Subthreshold oscillations generated by TTX-sensitive sodium currents in dorsal cochlear nucleus pyramidal cells

During intracellular recordings in rodent brainstem slice preparations, dorsal cochlear nucleus (DCN) pyramidal cells (PCs) exhibit characteristic discharge patterns to depolarizing current injection that depend on the membrane potential from which the responses are evoked. When depolarized from hyperpolarized potentials, PCs can respond with a short-latency action potential followed by a long silent interval (pauser) or a train of action potentials with a long latency (buildup). During the silent intervals in a pauser or a buildup response, the membrane potential slowly depolarizes towards spike threshold, often exhibiting distinct voltage oscillations of 1–2 mV before the first spike. The subthreshold voltage oscillations were investigated using whole cell recordings from DCN PCs in rat pup (P10–14) brainstem slices. The oscillations were unaffected by excitatory and inhibitory neurotransmitter antagonists, and were not temporally locked to the onset of the depolarization. The oscillations typically became larger as spike threshold was approached, and had a characteristic frequency between 40 and 100 Hz. In the presence of tetrodotoxin (TTX, 500 nM), the oscillations were significantly suppressed, and could not be evoked at any voltage below or above spike threshold. The oscillations were not blocked by phenytoin or Cd²⁺, but they were affected by prior activity in the neuron for approximately 1 s. We conclude that voltage-gated Na⁺ channels are required to generate membrane oscillations during the buildup phase. We suggest that the subthreshold oscillations play a role in controlling spike timing in PCs when the membrane potential slowly approaches, or hovers near, spike threshold.     

Biochemical characterization of I-Ak proteins from mutant antigen-presenting B-cell--B-lymphoma hybridomas

Chemically induced mutants of an I-Ak,d expressing antigen-presenting B-cell--B-lymphoma hybridoma have recently been generated by immunoselection in vitro and were found to possess alterations in some of their serologically and functionally defined I-Ak region dependent functions. In order to identify at the structural level the origin of the differences in serological and functional properties of these mutants, I-Ak molecules from several of these mutant hybridomas were compared biochemically to wild-type I-Ak polypeptides by two-dimensional gel electrophoresis and high-pressure liquid chromatographic tryptic peptide analyses. Two-dimensional gel electrophoresis indicated that no major structural alterations, resulting in changes in mol. wt or charge, had occurred in the Ak alpha or Ak beta polypeptides from the mutant cells. Likewise, Ak alpha peptide maps of the mutants were indistinguishable from the normal Ak alpha peptide maps. However, two of the three mutants studied did exhibit one additional peptide in their Ak beta peptide maps. These results suggest that the major deficiencies in T-cell-activating functions of these mutants are a result of a limited alteration in the Ak beta polypeptide primary structure.     

Effects of Culture Medium on Cystic Fibrosis and Normal Fibroblasts Studied by X-Ray Microanalysis

The effect of culture medium from fibroblast cultures of cystic fibrosis (CF) patients and healthy controls on the elemental composition of fibroblasts was investigated by X-ray microanalysis. Exposure of normal fibroblasts to culture medium from CF fibroblasts caused an increase in calcium level. Exposure of CF fibroblasts to culture medium from normal cells caused an increase of the sodium content of CF cells to approximately normal levels; the calcium level of the CF fibroblasts, however, remained abnormally high. The results may indicate that CF fibroblasts lack a factor needed for the regulation of sodium transport. CF fibroblast medium apparently contains a factor that interferes with the regulation of calcium transport.     

Endogenous interleukin-18 is involved in immunity to Leishmania donovani but its absence does not adversely influence the therapeutic activity of sodium stibogluconate

Immunity to Leishmania donovani is associated with an interleukin (IL)-12 driven T helper 1 (Th1) response. In addition, the ability to respond to chemotherapy with sodium stibogluconate (SSG) requires a fully competent immune response and both Th1 and Th2 responses have been shown to positively influence the outcome of drug treatment. In the present study, the influence of IL-18, which can modulate both interferon (IFN)-gamma and IL-4 production, on the outcome of primary L. donovani infection and SSG therapy following infection was assessed using BALB/c IL-18-deficient and wild type mice. IL-18 deficiency was associated with an increased susceptibility to L. donovani infection, evident by day 40 post infection, resulting in higher parasite burdens in the spleen, liver, and bone marrow compared with wild type control animals. Infected IL-18-deficient mice had significantly lower splenocyte concanavalin A (ConA) induced IFN-gamma production as well as lower serum IL-12 and IFN-gamma levels, indicating a reduced Th1 response. However, drug treatment was equally effective in both mouse strains and restored serum IL-12 and IFN-gamma levels, and IFN-gamma production by ConA stimulated splenocytes of IL-18-deficient mice, to levels equivalent to similarly treated wild type mice.     

High throughput ranking of recombinant avian scFv antibody fragments from crude lysates using the Biacore A100

Advances in molecular evolution strategies have made it possible to identify antibodies with exquisite specificities and also to fine-tune their biophysical properties for practically any specified application. Depending on the desired function, antibody/antigen interactions can be long-lived or short-lived and, therefore, particular attention is needed when seeking to identify antibodies with specific reaction-rate and affinity properties. Surface plasmon resonance (SPR) biosensors routinely generate sensitive and reliable kinetic data from antibody/antigen interactions for both therapeutic and diagnostic applications. However, many kinetic-based screening assays require rigorous sample preparation and purification prior to analysis. To ameliorate this problem, we developed a rapid and reliable assay for characterising recombinant scFv antibody fragments, directly from crude bacterial lysates. Ninety-six scFv antibodies derived from chickens immunised with C-reactive protein (CRP) were selected by phage display and evaluated using the Biacore A100 protein interaction array system. Antibodies were captured from crude bacterial extracts on the sensor chip surface and ranked based on the percentage of the complex left (% left) after dissociation in buffer. Kinetic rate constants (k(a) and k(d)) and affinity (K(D)) data were obtained for six clones that bound monomeric CRP across a broad affinity range (2.54 x 10(-8) to 3.53 x 10(-10) M). Using this assay format the A100 biosensor yielded high quality kinetic data, permitting the screening of nearly 400 antibody clones per day.     

The effect of sodium lauryl sulphate and triclosan on hamster cheek pouch mucosa

It has recently been shown that triclosan protects the human skin from the inflammation that may be caused by exposure to sodium lauryl sulphate (SLS). The aim of the present study was to examine whether triclosan can protect the hamster cheek pouch mucosa from the irritation caused by exposure to SLS. After four daily applications of a paste containing SLS, the epithelium of the hamster cheek pouch showed consistently prominent structural changes, especially basal hyperplasia, acanthosis, hypergranulosis, and hyperkeratosis. Identical morphological changes were also observed after applications of a paste containing SLS together with triclosan. In contrast, after applications of a paste containing triclosan alone, the cheek pouch mucosa revealed a histological structure essentially similar to the non-treated control mucosa. From these results, we may conclude that SLS, but not triclosan, irritates the hamster cheek pouch epithelium. Moreover, triclosan does not protect the cheek pouch mucosa against structural changes induced by SLS. It must be taken into account that triclosan does not always offer protection against the side-effects of SLS.     

Differentiating embryonic stem-derived neural stem cells show a maturation-dependent pattern of voltage-gated sodium current expression and graded action potentials

A population of mouse embryonic stem (ES)-derived neural stem cells (named NS cells) that exhibits traits reminiscent of radial glia-like cell population and that can be homogeneously expanded in monolayer while remaining stable and highly neurogenic over multiple passages has been recently discovered. This novel population has provided a unique in vitro system in which to investigate physiological events occurring as stem cells lose multipotency and terminally differentiate. Here we analysed the timing, quality and quantity of the appearance of the excitability properties of differentiating NS cells which have been long-term expanded in vitro. To this end, we studied the biophysical properties of voltage-dependent Na(+) currents as an electrophysiological readout for neuronal maturation stages of differentiating NS cells toward the generation of fully functional neurons, since the expression of neuronal voltage-gated Na(+) channels is an essential hallmark of neuronal differentiation and crucial for signal transmission in the nervous system. Using the whole cell and single-channel cell-attached variations of the patch-clamp technique we found that the Na(+) currents in NS cells showed substantial electrophysiological changes during in vitro neuronal differentiation, consisting mainly in an increase of Na(+) current density and in a shift of the steady-state activation and inactivation curves toward more negative and more positive potentials respectively. The changes in the Na(+) channel system were closely related with the ability of differentiating NS cells to generate action potentials, and could therefore be exploited as an appropriate electrophysiological marker of ES-derived NS cells undergoing functional neuronal maturation.     

The use of somatic antigen of Haemophilus influenzae for the monitoring of T cell-mediated skin test reactivity in man

To investigate its usefulness as a skin test antigen, Haemophilus influenzae somatic antigen was tested in 28 healthy individuals, both in soluble and aggregated form. All subjects were found to possess specific antibodies against H. influenzae of both IgG and IgM subclass, thus showing their previous exposure to this commensal micro-organism. The somatic antigen in solution was found to be a poor antigen for eliciting a delayed hypersensitivity skin response: only 2 out of 16 subjects reacted with a positive DTH pattern. In contrast, 25 out of 28 persons showed a positive DTH pattern when somatic antigen was used in aggregated form. Two types of DTH reaction patterns could be detected (in a ratio of approximately 3:2), viz. those with an early (24 h) and those with a late (48 h) maximal swelling. Histology of 3 early and 1 late DTH reaction showed perivascular infiltrates of mainly Thelper/Tinducer lymphocytes. Hardly any basophils were seen. One negative skin test, biopsied at 6 h, showed no signs of Arthus reactivity. It can be concluded that skin tests using the aggregated form of the somatic antigen of H. influenzae are useful for assaying specific T-cell-mediated reactivity in man.     

Calmodulin and Ca2+-dependent cyclic AMP phosphodiesterase activity in Trypanosoma cruzi

Calmodulin has been purified from Trypanosoma cruzi epimastigote forms by ion-exchange chromatography, gel filtration and affinity chromatography on 2-chloro-10-(3-aminopropyl)phenotiazine-Sepharose. Upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the factor showed a polypeptide band with an apparent molecular weight of 16 000. In addition, cyclic AMP phosphodiesterase activity from T. cruzi epimastigote forms was purified by ion-exchange chromatography and affinity chromatography on a brain calmodulin-Sepharose column. The enzyme was activated by homologous calmodulin as well as by bovine brain and Neurospora crassa calmodulins. The activation required micromolar concentrations of Ca2+ and it was blocked by EGTA and by some neuroleptic drugs such as chlorpromazine, fluphenazine and compound 48/80. Activations were observed at micromolar concentrations of cyclic AMP as substrate. In addition, T. cruzi calmodulin was also active in bringing about the stimulation of brain phosphodiesterase.     

Biosynthesis of Trypanosoma brucei variant surface glycoproteins — Analysis of carbohydrate heterogeneity and timing of post-translational modifications

The variant surface glycoproteins from two cloned populations of Trypanosoma brucei brucei which were known to migrate as multiple bands on SDS gels have been studied. The heterogeneity present was located in those oligosaccharide side chains the addition of which is tunicamycin-sensitive. The time required for the trypanosome to synthesize and express a variant surface glycoprotein molecule in vitro was found, from pulse-chase and limited trypsinisation experiments, to be approximately 40 min. In the light of these data, pulse-chase experiments on the two antigens known to have heterogeneity in their oligosaccharide side chains demonstrated that the heterogeneity probably arose by two different mechanisms. Pulse-chase experiments on three different clones of trypanosomes have also been used to investigate the timing of cleavage of the carboxyl-terminal extension, known to be encoded on variant surface glycoprotein mRNA. Similar pulse-chase experiments followed by immunoprecipitation using affinity purified antiserum have been used to investigate the addition of the cross-reacting determinant. The timing of both these events has been discussed in relation to the time necessary for the synthesis and expression of the variant surface glycoprotein on the surface of the trypanosome.