problems were presented by the university; one in which the responses (diseased or healthy) of the patients were not known and second, when the responses were known. In the latter case, the data can be used as a 'training' set. We attempted both problems. In particular, we use principle component analysis along with clustering methods to discriminate for the first problem set and partial least squares coupled with logistic and discriminant methods when the responses were known. In addition, we were able to detect regions of interest in the spectrum where there were differences in the protein patterns between healthy and diseased patients. There was considerable effort involved in the preprocessing of the data. We used a binning approach to reduce the number of variables rather than peak heights or peak areas. We performed a square root transformation on the data to help stabilize the variance; this in turn made a significant improvement in clustering results.     

Genetic diversity and relationships among germplasm of Jatropha curcas L. revealed by RAPDs

Variation is the phenomenon where individuals of a population differ from each other. The variation or diversity can have morphological manifestation or genetic basis. Individual identification based on morphological record is the most common practice in Jatropha. Therefore, in order to find a proper method for estimation of genetic diversity and genetic relationships among different germplasm of Jatropha curcas L., random amplified polymorphic DNA (RAPD) technique based on polymerase chain reaction (PCR) was used for described purpose. Out of 55 decamer primers tested, 26 primers produced good amplification products. A total of 6,011 amplification products were scored from which only 1,859 bands (30.92%) were found to be polymorphic and the size of bands ranged from 300 to 2,500 bp. Unweighted pair group method using arithmetic average cluster analysis revealed clear genetic difference among J. curcas germplasm. The scientific data presented in this study suggests that RAPD-PCR could be used as a valuable tool for estimation of genetic diversity and genetic relationship among germplasm of J. curcas L.     

Phosphorylation on Ser-279 and Ser-282 of connexin43 regulates endocytosis and gap junction assembly in pancreatic cancer cells

The molecular mechanisms regulating the assembly of connexins (Cxs) into gap junctions are poorly understood. Using human pancreatic tumor cell lines BxPC3 and Capan-1, which express Cx26 and Cx43, we show that, upon arrival at the cell surface, the assembly of Cx43 is impaired. Connexin43 fails to assemble, because it is internalized by clathrin-mediated endocytosis. Assembly is restored upon expressing a sorting-motif mutant of Cx43, which does not interact with the AP2 complex, and by expressing mutants that cannot be phosphorylated on Ser-279 and Ser-282. The mutants restore assembly by preventing clathrin-mediated endocytosis of Cx43. Our results also document that the sorting-motif mutant is assembled into gap junctions in cells in which the expression of endogenous Cx43 has been knocked down. Remarkably, Cx43 mutants that cannot be phosphorylated on Ser-279 or Ser-282 are assembled into gap junctions only when connexons are composed of Cx43 forms that can be phosphorylated on these serines and forms in which phosphorylation on these serines is abolished. Based on the subcellular fate of Cx43 in single and contacting cells, our results document that the endocytic itinerary of Cx43 is altered upon cell–cell contact, which causes Cx43 to traffic by EEA1-negative endosomes en route to lysosomes. Our results further show that gap-junctional plaques formed of a sorting motif–deficient mutant of Cx43, which is unable to be internalized by the clathrin-mediated pathway, are predominantly endocytosed in the form of annular junctions. Thus the differential phosphorylation of Cx43 on Ser-279 and Ser-282 is fine-tuned to control Cx43’s endocytosis and assembly into gap junctions.