Hyperparasitism of the cryptoniscid isopod Liriopsis pygmaea on the lithodid Paralomis granulosa from the Beagle Channel, Argentina

A total of 29,570 false king crab Paralomis granulosa were sampled from the Beagle Channel (54 degrees 51'S, 68 degrees 12'W), Tierra del Fuego, Argentina, between July 1996 and August 1998. Crab size varied from 6.8 to 111.2 mm carapace length (CL). A few crabs parasitized by the rhizocephalan Briarosaccus callosus were found; prevalences of externae (the rhizocephalan reproductive body) and scars (the mark left on the host after the death of the parasite) were 0.28 and 0.16%, respectively. Of 85 externae examined, 55 were non-ovigerous and 30 ovigerous. The cryptoniscid isopod Liriopsis pygmaea infested 36.5% of the B. callosus examined. The most abundant stage was the cryptonicus larva, accounting for 208 of the 238 L. pygmaea recovered. Cryptonisci showed a highly aggregated distribution. A total of 92.7% of cryptonicsci were recovered inside empty externae, suggesting that the latter were attractive to cryptonisci. Early subadult females of L. pygmaea were rare; only 3 individuals occurred inside 1 ovigerous externa. Eight late subadult and 18 adult females were found on 3 and 7 non-ovigerous externae, respectively; in addition, 1 aberrant late subadult was found on 1 ovigerous externa. In the Beagle Channel, the population of P. granulosa harbours 3 different parasites: the bopyrid isopod Pseudione tuberculata, which reaches highest prevalence at 10 to 20 mm CL, the rhizocephalan B. callosus, with highest prevalence at 20 to 40 mm CL, and the cryptoniscid isopod L. pygmaea, which mainly infests rhizocephalan on crabs >40 mm CL.     

A novel CDK5-dependent pathway for regulating GSK3 activity and kinesin-driven motility in neurons

Neuronal transmission of information requires polarized distribution of membrane proteins within axonal compartments. Membrane proteins are synthesized and packaged in membrane-bounded organelles (MBOs) in neuronal cell bodies and later transported to axons by microtubule-dependent motor proteins. Molecular mechanisms underlying targeted delivery of MBOs to discrete axonal subdomains (i.e. nodes of Ranvier or presynaptic terminals) are poorly understood, but regulatory pathways for microtubule motors may be an essential step. In this work, pharmacological, biochemical and in vivo experiments define a novel regulatory pathway for kinesin-driven motility in axons. This pathway involves enzymatic activities of cyclin-dependent kinase 5 (CDK5), protein phosphatase 1 (PP1) and glycogen synthase kinase-3 (GSK3). Inhibition of CDK5 activity in axons leads to activation of GSK3 by PP1, phosphorylation of kinesin light chains by GSK3 and detachment of kinesin from transported cargoes. We propose that regulating the activity and localization of components in this pathway allows nerve cells to target organelle delivery to specific subcellular compartments. Implications of these findings for pathogenesis of neurodegenerative diseases such as Alzheimer's disease are discussed.     

Lisinopril quantification in human plasma by liquid chromatography-electrospray tandem mass spectrometry

An analytical method based on liquid chromatography with positive ion electrospray ionization (ESI) coupled to tandem mass spectrometry detection was developed for the determination of Lisinopril in human plasma using Enalaprilat as internal standard. The analyte and internal standard were extracted from the plasma samples by solid-phase extraction using Waters HLB Oasis SPE cartridges and chromatographed on a C8 analytical column. The mobile phase consisted of acetonitrile/water (60:40, v/v) + 20 mM acetic acid + 4.3 mM of triethylamine. The method had a chromatographic total run-time of 6.5 min and was linear within the range 2.00-200 ng/ml. Detection was carried out on a Micromass triple quadrupole tandem mass spectrometer by multiple reaction monitoring (MRM). The precision (CV%) and accuracy, calculated from limit of quantification (LOQ) samples (n = 8), were 8.9 and 98.9%, respectively. The method herein described was employed in a bioequivalence study of two tablet formulations of Lisinopril 20mg.     

StCDPK1 is expressed in potato stolon tips and is induced by high sucrose concentration

StCDPK1 encodes a calcium-dependent protein kinase (CDPK) from Solanum tuberosum, which is transiently induced upon tuberization in swelling stolons. In situ hybridization determined that StCDPK1 mRNA is localized in the apical dome of tuberizing stolon tips, close to the region where sucrose was reported to accumulate. The expression of StCDPK1, and other tuber-specific genes was enhanced when in vitro-cultured potato plants were transferred to high sucrose or high sorbitol containing media. Glucose, fructose or a mixture of both showed no effect on CDPK expression. Okadaic acid blocked sucrose-inducible gene expression, suggesting that phosphatases from the PP1/PP2A family could also participate in the regulation of StCDPK1 and other tuberization-related genes.     

Alternative mRNAs arising from trans-splicing code for mitochondrial and cytosolic variants of Echinococcus granulosus thioredoxin Glutathione reductase

Thioredoxin and glutathione systems are the major thiol-dependent redox systems in animal cells. They transfer via the reversible oxidoreduction of thiols the reducing equivalents of NADPH to numerous substrates and substrate reductases and constitute major defenses against oxidative stress. In this study, we cloned from the helminth parasite Echinococcus granulosus two trans-spliced mRNA variants that encode thioredoxin glutathione reductases (TGR). These variants code for mitochondrial and cytosolic selenocysteine-containing isoforms that possess identical glutaredoxin (Grx) and thioredoxin reductase (TR) domains and differ exclusively in their N termini. Western blot analysis of subcellular fractions with specific anti-TGR antibodies showed that TGR is present in both compartments. The biochemical characterization of the native purified TGR suggests that the Grx and TR domains of the enzyme can function either coupled or independently of each other, because the Grx domain can accept electrons from either TR domains or the glutathione system and the TR domains can transfer electrons to either the fused Grx domain or to E. granulosus thioredoxin.     

20 S proteasome from Saccharomyces cerevisiae is responsive to redox modifications and is S-glutathionylated

The 20 S proteasome core purified from Saccharomyces cerevisiae is inhibited by reduced glutathione (GSH), cysteine (Cys), or the GSH precursor gamma-glutamylcysteine. Chymotrypsin-like activity was more affected by GSH than trypsin-like activity, whereas the peptidylglutamyl-hydrolyzing activity (caspase-like) was not inhibited by GSH. Cys-sulfenic acid formation in the 20 S core was demonstrated by spectral characterization of the Cys-S(O)-4-nitrobenzo-2-oxa-1,3-diazole adduct, indicating that 20 S proteasome Cys residues might react with reduced sulfhydryls (GSH, Cys, and gamma-glutamylcysteine) through the oxidized Cys-sulfenic acid form. S-Glutahionylation of the 20 S core was demonstrated in vitro by GSH-biotin incorporation and by decreased alkylation with monobromobimane. Compounds such as N-ethylmaleimide (-S-sulfhydril H alkylating), dimedone (-SO sulfenic acid H reactant), or 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (either -SH or -SOH reactant) highly inhibited proteasomal chymotrypsin-like activity. In vivo experiments revealed that 20 S proteasome extracted from H(2)O(2)-treated cells showed decreased chymotrypsin-like activity accompanied by S-glutathionylation as demonstrated by GSH release from the 20 S core after reduction with NaBH(4). Moreover, cells pretreated with H(2)O(2) showed decreased reductive capacity assessed by determination of the GSH/oxidized glutathione ratio and increased protein carbonyl levels. The present results indicate that at the physiological level the yeast 20 S proteasome is regulated by its sulfhydryl content, thereby coupling intracellular redox signaling to proteasome-mediated proteolysis.     

Polycystin-1 activates and stabilizes the polycystin-2 channel

Autosomal dominant polycystic kidney disease (ADPKD) is a prevalent genetic disorder largely caused by mutations in the PKD1 and PKD2 genes that encode the transmembrane proteins polycystin-1 and -2, respectively. Both proteins appear to be involved in the regulation of cell growth and maturation, but the precise mechanisms are not yet well defined. Polycystin-2 has recently been shown to function as a Ca(2+)-permeable, non-selective cation channel. Polycystin-2 interacts through its cytoplasmic carboxyl-terminal region with a coiled-coil motif in the cytoplasmic tail of polycystin-1 (P1CC). The functional consequences of this interaction on its channel activity, however, are unknown. In this report, we show that P1CC enhanced the channel activity of polycystin-2. R742X, a disease-causing polycystin-2 mutant lacking the polycystin-1 interacting region, fails to respond to P1CC. Also, P1CC containing a disease-causing mutation in its coiled-coil motif loses its stimulatory effect on wild-type polycystin-2 channel activity. The modulation of polycystin-2 channel activity by polycystin-1 may be important for the various biological processes mediated by this molecular complex.