Activation of a Ca2+-dependent K+ current by the oncogenic receptor protein tyrosine kinase v-Fms in mouse fibroblasts

We investigated the effects of the receptor-coupled protein tyrosine kinase (RTK) v-Fms on the membrane current properties of NIH3T3 mouse fibroblasts. We found that v-Fms, the oncogenic variant of the macrophage colony-stimulating factor receptor c-Fms, activates a K⁺ current that is absent in control cells. The activation of the K⁺ current was Ca²⁺-dependent, voltage-independent, and was completely blocked by the K⁺ channel blockers charybdotoxin, margatoxin and iberiotoxin with IC₅₀ values of 3nM, 18 nM and 76nM, respectively. To identify signalling components that mediate the activation of this K⁺ current, NIH3T3 cells that express different mutants of the wildtype v-Fms receptor were examined. Mutation of the binding site for the Ras-GTPase-activating protein led to a complete abolishment of the K⁺ current. A reduction of 76% and 63%, respectively, was observed upon mutation of either of the two binding sites for the growth factor receptor binding protein 2. Mutation of the ATP binding lobe, which disrupts the protein tyrosine kinase activity of v-Fms, led to a 55% reduction of the K⁺ current. Treatment of wild-type v-Fms cells with Clostridium sordellii lethal toxin or a farnesyl protein transferase inhibitor, both known to inhibit the biological function of Ras, reduced the K⁺ current amplitude to 17% and 6% of the control value, respectively. This is the first report showing that an oncogenic RTK can modulate K⁺ channel activity. Our results indicate that this effect is dependent on the binding of certain Ras-regulating proteins to the v-Fms receptor and is not abolished by disruption of its intrinsic protein tyrosine kinase activity. Furthermore, our data suggest that Ras plays a key role for K⁺ channel activation by the oncogenic RTK v-Fms. Received: 19 November 1997 / Accepted: 21 January 1998     

Low T3 syndrome and chronic inflammatory rheumatism

For the clarification of pathogenesis and clinical relevance of decreases of the triiodothyronine (T3) level in patients with chronic inflammatory rheumatism in a group of 63 patients with clinically, paraclinically and roentgenologically diagnosed rheumatoid arthritis (59 times) and with SLE (4 times), respectively, parallel were determined parameters of the thyroid gland function and of the rheumatic activity as well as a subtile drug anamnesis for the medication of antirheumatic drugs was established. In 33 of the 63 patients who were included into the study decreases and low normal values, respectively, for the total T3 (TT3 less than 1.5 nmol/l) were found. In comparison to the remaining 30 patients with normal TT3 a typical constellation of paraclinical parameters of the thyroid gland with distinct reduction of TT3 and free T3 (FT3), low normal total T4 (TT4), slight increase of the reverse T3 (rT3), moderate decrease of the basal and stimulated TSH and an only very small restriction of the binding capacity of the thyroid hormone (TBG) were found. A clinically relevant hypothyroidism is thus to be excluded with certainty. Antirheumatic drugs, in particular steroidal ones (glucocorticoids) may on principle also induce such paraclinical constellations, related to the thyroid gland. In our investigations a therapy with antirheumatic drugs is causally scarcely considered, since both in the group of patients with decrease of T3 and without decrease comparable quantities of antirheumatic drugs including glucocorticoids were administered and the cortisol values in the plasma do not differ. The investigations confirm our already formerly expressed supposition that also in rheumatics a "low-T3-syndrome" is existing as it is otherwise described in consumptive extrathyroidal diseases (NTI).(ABSTRACT TRUNCATED AT 250 WORDS)     

Radiologic search for embolized leaflets of prosthetic heart valves: A report of two cases

In a radiologic search for embolized leaflets of Edwards-Duromedics bileaflet valves in 2 patients, the embolized fragments were localized in the iliac vessels using computed tomography. Sonography was successful in one case and standard X-ray films of the abdomen were negative in both cases.In vitro investigations with Björk-Shiley and Edwards-Duromedics leaflets suggested that standard X-ray films of the abdomen and pelvis should be considered as the first investigational technique. If negative, computed tomography of the lower abdomen should be done.     

Generation of targets for alloreactive CTL using purified H-2K in liposomes and polyethylene glycol

The ability of a purified major histocompatibility antigen to serve as the target cell antigen for alloreactive CTL (H-2^d anti-H-2^k) was examined. Tumor cells syngeneic with responding CTL were used as targets following modification with purified alloantigen (H-2K^k). A short incubation of tumor cells with H-2K^k liposomes followed by the addition of polyethylene glycol (PEG) yielded modified tumor cells that were recognized and lysed by CTL. The macrophage-like cell line P388D1 was readily recognized following liposome and PEG modification; apparently because these cells can withstand PEG mediated insertion of H-2K^k and lipid into their membrane. The generation of targets by PEG mediated modification was most efficient using liposomes prepared with an H-2K^k:lipid ratio of about 1:500. H-2K^k containing liposomes prepared with negatively charged phospholipids readily attached to P388D1 cells, however these cells were not targets for CTL unless PEG was added. The specificity of CTL recognition and lysis of liposome modified cells was shown by the reactivity of CTL primed against alloantigens other than H-2K^k and by antibody (anti-H-2K^k) blocking of recognition and lysis. These results demonstrate that purified H-2K^k can serve as the alloantigen for CTL lysis and suggest that the H-2 must be oriented in the target cell lipid bilayer to serve as the alloantigen for CTL mediated target cell lysis.     

Human achaete-scute homologue (hASH1) mRNA level as a diagnostic marker to distinguish esthesioneuroblastoma from poorly differentiated tumors arising in the sinonasal tract

Distinction of high-grade esthesioneuroblastomas from other poorly differentiated tumors arising in the nasal cavity is an important diagnostic challenge because it determines patient management and prognosis. The human achaete-scute homologue (hASH1) gene is critical in olfactory neuronal differentiation and is expressed in immature olfactory cells; therefore, it could have potential use as a diagnostic marker The aim of the present study was to determine the value of hASH1 messenger RNA (mRNA) levels in differentiating esthesioneuroblastoma from other poorly differentiated tumors. A real-time polymerase chain reaction assay was developed, permitting the comparative determination of hASH1 mRNA levels in triplicate in a double-blind pilot study including 24 frozen cases of esthesioneuroblastoma and poorly differentiated tumors. All 4 positive cases were esthesioneuroblastomas, and all 19 poorly differentiated tumors were negative. In addition, there was an inverse association between the grade of esthesioneuroblastomas and hASH1 mRNA levels. The hASH1 mRNA level might represent a useful tool for distinguishing esthesioneuroblastoma from poorly differentiated tumors of the sinonasal region.     

Identification of a naturally processed cyclin D1 T-helper epitope by a novel combination of HLA class II targeting and differential mass spectrometry

T-helper (Th) cells play an important role in orchestrating the effector function of CTL in anti-tumor immunity. However, only a limited number of Th cell epitopes has been characterized. Here we describe a novel approach for identifying naturally processed and presented peptides derived from chosen antigens. This method combines a transfection step of antigen-presenting cells with a vector encoding a fusion protein between the Ii chain and the antigen of interest, elution of the HLA-bound peptides and identification of the antigen-derived peptides by mass spectrometric comparison to the non-transfected cells. In vitro-stimulated Th cells against the identified peptide of interest specifically recognize transfectants overexpressing the cognate antigen. Using this approach, we were able to identify the HLA-DR4-restricted Th cell epitope NPPSMVAAGSVVAAV derived from cyclin D1, which is frequently overexpressed in tumors. This method will help in identifying peptide candidates for vaccination studies for tumor immunotherapy.     

PCR analysis of pulsed-field gel electrophoresis-purified plastid DNA,a sensitive tool to judge the hetero-/homoplastomic status of plastid transformants

The genetic transformation of plastids of higher plants has developed into a powerful approach for both basic research and biotechnology. Due to the high copy number of the plastid genome per plastid and per cell, repeated cycles of shoot regeneration under conditions selective for the modified plastid chromosome are required to obtain transformants entirely lacking wild-type plastid genomes. The presence of promiscuous plastid DNA in nuclear and/or mitochondrial genomes that generally contaminate even gradient-purified plastid fractions reduces the applicability of the highly sensitive PCR approach to monitor the absence of residual wild-type plastid chromosomes in transformed lines. It is therefore difficult, or even impossible, to assess reliably the hetero- or homoplastomic state of plastid transformants in this manner. By analysing wild-type and transplastomic mutants of tobacco, we demonstrate that separation of plastid chromosomes isolated from gradient-purified plastid fractions by pulsed-field gel electrophoresis can overcome the problem of (co)amplification of interfering promiscuous plastid DNA. PCR analyses with primers specific for plastid, mitochondrial and nuclear genes reveal an impressive purity of such plastid DNA fractions at a detection limit of less than one wild-type plastid chromosome copy per ten transplastomic cells.     

Gender and smoking-related risk reduction of periodontal disease with variant myeloperoxidase alleles

Myeloperoxidase (MPO) is an oxidative enzyme expressed in polymorphonuclear leukocytes. It is involved in the defence against periodontal bacteria, and is also able to mediate inflammatory tissue destruction in periodontal disease. A G/A polymorphism in the promoter region of the MPO gene at position -463 has been assumed to exert profound effects on the expression of the enzyme. It is the aim of this study to evaluate whether this polymorphism may influence the risk of periodontal diseases. A total of 3148 subjects were randomly selected from the general population in the SHIP study (Study of Health in Pomerania). Periodontal status, health-related and socio-economic items were assessed. All subjects aged 40-60 years (n = 1103) were included in this study, and 1083 genotyped for the MPO -463 G/A polymorphism by PCR and RFLP methods. The genotype frequencies determined were homozygous wild type G/G 65.9% (95% CI 63.5-68.6), heterozygous A/G 31.4% (28.8-34.4), and homozygous variant A/A 2.7% (2.0-3.8). Only female subjects have a significantly reduced risk of severe periodontal disease when bearing the variant genotypes A/G or A/A. In female subjects the reduction in periodontal risk was significant for non-smokers (OR = 0.48; 95% CI 0.23-0.96); the smoke-related increase in risk was also reduced (OR = 0.50; 95% CI 0.22-1.10). When adjusted for age, smoking, and education the odds ratios were calculated as 0.52 (P = 0.01) and 0.97 (P = 0.90) for female and male subjects, respectively. The results of this study confirm the assumption that the MPO -463A allele variants are protective in the pathogenesis of periodontal diseases. This holds true only with women but not with men. The results are discussed with respect to the known influences of sexual hormones on MPO activity.     

Stability of Late Event-Related Potentials: Topographical Descriptors of Motor Control Compared with the P300 Amplitude

The P300-amplitude evoked with an acoustic oddball-paradigm is considered the most stable late event-related potential (ERP). This amplitude-index has become a standard parameter in electrophysiology. Recently, a robust ERP-parameter (NoGo-anteriorization, NGA) has been introduced, which reflects spatial brain electrical changes in relation to execution and inhibition of a motor response elicited with a Continuous Performance Test (CPT). The current study refers to the stability of this new topographical ERP-parameter compared to the stability of the classical P300-amplitude. For that purpose, 12 healthy subjects were investigated with both paradigms during recording of a 21-channel EEG. Analysis of the resulting ERPs revealed a very high stability for both, topographical and amplitude index: In every single subject, the brain electrical fields were characterized by a more anterior location in the NoGo- compared to the Go-condition (=NGA) and by higher amplitudes after target compared to distractor condition. T-tests, analyses of the effect size and of the power revealed equivalent differences between the two contrasting conditions for the topographical compared to the amplitude index. These results indicate that the stability of the topographical ERP-parameters elicited with the CPT is sufficient for an electrophysiological standard-index. The possibility to elicit a robust and specific spatial brain activation with the CPT is an ideal completion to the classical P300 amplitude effect and, therefore, hopefully will be a useful expansion of the standard paradigms in electrophysiological laboratories.