Olivia Hanson-Painton

Protein kinases as therapeutic targets.

Protein kinases and phosphatases are likely targets for the development of therapeutic drugs since they are involved in specific signaling pathways which regulate cell functions such as metabolism, cell cycle progression, cell adhesion, vascular function and angiogenesis. Protein phosphorylation and dephosphorylation serve as molecular switches for modulating these processes and the level and duration of each is a highly regulated process in normal cells. Several compounds that inhibit the activity of tyrosine kinases are being evaluated as cancer therapeutic agents in clinical trials. Diabetes and complications of diabetes also involve deregulated levels of protein kinases. New approaches for regulating kinase gene expression include specific antisense oligonucleotides for inhibiting post-transcriptional processing of the messenger RNA, naturally occurring products and their chemical derivatives to inhibit kinase activity, and monoclonal antibodies to inhibit receptor linked kinases. Inhibition of phosphatases also serves to alter the duration of phosphorylation by kinases. Considerations for development of effective inhibitors include non-specific actions of compounds, cellular uptake, multiple intracellular targets that can dilute the effective cellular concentration of an agent, and tissue specificity. Kinase inhibitors may allow other therapeutic agents additional time to become effective and they may act synergistically with current treatments.

Cerebral Microvessels in Alzheimer's Have Reduced Protein Kinase C Activity

Protein kinase C (PKC) is an important intracellular signalling enzyme. Numerous studies have suggested that alterations in this enzyme occur in aging and dementia. The objective of this study was to examine PKC in the cerebral microcirculation in aging and Alzheimers disease. PKC activity, amount, and isoform distribution were analyzed in microvessels from adult and aged rodents as well as from Alzheimer patients and nondemented elderly controls. PKC activity was lower in Alzheimer vessels than in vessels from control brains, despite the presence of similar levels of PKC enzyme. In contrast, both activity and enzyme levels in young and aged rats were comparable. The beta-isoform was present in both rat and human microvessels and there were no age- or disease-related alterations. The loss in activity in cerebromicrovascular PKC in Alzheimers suggest that perturbations in phosphorylation signalling cascades may exist at the Alzheimer blood-brain barrier.

Evidence for cytosolic benzo(a)pyrene carrier proteins which function in cytochrome P450 oxidation in rat liver

Abstract Solutions of cytosolic proteins from rat liver contain benzo(a)pyrene solubilizing activity capable of serving as a carrier between solid state benzo(a)pyrene and microsomal cytochrome P 450 . Fractionation of benzo(a)pyrene-saturated cytosolic proteins on a Sephadex G-100 column or by sucrose density gradients produced benzo(a)pyrene peaks of about 46,000 daltons and a very high molecular weight material. The protein-bound benzo(a)pyrene obtained in both peaks was oxidized rapidly by microsomes in the presence of NADPH, indicating that the benzo(a)pyrene carrier activity is capable of presenting the substrate to the cytochrome P 450 . Liver cytosolic proteins from rats receiving intraperitoneal injection of [ 14 C] benzo(a)pyrene was chromatographed on a column of Sephadex G-75. Radioactivity eluted at the same positions of the chromatogram as did the carrier activities described above. These results indicate that these benzo(a)pyrene carrier proteins may have an in vivo role in the metabolism of benzo(a)pyrene.

Protein kinase C in rat cerebral microvessels

Activation of protein kinase C is a key event in the transduction of receptor-mediated extracellular signals. Little is known about the role of protein kinase C in the microcirculation of the brain. In this study, we examined protein kinase C in isolated cerebral microvessels. A technique for partial purification of protein kinase C from microvessels was employed, using Q-Sepharose batch adsorption and single-step salt elution in microfuge tubes. This procedure greatly reduced variability and increased protein kinase C specific activity in both the cytosolic and particulate fractions by nearly 50-fold. The identity of the enzyme was confirmed by its inhibition by staurosporine and bisindolylmaleimide and by its translocation in response to phorbol ester. The level of protein kinase C was assessed by [3H]phorbol ester binding and the endogenous substrates evaluated by in vitro phosphorylation studies. Finally, western blot analysis of protein kinase C isoforms indicated that the beta-isoform was present in both cytosolic and particulate fractions. The alpha-isoform was present at low levels in the cytosolic fraction, whereas the gamma-isoform was not detected.

In vivo kinetic study on uptake and distribution of intramuscular tritium-labeled polysulfated glycosaminoglycan in equine body fluid compartments and articular cartilage in an osteochondrial defect model

Summary The uptake and distribution of intramuscularly (IM) administered tritium-labeled polysulfated glycosaminoglycan ( 3 H-PSGAG) in serum, synovial fluid, and articular cartilage of eight horses was quantitated, and hyaluronic acid (HA) concentration of the middle carpal joint was evaluated in a pharmacokinetic study. A full-thickness articular cartilage defect, created on the distal articular surface of the left radial carpal bone of each horse served as an osteochondral defect model. 3 H-PSGAG (500 mg) was injected IM, between 14 and 35 days after creation of the defects. Scintillation analysis of serum and synovial fluid, collected from both middle carpal joints at specific predetermined times up to 96 hours post-injection, revealed mean 3 H-PSGAG concentrations peaked at 2 hours post-injection. 3 H-PSGAG was detected in cartilage and subchondral bone 96 hours post-injection in samples from all eight horses. There were no statistically significant differences in 3 H-PSGAG concentration of synovial fluid or cartilage between cartilage defect and control (right middle carpal) joints. HA assay of synovial fluid revealed concentrations significantly increased at 24, 48, and 96 hours post-injection in both joints. The concentration nearly doubled 48 hours post-injection. However, no statistically significant differences were found between synovial concentrations of HA in cartilage defect and control joints. 3 H-PSGAG administered IM to horses, was distributed in the blood, synovial fluid, and articular cartilage. HA concentrations in synovial fluid increased after IM administration of polysulfated glycosaminoglycan.

Cell surface γ-glutamyl transpeptidase in live cultures☆

A physiological assay for measuring surface accessible gamma-glutamyl transpeptidase activity in adherent, living cultures is described. Cell surface transpeptidase activity remained linear throughout a 60-min time course over a wide range of cell densities. In addition, the assay conditions have neither acute nor long-term effects on cell growth potential, cellular morphology, or cell surface transpeptidase activity levels. As a result, cell surface transpeptidase activity can be continually evaluated in the same cultures during proliferation. The assay appears to be specific for cell surface transpeptidase and can be used to study the partitioning of the enzyme between substrate-accessible and substrate-inaccessible pools. This method utilizes an automated microtiter plate reader for the spectrophotometric quantification of small aliquots removed from cultures incubated with the chromogenic substrate L-gamma-glutamyl-p-nitroanilide. The use of a microtiter plate autoreader and the minimal handling of the cells permit a large number of cultures to be assayed with a substantial reduction in the time required to measure surface transpeptidase activity. The assay described is a nondestructive means for studying cell surface-accessible gamma-glutamyl transpeptidase catalytic activity within the microenvironment of the living culture.

Regulation of protein kinase C activity in cerebral microvessels.

Regulation of protein kinase C (PKC)-mediated responses may occur by inhibition of PKC-dependent phosphorylation or by dephosphorylation of targets by specific phosphatases. Mechanisms for the regulation of PKC were examined in isolated cerebral microvessels and compared to those in brain. The data demonstrated that inhibitors of phosphorylation are responsible for the regulation in brain microvessels while dephosphorylation by protein phosphatases accounts for a substantial portion of the regulation of the PKC response in brain. In addition, the inhibitory activity apparently increases with age. These results suggest that the control of PKC may be cell-type specific and developmentally regulated.

STRUCTURE OF THE Drosophila CALMODULIN GENE

Publisher Summary This chapter reviews the structure of Drosophila calmodulin gene. Calmodulin acts as a regulatory agent in many cellular functions via its regulation of numerous other proteins. The gene was isolated from a D. melanogaster genomic library with an eel calmodulin cDNA clone. Nucleotide sequence analysis confirmed that the fragment contained an open reading frame corresponding to amino acids 59–139 of bovine calmodulin. Comparison of the hybridization signals indicated that the gene is present only once per haploid genome. Consensus splice junction sequences were present at the ends of the open reading frame located in p500. The derived amino acid sequence of Drosophila calmodulin differs from the bovine sequence at three positions: Phe is substituted for Tyr at residue 99, Thr for Glu at 143 and Ser for Ala at 147. The single Drosophila calmodulin gene spans over 10 kb of DNA and contains at least three introns. The locations of the three known intervening sequences are in the same position relative to the coding sequence as three of the six chicken calmodulin introns.