Enhanced production of mouse hybridomas to picomoles of antigen using EL-4 conditioned media with an in vitro immunization protocol

Summary We report here that the use of murine thymoma cell EL-4 conditioned medium enhances hybridoma yield in a low-antigen dose in vitro immunization protocol. This improved protocol allowed the production of a panel of monoclonal antibodies toDrosophila yolk proteins using less than 1 nanomole of antigen. We believe this refinement will be valuable for the application of hybridoma technology to biologically active materials that are hard to isolate and purify due to their low concentration in the biological fluids. This research was supported by the College of Agriculture and Life Sciences, University of Maryland, USDA-University of Maryland Cooperative Editor's Statement This observation should simplify in vitro immunization approaches and shed new light on the factors required for the in vitro immune response. Wallace L. McKeehan     

Generation of grouped discharges by tectal projection cells

In this paper we briefly summarize our recent data on the transduction properties of tecto-bulbo-spinal neurons (TBSN) in the cat. These neurons from an important link between the superior colliculus and the "premotor" structures of the brain stem and the cervical spinal cord. They are closely similar to spinal alpha-motoneurons, as concerns the soma-dendritic geometry and electrotonic parameters. In contrast, their rhythmic firing behavior is characterized by much higher sensitivity to depolarizing currents and by the capability ot generate extraspikes ("regenerative firing mode"). This results in an abrupt increase of sensitivity when a certain limit of depolarization is surpassed. Membrane parameters of TBSN which are responsible for their characteristic transduction properties are presented. We forward an hypothesis that different modes of rhythmic firing, as dependent on behavioral situation, play a role in the distribution of efferent signals among many different target areas of TBSNs.     

Ceruloplasmin and myeloperoxidase in complex affect the enzymatic properties of each other

Ceruloplasmin (CP), the multicopper oxidase of plasma, interacts with myeloperoxidase (MPO), an enzyme of leukocytes, and inhibits its peroxidase and chlorinating activity. Studies on the enzymatic properties shows that CP behaves as a competitive inhibitor impeding the binding of aromatic substrates to the active centre of MPO. The contact between CP and MPO probably entails conformational changes close to the p-phenylenediamine binding site in CP, which explains the observed activation by MPO of the substrate's oxidation. CP subjected to partial proteolysis was virtually unable to inhibit activity of MPO. The possible protein–protein interface is comprised of the area near active site of MPO and the loop linking domains 5 and 6 in CP. One of the outcomes of this study is the finding of a new link between antioxidant properties of CP and its susceptibility to proteolysis.     

Ion conductances related to development of repetitive firing in mouse retinal ganglion neurons in situ

ABSTRACT: In the retina, the ability to encode graded depolarizations into spike trains of variable frequency appears to be a specific property of retinal ganglion neurons (RGNs). To deduce the developmental changes in ion conductances underlying the transition from single to repetitive firing, patch-clamp recordings were performed in the isolated mouse retina between embryonic day 15 (E15) and postnatal day 5 (P5). Immature neurons of the E15 retina were selected according to their capacity to generate voltage-activated Na+ currents (I(Na)(v)). Identification of P5 RGNs was based on retrograde labeling, visualization of the axon, or the amplitude of I(Na)(v). At E15, half of the cells were excitable but none of them generated more than one spike. At P5, all cells were excitable and a majority discharged in tonic fashion. Ion conductances subserving maintenance of repetitive discharge were identified at P5 by exposure to low extracellular Ca2+, Cd2+, and charybdotoxin, all of which suppressed repetitive discharge. omega-Conotoxin GVIA and nifedipine had no effect. We compared passive membrane properties and a variety of voltage-activated ion channels at E15 and P5. It was found that the density of high voltage-activated (HVA) Ca2+ currents increased in parallel with the development of repetitive firing, while the density of Ni2+-sensitive low voltage-activated (LVA) Ca2+ currents decreased. Changes in density and activation kinetics of tetrodotoxin-sensitive Na+ currents paralleled changes in firing thresholds and size of action potentials, but seemed to be unrelated to maintenance of repetitive firing. Densities of A-type K+ currents and delayed rectifier currents did not change. The results suggest that HVA Ca2+ channels, and among them a toxin-resistant subtype, are specifically engaged in activation of Ca2+-sensitive K+ conductance and thereby account for frequency coding in postnatal RGNs.     

Functional specialization of presynaptic Cav2.3 Ca2+ channels

Ca2+ influx into presynaptic terminals via voltage-dependent Ca2+ channels triggers fast neurotransmitter release as well as different forms of synaptic plasticity. Using electrophysiological and genetic techniques we demonstrate that presynaptic Ca2+ entry through Cav2.3 subunits contributes to the induction of mossy fiber LTP and posttetanic potentiation by brief trains of presynaptic action potentials while they do not play a role in fast synaptic transmission, paired-pulse facilitation, or frequency facilitation. This functional specialization is most likely achieved by a localization remote from the release machinery and by a Cav2.3 channel-dependent facilitation of presynaptic Ca2+ influx. Thus, the presence of Cav2.3 channels boosts the accumulation of presynaptic Ca2+ triggering presynaptic LTP and posttetanic potentiation without affecting the low release probability that is a prerequisite for the enormous plasticity displayed by mossy fiber synapses.     

Identification and Characterization of Two Quiescent Porin Genes, nmpC and ompN, in Escherichia coli BE

The genomic DNA of the B^E strain of Escherichia coli has been scrutinized to detect porin genes that have not been identified so far. Southern blot analysis yielded two DNA segments which proved highly homologous to, yet distinct from, the ompC, ompF, and phoE porin genes. The two genes were cloned and sequenced. One of them, designated ompN, encodes a porin which, due to low levels of expression, has eluded prior identification. The functional properties (single-channel conductance) of the OmpN porin, purified to homogeneity, closely resemble those of the OmpC porin from E. coli K-12. The second DNA fragment detected corresponds to the nmpC gene, which, due to an insertion of an IS1 element in its coding region, is not expressed in E. coli B^E.     

UmuD and RecA directly modulate the mutagenic potential of the Y family DNA polymerase DinB

DinB is the only translesion Y family DNA polymerase conserved among bacteria, archaea, and eukaryotes. DinB and its orthologs possess a specialized lesion bypass function but also display potentially deleterious -1 frameshift mutagenic phenotypes when overproduced. We show that the DNA damage-inducible proteins UmuD(2) and RecA act in concert to modulate this mutagenic activity. Structural modeling suggests that the relatively open active site of DinB is enclosed by interaction with these proteins, thereby preventing the template bulging responsible for -1 frameshift mutagenesis. Intriguingly, residues that define the UmuD(2)-interacting surface on DinB statistically covary throughout evolution, suggesting a driving force for the maintenance of a regulatory protein-protein interaction at this site. Together, these observations indicate that proteins like RecA and UmuD(2) may be responsible for managing the mutagenic potential of DinB orthologs throughout evolution.     

NOX5 variants are functionally active in endothelial cells

NADPH oxidases have been identified as sources of reactive oxygen species (ROS) in vascular cells. In addition to the initially described enzyme containing gp91phox (NOX2), several homologues to NOX2 have been identified. Whereas NOX1, NOX2, and NOX4 are expressed in endothelial cells, a functional role of NOX5 containing additional N-terminal calcium-binding domains of varying sequences has not been reported in these cells. NOX5 protein was found in the endoplasmic reticulum of human microvascular endothelial cells (HMEC-1) and in the vascular wall. HMEC-1 cells expressed NOX5beta and NOX5delta as well as a variant lacking calcium-binding domains (NOX5S). NOX5beta and NOX5S increased basal ROS levels. Ionomycin exclusively enhanced NOX5beta-mediated ROS production. Although p22phox, when overexpressed, interacted with both NOX5 proteins, it was not essential for NOX5-mediated ROS production. NOX5 proteins stimulated endothelial cell proliferation and the formation of capillary-like structures whereas depletion of NOX5 by siRNA prevented these responses to thrombin. These data show that endothelial cells express different NOX5 variants including NOX5S lacking calcium-binding domains. NOX5 proteins are functional, promoting endothelial ROS production, proliferation, and the formation of capillary-like structures and contribute to the endothelial response to thrombin. These findings suggest that NOX5 variants play a novel role in controlling ROS-dependent processes in the vasculature.     

Role of Nucleotide Binding and GTPase Domain Dimerization in Dynamin-like Myxovirus Resistance Protein A for GTPase Activation and Antiviral Activity

Myxovirus resistance (Mx) GTPases are induced by interferon and inhibit multiple viruses, including influenza and human immunodeficiency viruses. They have the characteristic domain architecture of dynamin-related proteins with an N-terminal GTPase (G) domain, a bundle signaling element, and a C-terminal stalk responsible for self-assembly and effector functions. Human MxA (also called MX1) is expressed in the cytoplasm and is partly associated with membranes of the smooth endoplasmic reticulum. It shows a protein concentration-dependent increase in GTPase activity, indicating regulation of GTP hydrolysis via G domain dimerization. Here, we characterized a panel of G domain mutants in MxA to clarify the role of GTP binding and the importance of the G domain interface for the catalytic and antiviral function of MxA. Residues in the catalytic center of MxA and the nucleotide itself were essential for G domain dimerization and catalytic activation. In pulldown experiments, MxA recognized Thogoto virus nucleocapsid proteins independently of nucleotide binding. However, both nucleotide binding and hydrolysis were required for the antiviral activity against Thogoto, influenza, and La Crosse viruses. We further demonstrate that GTP binding facilitates formation of stable MxA assemblies associated with endoplasmic reticulum membranes, whereas nucleotide hydrolysis promotes dynamic redistribution of MxA from cellular membranes to viral targets. Our study highlights the role of nucleotide binding and hydrolysis for the intracellular dynamics of MxA during its antiviral action.