Alternative non-coding exons support serotonin transporter mRNA expression in the brain and gut.

A number of studies in recent years have linked polymorphisms within the serotonin transporter (5HTT) gene to affective disorders and anxiety traits. The human 5HTT mRNA is alternatively spliced, and the splice variants are equally expressed in the human placental cell line and dorsal raphe. In this study, using 5' rapid amplification of cDNA ends, we show that the rat 5HTT mRNA is alternatively spliced, leading to three distinct mRNAs differing in the 5' untranslated region. To determine whether the three alternatively spliced mRNA species that contain one of the following untranslated regions (i) exon 1A, 63 bp (ii) exon 1A + 1B, 125 bp or (iii) exon 1C, 101 bp, were expressed in a tissue-specific manner, we used RT-PCR and exon-specific oligonucleotide hybridization. Our results suggest two of the variants (1A + 1B and 1A) may utilize the same promoter; however, they are not equally expressed. While in the adult CNS and adrenal medulla, the shorter mRNA consisting of exon 1A was considerably more abundant, in the stomach and heart, the two variants were equally expressed. The third splice variant exon 1C is only expressed in the gut and to a lesser extent in the heart. The data from this study suggest the splice variant consisting of exon 1C may utilize a distinct promoter compared to the other two.

Platelet-activating factor evokes Ca2+ transients after the blockade of ryanodine receptor by dantrolene in RAW 264.7 macrophages.

In the present study we studied platelet-activating factor (PAF)-, and ATP-induced increases in intracellular Ca2+ concentration ([Ca2+]i) using RAW 264.7 macrophages filled with fura-2/AM and imaged with fluorescence video microscopy. We found that the prevalence of detectable [Ca2+]i responses to PAF application was significantly higher in the presence of dantrolene. Dantrolene itself significantly decreased basal [Ca2+]i of macrophages compared to control cases after a 20-min incubation period. In the dantrolene-treated cells even the peak [Ca2+]i in response to PAF (as an average of all cells) was below the baseline of control suggesting that decreased [Ca2+]i plays a permissive role in the Ca2+ rise induced by PAF in macrophages. In contrast to the effect of PAF, neither the amplitude of response to ATP nor the frequency of responding cells changed significantly during dantrolene treatment in our experiments. These cells were able to respond to a standard immune stimulus as well: lipopolysaccharide (LPS) was able to increase [Ca2+]i. Our data indicate that the effectiveness of PAF to increase [Ca2+]i in RAW 264.7 macrophages depends on the resting [Ca2+]i. It has also been shown in this study that PAF and ATP differently regulate Ca2+ homeostasis in macrophages during inflammatory response and therefore they possibly differently modulate cytokine production by macrophages.

Hydrophilic, pro-drug analogues of T138067 are efficacious in controlling tumor growth in vivo and show a decreased ability to cross the blood brain barrier.

The novel anticancer compound T138067 is an irreversible inhibitor of tubulin polymerization. Amides 3-6 were synthesized using standard methodologies and determined to be significantly less lipophilic than T138067 based on logP calculations. Tubulin polymerization and [(3)H]-T138067 competition assays revealed that these amides are pro-drugs for parent aniline 2. Amides 3-5 showed no detectable signs of crossing the blood brain barrier, while amide 6 was found in extremely small amounts (12 ng/g of brain tissue). Aniline 2, which was formed in vivo from these amides, was found in significantly smaller amounts (approximately 20 to >5000 times) in the brain than when 2 was administered directly. The in vivo efficacy of amide 6 approached that of T138067 and was better tolerated when administered to athymic nude mice bearing MX-1 human mammary tumor xenografts.

Low temperature prevents potentiation of norepinephrine release by phenylephrine.

The effect of 1-phenylephrine (1-PE), an alpha(1)-receptor agonist, was investigated on the release of tritiated norepinephrine ([3H]NE). Pairs of guinea pig vasa deferentia were loaded with [3H]NE, superfused continuously, and stimulated electrically. 1-PE (10, 100 microM) enhanced the basal release of tritium in concentration-dependent manner. The stimulation-evoked release of radioactivity was significantly increased by 100 microM 1-PE. Both basal and stimulation-evoked release by 1-PE were reduced by desipramine (10 microM), a monoamine uptake inhibitor. The effect of 1-PE on basal release was independent on extracellular Ca(2+) concentration ([Ca(2+)](o)) and alpha(1)-adrenoceptor blockade. However, the 1-PE-induced release was temperature dependent: at low temperature 1-PE failed to increase either basal or stimulation-evoked release of NE. Using three different temperatures (7, 12, 17 degrees C, respectively), it was found that basal release was blocked at all three temperature values but the stimulation-evoked release was inhibited only at the lower values. The effect of 1-PE on the NE release appears to involve a desipramine-, and temperature-sensitive process. These results suggest that a non-receptorial and direct carrier-mediated mechanism is involved in NE releasing effect of 1-PE.

GABAergic interneurons are the targets of cannabinoid actions in the human hippocampus.

Cannabinoids have been shown to disrupt memory processes in mammals including humans. Although the CB1 neuronal cannabinoid receptor was identified several years ago, neuronal network mechanisms mediating cannabinoid effects are still controversial in animals, and even more obscure in humans. In the present study, the localization of CB1 receptors was investigated at the cellular and subcellular levels in the human hippocampus, using control post mortem and epileptic lobectomy tissue. The latter tissue was also used for [3H]GABA release experiments, testing the predictions of the anatomical data. Detectable expression of CB1 was confined to interneurons, most of which were found to be cholecystokinin-containing basket cells. CB1-positive cell bodies showed immunostaining in their perinuclear cytoplasm, but not in their somadendritic plasmamembrane. CB1-immunoreactive axon terminals densely covered the entire hippocampus, forming symmetrical synapses characteristic of GABAergic boutons. Human temporal lobectomy samples were used in the release experiments, as they were similar to the controls regarding cellular and subcellular distribution of CB1 receptors. We found that the CB1 receptor agonist, WIN 55,212-2, strongly reduced [3H]GABA release, and this effect was fully prevented by the specific CB1 receptor antagonist SR 141716A. This unique expression pattern and the presynaptic modulation of GABA release suggests a conserved role for CB1 receptors in controlling inhibitory networks of the hippocampus that are responsible for the generation and maintenance of fast and slow oscillatory patterns. Therefore, a likely mechanism by which cannabinoids may impair memory and associational processes is an alteration of the fine-tuning of synchronized, rhythmic population events.

Multiple cellular mechanisms mediate the effect of lobeline on the release of norepinephrine.

The complex effect of lobeline on [(3)H]norepinephrine ([(3)H]NE) release was investigated in this study. Lobeline-induced release of [(3)H]NE from the vas deferens was strictly concentration-dependent. In contrast, electrical stimulation-evoked release was characterized by diverse effects of lobeline depending on the concentration used: at lower concentration (10 microM), it increased the release and at high concentration (100 and 300 microM), the evoked release of [(3)H]NE was abolished. The effect of lobeline on the basal release was [Ca(2+)]-independent, insensitive to mecamylamine, a nicotinic acetylcholine receptor antagonist, and to desipramine, a noradrenaline uptake inhibitor. However, lobeline-induced release was temperature-dependent: at low temperature (12 degrees C), at which the membrane carrier proteins are inhibited, lobeline failed to increase the basal release. Lobeline dose dependently inhibited the uptake of [(3)H]NE into rat hippocampal synaptic vesicles and purified synaptosomes with IC(50) values of 1.19 +/- 0.11 and 6.53 +/- 1.37 microM, respectively. Lobeline also inhibited Ca(2+) influx induced by KCl depolarization in sympathetic neurons measured with the Fura-2 technique. In addition, phenylephrine, an alpha(1)-adrenoceptor agonist, contracted the smooth muscle of the vas deferens and enhanced stimulation-evoked contraction. Both effects were inhibited by lobeline. Our results can be best explained as a reversal of the monoamine uptake by lobeline that is facilitated by the increased intracellular NE level after lobeline blocks vesicular uptake. At high concentrations, lobeline acts as a nonselective Ca(2+) channel antagonist blocking pre- and postjunctional Ca(2+) channels serving as a counterbalance for the multiple transmitter releasing actions.

Modulation of norepinephrine release by ATP-dependent K(+)-channel activators and inhibitors in guinea-pig and human isolated right atrium.

OBJECTIVE: The aim of this study was to show, whether ATP sensitive K+ channels (KATP channels), are involved in the modulation of norepinephrine (NE) release from the sympathetic nerves innervating the guinea-pig and human right atrium. METHODS: The resting and stimulation-evoked release of [3H]norepinephrine ([3H]NE) was measured from the isolated guinea-pig and human right atrium and the effect of activators and inhibitors of ATP sensitive K+ channels was studied. RESULTS: Cromakalim (30-300 microM), a KATP channel-agonist decreased concentration-dependently the stimulation-evoked release of NE from the guinea-pig atrium, an effect, antagonized by glibenclamide, a KATP channel-antagonist (30 microM). Diazoxide (30-300 microM), another activator of the KATP channels reduced the resting release of NE, and also attenuated the evoked release at a single concentration (100 microM), and this latter action was also counteracted by glibenclamide (30 microM). Pinacidil, increased dose-dependently the resting and stimulation-evoked release of NE in a glibenclamide-sensitive manner and reversed the inhibitory effect of cromakalim (100 microM), suggesting that it acts as an antagonist. Glibenclamide (30-300 microM), by itself enhanced the stimulation-evoked release of [3H]NE, and also increased the resting release of NE. On the other hand, 5-hydroxydecanoate, an ischemia-selective inhibitor of cardiac KATP channels did not change NE release. Adenosine, (30-300 microM), an A1-receptor agonist, clonidine (3 microM), an alpha 2-adrenoceptor agonist and oxotremorine, a muscarinic receptor agonist (30 microM) all reduced the evoked release of [3H]NE, but these effects were not modified by glibenclamide (300 microM), indicating that neuronal adenosine (A1), adrenergic (alpha 2) and muscarinic (M3) receptors do not act on KATP channels. In the human right atrium, cromakalim, and diazoxide did not affect significantly the release of [3H]NE. However, glibenclamide (30-300 microM) and pinacidil (30-300 microM) enhanced dose-dependently the evoked-release of NE, and pinacidil also augmented the resting release. CONCLUSIONS: Our results indicate that sympathetic nerve endings of the human and guinea-pig atrium are endowed with ATP-sensitive K+ channels. These channels responded to agonists and antagonists under the experimental conditions applied and they could modulate the release of NE thereby affecting the autonomic control of cardiac function under various physiological and pathophysiological conditions.

Nitric oxide (NO) increases acetylcholine release from and inhibits smooth muscle contraction of guinea-pig gastric fundus.

Experiments were carried out to investigate the interaction between nitric oxide (NO) and cholinergic neurotransmission in smooth muscle strips of guinea-pig gastric fundus. Electrical field stimulation (2 Hz, 1 ms, 360 shocks) evoked atropine-sensitive contractions. Dimethylphenylpiperazinium (DMPP) (100 microM), a nicotinic receptor agonist, reversed the stimulation-evoked contraction and resulted in relaxation. Nomega-nitro-L-arginine (L-NNA) (100 microM), an NO synthase inhibitor, significantly increased the amplitude of stimulation-evoked contraction and abolished the effect of DMPP. Electrical stimulation increased the release of [3H]acetylcholine ([3H]ACh) from the tissue strips above the basal levels. Neither L-NNA (100 microM) nor DMPP (100 microM) alone influenced the basal release of [3H]ACh. Nomega-nitro-L-arginine (100 microM) decreased the electrical stimulation-evoked release of [3H]ACh. Dimethylphenylpiperazinium increased the stimulation-evoked release of [3H]ACh but had no effect in the presence of L-NNA. It is suggested that in guinea-pig gastric fundus, endogenous NO released in response to field stimulation has an opposite effect at the pre- and postsynaptic sites: it increases the release of ACh from cholinergic nerve terminals but reduces smooth muscle responses to ACh.

Selective, covalent modification of beta-tubulin residue Cys-239 by T138067, an antitumor agent with in vivo efficacy against multidrug-resistant tumors.

Microtubules are linear polymers of alpha- and beta-tubulin heterodimers and are the major constituents of mitotic spindles, which are essential for the separation of chromosomes during mitosis. Here we describe a synthetic compound, 2-fluoro-1-methoxy-4-pentafluorophenylsulfonamidobenzene (T138067), which covalently and selectively modifies the beta1, beta2, and beta4 isotypes of beta-tubulin at a conserved cysteine residue, thereby disrupting microtubule polymerization. Cells exposed to T138067 become altered in shape, indicating a collapse of the cytoskeleton, and show an increase in chromosomal ploidy. Subsequently, these cells undergo apoptosis. Furthermore, T138067 exhibits cytotoxicity against tumor cell lines that exhibit substantial resistance to vinblastine, paclitaxel, doxorubicin, and actinomycin D. T138067 is also equally efficacious in inhibiting the growth of sensitive and multidrug-resistant human tumor xenografts in athymic nude mice. These observations suggest that T138067 may be clinically useful for the treatment of multidrug-resistant tumors.

[Active movement therapy after flexor tendon suture using a new dynamic control splint]. / Aktive Bewegungstherapie nach Beugesehnennähten mittels einer neuen dynamischen Führungsschiene.

The authors constructed a new dynamic guiding splint assisting the active mobilisation after flexor tendon repair distal to the wrist. In these cases, the "inverse" wrist position seems to be the best position for mobilisation. This means that finger flexion should be carried out during wrist extension, and finger extension during wrist flexion. The splint guides and co-ordinates the movements of the wrist and the fingers, and it limits the free usage of the hand.

Isolation and structural and genetic analysis of the mouse enkephalin gene and its d(AC/TG)n repeats.

Enkephalins, the endogenous opioids, mediate a wide variety of intercellular communications through ontogeny and their involvement has been suggested in drug addiction and alcohol abuse as well as in various neuropsychiatric disorders. In order to generate a genetic model, we have isolated the mouse enkephalin (mENK) gene, analyzed its regulatory region and compared its structure to the well characterized rat ENK (rENK) gene. We analyzed 2600 bp and found 3 highly homologous regions: The highest level (98%) of positional and sequence homology between mice and rats was in the TATA/proximal regulatory region. This region contains all the inducible regulatory elements (enkCRE1, NF1, AP-2, NFkappaB, etc.) and also an octamer-like element at -543 bp. This high homology is interrupted in both mice and rats by the typically polymorphic d(AC/TG)n and d(TC/GA)n dinucleotide repeats positioned between nucleotides -670 and -950. The position and orientation of these repetitive elements differ substantially in the two species. Genomic PCR analysis of the d(AC/TG)n repeat in various mouse strains, including aberrant behavioral or neurological phenotypes, showed lack of polymorphism at this repeat. The positional and sequence homologies between the rat and the mouse ENK genes decrease in more upstream regions due to the presence of nonhomologues repetititve DNA sequences.

Differential mechanisms involved in the effect of nicotinic agonists DMPP and lobeline to release [3H]5-HT from rat hippocampal slices.

In the present study we investigated the effect of different nicotinic agonists (dimethylphenyl-piperazinium-iodide (DMPP), (-)nicotine, cytisine, (-)-lobeline, and (-)epibatidine) and antagonists (mecamylamine and dihydro-beta-erythroidine) on the release of [3H]5-HT from hippocampal slices. The nicotinic agonists DMPP and lobeline and electrical field stimulation, released [3H]5-HT from the hippocampus; other nicotinic agonists, such as (-)-nicotine, cytisine, and (-)-epibatidine had no effect. Unlike lobeline-induced release of [3H]5-HT, the effect of DMPP (10 and 40 microM) was antagonized by mecamylamine (20 and 10 microM). The effect of DMPP was [Ca2+]o-independent. In experiments carried out at 7 degrees C, i.e. the membrane carrier proteins are inhibited and the release by lobeline was abolished while the DMPP-induced release of 5-HT was rather potentiated. It is proposed that the effect of DMPP and lobeline, to enhance the release of [3H]5-HT from the hippocampus, was mediated by two different mechanisms. While DMPP-induced 5-HT release can be linked to a non-classical nAChR activation ([Ca2+]o-independence), the effect of lobeline was likely mediated by uptake carriers.

Protein-DNA interactions during phenotypic differentiation.

We have been studying the molecular mechanism of neuronal differentiation through which the multipotent precursor becomes limited to the final transmitter phenotype. Here we focused on the role of the 5' proximal regulatory cassette (-190; +53 bp) of the rat enkephalin (rENK) gene in the developmental regulation of the enkephalin phenotype. Several well characterized cis-elements, including AP2, CREB, NF1, and NFkB, reside on this region of the rENK gene. These motifs were sufficient to confer activity-dependent expression of the gene during neurodifferentiation when it was tested using transient transfection assays of primary developing spinal cord neurons treated with tetrodotoxin (TTX). This region was then used as a DNA probe in mobility shift assays, with nuclear proteins derived from phenotypically and ontogenetically distinct brain regions. Only a few low abundance protein-DNA complexes were detected and only with nuclear proteins derived from developing but not from adult brain. The spatiotemporal pattern of these complexes did not show correlation with enkephalin expression which was assessed by RT-PCR. We employed synthetic probes corresponding to consensus as well as ENK-specific sequences of the individual motifs to identify the nature of the observed bands. Although both consensus NF1 and enkCRE1(NF1) formed complexes with nuclear proteins derived from the striatum and cortex at various ages, the appearance of the bands was not correlated with ENK expression. Surprisingly, no complexes were detected if other ENK-specific motifs were used as probes. We also tested nuclear extracts derived from forskolin-induced and control C6 glioma cells, again using the whole proximal regulatory cassette as well as individual motifs. These experiments showed the formation of elaborate protein-DNA bands. There was no direct correlation between the appearance of bands and forskolin-induced ENK expression. Unexpectedly, all ENK-specific motifs formed specific and highly abundant protein-DNA complexes when nuclear extracts from the human tumor cell line (HeLa), which does not express ENK, were used. Based on these observations, we concluded that: 1. Interactions between the proximal regulatory cassette and additional probably far distant regions of the rENK gene and their binding proteins may be necessary to confer developmentally regulated, cell-specific expression of the ENK gene; and 2. Inducibility of the gene by common cis-elements can be governed by this region; however, the cell-specificity of the induction remains elusive.

[Immediate full-range active movement after suturing of the flexor tendon. A new dynamic guiding splint]. / Azonnali, teljes terjedelmü, aktív mozgatás hajlítóín varrat után. Uj, dinamikus vezetösín.

Immediate full range active movement after suture of the flexor tendon. New dynamic guiding splintAuthors emphasize based on literary data, the advantages of the active postoperative movements. A technique for suture and mobilization protocol are offered. They constructed a new dynamic guiding splint assisting the active movement and describe its preparation in details. Their own material, treated with the method suggested, is analysed.

[Does the flexed position of the wrist releave the stress on the suture in the flexor tendon?]. / Tehermentesíti-e a flexióban rögzített csukló a hajlítóín varratát?

Authors have examined the optimal position of the wrist from the viewpoint of active motion during the postoperative care, after the suture of the flexor tendons of the hand. Based on clinical and cadaveric experiences a motion of the wrist (inverse) in contrary direction to the motion of the fingers is suggested i. e. flexion of the fingers in wrist extension and vice versa. The tendon injuries distally and proximally from the wrist are discussed separately.

[Tensile strength of new and modified flexor tendon sutures]. / Uj és módosított hajlítóín-varratok szakító szilárdsági vizsgálata.

Authors tested modified and new sutures and different sewing materials on the deep flexor tendons of the hand in cadavera. Tensile and pull strength examinations were performed. The results were compared with the Kirchmayr-Kessler suture. Suture types suitable for immediate postoperative active motion exercises are suggested.

[Primary tensile strength of newer and modified tendon sutures. A comparative in-vitro study]. / Primäre Zugfestigkeit neuer und modifizierter Beugesehnennähte. Eine vergleichende In-vitro-Untersuchung.

Modified with new methods of flexor tendon sutures compared with the Kessler technique on cadaveric tendons are reported. The authors offer some suturing methods which allow active movement of the reconstructed flexor tendons.

[Measurement of the tensile force exerted on the flexor tendons of the hand]. / Die Messung der auf die Beugesehnen der Hand wirkenden Kraft.

Immediate postoperative active motion after flexor tendon repair is a recurring theme in hand surgery. In the present study the authors determined the tensile stress of the sutured tendon subjected to active motion by the use of cadaveric hands and a tensiometer. The force necessary to draw forth the proximal stump was also measured. A tendon suture which can resist two kilo-ponds tensile force seems to allow active motion of the reconstructed flexor tendon.