Effects of foscarnet on the cell kinetics of Madin-Darby canine kidney cells

The antiherpes compound, foscarnet (trisodium phosphonoformate), showed concentration-dependent effects on the cell kinetics of Madin-Darby canine kidney cells. At 1 mM, only minor effects could be seen on cell proliferation and cell cycle distribution, as measured by flow cytometry DNA analysis. Treatment with 5 mM foscarnet resulted in an accumulation of cells in the S-phase although no complete cell cycle block was evident. At 10 mM foscarnet, cells accumulated earlier in the S phase, probably at the G1/S border. However, at both 5 and 10 mM foscarnet the block was not established until after 15 h incubation. Upon removing 10 mM foscarnet after 24 h incubation, G1 cells rapidly entered the S phase, whereas the progression through S and G2 + M was delayed considerably. The DNA synthesizing S phase seems, therefore, to be the main cell cycle phase affected by foscarnet.     

Characterization of 2'':3''-Cyclic Nucleotide 3''-Phosphodiesterase: Rapid Isolation, Native Enzyme Analysis, Identification of a Serum-Soluble Activity, and Kinetics

The enzyme 2':3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) was isolated from bovine brain white matter by a rapid (72 h) procedure. The minimum molecular weight (MW) of the enzyme was approximately 52,500 as estimated by sucrose density gradient analysis. When this isolated enzyme was stimulated with bovine serum albumin (BSA), the peak of activity was shifted to approximately 90,000 MW. Prior treatment by trypsin blocked the expression of the higher MW form of CNPase, but not the BSA activation of the enzyme. If the trypsin digestion was allowed to progress, the MW was gradually lowered to a broad peak sedimenting between 20,000 and 50,000 MW. An apparently soluble form of CNPase found in serum is described. Kinetic and MW comparisons between the serum soluble enzyme and CNPase isolated from bovine brain, as well as an analysis of substrate specificity, were made and it was concluded that the two enzymes were identical.     

Characterization of 2'':3''-Cyclic Nucleotide 3''-Phosphodiesterase: Limited Proteolytic Digestion, Plant Lectin Affinity Chromatography, and Immunological Identification

Purified bovine brain 2':3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) migrates as a protein double band in SDS-polyacrylamide gel electrophoresis. The positions of the two protein bands correspond to approximate molecular weights (MW) of 56,000 and 53,000. Limited protease treatment of isolated CNPase leads to subsequent degradation of the enzyme into smaller polypeptides having MWs of approximately 40,000, 30,000, and 20,000. During proteolytic digestion CNPase remains enzymatically active. Binding studies with several immobilized plant lectins as well as periodic acid-Schiff reagent (PAS) staining of SDS gels indicate that CNPase is a glycoprotein. An antiserum against purified CNPase, prepared in rabbits, was used to confirm the immunological identity of various CNPase preparations obtained in our laboratory.     

Intracellular Localization of 2'',3''-Cyclic Nucleotide 3''-Phosphodiesterase in a Neuronal Cell Line as Examined by Immunofluorescence and Cell Fractionation

The enzyme 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase, EC 3.1.4.37) occurs not only in myelin fractions and glial cells, but can also be shown to be present in a CNS cell line of neuronal origin (B104). Direct immunofluorescence microscopy of B104 cells with fluorescein isothiocyanate-conjugated rabbit anti-CNPase antibodies shows a discrete and specific intracytoplasmic location of CNPase. Fractionation of the cells was performed by differential centrifugation of a cell homogenate and continuous sucrose density-gradient centrifugation. As monitored by marker enzyme activities, CNPase seems to be associated with endoplasmic reticulum membranes.     

Optimal techniques for the immunocytochemical demonstration of beta-thromboglobulin, platelet factor 4, and fibrinogen in the alpha granules of unstimulated platelets

Summary The distribution of -thromboglobulin, platelet factor 4, and fibrinogen in unstimulated platelets was investigated by several immunocytochemical techniques. All three substances were found to be localized in the majority of platelet alpha granules either by immunoperoxidase methods on saponin-treated platelets or by colloidal gold immunoconjugates on frozen thin sections. The optimal conditions for preparing and fixing platelets for immunocytochemistry were also determined. Platelets obtained from blood dripped directly into fixative or anticoagulated blood were compared systematically with respect to shape. Temperature was found to be the most important variable. Immediately fixed platelets were generally disc-shaped, regardless of the temperature of the fixative. Reducing the temperature of blood (stored with anticoagulant) before fixation resulted in more swollen and fewer disc-shaped platelets. However, if the blood was mixed with an anticoagulant and maintained at 37° C for 1 h before fixation, the same number of disc-shaped platelets were present as in samples from blood fixed immediately. The intracellular localization of -thromboglobulin, platelet factor 4, and fibrinogen was consistent regardless of platelet preparatory procedure, but several technical problems were encountered with respect to plasma membrane labelling when control experiments were analysed. Immediately fixed, non-permeabilized platelet plasma membranes were always labelled, no matter which control substances or immunoperoxidase markers were used. However, when platelets were washed by centrifugation, the plasma membranes were negative. Exposure to saponin markedly diminished labelling of the plasma membranes. Optimal techniques for the immunocytochemical demonstration of these alpha granule proteins in platelets are presented in this report.This paper is based in part on theHistochemical Journal Lecture for 1983 given by Dr Bainton at a Symposium on Haematological Cytochemistry in Cambridge on 29 September 1983 at the invitation of the Royal Microscopical Society.     

Ultrastructural analysis of acute immune thrombocytopenia in mice: dissociation between alterations in megakaryocytes and platelets

Thrombopoiesis was studied in mice after the induction of acute immune thrombocytopenia with platelet antiserum (PAS). Utilizing electron microscopy, we examined platelets and megakaryocytes (MK) obtained 4, 8, 12, 24, 48, 72, and 120 hr after administration of PAS. Four to 24 hr after injection of PAS, the majority of bone marrow MK were normal in size and organelle distribution. The demarcation membrane system (DMS) extended normally throughout the mature cell cytoplasm at these times. However, approximately 50% of MK observed 48 hr and 72 hr after injection of PAS were significantly larger than normal, and often had demarcation membranes confined to an area between a peripheral organelle-deficient zone and a central nuclear zone. The median values for sectional areas of platelets obtained 8-72 hr after administration of PAS were significantly greater than the median value for sectional areas of platelets in a pooled control sample. The proportion of cytoplasm to surface-connected canalicular system appeared greater than normal in most large platelets from the PAS samples; and increased numbers of profiles of Golgi complex and endoplasmic reticulum were observed. By 48 hr post-injection of PAS (at which time the modal ploidy class of MK has shifted from 16N to 32N; Corash et al., Blood 70:177, 1987), most platelets were normal in size and cytoplasmic appearance. At 120 hr post-injection of PAS, virtually all platelets exhibited a normal size and complement of organelles, and MK also had returned to normal. Our data indicate that in response to acute thrombocytopenia, MK prematurely release platelets which differ from normal platelets in size and cytoplasmic appearance. There was a marked dissociation between alterations in platelets and MK, since a statistically significant increase in platelet sectional area occurred 40 hr before the shift in modal ploidy class of MK, and platelet size subsequently decreased toward normal during the period that has been shown to be associated with the maximum shift in MK ploidy. These results strongly suggest that the characteristics of platelet release do not depend on the ploidy or cytoplasmic characteristics of MK.     

Presence of Lythrum salicaria enhances the bodyguard effects of the parasitoid Asecodes mento for Filipendula ulmaria

This paper reports significant effects of a co‐occurring plant species (Lythrum salicaria, Lythraceae) on the reproductive success of the perennial herb Filipendula ulmaria (Rosaceae). We studied 15 Filipendula populations in the Skeppsvik Archipelago; seven of which were monospecific and eight mixed with Lythrum. All the Filipendula populations studied harbored the chrysomelid beetle Galerucella tenella, and in 2005 seed set was strongly negatively correlated with the percentage leaf area consumed. Moreover, data from 2004 showed that 25–100% of the G. tenella larvae were parasitized by the hymenopteran parasitoid Asecodes mento, and we found a strong cascading top‐down effect of parasitism in 2004 on Filipendula seed set in 2005. In 2004, parasitism (at the population level) was negatively correlated with percentage leaf area consumed and positively correlated with seed set in 2005. The parasitoid Asecodes also parasitized G. calmariensis, which is monophagous on Lythrum. Mixed populations of Filipendula and Lythrum supported higher densities of their shared ‘bodyguard’Asecodes. Further, Y‐tube bioassays showed that floriferous Filipendula attracted more than twice as many gravid Asecodes females as floriferous Lythrum. Taken together, these findings suggest that coexistence of the two plants results in ‘associational resistance’ for Filipendula and ‘associational susceptibility’ for Lythrum. This scenario was supported for Filipendula since, for this species, we found lower leaf consumption followed by higher seed production in mixed than in monospecific populations. Considered together, our results show that bodyguards may increase the reproductive fitness of a perennial herb, and that the strength of the cascading ‘bodyguard’ effect can be strongly influenced by co‐occurring plants through ‘apparent competition’. This is the first paper to demonstrate that, in the wild, plant species may use odors to compete for ‘bodyguards’, thereby causing asymmetrical ‘apparent competition’ between the herbivores involved. Our data emphasize the need to consider community factors in studies of trophic interactions.     

The folding and stability of human alpha class glutathione transferase A1-1 depend on distinct roles of a conserved N-capping box and hydrophobic staple motif.

An N-capping box and a hydrophobic staple motif are strictly conserved in the core of all known glutathione S-transferases (GST). In the present work, mutations of hGSTA1-1 enzyme residues forming these motifs have been generated. The analysis of S154A, D157A, and S154A/D157A capping mutants indicate that the removal of this local signal destabilizes the protein. The fact that the third helical residue D157A mutation (N-3) was much more destabilizing than the first helical residue S154A mutation (N-cap) suggests that the appropriate conformation of the conserved substructure formed by the alpha 6-helix and preceding loop (GST motif II) is crucial for the overall protein stability. The refolding study of GSTA1-1 variants supports the prediction that this subdomain could represent a nucleation site of refolding. The analysis of L153A, I158A, L153G, and L153A/I158A hydrophobic staple mutants indicate that the removal of this motif destabilizes the GSTA1-1 structure as well as its refolding transition state. The hydrophobic staple interaction favors essential inter-domain contacts and, thereby, in contrast to capping interactions, accelerates the enzyme reactivation. Its strict conservation in the GST system supports the suggestion that this local signal could represent an evolutionarily conserved determinant for rapid folding.