Direct duplication of the Y chromosome with normal phenotype - incidental finding in two cases.

Structural rearrangement in the Y chromosome is closely involved in spermatogenesis. However, several Y chromosome variants may have no deleterious effects on male reproduction. Here, we report two cases of Y chromosomal duplication from incidental findings. Their FISH analysis revealed direct duplication of large segments of short and long arms of the Y chromosome. Nearly two intact Y chromosomes were carried in these two cases with normal phenotype.

Genetic testing in spinocerebellar ataxia in Taiwan: expansions of trinucleotide repeats in SCA8 and SCA17 are associated with typical Parkinson's disease.

DNA tests in normal subjects and patients with ataxia and Parkinson's disease (PD) were carried out to assess the frequency of spinocerebellar ataxia (SCA) and to document the distribution of SCA mutations underlying ethnic Chinese in Taiwan. MJD/SCA3 (46%) was the most common autosomal dominant SCA in the Taiwanese cohort, followed by SCA6 (18%) and SCA1 (3%). No expansions of SCA types 2, 10, 12, or dentatorubropallidoluysian atrophy (DRPLA) were detected. The clinical phenotypes of these affected SCA patients were very heterogeneous. All of them showed clinical symptoms of cerebellar ataxia, with or without other associated features. The frequencies of large normal alleles are closely associated with the prevalence of SCA1, SCA2, MJD/SCA3, SCA6, and DRPLA among Taiwanese, Japanese, and Caucasians. Interestingly, abnormal expansions of SCA8 and SCA17 genes were detected in patients with PD. The clinical presentation for these patients is typical of idiopathic PD with the following characteristics: late onset of disease, resting tremor in the limbs, rigidity, bradykinesia, and a good response to levodopa. This study appears to be the first report describing the PD phenotype in association with an expanded allele in the TATA-binding protein gene and suggests that SCA8 may also be a cause of typical PD.

The role of nitric oxide in the development of morphine tolerance in rat hippocampal slices.

In the present study, we investigated the effects of a nitric oxide (NO) precursor, L-arginine, on the effect of different drugs, [trans-3, 4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl]-benzeneacetamid e hydrochloride] (U-50,488, a kappa-opioid receptor agonist); dPTyr(Me)AVP (a vasopressin receptor antagonist); dizocilpine (MK-801, a N-methyl-D-aspartate (NMDA) receptor antagonist), to block the development of morphine tolerance or NO release in Sprague-Dawley rat hippocampal slices (450 microm). Slices were continuously superfused with artificial cerebrospinal fluid (ACSF) or drugs at 1 ml/min. Nichrome wire electrodes were placed in the Schaffer-collateral pathway and used to deliver biphasic 0.2-ms pulses of 5-30 V (0.033 Hz). A glass microelectrode was placed in the CA1 area to record population spikes. The amount of NO released in the superfusate was measured as nitrite formation. When the slices were superfused with 10 microM morphine, the amplitude of population spikes increased 200%-300% in 30-40 min. However, this effect of morphine decreased, i.e., tolerance developed, after continuous superfusion of morphine for 2-6 h. On the other hand, the nitrite level was increased about 250% of the control level through 6 h of morphine superfusion. Co-superfusion of L-arginine with morphine could further increase the nitrite level and also facilitate the development of morphine tolerance. On the other hand, 3-Br-7-nitroindazole (a neuronal NO synthase inhibitor) decreased the nitrite level significantly and blocked the development of morphine tolerance. When either U-50,488 (200 nM) or dPTyr(Me)AVP (500 pM) or MK-801 (500 pM) was co-superfused with morphine (10 microM), the development of morphine tolerance was blocked significantly and the nitrite level decreased to 100%-150% of the control level. L-arginine (500 nM) significantly reversed the effect of these drugs to block the development of morphine tolerance or to decrease the nitrite level through 6 h of superfusion. These data suggest that NO may play a key role in the development of morphine tolerance. Drugs which suppress the synthesis or release of NO would be expected to block the development of morphine tolerance.

SIP1, a novel zinc finger/homeodomain repressor, interacts with Smad proteins and binds to 5'-CACCT sequences in candidate target genes.

Activation of transforming growth factor beta receptors causes the phosphorylation and nuclear translocation of Smad proteins, which then participate in the regulation of expression of target genes. We describe a novel Smad-interacting protein, SIP1, which was identified using the yeast two-hybrid system. Although SIP1 interacts with the MH2 domain of receptor-regulated Smads in yeast and in vitro, its interaction with full-length Smads in mammalian cells requires receptor-mediated Smad activation. SIP1 is a new member of the deltaEF1/Zfh-1 family of two-handed zinc finger/homeodomain proteins. Like deltaEF1, SIP1 binds to 5'-CACCT sequences in different promoters, including the Xenopus brachyury promoter. Overexpression of either full-length SIP1 or its C-terminal zinc finger cluster, which bind to the Xbra2 promoter in vitro, prevented expression of the endogenous Xbra gene in early Xenopus embryos. Therefore, SIP1, like deltaEF1, is likely to be a transcriptional repressor, which may be involved in the regulation of at least one immediate response gene for activin-dependent signal transduction pathways. The identification of this Smad-interacting protein opens new routes to investigate the mechanisms by which transforming growth factor beta members exert their effects on expression of target genes in responsive cells and in the vertebrate embryo.

The Drosophila homeobox genes zfh-1 and even-skipped are required for cardiac-specific differentiation of a numb-dependent lineage decision.

A series of inductive signals are necessary to subdivide the mesoderm in order to allow the formation of the progenitor cells of the heart. Mesoderm-endogenous transcription factors, such as those encoded by twist and tinman, seem to cooperate with these signals to confer correct context and competence for a cardiac cell fate. Additional factors are likely to be required for the appropriate specification of individual cell types within the forming heart. Similar to tinman, the zinc finger- and homeobox-containing gene, zfh-1, is expressed in the early mesoderm and later in the forming heart, suggesting a possible role in heart development. Here, we show that zfh-1 is specifically required for formation of the even-skipped (eve)-expressing subset of pericardial cells (EPCs), without affecting the formation of their siblings, the founders of a dorsal body wall muscle (DA1). In addition to zfh-1, mesodermal eve itself appears to be needed for correct EPC differentiation, possibly as a direct target of zfh-1. Epistasis experiments show that zfh-1 specifies EPC development independently of numb, the lineage gene that controls DA1 founder versus EPC cell fate. We discuss the combinatorial control mechanisms that specify the EPC cell fate in a spatially precise pattern within the embryo.

Sequence and developmental expression of amphioxus AmphiNk2-1: insights into the evolutionary origin of the vertebrate thyroid gland and forebrain.

We characterized an amphioxus NK-2 homeobox gene (AmphiNk2-1), a homologue of vertebrate Nkx2-1, which is involved in the development of the central nervous system and thyroid gland. At the early neurula stage of amphioxus, AmphiNk2-1 expression is first detected medially in the neural plate. By the mid-neurula stage, expression is localized ventrally in the nerve cord and also begins in the endoderm. During the late neurula stage, the ventral neural expression becomes transiently segmented posteriorly and is then down-regulated except in the cerebral vesicle at the anterior end of the central nervous system. Within the cerebral vesicle AmphiNk2-1 is expressed in a broad ventral domain, probably comprising both the floor plate and basal plate regions; this pattern is comparable to Nkx2-1 expression in the mouse diencephalon. In the anterior part of the gut, expression becomes intense in the endostyle (the right wall of the pharynx), which is the presumed homologue of the vertebrate thyroid gland. More posteriorly, there is transitory expression in the midgut and hindgut. In sum, the present results help to support homologies (1) between the amphioxus endostyle and the vertebrate thyroid gland and (2) between the amphioxus cerebral vesicle and the vertebrate diencephalic forebrain.

The pioneer gene, apontic, is required for morphogenesis and function of the Drosophila heart.

In an effort to isolate genes required for heart development and to further our understanding of cardiac specification at the molecular level, we screened PlacZ enhancer trap lines for expression in the Drosophila heart. One of the lines generated in this screen, designated B2-2-15, was particularly interesting because of its early pattern of expression in cardiac precursor cells, which is dependent on the homeobox gene tinman, a key determinant of heart development in Drosophila. We isolated and characterized a gene in the vicinity of B2-2-15 that exhibits an identical expression pattern than the reporter gene of the enhancer trap. The product of his gene, apontic (apt; see also "Gellon et al., 1997"), does not appear to have any homology with known genes. apt mutant embryos show distinct abnormalities in heart morphology as early as mid-embryonic stages when the heart tube assembles, in that segments of heart cells (those of myocardial and pericardial identity) are often missing. Most strikingly, however, apt mutant embryos or larvae only develop a much reduced heart rate, perhaps because of defects in the assembly of an intact heart tube and/or because of defects in the function or physiological control of the myocardial cells, which normally mediate heart contractions. These cardiac defects may be the cause of death of these mutants during late embryonic or early larval stages.

Blockade of the development of morphine tolerance by U-50,488, an AVP antagonist or MK-801 in the rat hippocampal slice.

1. In this study, we investigated the effects of different drugs (a kappa-opioid receptor agonist U-50,488, a vasopressin receptor antagonist dPTyr(Me)AVP or an N-methyl-D-aspartate (NMDA) receptor antagonist MK-801) on the development of morphine tolerance in rat hippocampal slices. 2. Hippocampal slices (450 microm) of Sprague-Dawley rats (250-300 g) were used. Slices were continuously superfused with artificial CSF or drugs at 1 ml min(-1). Nichrome wire electrodes were placed in the Schaffer-collateral pathway and used to deliver biphasic 0.2 ms pulses of 5-30 V (0.033 Hz). A glass microelectrode was placed in the CA1 area to record population spikes. 3. When the slices were superfused with 10 microM morphine, the amplitude of population spikes increased 2-3 fold in 30-40 min. However, this effect of morphine decreased, i.e. tolerance developed after continuous superfusion of morphine for 2-6 h. 4. When either U-50,488 (200 nM) or dPTyr(Me) AVP (500 pM) or MK-801 (500 pM) was co-superfused with morphine (10 microM), it significantly blocked the development of morphine tolerance. Nor-BNI (a kappa-opioid receptor antagonist, 200 nM) significantly reversed the inhibitory effect of U-50,488 but not those of dPTyr(Me)AVP or MK-801 on the development of morphine tolerance. 5. These data indicate that kappa-opioid receptors, AVP receptors and NMDA receptors are all involved in the development of morphine tolerance. The suppression of kappa-opioid receptor activity after chronic morphine may occur before the activation of AVP receptors or NMDA receptors during the development of morphine tolerance.

L-deprenyl (selegiline) decreases excitatory synaptic transmission in the rat hippocampus via a dopaminergic mechanism.

The effect of L-deprenyl (selegiline) on the excitatory synaptic transmission was characterized in the CA1 neurons of rat hippocampal slices by using a intracellular recording technique. Superfusion of L-deprenyl (0.1-10 microM) reversibly decreased the EPSP, which was evoked by orthodromic stimulation of the Schaffer collateral-commissural afferent pathway in a concentration-dependent manner. The sensitivity of postsynaptic neurons to the glutamate receptor agonists, alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid or N-methyl-D-aspartate, was not affected by L-deprenyl (1 microM) pretreatment. In addition, L-deprenyl (1 microM) clearly increased the magnitude of paired-pulse facilitation regardless of the interstimulus intervals of 20 to 300 msec used. The ability of L-deprenyl to decrease the EPSP amplitude was not observed in the dopamine-depleted rats. Pargyline and 4-phenylpyridine, the monoamine oxidase type B inhibitors, mimicked the depressant effect of L-deprenyl on the EPSP. Moreover, the reduction of L-deprenyl (1 microM) on the EPSP amplitude was specifically antagonized by sulpiride (0.01-0.1 microM), a selective dopamine D2 receptor antagonist. However, the dopamine D1 receptor antagonist, SKF-83566 (1-10 microM), did not significantly affect L-deprenyl's action. These results indicate that the monoamine oxidase type B inhibitory ability leading to an increase of the dopaminergic tonus in the hippocampus is involved in the L-deprenyl-induced depression of excitatory synaptic transmission in the CA1 region of the rat hippocampus. Moreover, application of L-deprenyl (1 and 10 microM) also reversibly suppressed the epileptiform activity evoked by picrotoxin.

Combined electrochemical and electrophysiological studies of monoamine overflow in rat hippocampal slices.

In vivo electrochemical measurements of chronoamperometric recordings from Nafion-coated electrodes were used to investigate monoamine overflow from selected regions of the rat hippocampal slice. Concurrent electrophysiological measurements of evoked CA1 pyramidal cell population spike responses were used to characterize changes in the electrical activity in the slices that occur during potassium-induced neurotransmitter overflow. Superfusion with elevated K+ (10-50 mM, 5 min) elicited consistent concentration-dependent increases in the electrochemical responses recorded from the dentate gyrus. At the onset of K+ perfusion, there was an initial increase in the population spike response, followed by electrical silence, which usually lasted 5-10 min following the return to normal medium, and required 20-30 min for complete recovery of the response. The potassium-induced electrochemical signal always increased following the decline in the electrophysiological response. Although the electrochemical signal usually returned to baseline much before the electrophysiological response (usually within 5 min), both signals remained refractory for some time. Cocaine pretreatment (10-50 microM) caused a dose-dependent augmentation of the electrochemical responses. Local pressure ejection of K+ via a micropipette elicited dose-dependent increases in the electrochemical signals that were of relativity brief duration as compared to superfusion with K+. Such potassium-evoked responses were highly localized, and were attenuated in amplitude in animals that had been previously treated with the selective noradrenergic neurotoxin, DSP-4. In addition to K+, local applications of methyl-amphetamine, tyramine and veratridine also elicited electrochemical signals, and the time courses of these responses were specific to the releasing agent that was used. Preliminary data obtained using high-speed electrochemical recordings of both oxidation and reduction current suggested that tyramine ejections evoked primarily norepinephrine overflow, while K+ evoked the overflow of both norepinephrine and serotonin. The present experiments demonstrate that simultaneous electrophysiological and electrochemical experiments can be used in an isolated preparation of brain such as the hippocampal slice to characterize the electrophysiological events that occur during stimulated transmitter release.

Electrochemical characterization of stimulated norepinephrine overflow in locus coeruleus-hippocampus double brain grafts grown in oculo.

Rat locus coeruleus-hippocampus double in oculo brain grafts were studied with high-speed electrochemical techniques to characterize stimulus-evoked overflow of norepinephrine. Local pressure ejections of KCl into the locus coeruleus (LC) portion of the double graft elicited electrochemical signals in the hippocampal portion that had NE-like reduction/oxidation current ratios. In contrast, electrical stimulation of the LC part of the double graft did not lead to consistently detectable signals in the hippocampal portion of the transplant. These data demonstrate that LC graft neurons are able to release NE in target tissues when stimulated in an appropriate manner, and that the time course and magnitude of the overflow of NE can be detected with sensitive high-speed electrochemical recording techniques.

Pertussis toxin pretreatment antagonizes the actions of mu- and delta-opiate agonists in hippocampal slices.

The effect of pertussis toxin pretreatment on electrophysiological responses to selective delta- and mu-opioid receptor agonists was examined in rat hippocampal brain slices. The evoked population spike response in the CA1 region following activation of the Schaffer collateral and commissural afferents was increased following perfusion with the delta-selective agonist [D-Pen2,D-Pen5]enkephalin (DPDPE), and with the mu-selective agonist [D-Ala2,NMe-Phe4,Gly(O)5ol]enkephalin (DAGO). Both effects were significantly reduced or abolished in brain slices obtained from animals that had been pretreated with pertussis toxin 2-3 days earlier. These findings suggest that the excitatory responses to opioid agonists in hippocampus are the result of interactions with receptors that are coupled via pertussis toxin sensitive GTP-binding proteins to their respective effector mechanisms.

Suppressive effects of somatostatin in dog Purkinje fibres.

1. The effects of somatostatin (SS, 1 nM-3 microM) on the electrical and mechanical activities of isolated Purkinje fibres of the dog were studied. 2. In most Purkinje fibres driven electrically in normal [K]o Tyrode solution, SS decreased the force of contraction slightly and had very little effect on the fast response action potential. However, in sensitive fibres SS induced a moderate reduction of action potential duration and contractile force in normal [K]o and depressed the slow response action potentials in high [K]o. 3. In spontaneously beating Purkinje fibres, SS decreased the regular rhythms slightly but abolished bursts of fast rhythms at a concentration as low as 1 nM. 4. When the fibres were depolarized in the presence of 0.2 mM barium or in Na-free solution, SS suppressed the Ca-dependent slow response action potentials. 5. These findings suggest that SS may suppress abnormal automatic activity of dog Purkinje fibres through a reduction of transmembrane Ca influx or a modulation of intracellular calcium.