Differential expression of sodium channel mRNAs in rat peripheral nervous system and innervated tissues.

RNA blot hybridization analyses using probes specific for sodium channels I, II and III revealed high levels of sodium channel I mRNA and low levels of sodium channel II and III mRNAs in peripheral nervous system (PNS) tissues. The developmental expression patterns of these mRNAs were generally similar to those described for the central nervous system. The small amounts of sodium channel I and III mRNAs present in tongue muscle were greatly reduced after partial denervation. Expression of the three sodium channels thus appears to be restricted to the nervous system. Putative novel additional mRNAs, specifically expressed in the PNS, were detected with a probe that recognizes nucleotide sequences common to sodium channels I, II and III.

Functional expression of cloned cDNA encoding sodium channel III.

mRNA synthesized by transcription in vitro of the cloned cDNA encoding rat brain sodium channel III directs the formation of a functional sodium channel in Xenopus oocytes. The tissue distribution of the mRNAs encoding sodium channels I, II and III has been studied by blot hybridization analysis with specific probes.

A method of in situ hybridization combined with immunocytochemistry, histochemistry, and tract tracing to characterize the mRNA expressing cell types in heterogeneous neuronal populations.

A rapid, sensitive, non-isotopic in situ hybridization histochemistry protocol is presented to study the expression of mRNA at the single cell level in anatomically complex structures of the mammalian central nervous system. The protocol uses digoxigenin-UTP-labeled riboprobes, freefloating sections, and alkaline phosphatase and horseradish peroxidase detection. Modifications have been introduced which preserve the integrity of marker molecules, and as such enable the simultaneous identification and characterization of CNS cell types by tract tracing, histochemical, and immunocytochemical detection of intra- and extracellular markers. All pretreatments that enhance probe penetration have been omitted without substantial loss in sensitivity. The protocol has been successfully extended to vibratome sections with subsequent plastic-embedding and semithin sectioning, which considerably broadens the general applicability of this fast and easy ISHH method.

Molecular properties of the glutamate receptor mediating synaptic excitation in rat hypothalamic neurons.

AMPA-type glutamate receptors (GluRs) mediate synaptic excitation in networks of cultured rat hypothalamic neurons [18, 25]. Under voltage clamp the agonists quisqualate and AMPA induce current responses which consist of a maintained and/or transient component depending on the concentrations applied. The current-voltage relationship for both components is linear. The biphasic response patterns are due to receptor desensitization which is fast and does not require intracellular second messengers for its activation. Several GluR-subtype-encoding transcripts were found in these neurons using polymerase chain reaction (PCR) methods. While mRNAs encoding the GluR2 and 3 flip forms are expressed early, mRNAs encoding the GluR1, 2 and 3 flop forms and the GluR4 flip form appear only in cultures older than 3 weeks. By comparison to recombinant receptors, the properties of the native receptor can be accommodated by a heteromeric receptor containing GluR2 as one of the subunits.

Members of the RCK potassium channel family are differentially expressed in the rat nervous system.

mRNAs encoding four members of the RCK potassium channel family, named RCK1, RCK3, RCK4 and RCK5 have been analyzed by RNA blot hybridization experiments using specific RNA probes. Each probe recognizes a single mRNA species, their sizes ranging from approximately 4600 nucleotides up to approximately 11,000 nucleotides. The expression of RCK mRNAs as well as their developmental appearance in different regions of the central and peripheral rat nervous system has been investigated. The two most abundant RCK potassium channel mRNAs (RCK1 and RCK5) are predominantly expressed in the adult nervous system. RCK3 and RCK4 mRNAs are present throughout all developmental stages studied. The temporal and regional patterns observed are specific for each RCK potassium channel mRNA indicating that specific regulation of expression occurs. Differential mRNA expression might provide one mechanism for the generation of potassium channel diversity in vivo.

[Biopsy of thoracic space-occupying lesions--from computerized tomography to ultrasound-controlled puncture]. / Biopsie thorakaler Raumforderungen--Von der computertomographischen zur ultraschallgezielten Punktion.

PURPOSE: Having practised CT-controlled biopsies only, we introduced sonographically guided punctures since 1993 for pulmonary diagnoses. The effects are studied. METHOD: In a retrospective study 166 CT-guided biopsies from 1/89 to 12/95 and 50 sonographically guided biopsies from 7/93 to 12/95 were analysed. RESULTS: By CT, 67% intrapulmonary, 22% peripheral pulmonary and 11% pleuropulmonary, pleural and chest wall lesions were punctured. In 66% a diagnosis could be made. 13% of the patients experienced complications, most of them pneumothorax. Half of the patients subjected to sonographic biopsy showed peripheral pulmonary lesions, the other half tumours of the pleura, pleura and lung, mediastinum and chest wall. In 92% a positive result was obtained, whereas pneumothorax occurred in 2%. Leaving aside the intrapulmonary lesions, which would not have been visible by ultrasound, diagnosis with CT could be achieved in only 56% of the cases. CONCLUSION: In diagnosis of pleural, peripheral pulmonary and chest wall lesions, ultrasound guided biopsy is a safe, cost-effective, convenient and accurate method without exposure to x-rays.

Differential regulation of three sodium channel messenger RNAs in the rat central nervous system during development.

The levels of the mRNAs encoding sodium channels I, II and III in various regions of the developing rat central nervous system (from embryonal day 10 to postnatal day 90) have been examined by blot hybridization analysis with specific probes. The three sodium channel mRNAs exhibit different temporal and regional expression patterns. The expression of sodium channel I mRNA rises after a lag phase to adult levels during the second and third postnatal weeks with stronger increases in caudal regions of the brain and in spinal cord. Sodium channel II mRNA increases steadily until the first postnatal week, keeping high adult levels in rostral regions of the brain or reaching low adult levels after the second postnatal week in most caudal regions of the brain and in spinal cord; cerebellum shows low levels during the first two postnatal weeks but high adult levels. In all regions, sodium channel III mRNA attains maximum levels around birth and decreases during the first and second postnatal weeks to reach variable low adult levels. These results suggest that sodium channel III is expressed predominantly at fetal and early postnatal stages and sodium channel I predominantly at late postnatal stages, whereas sodium channel II is expressed throughout the developmental stages studied with greater regional variability.

Neurotrophic factor for central neurons.

A serum-free culture of dissociated neurons from embryonic rat hippocampus has been established as a rapid and quantitative in vitro test system for neurotrophic signals in the mammalian brain. By means of this cell culture bioassay, a novel low molecular weight neurotrophic factor (NTF) could be identified. NTF is essential for in vitro brain neuron development, promoting survival and neurite outgrowth. The diffusible factor is synthesized and secreted into serum-free defined medium by cultured astrocytes from rat cerebral hemispheres. The number of viable neurons responding to NTF by neurite outgrowth is dependent on the concentration of the factor. Fractionation of astroglial conditioned medium by gel filtration on columns of Sephadex G-10 recovered biological activity of NTF in a single sharp peak corresponding to an apparent molecular weight of approximately equal to 500. NTF is stable to heat and cold and resistant to trypsin and pronase. Unlike nerve growth factor, NTF has no apparent effect on the neurite outgrowth of peripheral neurons. NTF-like activity is present in situ in the mammalian brain, in certain other nonneural tissues, and in C6 and B12 glioma cell conditioned media.

[Severe pulmonary hypertension in systemic lupus erythematosus. The successful therapy of an unusual manifestation]. / Schwere pulmonale Hypertonie bei systemischem Lupus erythematodes. Erfolgreiche Therapie bei einer ungewöhnlichen Manifestation.

UNLABELLED: OBJECTIVE AND CLINICAL FINDINGS: A 48-year-old woman was hospitalized because of haemoptysis. Until shortly before admission she had been on phenprocoumon after pulmonary embolism sustained 18 months previously. Six months before admission systemic lupus erythematodes (SLE) had been diagnosed and treatment with cortisone initiated. Physical examination revealed jugular venous congestion, tachycardia, dyspnoea on even minimal physical activity and pretibial oedema. INVESTIGATIONS: Lung scintigraphy showed a perfusion deficiency in the right lung, unchanged since a test 18 month before. Doppler echocardiography recorded an estimated pulmonary artery systolic pressure of 110 mm Hg. Angiography showed a fully patent superior vena cava and nearly complete occlusion of the main right pulmonary artery by a thrombus. DIAGNOSIS, TREATMENT AND COURSE: The haemoptyses ceased after 5 days of treatment with methylprednisolone, 130 mg daily for 5 days, reduced after 5 days to 80 mg i.v. every other day, plus cyclophosphamide, 50 mg daily by mouth. The pulmonary hypertension remained unchanged so that pulmonary thrombendarterectomy was indicated. Surgery revealed extensive mediastinal fibrosis and almost complete occlusion of the thick-walled right pulmonary artery by thrombus adherent to the wall. Histology showed vasculitis of the pulmonary arterial intima and of the small pulmonary vessels. After thrombectomy the pulmonary arterial systolic pressure fell to an remained at below 40 mm Hg. Phenprocoumon was continued (at an INR of 2.5-3.5) as was immunosuppressive treatment. The patient has remained free of symptoms and is able to be physically active. CONCLUSION: Pulmonary hypertension is a serious complication of SLE. Echocardiography is recommended for both the original diagnosis and serial follow-up, complemented by other imaging methods if indicated.

Cloning and expression of a human voltage-gated potassium channel. A novel member of the RCK potassium channel family.

We have isolated and characterized a human cDNA (HBK2) that is homologous to novel member (RCK2) of the K+ channel RCK gene family expressed in rat brain. RCK2 mRNA was detected predominantly in midbrain areas and brainstem. The primary sequences of the HBK2/RCK2 K+ channel proteins exhibit major differences to other members of the RCK gene family. The bend region between segments S1 and S2 is unusually long and does not contain the N-glycosylation site commonly found in this region. They might be O-glycosylated instead. Functional characterization of the HBK2/RCK2 K+ channels in Xenopus laevis oocytes following micro-injection in in vitro transcribed HBK2 or RCK2 cRNA showed that the HBK2/RCK2 proteins form voltage-gated K+ channels with novel functional and pharmacological properties. These channels are different to RCK1, RCK3, RCK4 and RCK5 K+ channels.

Oncogenic potential of EAG K(+) channels.

We have investigated the possible implication of the cell cycle-regulated K(+) channel ether à go-go (EAG) in cell proliferation and transformation. We show that transfection of EAG into mammalian cells confers a transformed phenotype. In addition, human EAG mRNA is detected in several somatic cancer cell lines, despite being preferentially expressed in brain among normal tissues. Inhibition of EAG expression in several of these cancer cell lines causes a significant reduction of cell proliferation. Moreover, the expression of EAG favours tumour progression when transfected cells are injected into immune-depressed mice. These data provide evidence for the oncogenic potential of EAG.

Characterization of a Shaw-related potassium channel family in rat brain.

Previously, we characterized a Shaker-related family of voltage-gated potassium channels (RCK) in rat brain. Now, we describe a second family of voltage-gated potassium channels in the rat nervous system. This family is related to the Drosophila Shaw gene and has been dubbed Raw. In contrast to the RCK potassium channel family the Raw family utilizes extensive alternative splicing for expressing potassium channel subunits with variant C-termini. These alternative C-termini do not appear to influence the electrophysiological and pharmacological properties as studied in the Xenopus oocyte expression system. In situ hybridizations to sections of rat brain indicate that members of the Raw family are expressed in distinct areas of the central nervous system. Probably, Raw channels are expressed predominantly as homomultimers. Immunocytochemical experiments with antibodies against Raw3 and RCK4 proteins which form two distinct A-type potassium channels indicate that in hippocampus the two channels are expressed both in different neurons and in the same ones. In general, properties of Raw potassium channels appeared to be similar to RCK channels. However, Raw outward currents, in contrast to RCK currents, exhibit an intense rectification at test potentials higher than +20 to +40 mV. RCK and Raw channel subunits did not measurably coassemble into RCK/Raw heteromultimers after coinjecting RCK and Raw cRNA into Xenopus oocytes. These results suggest that members of the RCK and the Raw potassium channel families express potassium channels which form independent outward current systems. Combining the results of in situ hybridizations, immunocytochemical staining and expression of the cloned potassium channels in Xenopus oocytes demonstrates that unrestrained mixing of potassium channel subunits to form hybrid channels does not occur in the rat central nervous system. A single neuron is able to express multiple, independently assembled potassium channels.