Gating in iodate-modified single cardiac Na+ channels

Summary Elementary Na⁺ currents were recorded at 19°C during 220-msec lasting step depolarizations in cell-attached and inside-out patches from cultured neonatal rat cardiocytes in order to study the modifying influence of iodate, bromate and glutaraldehyde on single cardiac Na⁺ channels.Iodate (10 mmol/liter) removed Na⁺ inactivation and caused repetitive, burst-like channel activity after treating the cytoplasmic channel surface. In contrast to normal Na⁺ channels under control conditions, iodate-modified Na⁺ channels attain two conducting states, a short-lasting one with a voltage-independent lifetime close to 1 msec and, likewise tested between –50 and +10 mV, a long-lasting one being apparently exponentially dependent on voltage. Channel modification by bromate (10 mmol/liter) and glutaraldehyde (0.5 mmol/liter) also included the occurrence of two open states. Also, burst duration depended apparently exponentially on voltage and increased when shifting the membrane in the positive direction, but there was no evidence for two bursting states. Chemically modified Na⁺ channels retain an apparently normal unitary conductance (12.8±0.5 pS). Of the two substates observed, one of them is remarkable in that it is mostly attained from full-state openings and is very short living in nature; the voltage-independent lifetime was close to 2 msec. Despite removal of inactivation, open probability progressively declined during membrane depolarization. The underlying deactivation process is strongly voltage sensitive but, in contrast to slow Na⁺ inactivation, responds to a voltage shift in the positive direction with a retardation in kinetics. Chemically modified Na⁺ channels exhibit a characteristic bursting state much shorter than in DPI-modified Na⁺ channels, a difference not consistent with the hypothesis of common kinetic properties in noninactivating Na⁺ channels.     

Crystal structure of thermitase and stability of subtilisins

Crystal structure of thermitase, a serine proteinase from Thermoactinomyces vulgaris, has been determined by X-ray diffraction at 1.4 A resolution. The atomic model of thermitase refined to an R-factor of 0.149 contains 1997 protein atoms, 182 water molecules and 2 Ca2+ ions. The tertiary structure of thermitase is similar to that of subtilisin BPN'. The greatest variations are connected with insertions and deletions in the amino acid sequence, which are located on the surface of the molecule. Higher thermostability of thermitase can be explained in terms of the three-dimensional structure. The Ca2+ ions, bound to the protein molecule, as well as the ionic and hydrophobic interactions are supposed to give the main contribution to the stabilization of the structure.     

Humoral anti-idiotypic and anti-anti-idiotypic immune response in cancer patients treated with monoclonal antibody 17-1A

 A group of 96 patients with advanced colorectal carcinoma were treated with the mouse (m) or chimeric (c) (mouse variable regions × human IgG1 constant regions) monoclonal antibody (mAb) 17-1A recognizing the tumour-associated antigen GA733-2. Eighty-two of the 83 patients treated with mmAb17-1A and 69% of the patients given cmAb17-1A (n = 13) developed anti-idiotypic antibodies (ab₂). Auto-antibodies binding to tumour cells expressing GA733-2 were found in 7% of the patients. In a further 38 patients (40%) antitumour-cell antibodies, i.e. anti-anti-idiotypic antibodies (ab₃), were induced by the mAb17-1A therapy. Patients with detectable ab₃ after treatment had significantly higher ab₂ levels than those not developing ab₃. Addition of granulocyte/macrophage-colony-stimulating factor (GM-CSF) to mmAb17-1A significantly enhanced the induction of ab₂ as well as induction of anti-anti-idiotypic antibodies (ab₃), compared to mmAb17-1A alone. Patients with a high increase in antitumour-cell antibodies (ab₃) induced by the therapy lived significantly longer than patients with no or a low level of induction of ab₃ (P = 0.016). The results indicate that induction of an idiotypic network response might be an important effector mechanism in mAb therapy. Received: 20 October 1995 / Accepted: 18 December 1995     

The effect of virus infection on morphology and protein components of pollen grains

Morphology of pollen grains collected from healthy and virus infected plants ofChenopodium quinoa L.,Chenopodium album L. andNicotiana tabacum L. cv. Samsun was investigated using scanning electron microscopy (SEM). Pollen grains from tobacco plans infected with tobacco mosaic virus (TMV) were smaller, with rounded shape and conspicuous deformation of aperture unlike oval and smooth pollen grains from healthy plants. No morphological alterations were observed inC. quinoa andC. album plants infected with TMV and cucumber mosaic virus (CMV). Polyacrylamide gel electrophoresis of pollen proteins revealed substantial quantitative and qualitative differences in protein components of pollen grains collected from healthy and virus infected plants     

Influence of protein conditioning films on binding of a bacterial polysaccharide adhesin from Hyphomonas MHS-3

A putative polysaccharide adhesin which mediates non-specific attachment of Hyphomonas MHS-3 (MHS-3) to hydrophilic substrata has been isolated and partially characterized. A polysaccharide-enriched portion of the extracellular polymeric substance (EPS(P)) from MHS-3 was separated into four fractions using high performance size exclusion chromatography (HPSEC). Comparison of chromatograms of EPS(P) from MHS-3 and a reduced adhesion strain (MHS-3 rad) suggested that one EPS(P) fraction, which consisted of carbohydrate, served as an adhesin. Adsorption of this fraction to germanium (Ge) was investigated using attenuated total reflection Fourier transform infrared (ATR/FT-IR) spectrometry. Binding curves indicated that the isolated fraction had a relatively high affinity for Ge when ranked against an adhesive protein from Mytilis edulis, mussel adhesive protein (MAP) and an acidic polysaccharide (alginate from Macrocystis pyrifera). Spectral features were used to identify the fraction as a polysaccharide previously reported to adsorb preferentially out of the EPS(P) mixture. Conditioning the Ge substratum with either bovine serum albumin (BSA) or MAP decreased the adsorption of the adhesive polysaccharide significantly. Conditioning Ge with these proteins also decreased adhesion of whole cells.     

Displacement of housekeeping proteasome subunits by MHC-encoded LMPs: a newly discovered mechanism for modulating the multicatalytic proteinase complex.

The degradation of cytoplasmic antigens to peptides presented by class I MHC molecules is thought to be mediated by the ubiquitin/proteasome pathway. Support for this view came from our observation that the subunit composition of proteasomes can be changed by interferon-gamma (IFN-gamma) treatment. Thereby two subunits, LMP2 and LMP7, which are encoded in the MHC class II region, are incorporated into the proteasomal complex, whereas other subunits disappear. In the experiments reported in this communication we studied the subunit changes occurring in cell lines where the expression of LMP2 or LMP7 can be regulated individually either by IFN-gamma induction or by applying a new system to control the expression of transfected LMPs. In both situations LMP2 induction leads exclusively to the disappearance of housekeeping subunit 2, whereas LMP7 affects only subunit 10. Subunit 2 was found to be 76% homologous to LMP2. Since incorporation of LMP2 into the proteasomal complex prevents processing of the subunit 2 precursor, we conclude that LMP2 displaces subunit 2 during assembly. Subunit displacement is most likely a general mechanism to modulate the catalytic activity of the proteasomal complex without changing its structure. Furthermore, the controlled incorporation of transfected subunits into the complex offers a new approach to study proteasome function in vivo.     

Herbivory on calcicolous lichens: different food preferences and growth rates in two co-existing land snails

A total of 32 calcicolous lichen species, one alga and one bryophyte were recorded on a limestone wall in the grassland Great Alvar on the Baltic island of Öland, Sweden. Fourteen (41%) of these 34 species and free-living cyanobacteria showed herbivore damage, most probably due to grazing by the land snails Chondrina clienta and Balea perversa which inhabited the limestone wall. Three laboratory experiments were conducted to examine the food preferences of individuals of C. clienta and B. perversa collected at this site and to evaluate any association between their preference and the net food quality of the lichens to the snails. Chondrina clienta and B. perversa exhibited food preferences, which differed significantly between species. Within species, variation in food choice was similar among individuals. This indicates that snail populations may be composed of polyphagous individuals with similar food preferences. Different lichen species were of different net food quality to the snails as indicated by growth rate differences. In both snail species the most preferred lichen species of the choice experiment caused the largest weight increase in juveniles, viz. Caloplaca flavovirescens for C. clienta and Aspicilia calcarea for B. perversa. This suggest that the snail species studied differ in their abilities to deal with secondary compounds and physical characteristics of certain lichens or that they can utilize the energy and nutrients of these lichens to a different extent. It is suggested that differential food preferences might reduce the intensity of interspecific competition for resources (lichens) between C. clienta and B. perversa.     

Skewed inactivation of an X chromosome deleted at the dystrophin gene in an asymptomatic mother and her affected daughter

A girl with severe Becker muscular dystrophy and apparently normal chromosomes had a heterozygous deletion for exons 51, 52, and 53 of the dystrophin gene. This deletion was transmitted by her mother, who was unaffected. To differentiate the normal and the deleted X chromosomes, fluorescence in situ hybridization (FISH) was applied to metaphase chromosomes, using probes for both exons 51 and 52, which are only 388 and 113 base pairs long, respectively. FISH signals were observed in one or both chromatids of one chromosome, but never on both chromosomes, suggesting the lack of hybridization on the deleted X chromosome. Using 5-bromodeoxyuridine incorporation to differentiate the late (inactive) and the early replicating (active) X chromosomes, 77% of the signals were observed on the active X chromosomes in the mother. This percentage was only 18% in the daughter, suggesting that skewed inactivation of the X chromosomes was responsible for the phenotypic differences.     

Induction of an immune network cascade in cancer patients treated with monoclonal antibodies (ab1)

The antitumor effector functions of unconjugated monoclonal antibodies (mAb) in cancer therapy are not fully understood. Direct cytotoxic mechanisms such as antibody-dependent cellular cytotoxicity, complement-dependent cytolysis and apoptosis have been suggested. Induction of anti-idiotypic (ab₂) and anti-anti-idiotypic (ab₃) antibodies as well as the corresponding T cells (T₂ and T₃) has also been proposed to be of therapeutic significance. In this study induction of an immune network cascade in ten patients with colorectal carcinoma, treated with mAb 17-1A (ab₁) was assessed. After treatment, all ten patients had anti-idiotypic antibodies and anti-anti-idiotypic antibodies with ab₁-like binding specificity while only five of ten patients had T cells corresponding to ab₃ (T₃) as assessed by a proliferation assay (DNA synthesis), and an assay of interferon production (ELISPOT) (Enzyme-linked immuno SPOT) in vitro or by a delayed-type hypersensitivity reaction in vivo. Purified T cells from four of the five patients with a positive T₃ test responded with DNA synthesis after stimulation using human anti-mAb 17-1A anti-idiotypic monoclonal antibodies. These four patients had a clinical response showing a tumor reduction after therapy, while all six patients lacking a proliferative response failed to show tumor regression. Induction of a cell-mediated immune network cascade might accordingly be an important anti-tumor effector function of mAb and should be considered in the future design of mAb-based therapy protocols in cancer patients.