In Vitro Interactions of Asian Freshwater Clam (Corbicula fluminea) Hemocytes and Cryptosporidium parvum Oocysts

Corbicula fluminea hemocytes phagocytosed infectious oocysts of Cryptosporidium parvum in vitro. After 15, 30, 60, 90, and 120 min of incubation, averages of 35.8, 58.0, 69.7, 77.7, and 81.6% of the oocysts were phagocytosed by 24.3, 70.0, 78.5, 87.3, and 93.0% of the hemocytes, respectively. A single clam can retain by phagocytosis an average of 1.84 x 10(sup6) oocysts per ml of hemolymph. C. fluminea bivalves can serve as biological indicators of contamination of wastewaters and agricultural drainages with Cryptosporidium.     

Purification and characterization of an inducible aromatic amino acid-sensitive form of chorismate mutase from Solanum tuberosum L. tubers

An amino acid-sensitive form of chorismate mutase (CM) has been purified over 1000-fold from disks excised from tubers of Solanum tuberosum L. cv White Rose. Purification was accomplished by chromatography on Matrix Blue A followed by affinity chromatography with tryptophan as ligand. CM assays performed in the absence of tryptophan yielded pH-dependent sigmoidal kinetics. At pH 8.0, sigmoidal kinetics were observed with a Hill coefficient of 1.66 (S0.5 = 188 microM). However, a shift from sigmoidal to hyperbolic kinetics was observed when assays were performed at pH 8.5. Addition of 9 microM tryptophan to the assay resulted in maximum activation of the enzyme with a Ka of 1.2 microM. When assayed in the presence of tryptophan, hyperbolic kinetics were observed over the pH range 6.0-8.0. Addition of tryptophan also decreased the Km for chorismate from 185 to 45 microM. Tryptophan (0.1 mM) completely protected CM from inhibition by phenylalanine (1.8 mM) and tyrosine (1.8 mM). However, in the absence of the activator, phenylalanine and tyrosine exhibited 50% inhibition at 0.80 and 0.68 mM concentrations, respectively. Both phenylalanine and tyrosine competitively inhibited CM activity with Ki values of 550 and 440 mM, respectively. Arogenate (1.0 mM) had no effect on CM activity in either the presence or absence of tryptophan. Analytical isoelectric focusing yielded an isoelectric point of 4.73.     

Protein folding as posttranslational regulation: evolution of a mechanism for controlled plasma membrane expression of a G protein-coupled receptor

Recent studies reveal that a number of G protein-coupled receptors (GPCRs) and other proteins are expressed inefficiently at the site normally associated with their biological action. In the case of some GPCRs, large amounts of receptor (perhaps more than half) may be destroyed without ever binding ligand or even arriving at the plasma membrane. For the human GnRH receptor (GnRHR), this apparent inefficiency has evolved under strong and convergent evolutionary pressure. The result is a human GnRHR molecule that is delicately balanced between either expression at the plasma membrane (PM) or retention/degradation in the endoplasmic reticulum, an effect mediated by engagement with the cellular quality control system. This balance appears to be the reason that the human receptor, but not the rat or mouse counterpart (which are more robustly routed to the PM), is highly susceptible to single-point mutations that result in disease. A single change in net charge is sufficient to tip the balance in favor of the endoplasmic reticulum and diminish GnRHR available at the PM. The apparent paradox that results from observing convergent pressure for evolution of a receptor that is both inefficiently produced and highly susceptible to mutational disease suggests that this approach must offer a strong advantage. This review focuses on the evolved mechanisms and considers that this is an underappreciated mechanism by which the cell controls functional levels of receptors and other proteins at the posttranslational level.     

Activation of Metabotropic Glutamate Receptors in the Hippocampus Increases Cyclic AMP Accumulation

The selective metabotropic glutamate receptor agonist trans-1-aminocyclopentane-1,3-dicarboxylic acid (trans-ACPD) stimulates phosphoinositide hydrolysis and elicits several physiological responses in rat hippocampal slices. However, recent studies suggest that the physiological effects of trans-ACPD in the hippocampus are mediated by activation of a receptor that is distinct from the phosphoinositide hydrolysis-linked receptor. Previous experiments indicate that cyclic AMP mimics many of the physiological effects of trans-ACPD in hippocampal slices. Furthermore, recent cloning and biochemistry experiments indicate that multiple metabotropic glutamate receptor subtypes exist, some of which are coupled to yet unidentified effector systems. Thus, we performed a series of experiments to test the hypothesis that ACPD increases cyclic AMP levels in hippocampal slices. We report that 1S,3R- and 1S,3S-ACPD (but not 1R,3S-ACPD) induce a concentration-dependent increase in cyclic AMP accumulation in hippocampal slices. This effect was blocked by the metabotropic glutamate receptor antagonist L-2-amino-3-phosphonoproprionic acid but not by selective antagonists of ionotropic glutamate receptors. Furthermore, our results suggest that 1S,3R-ACPD-stimulated increases in cyclic AMP accumulation are not secondary to increases in cell firing or to activation of phosphoinositide hydrolysis.     

Normal and aberrant Mesocestoides tetrathyridia from Crocidura spp. (Soricimorpha) in Corsica and Spain

Tetrathyridia of Mesocestoides sp. were collected from the body cavities of the shrews (Insectivora), Crocidura russula, in Valencia, Spain and Crocidura suaveolens on the Mediterranean island of Corsica, France. Specimens were processed by routine microscopic and histological techniques, including examination with brightfield, phase-contrast, and differential-interference-contrast optics. Most tetrathyridia were clustered together inside host-derived fibrotic capsules, but some occurred free in the body cavity. All specimens examined from both locations had solid hindbodies, i.e., lacking a primary lacuna, thus conforming to the plerocercoid metacestode type; all possessed a single normal tetra-acetabulate scolex. All metacestodes from C. russula in Valencia were normal tetrathyridia. Those from C. suaveolens in Corsica were either normal tetrathyridia or had aberrant deep convolutions of an unusually elongated hindbody. No tetrathyridium from either location or host showed tegumental or excretory duct anomalies such as those reported by several authors from aberrant tetrathyridia and spargana in some other locations. No definitive evidence of asexual proliferation was visible in any of the tetrathyridia, but those with abnormally convoluted hindbodies from a single C. suaveolens in Corsica suggest the potential for asexuality by fission of the hindbody. These results add to our understanding of morphological and developmental variation among metacestodes in this widespread and variable genus.     

Presence of the cyanogenic glucoside dhurrin in isolated vacuoles from sorghum

Large numbers of vacuoles (10⁶-10⁷) have been isolated from Sorghum bicolor protoplasts and analyzed for the cyanogenic glucoside dhurrin. Leaves from light-grown seedlings were incubated for 4 hours in 1.5% cellulysin and 0.5% macerase to yield mesophyll protoplasts which then were recovered by centrifugation, quantitated by a hemocytometer, and assayed for cyanogenic glucosides. Mature vacuoles, released from the protoplasts by osmotic shock, were purified on a discontinuous Ficoll gradient and monitored for intactness by their ability to maintain a slightly acid interior while suspended in an alkaline buffer as indicated by neutral red stain. Cyanide analysis of the protoplasts and the vacuoles obtained there from yielded equivalent values of 11 μmoles of cyanogenic glucoside dhurrin per 10⁷ protoplasts or 10⁷ vacuoles. This work supports an earlier study from this laboratory which demonstrated that the vacuole is the site of accumulation of the cyanogenic glucoside in Sorghum.     

Comparison of the electrophoretic and hydrodynamic properties of DNA and RNA oligonucleotide duplexes.

The electrophoretic behavior of defined DNA and RNA oligonucleotide duplexes from 10 to 20 bp in length has been investigated as a function of salt conditions, gel concentration, and temperature. The RNA oligomers migrated much more slowly than the DNA oligomers of the same sequence under all conditions. From sedimentation equilibrium and velocity measurements, the apparent partial specific volume in 0.1 M KCI, 20 mM NaPi, pH 7, was determined as 0.56 +/- 0.015 ml g(-1) for DNA and 0.508 ml g(-1) for RNA. The translational friction coefficients were determined and compared with the values calculated for cylinders. Taking into account the shape factors, the solution density, and partial specific volumes, the effective degree of hydration was estimated as 0.8-1 g g(-1) DNA. There was no significant difference in the frictional coefficients of the DNA and RNA oligomers, indicating that the effective sizes of DNA and RNA are very similar in solution. The differential electrophoretic mobility of DNA and RNA must arise from the differences in interaction with counterions, which is probably a global property of the oligonucleotides.     

Monitoring EDTA process residuals in recombinant protein manufacturing using liquid chromatography

We have developed a chromatographic method for the high sensitivity quantitation of EDTA process residuals in recombinant protein manufacturing validation studies. The reversed-phase HPLC method is based upon the detection of Cu(2+)/EDTA complexes at 254 nm, and has been qualified for use on intermediates from a purification process for a recombinant protein expressed in E. coli. Quantitation of EDTA in recombinant protein process intermediates is linear in the range of 0.2 to 64 microM with LOD/LOQ values below 2.0 microM. The assay is suitable for use in process backgrounds containing Tris, HEPES, MES, NaCl, hexanediol, NH(4)SO(4), and PEG. EDTA spike recovery values in all process samples tested were greater than 90% at the 4.0 microM concentration. System suitability parameters for the chromatographic method were developed based upon peak area and retention time precision, column efficiency and USP tailing. Peak area precision and intermediate precision values across the linear range of the assay exhibited C.V. values less than 15% at any concentration tested in all sample backgrounds. The assay robustness was tested by transfer of the assay to a second laboratory and analyst with use of multiple process intermediate lots, reagent/column lots, and HPLC systems.     

Differential regulation of metabotropic glutamate receptor 5-mediated phosphoinositide hydrolysis and extracellular signal-regulated kinase responses by protein kinase C in cultured astrocytes

The metabotropic glutamate receptor 5 (mGluR5) exhibits a rapid loss of receptor responsiveness to prolonged or repeated agonist exposure. This receptor desensitization has been seen in a variety of native and recombinant systems, and is thought to result from receptor-mediated, protein kinase C (PKC)-dependent phosphorylation of the receptor, uncoupling it from the G protein in a negative feedback regulation. We have investigated the rapid PKC-mediated desensitization of mGluR5 in cortical cultured astrocytes by measuring downstream signals from activation of mGluR5. These include activation of phosphoinositide (PI) hydrolysis, intracellular calcium transients, and extracellular signal-regulated kinase 2 (ERK2) phosphorylation. We present evidence that PKC plays an important role in rapid desensitization of PI hydrolysis and calcium signaling, but not in ERK2 phosphorylation. This differential regulation of mGluR5-mediated responses suggests divergent signaling and regulatory pathways which may be important mechanisms for dynamic integration of signal cascades.     

Amygdala Kindling Alters Protein Kinase C Activity in Dentate Gyrus

Kindling is a use-dependent form of synaptic plasticity and a widely used model of epilepsy. Although kindling has been widely studied, the molecular mechanisms underlying induction of this phenomenon are not well understood. We determined the effect of amygdala kindling on protein kinase C (PKC) activity in various regions of rat brain. Kindling stimulation markedly elevated basal (Ca(2+)-independent) and Ca(2+)-stimulated phosphorylation of an endogenous PKC substrate (which we have termed P17) in homogenates of dentate gyrus, assayed 2 h after kindling stimulation. The increase in P17 phosphorylation appeared to be due at least in part to persistent PKC activation, as basal PKC activity assayed in vitro using an exogenous peptide substrate was increased in kindled dentate gyrus 2 h after the last kindling stimulation. A similar increase in basal PKC activity was observed in dentate gyrus 2 h after the first kindling stimulation. These results document a kindling-associated persistent PKC activation and suggest that the increased activity of PKC could play a role in the induction of the kindling effect.