Intracellular Synthesis of Myxovirus Neuraminidase in Chick Embryo Cell Monolayer Culture I. Neuraminidase Activity, Hemagglutinin Synthesis, and Content of Cell-bound Sialic Acid in Newcastle Disease Virus-infected Cells

Neuraminidase (Nase) activity of chick embryo monolayer cell homogenates was determined by its rate of splitting of neuraminlactose, free neuraminic acid (NA) being determined by the thiobarbituric acid assay. Noninfected cells were found to have no detectable amount of Nase activity. Newcastle disease virus (NDV)-infected cells (multiplicity of infection, 20 to 75 plaque-forming units per cell) displayed a high level of Nase synthesis, the rate of synthesis being parallel to that of hemagglutinin (HA) synthesis (with a 1.5 hr delay in the latter). An "eclipse" of the Nase and HA activities associated with the virus that was adsorbed onto cells was observed. The data provide evidence that the Nase is not incorporated into the viral envelope from a pre-existing cell supply but that its synthesis is coded by the viral genome. The content of cell-bound sialic acid, determined simultaneously in infected-cell homogenates, showed characteristic features allowing certain conclusions concerning the renewal of NA-terminating cell receptors during the course of infection, and the intracellular action of the Nase of the virus introduced into cells by the inoculum and that of the newly synthesized Nase at different stages of infection.     

Osmotic water permeability of cell membranes from Mesembryanthemum crystallinum leaves: effects of age and salinity

The osmotic water permeability ( P _os) of cell membranes isolated from leaves of 40-, 50- and 60-day-old Mesembryanthemum crystallinum plants was estimated by measuring light-scattering kinetics using stopped-flow spectrophotometry. The measurements were performed on the plasma membrane (PM), purified tonoplast (TP), and TP-enriched vesicles. The PM and TP-enriched vesicles were obtained by partitioning the microsomal fraction in an aqueous polymer two-phase system, whereas the purified TP vesicles were prepared by microsomal vesicle flotation on a sucrose cushion. The P _os of isolated membranes declined with plant age. The kinetic experiments showed that there was no difference between the P _os of the PM and TP isolated from plants of all ages. A 24-h exposure of plants to 400 m M NaCl caused a decline in the P _os as well. These findings suggest that, during M. crystallinum transition to CAM, which was induced by plant ageing or salinity, plant osmoregulatory responses included changes in the P _os of the leaf-cell membranes. These variations in the P _os are discussed in the context of adaptive mechanisms responsible for the maintenance of the water balance in the common ice plant.     

Backbone Cyclization of the C-terminal Part of Substance P. Part 1: The Important Role of the Sulphur in Position 11

Novel backbone-to-side chain and backbone-to-backbone cyclic analogues of substance P (SP) were prepared by solid-phase synthesis and screened for biological activity. An analogue containing a thioether- lactam ring between positions 9 and 11 showed an EC₅₀ value of 20nM toward the neurokinin 1 (NK-1) and was inactive toward the NK-2 and NK-3 receptors. On the other hand, in a multiple backbone cyclic peptide library of similar analogues, in which the sulphur was excluded from the ring, very low activity was detected. The activity was re-evaluated and was found to be even lower (EC₅₀=0.11 mM ) than the previously published data. These results indicate that the thioether moiety has a crucial role in receptor activation. The results also show tolerance of the NK-1 receptor, but not NK-2 or NK-3, to cyclization of the C-terminal portion of the SP_6–11 hexapeptide.     

Nystatin-induced increase in photocurrent in the system ‘bacteriorhodopsin proteoliposome/bilayer planar membrane’

Proteoliposomes were reconstituted from bacteriorhodopsin sheets, asolectin and cholesterol with or without nystatin. Bacteriorhodopsin-mediated electrogenesis was monitored using (1) a proteoliposome suspension and phenyldicarbaundecaborane (PCB^?) probe or (2) proteoliposomes associated with planar bilayer membrane and orthodox electrometer techniques. In the light, PCB^? was shown to be taken up by proteoliposomes. The PCB^? uptake was inhibited by addition of nystatin to an incubation mixture with proteoliposomes if they were reconstituted in the presence of nystatin. Extraproteoliposomal nystatin was without influence if nystatin was omitted from the reconstitution mixture. The nystatin-containing proteoliposomes were associated with a planar bilayer asolectin membrane in the presence of Ca²⁺. It was found that in such a system, bacteriorhodopsin generated a photocurrent charging the proteoliposome-containing (cis-side) compartment negatively and the trans-side compartment positively. The photoresponse was shown to be increased several-fold by addition of nystatin to the trans-side solution. Nystatin addition was ineffective if proteoliposomes were reconstituted without nystatin. Taking into account that nystatin forms ion-permeable pores in a membrane only if present on both sides of the membrane and that this membrane is bilayer, one can explain the above data assuming that (1) the intraproteoliposomal solution does not mix with the extraproteoliposomal one when proteoliposomes are attached to a planar black membrane and (2) the attached proteoliposomes are separated from the trans-side bathing solution by a bimolecular membrane. If this is the case, nystatin in the trans-side bathing solution and inside the attached proteoliposome can form pores across that part of the planar membrane which separates the proteoliposome interior from the trans-side solution. Through these pores, H⁺ (pumped by bacteriorhodopsin from the cis-side solution into the proteoliposome interior) or some other intraproteoliposomal ions can be equilibrated with those in the trans-side solution. As a result, the bacteriorhodopsin-generated photocurrent increases.     

Glutamate dehydrogenase deficiency disrupts glutamate homeostasis in hippocampus and prefrontal cortex and impairs recognition memory

Glutamate Dehydrogenase 1 (GDH), encoded by the Glud1 gene in rodents, is a mitochondrial enzyme critical for maintaining glutamate homeostasis at the tripartite synapse. Our previous studies indicate that the hippocampus may be particularly vulnerable to GDH deficiency in central nervous system (CNS). Here, we first asked whether mice with a homozygous deletion of Glud1 in CNS (CNS‐Glud1 ?/? mice) express different levels of glutamate in hippocampus, and found elevated glutamate as well as glutamine in dorsal and ventral hippocampus, and increased glutamine in medial prefrontal cortex (mPFC). l ‐serine and d ‐serine, which contribute to glutamate homeostasis and NMDA receptor function, are increased in ventral but not dorsal hippocampus, and in mPFC. Protein expression levels of the GABA synthesis enzyme glutamate decarboxylase (GAD) GAD67 were decreased in the ventral hippocampus as well. Behavioral analysis revealed deficits in visual, spatial and social novelty recognition abilities, which require intact hippocampal‐prefrontal cortex circuitry. Finally, hippocampus‐dependent contextual fear retrieval was deficient in CNS‐Glud1 ?/? mice, and c‐Fos expression (indicative of neuronal activation) in the CA1 pyramidal layer was reduced immediately following this task. These data point to hippocampal subregion‐dependent disruption in glutamate homeostasis and excitatory/inhibitory balance, and to behavioral deficits that support a decline in hippocampal‐prefrontal cortex connectivity. Together with our previous data, these findings also point to different patterns of basal and activity‐induced hippocampal abnormalities in these mice. In sum, GDH contributes to healthy hippocampal and PFC function; disturbed GDH function is relevant to several psychiatric and neurological disorders.     

Cytotoxic Effects of the Selective Ligands of Membrane Progesterone Receptors in Human Pancreatic Adenocarcinoma Cells BxPC3

Biochemistry (Moscow) - Progesterone and its synthetic analogues act on cells through different types of receptors, affecting proliferation and apoptosis. These compounds exert their effect through...     

Interactive effects of elevated temperature and CO2 levels on metabolism and oxidative stress in two common marine bivalves (Crassostrea virginica and Mercenaria mercenaria)

Marine bivalves such as the hard shell clams Mercenaria mercenaria and eastern oysters Crassostrea virginica are affected by multiple stressors, including fluctuations in temperature and CO₂ levels in estuaries, and these stresses are expected to be exacerbated by ongoing global climate change. Hypercapnia (elevated CO₂ levels) and temperature stress can affect survival, growth and development of marine bivalves, but the cellular mechanisms of these effects are not yet fully understood. In this study, we investigated whether oxidative stress is implicated in cellular responses to elevated temperature and CO₂ levels in marine bivalves. We measured the whole-organism standard metabolic rate (SMR), total antioxidant capacity (TAOC), and levels of oxidative stress biomarkers in the muscle tissues of clams and oysters exposed to different temperatures (22 and 27 °C) and CO₂ levels (the present day conditions of ~ 400 ppm CO₂ and 800 ppm CO₂ predicted by a consensus business-as-usual IPCC emission scenario for the year 2100). SMR was significantly higher and the antioxidant capacity was lower in oysters than in clams. Aerobic metabolism was largely temperature-independent in these two species in the studied temperature range (22–27 °C). However, the combined exposure to elevated temperature and hypercapnia led to elevated SMR in clams indicating elevated costs of basal maintenance. No persistent oxidative stress signal (measured by the levels of protein carbonyls, and protein conjugates with malondialdehyde and 4-hydroxynonenal) was observed during the long-term exposure to moderate warming (+ 5 °C) and hypercapnia (~ 800 ppm CO₂). This indicates that long-term exposure to moderately elevated CO₂ and temperature minimally affects the cellular redox status in these bivalve species and that the earlier observed negative physiological effects of elevated CO₂ and temperature must be explained by other cellular mechanisms.     

DNA damage causes TP53-dependent coupling of self-renewal and senescence pathways in embryonal carcinoma cells

Comment on: Wu R, et al. Clin Cancer Res 2011; 17:7359–72