The sodium channels of the neuroblastoma x glioma 108 CC 15 hybrid cell change their sensitivity for volatile and local anesthetics upon continuous passage

Summary We have studied the ion flux through the sodium channels of low passage number (<50p.) and high passage number (>150p.) neuroblastoma x glioma hybrid cells using [¹⁴C] guanidinium and specific neurotoxins to induce channel opening and closing. The sodium channels of low passage number hybrid cells could be opened by veratridine alone, suggesting the presence of voltage dependent channels in agreement with electrophysiological studies reported in the literature. The sodium channels of the high passage number hybrid cells, however, needed the synergistic action of veratridine and scorpion toxin for activation suggesting that these channels are silent. The [¹⁴C] guanidinium ion flux through the sodium channels of the high passage number hybrid cells was inhibited by significantly lower concentrations of the volatile anesthetics (halothane, isoflurane and enflurane) and the lcoal anesthetics (tetracaine and bupivacaine) than the comparable flux through the sodium channels of the low passage number hybrid cells.Dedicated to Prof. Dr. E. Wecker on the occasion of his 65th birthday.     

Down-regulation of 3H-lofentanil binding to opiate receptors in different cultured neuronal cells

Summary There was stereospecific binding of ³H-lofentanil (K _D value = 1.53 nM) to membranes of neuroblastoma-glioma NG 108-15 cells which are known to bear high affinity binding sites for enkephalin derivatives (-opiate receptor subtype). There was no high affinity specific binding of the -opiate specific ligand ³H-sufentanil. The specific binding of ³H-lofentanil to -opiate receptor subtype was down-regulated (decrease in B _max value without change in the K _D value) after prolonged incubation of the cells in the presence of leu- and met- enkephalin (0.1 M). There was no down-regulation of the opiate receptors (³H-lofentanil and ³H-d-ala-d-leu-enkephalin specific binding) after incubation of NG 108-15 cells with drugs from the fentanyl series (alfentanil or sufentanil).In cultured neurones from rat forebrain (15 day old embryos), the ³H-lofentanil binding was specific with high affinity (K _D: 0.048 nM) and a slow dissociation rate similar to that in adult rat cortex. Drugs of the fentanyl series (4-anilino-piperidines) were potent displacers whereas agonists of the - (enkephalin derivatives), (phencyclidine, haloperidol, 3-hydroxyphenyl-propylpiperidine) or K- (U 50488) opiate sites had a low affinity (K _i > 0.5 M) for ³H-lofentanil specific binding sites. Since there was also specific binding of ³H-sufentanil, the opiate receptors in cultured neurones seem to be mainly of the -subtype and this is consistent with the ontogeny of opiate receptors subtypes. These receptors were down-regulated after incubation in the presence of etorphine, sufentanil and alfentanil but not enkephalin derivatives.These results strongly suggest specific binding of ³H-sufentanil and ³H-lofentanil mainly to the so-called -opiate receptors in cultured neurones and a specific binding of ³H-lofentanil to lower affinity -opiate receptors in neuroblastoma-glioma cells. The down-regulation of the -opiate binding sites in cultured neurones and that of the -site in neuroblastoma × glioma hybrid cells were dose-and temperature-dependent, induced by the corresponding high affinity agonists and prevented by naloxone. Morphine did not induce down-regulation of or receptor sites, possibly because of a partial antagonist effect on both receptor subtypes. Send offprint requests to J. M. Maloteaux at the above address     

Human isolated small intestine: motor responses of the longitudinal muscle to field stimulation and exogenous neuropeptides

Summary (1) Longitudinal muscle strips from the human small intestine (jejunum/ileum) responded to electrical field stimulation (1–50 Hz) with frequency-related primary contractions which were largely atropine- (3 M) sensitive. When the tone was raised by addition of galanin (0.3 – 1 M), prostaglandin (PG) E₂ (1–10 M) or neurokinin A (NKA, 0.1 M), a frequency-related relaxation was evident which was potentiated by atropine. All the responses to field stimulation were abolished by tetrodotoxin (1 M), thus indicating their neural origin. (2) The atropine-sensitive primary contraction to field stimulation was virtually abolished by omega conotoxin fraction GVIA (CTX, 0.1–0.3 M) while the relaxations were CTX-resistant. The field stimulation-induced relaxations, which were observed in the presence of atropine and guanethidine (3 M), were also unaffected by apamin (0.1 M). (3) NKA and substance P (SP) produced a concentration- (1 nM–1 M for both peptides) related contraction, NKA being about 53 times more potent than SP. [Pro⁹]SP sulphone and [MePhe⁷]-NKB, selective agonists of the NK-1 and NK-3 receptor, respectively, were barely effective. On the other hand, [\Ala⁸]NKA(4–10), a selective NK-2 receptor agonist, had a potent contractile activity, similar to that of NKA. (4) Galanin (1 nM–1M) produced an atropine- and tetrodotoxin-resistant concentration-related contraction of longitudinal muscle of human isolated small intestine. The response to galanin did not show any sign of fading and was particularly suitable to study the evoked relaxations. (5) Calcitonin gene-related peptide (CGRP) (10–100 nM) consistently inhibited the nerve-mediated contractions of strips from the ileum while the effect on the jejunum was less pronounced. Vasoactive intestinal polypeptide (VIP, 0.1–1 M) inhibited nerve-mediated contractions both in the ileum and the jejunum. (6) These experiments indicate that both cholinergic excitatory and non-adrenergic non-cholinergic nerves affect motility of the longitudinal muscle of the human small intestine. Furthermore, several neuropeptides produce potent motor effects, the contractile response to tachykinins being apparently mediated by activation of NK-2 receptors.     

The potential role of lonidamine (LND) in the treatment of malignant glioma

Summary Up-to-date unsatisfactory results obtained in multimodality treatments of malignant glioma have prompted the research of new therapeutic modalities with unconventional modes of action. Lonidamine (LND) is a drug which reduces aerobic glycolytic activity in both human and experimental tumors. This effect mainly depends on the inhibition of mitochondrially-bound hexokinase (HK) which is present in large amounts in malignant cells. A Phase II study was conducted on patients with recurrent glioma; 12 patients were admitted to the study. Clinical side effects were moderate, necessitating a reduction of the dosage in only 1 case. The objective results were evaluated according to the indications of Levin. 2 responders and 3 cases of stable disease were observed out of 10 evaluable patients. The potential value of this new drug is discussed.     

A two-dimensional electrophoresis reference map of human ovary

The ovary plays a central role in oogenesis and gonadal hormone secretion. Proteomic analysis is a valuable approach for gaining an increased understanding of the molecular nature of the ovary. In this work, two-dimensional electrophoresis for protein separation followed by matrix-assisted laser desorption/ionization mass spectrometry and database searches, identified 231 protein spots corresponding to 138 individual proteins that were found in gels representing both the follicular and luteal phases. The data were used to construct a database online (). The identified proteins were functionally classified into seven groups: (1) cell signaling/communication, (2) cell division, (3) gene/protein expression, (4) metabolism, (5) cell structure and motility, (6) cell/organism defense, and (7) unclassified. Among the proteins identified, 47% had not been previously reported in the human ovary. In addition, a number of disease-related proteins were identified in this protein map, including some cancer- and polycystic ovarian syndrome-related proteins. Two proteins with phosphorylation were verified by Western blot analysis. Comparison of protein abundance between follicular and luteal stages produced seven protein spots that had been identified in our database. This study provides a preliminary reference map of normal human ovary that will form a basis for comparative studies on normal and pathological conditions of the human ovary and may serve as a potential tool for clinical diagnosis, therapeutics, and prognosis.Electronic Supplementary Material Supplementary material is available in the online version of this article at L. Wang and Y.-F. Zhu contributed equally to this work     

Preservation and redirection of HPV16E7-specific T cell receptors for immunotherapy of cervical cancer

Human papilloma virus (HPV) type 16 infections of the genital tract are associated with the development of cervical cancer (CxCa) in women. HPV16-derived oncoproteins E6 and E7 are expressed constitutively in these lesions and might therefore be attractive candidates for T-cell-mediated adoptive immunotherapy. However, the low precursor frequency of HPV16E7-specific T cells in patients and healthy donors hampers routine isolation of these cells for adoptive transfer. To overcome this problem, we have isolated T cell receptor (TCR) genes from four different HPV16E7-specific healthy donor and patient-derived human cytotoxic T lymphocyte (CTL) clones. We examined whether genetic engineering of peripheral blood-derived CD8+ T cells in order to express HPV16E711-20-specific TCRs is feasible for adoptive transfer purposes. Reporter cells (Jurkat/MA) carrying a transgenic TCR were shown to bind relevant but not irrelevant tetramers. Moreover, these TCR-transgenic Jurkat/MA cells showed reactivity towards relevant target cells, indicating proper functional activity of the TCRs isolated from already available T cell clones. We next introduced an HPV16E711-20-specific TCR into blood-derived, CD8+ recipient T cells. Transgenic CTL clones stained positive for tetramers presenting the relevant HPV16E711-20 epitope and biological activity of the TCR in transduced CTL was confirmed by lytic activity and by interferon (IFN)-gamma secretion upon antigen-specific stimulation. Importantly, we show recognition of the endogenously processed and HLA-A2 presented HPV16E711-20 CTL epitope by A9-TCR-transgenic T cells. Collectively, our data indicate that HPV16E7 TCR gene transfer is feasible as an alternative strategy to generate human HPV16E7-specific T cells for the treatment of patients suffering from cervical cancer and other HPV16-induced malignancies.     

Modulation of heteronymous reflexes from ankle dorsiflexors to hamstring muscles during human walking

In 16 human subjects, stimulation of the common peroneal nerve (CPN) was applied during walking and standing. The effect of the stimulation was evaluated from the rectified and averaged biceps femoris (BF) electromyographic (EMG) activity. In the swing phase of walking, the CPN stimulation evoked a suppression in the BF EMG in 12 of the subjects. In the early stance phase, the suppression was replaced by facilitation at a similar latency in 9 of the subjects. Of the other 3 subjects, in whom a suppression was observed during swing, a decrease in the suppression was observed in the stance phase in two of them. During a voluntary co-contraction of BF and tibialis anterior while standing, a suppression similar to that observed in the swing phase was observed. The thresholds of the suppression and facilitation were identical, suggesting that afferents of similar diameter were responsible. Cutaneous stimuli, which mimicked the sensation evoked by the CPN stimulation, but without activation of muscle afferents, did not produce similar effects in the BF EMG activity. It is suggested that the observed response and reflex reversal may reflect opening of an excitatory group I pathway in the early stance phase of walking with a concomitant shut-down of heteronymous group I inhibition.     

The breakdown of Fitts' law in rapid,reciprocal aiming movements

According to Fitts' law, there is speed-accuracy trade-off in a wide variety of discrete aiming movements. However, it is unknown whether the same law applies to cyclic aiming movements. In the present study, a comparison is made between discrete versus cyclic aiming movements. A group of 24 healthy participants made graphical pen movements in 12 different aiming tasks in which successive finger and wrist movements were emphasized, consecutively executed as discrete and cyclic movements and varying in three target widths. In the cyclic condition, aiming movements consisted of back-and-forth movements that were performed in immediate succession for 20 s. In the discrete condition, back-and-forth aiming movements were drawn as 20 single strokes, starting after a go signal and stopping after reaching the target area. The targets had various levels of spatial accuracy and the movements had different directions (from lower left to upper right; from lower right to upper left) elicit either predominantly wrist or finger movements. The amount of information processed per unit of time (bits per second; index of performance, IP), tangential velocity, the pen pressure, and the ratio of peak-over-mean velocity were studied to gain understanding about the differences in control between discrete and cyclic movements. It was found that the IP and movement velocity were almost twice as large in cyclic versus discrete movements. In contrast, the axial pen pressure and the ratios of peak-over-mean velocity were much lower in cyclic movements (1.24 N versus 0.94 N; 2.26 N versus 1.81 N). The results of our study indicate that the predicted constant IP does not hold for rapid cyclic aiming movements and that speed-accuracy trade-off is different. It is concluded that cyclic movements exploit the energetic and physiological properties of the neuromotor system. Expected differences in brain activity related to discrete and cyclic aiming movements are discussed as well as several neurophysiological mechanisms, which predict more economic force recruitment and information processing in cyclic than in discrete movements.     

Elastic strain energy in the low back muscles during human walking

Summary A simple model of the thorax, pelvis and three columns of the intrinsic lumbar back muscles (=ILBM) was constructed. The model was used to study the length of the ILBM during the different stages of the walking cycle. The length of the right ILBM (especially the lateral column) was largest at right toe off, exactly the stage of the walking cycle in which most force was needed to prevent the torso from falling forwards and laterally.     

LSECtin interacts with filovirus glycoproteins and the spike protein of SARS coronavirus

Cellular attachment factors like the C-type lectins DC-SIGN and DC-SIGNR (collectively referred to as DC-SIGN/R) can augment viral infection and might promote viral dissemination in and between hosts. The lectin LSECtin is encoded in the same chromosomal locus as DC-SIGN/R and is coexpressed with DC-SIGNR on sinusoidal endothelial cells in liver and lymphnodes. Here, we show that LSECtin enhances infection driven by filovirus glycoproteins (GP) and the S protein of SARS coronavirus, but does not interact with human immunodeficiency virus type-1 and hepatitis C virus envelope proteins. Ligand binding to LSECtin was inhibited by EGTA but not by mannan, suggesting that LSECtin unlike DC-SIGN/R does not recognize high-mannose glycans on viral GPs. Finally, we demonstrate that LSECtin is N-linked glycosylated and that glycosylation is required for cell surface expression. In summary, we identified LSECtin as an attachment factor that in conjunction with DC-SIGNR might concentrate viral pathogens in liver and lymph nodes.