This article reviewed the beneficial effects of moderate voluntary physical exercise on brain health according to the studies on humans and animals, which includes improving psychological status and cognitive function...This article reviewed the beneficial effects of moderate voluntary physical exercise on brain health according to the studies on humans and animals, which includes improving psychological status and cognitive function, enhancing psychological well-being, decreasing the risks of Alzheimer＇s disease （AD） and dementia, and promoting the effects of antidepressant and anxiolytic. The possible underlying neurobiological mechanisms are involved up-active and down-active pathways. The up-active pathway is associated with enhancements of several neurotransmitters systems afferent to hippocampus, including norepinephrine （NE）, serotonin （5-Hydroxytryptamine, 5-HT）, acetylcholine （ACh） and γ-aminobutyric acid （GABA）. The down-active pathway is mainly concerned with up-regulation of the brain-derived neurotrophic factor （BDNF） and neurogenesis. It is suggested that NE activation via β-adrenergic receptors may be essential for exercise-induced BDNF up-regulation. The possible intracellular signaling pathways of NE-mediated BDNF up-expression may be involved in GPCR-MAPK-PI-3K crosstalk and positive feedback.展开更多
The sympathetic nervous system plays a cardinal role in regulating cardiac function through releasing the neurotransmitter norepinephrine (NE). In comparison with central nervous system, the molecular mechanism of NE ...The sympathetic nervous system plays a cardinal role in regulating cardiac function through releasing the neurotransmitter norepinephrine (NE). In comparison with central nervous system, the molecular mechanism of NE uptake in myocardium is not clear. In present study, we proved that in rat the CNS type of NE transporter (NET) was also expressed in middle cervical-stellate ganglion complex (MC-SG complex) which is considered to control the activity of heart, but not expressed in myocardium. The results also showed that NET expression level in right ganglion was significantly higher than in the left, rendering the greater capacity of NE uptake in right ventricle, a fact which may contribute to the maintenance of right ventricular function under pathologic state.展开更多
Functional dyspepsia is a symptom complex characterised by upper abdominal discomfort or pain, early satiety, motor abnormalities, abdominal bloating and nausea in the absence of organic disease. The central nervous s...Functional dyspepsia is a symptom complex characterised by upper abdominal discomfort or pain, early satiety, motor abnormalities, abdominal bloating and nausea in the absence of organic disease. The central nervous system plays an important role in the conducting and processing of visceral signals. Alterations in brain processing of pain, perception and affective responses may be key factors in the pathogenesis of functional dyspepsia. Central serotonergic and noradrenergic receptor systems are involved in the processing of motor, sensory and secretory activities of the gastrointestinal tract. Visceral hypersensitivity is currently regarded as the mechanism responsible for both motor alterations and abdominal pain in functional dyspepsia. Some studies suggest that there are alterations in central serotonergic and noradrenergic systems which may partially explain some of the symptoms of functional dyspepsia. Alterations in the autonomic nervous system may be implicated in the motor abnormalities and increases in visceral sensitivity in these patients. Noradrenaline is the main neurotransmitter in the sympathetic nervous system and again alterations in the functioning of this system may lead to changes in motor function. Functional dyspepsia causes considerable burden on the patient and society. The pathophysiology of functional dyspepsia is not fully understood but alterations in central processing by the serotonergic and noradrenergic systems may provide plausible explanations for at least some of the symptoms and offer possible treatment targets for the future.展开更多
AIM: To investigate the effects of adrenomedullin (AM) gene overexpression on the biological characteristics of human hepatic stellate cells (hHSCs) by stable transfection.METHODS: hHSCs which express low basal levels...AIM: To investigate the effects of adrenomedullin (AM) gene overexpression on the biological characteristics of human hepatic stellate cells (hHSCs) by stable transfection.METHODS: hHSCs which express low basal levels of AM were stably transfected with an expression construct containing rat AM gene or with an empty expression vector. Expression of AM in hHSCs was determined by reverse transcription (RT)-polymerase chain reaction (PCR) and radioimmunoassay (RIA). Cell proliferation was evaluated by 5-bromo-2'-deoxyuridine (BrdU) incorporation and immunocytochemistry. RT-PCR and Western blot were used to test the expression of procollagen types Ⅰ and Ⅲ. Protein expressions of interstitial collagenase (MMP-1), gelatinase (MMP-2) and tissue inhibitors of matrix metalloproteinases-2 (TIMP-2) were assessed by Western blot.RESULTS: Two cell clones (A-2, A-8) transfected withthe AM gene expressed higher levels of AM mRNA (nontransfected group: 0.86±0.11, empty vector group: 1.01±0.11, A-2 clone group: 1.44±0.08 and A-8 clone group: 1.36±0.05) and protein (12.31±0.17, 12.35±0.12,12.56±0.06 and 12.62±0.07) (P＜0.05). AM geneoverexpression had inhibitory effects on cell proliferation of hHSCs (29.6%, 30.9%, 18.9% and 21.8%, respectively. P＜0.05) and expression of procollagen type Ⅰ (0.58±0.1,0.48±0.11, 0.3±0.06 and 0.31±0.07 at mRNA level)(0.27±0.07, 0.3±0.06, 0.14±0.05 and 0.13±0.05 at protein level) (P＜0.05) and procollagen type Ⅲ (0.17±0.04, 0.15±0.03, 0.1±0.02 and 0.09±0.02 at mRNA level) (0.22±0.04, 0.2±0.03, 0.11±0.04 and 0.13±0.03 at protein level) (P＜0.05). Compared with cells non-transfected (TIMp2: 2.77±0.03, MMP-2: 0.5±0.04, MMP-1: 0.49±0.07) and transfected with empty vector (TIMP2: 2.79±0.04,MMP-2: 0.48±0.03, MMP-1: 0.45±0.09), these two clones had lower expression levels of TIMP2(A-2 clone group: 2.7±0.02 and A-8 clone group: 2.71±0.02) (P＜0.05) and MMP-2(A-2 clone group: 0.15±0.05 and A-8 clone group: 0.13±0.04) (P＜0.05) but displayed a higher expression level of MMP-1(A-2 clone group: 0.68±0.06 and A-8 clone group: 0.81±0.09) (P＜0.05).CONCLUSION: AM gene exerts negative influence to some extent on hHSCs by inhibiting proliferation and production of extracellular matrix (ECM) in addition to inducing MMP-1 expression.展开更多
Norepinephrine(NE)endogenously released following electrical field stimulation(prazosin and TTX sensitive responses),produced a biphasic contraction of the rat vas deferens(RVD).The initial transient contraction was d...Norepinephrine(NE)endogenously released following electrical field stimulation(prazosin and TTX sensitive responses),produced a biphasic contraction of the rat vas deferens(RVD).The initial transient contraction was decreased by 30μmol/L ryanodine andμmol/L nifedipine while the secondary component was abolished by 2μmol/L nifedipine but increased by 30μmol/L ryanodine.Exogenously added NE produced biphasic contractions of the RVD.These contractions were inhibited by 2μmol/L nifedipine.Ryanodine(30μmol/L)decreased both phases by about 50%.We conclude that ryanodine binding sites reside in RVD endoplasmic reticulum(ER).There was a lack of uniformity in the effect of ryanodine against different phases of alpha-adrenergic stimulation may be indicative of two modes of stimulation-contraction coupling process related to this stimulation.展开更多
The human norepinephrine transporter(NET) gene was cloned and structurally analyzed. The far 5’ fragment containing exon 1 (a non-coding exon) and exon 2 was sequenced. The transcription start site of the gene in hum...The human norepinephrine transporter(NET) gene was cloned and structurally analyzed. The far 5’ fragment containing exon 1 (a non-coding exon) and exon 2 was sequenced. The transcription start site of the gene in human brain stem tissue was determined by primer extension analysis. It was found that the gene could be transcribed from multiple starting points. The 5’ flanking sequence contains a proximal G-C rich region, one possible GSG elemeflt and several SP1 sites. However it does not contain TATA box and CAAT box motifS. Gel shift analysis with nuclear extracts from different tissues of mouse shows that the G-C rich region may be involved in tissue specific expression of the gene.展开更多
In this research, Lysolecithin-a substance made with 100% natural ingredients - was given to ICR mice as medication to measure its periodic effect on the noradrenalin (NA), dopamine (DA), and serotonin (5-HT) levels o...In this research, Lysolecithin-a substance made with 100% natural ingredients - was given to ICR mice as medication to measure its periodic effect on the noradrenalin (NA), dopamine (DA), and serotonin (5-HT) levels of the brain. Both ICR and SAM mice were separated into two groups - control group and Lysolecithin (K. Lysolecithin: hydrolytic lysolecithin) medicated group, and given 1-week preparation period. The K. Lysolecithin group was given 500mg/kg of K. Lysolecithin at 0.2mL per dosage for 4 weeks, and the control group was given the same amount of dosage of water during the same period. NA, DA and 5-HT concentrations were measured from the blood before medication and 8 weeks / 12 weeks / 16 weeks after the first medication. For the SAM mice, 8 weeks after they were medicated with K .Lysolecithin, Morris Water Maze Test was conducted for 7 consecutive days and then the concentrations were measured by drawing blood from the heart. The K. Lysolecithin medicated group showed a tendency to have a statistically significant higher concentrations of 5-HT and NA in the blood. Also, periodic examination showed that the monoamine levels were highest in the 12th week and declined thereafter.展开更多
The norepinephrine transporter(NET) is a member of the Na+/Cl- dependent neurotransmitter transporter family and constitutes the target of several clinically important antidepressants. To delineate the critical amino ...The norepinephrine transporter(NET) is a member of the Na+/Cl- dependent neurotransmitter transporter family and constitutes the target of several clinically important antidepressants. To delineate the critical amino acid residues and the function of C-terminal in regulating transport activity of NET, here we constructed two site mutants (V70F, F72V; V70I, F72V) and one C-terminal truncated mutant (△611-617). The wild type and mutants of NET were expressed in Xenopus oocytes by injection of their cRNA. We found that all of these mutants lost their transport activity. These results indicate that the amino acid residues of V70 and F72 3 and the last seven amino acids of C-terminal are essential to the transport activity of NET.展开更多
Venlafaxine, an SNRI （serotonin-norepinephrine reuptake inhibitor） drug, is commonly used to treat depression. Although it is very rare, venlafaxine may lead to addiction or abuse in some patients. To date, there ar...Venlafaxine, an SNRI （serotonin-norepinephrine reuptake inhibitor） drug, is commonly used to treat depression. Although it is very rare, venlafaxine may lead to addiction or abuse in some patients. To date, there are very limited data available on venlafaxine addiction. The aim of this article is to draw an attention to dependence of venlafaxine in clinical practice. Significant point of this article is： Our patient was addicted by ＂high＂ doses of venlafaxine for getting high effects. Overdose of venlafaxine produces an amphethamine-like ＂high＂ with experiences of euphoria by its ability to increase neurotransmitter levels in addiction literature [1-3]. We present a man aged 42 years, who was referred to our clinic for detoxification from venlafaxine addiction. He had been taking venlafaxine upto 1,950-2,100 mg/day to get high for the past 10 years. Physicians must be careful when prescribing antidepressants to patients, especially those with a history of alcohol and/or drug addictions or abuse.展开更多
The effects of four ions and eight neuroactive compounds on inducing larval settlement ofA. japonicus were assessed in the present study. All bioassays were conducted in 60 × 9mm Petri dishes, each contained 10mL...The effects of four ions and eight neuroactive compounds on inducing larval settlement ofA. japonicus were assessed in the present study. All bioassays were conducted in 60 × 9mm Petri dishes, each contained 10mL of the test solution and 10 doliolaria larvae. There were significant inductive effects of K＋ （10 mmol L-l）, NH4＋ （0.1 mmol L-l）, GABA （10-3 tool L-l）, acetylcholine （10-5 molL-l）, L-DOPA （10-SmolL-1）, norepinephrine （10-SmolL-1） and dopamine （10-TmolL-1 and 10-5 molL-1） on the settlement of sea cucumber larvae. L-DOPA and dopamine are the most efficient chemical cues to induce A.japonicus larvae to settle. The highest percentage of larval settlement was induced by 10-5 tool L-1 L-DOPA and dopamine （33% and 40%） compared to the control （7%）. However, Ca2＋, Mg2＋, choline, serotonin, and epinephrine were less effective on larval settlement at all tested concentrations. This study evaluated the stability and feasibility of chemical cues for larval settlement in different culture systems, which can be applied to improve the hatchery production of this valuable species.展开更多
A cDNA molecule encoding a major part of the hu-man Norepinephrine transporter(hNET) was synthesized by means of Polymerase Chain Reaction(PCR) technique and used as a probe for selecting the human genomic NET gene. A...A cDNA molecule encoding a major part of the hu-man Norepinephrine transporter(hNET) was synthesized by means of Polymerase Chain Reaction(PCR) technique and used as a probe for selecting the human genomic NET gene. A positive clone harbouring the whole gene was ob-tained from a human lymphocyte genomic library through utilizing the "genomic walking" technique. The clone, des-ignated as phNET, harbours a DNA fragment of about 59 kb in length inserted into BamH Ⅰ site in cosmid pWE15.The genomic clone contains 14 exons encoding all amino acid residues in the protein. A single exon encodes a dis-tinct transmembrane domaill, except for transmembrane domain 10 and 11, which are encoded by part of two ex-ons respectively, and exon 12, which encodes part of do-main 11 and all of domain 12. These results imply that there is a close relationship between exon splicing of a gene and structural domains of the protein, as is the case for the human γ-aminobutyric acid transporter(hGAT) and a number of other membrane proteins.展开更多
基金the National Natural Science Fundation of China (No. 30570895,No. 30700389)
文摘This article reviewed the beneficial effects of moderate voluntary physical exercise on brain health according to the studies on humans and animals, which includes improving psychological status and cognitive function, enhancing psychological well-being, decreasing the risks of Alzheimer＇s disease （AD） and dementia, and promoting the effects of antidepressant and anxiolytic. The possible underlying neurobiological mechanisms are involved up-active and down-active pathways. The up-active pathway is associated with enhancements of several neurotransmitters systems afferent to hippocampus, including norepinephrine （NE）, serotonin （5-Hydroxytryptamine, 5-HT）, acetylcholine （ACh） and γ-aminobutyric acid （GABA）. The down-active pathway is mainly concerned with up-regulation of the brain-derived neurotrophic factor （BDNF） and neurogenesis. It is suggested that NE activation via β-adrenergic receptors may be essential for exercise-induced BDNF up-regulation. The possible intracellular signaling pathways of NE-mediated BDNF up-expression may be involved in GPCR-MAPK-PI-3K crosstalk and positive feedback.
文摘The sympathetic nervous system plays a cardinal role in regulating cardiac function through releasing the neurotransmitter norepinephrine (NE). In comparison with central nervous system, the molecular mechanism of NE uptake in myocardium is not clear. In present study, we proved that in rat the CNS type of NE transporter (NET) was also expressed in middle cervical-stellate ganglion complex (MC-SG complex) which is considered to control the activity of heart, but not expressed in myocardium. The results also showed that NET expression level in right ganglion was significantly higher than in the left, rendering the greater capacity of NE uptake in right ventricle, a fact which may contribute to the maintenance of right ventricular function under pathologic state.
基金the Health Research Board(Ireland),Science Foundation Ireland through the Alimentary Pharmabiotic Centre and the Wellcome Trust.
文摘Functional dyspepsia is a symptom complex characterised by upper abdominal discomfort or pain, early satiety, motor abnormalities, abdominal bloating and nausea in the absence of organic disease. The central nervous system plays an important role in the conducting and processing of visceral signals. Alterations in brain processing of pain, perception and affective responses may be key factors in the pathogenesis of functional dyspepsia. Central serotonergic and noradrenergic receptor systems are involved in the processing of motor, sensory and secretory activities of the gastrointestinal tract. Visceral hypersensitivity is currently regarded as the mechanism responsible for both motor alterations and abdominal pain in functional dyspepsia. Some studies suggest that there are alterations in central serotonergic and noradrenergic systems which may partially explain some of the symptoms of functional dyspepsia. Alterations in the autonomic nervous system may be implicated in the motor abnormalities and increases in visceral sensitivity in these patients. Noradrenaline is the main neurotransmitter in the sympathetic nervous system and again alterations in the functioning of this system may lead to changes in motor function. Functional dyspepsia causes considerable burden on the patient and society. The pathophysiology of functional dyspepsia is not fully understood but alterations in central processing by the serotonergic and noradrenergic systems may provide plausible explanations for at least some of the symptoms and offer possible treatment targets for the future.
基金Supported by the National Natural Scientific Foundation of China,No.30170417
文摘AIM: To investigate the effects of adrenomedullin (AM) gene overexpression on the biological characteristics of human hepatic stellate cells (hHSCs) by stable transfection.METHODS: hHSCs which express low basal levels of AM were stably transfected with an expression construct containing rat AM gene or with an empty expression vector. Expression of AM in hHSCs was determined by reverse transcription (RT)-polymerase chain reaction (PCR) and radioimmunoassay (RIA). Cell proliferation was evaluated by 5-bromo-2'-deoxyuridine (BrdU) incorporation and immunocytochemistry. RT-PCR and Western blot were used to test the expression of procollagen types Ⅰ and Ⅲ. Protein expressions of interstitial collagenase (MMP-1), gelatinase (MMP-2) and tissue inhibitors of matrix metalloproteinases-2 (TIMP-2) were assessed by Western blot.RESULTS: Two cell clones (A-2, A-8) transfected withthe AM gene expressed higher levels of AM mRNA (nontransfected group: 0.86±0.11, empty vector group: 1.01±0.11, A-2 clone group: 1.44±0.08 and A-8 clone group: 1.36±0.05) and protein (12.31±0.17, 12.35±0.12,12.56±0.06 and 12.62±0.07) (P＜0.05). AM geneoverexpression had inhibitory effects on cell proliferation of hHSCs (29.6%, 30.9%, 18.9% and 21.8%, respectively. P＜0.05) and expression of procollagen type Ⅰ (0.58±0.1,0.48±0.11, 0.3±0.06 and 0.31±0.07 at mRNA level)(0.27±0.07, 0.3±0.06, 0.14±0.05 and 0.13±0.05 at protein level) (P＜0.05) and procollagen type Ⅲ (0.17±0.04, 0.15±0.03, 0.1±0.02 and 0.09±0.02 at mRNA level) (0.22±0.04, 0.2±0.03, 0.11±0.04 and 0.13±0.03 at protein level) (P＜0.05). Compared with cells non-transfected (TIMp2: 2.77±0.03, MMP-2: 0.5±0.04, MMP-1: 0.49±0.07) and transfected with empty vector (TIMP2: 2.79±0.04,MMP-2: 0.48±0.03, MMP-1: 0.45±0.09), these two clones had lower expression levels of TIMP2(A-2 clone group: 2.7±0.02 and A-8 clone group: 2.71±0.02) (P＜0.05) and MMP-2(A-2 clone group: 0.15±0.05 and A-8 clone group: 0.13±0.04) (P＜0.05) but displayed a higher expression level of MMP-1(A-2 clone group: 0.68±0.06 and A-8 clone group: 0.81±0.09) (P＜0.05).CONCLUSION: AM gene exerts negative influence to some extent on hHSCs by inhibiting proliferation and production of extracellular matrix (ECM) in addition to inducing MMP-1 expression.
基金This work was supported bv a granl-in-aid awarded by the Medical Re-search Council of Canadaa Career Investigator Awand(CY Kwan)from the Heart and Stroke Foun-dation of Ontario.
文摘Norepinephrine(NE)endogenously released following electrical field stimulation(prazosin and TTX sensitive responses),produced a biphasic contraction of the rat vas deferens(RVD).The initial transient contraction was decreased by 30μmol/L ryanodine andμmol/L nifedipine while the secondary component was abolished by 2μmol/L nifedipine but increased by 30μmol/L ryanodine.Exogenously added NE produced biphasic contractions of the RVD.These contractions were inhibited by 2μmol/L nifedipine.Ryanodine(30μmol/L)decreased both phases by about 50%.We conclude that ryanodine binding sites reside in RVD endoplasmic reticulum(ER).There was a lack of uniformity in the effect of ryanodine against different phases of alpha-adrenergic stimulation may be indicative of two modes of stimulation-contraction coupling process related to this stimulation.
文摘The human norepinephrine transporter(NET) gene was cloned and structurally analyzed. The far 5’ fragment containing exon 1 (a non-coding exon) and exon 2 was sequenced. The transcription start site of the gene in human brain stem tissue was determined by primer extension analysis. It was found that the gene could be transcribed from multiple starting points. The 5’ flanking sequence contains a proximal G-C rich region, one possible GSG elemeflt and several SP1 sites. However it does not contain TATA box and CAAT box motifS. Gel shift analysis with nuclear extracts from different tissues of mouse shows that the G-C rich region may be involved in tissue specific expression of the gene.
文摘In this research, Lysolecithin-a substance made with 100% natural ingredients - was given to ICR mice as medication to measure its periodic effect on the noradrenalin (NA), dopamine (DA), and serotonin (5-HT) levels of the brain. Both ICR and SAM mice were separated into two groups - control group and Lysolecithin (K. Lysolecithin: hydrolytic lysolecithin) medicated group, and given 1-week preparation period. The K. Lysolecithin group was given 500mg/kg of K. Lysolecithin at 0.2mL per dosage for 4 weeks, and the control group was given the same amount of dosage of water during the same period. NA, DA and 5-HT concentrations were measured from the blood before medication and 8 weeks / 12 weeks / 16 weeks after the first medication. For the SAM mice, 8 weeks after they were medicated with K .Lysolecithin, Morris Water Maze Test was conducted for 7 consecutive days and then the concentrations were measured by drawing blood from the heart. The K. Lysolecithin medicated group showed a tendency to have a statistically significant higher concentrations of 5-HT and NA in the blood. Also, periodic examination showed that the monoamine levels were highest in the 12th week and declined thereafter.
文摘The norepinephrine transporter(NET) is a member of the Na+/Cl- dependent neurotransmitter transporter family and constitutes the target of several clinically important antidepressants. To delineate the critical amino acid residues and the function of C-terminal in regulating transport activity of NET, here we constructed two site mutants (V70F, F72V; V70I, F72V) and one C-terminal truncated mutant (△611-617). The wild type and mutants of NET were expressed in Xenopus oocytes by injection of their cRNA. We found that all of these mutants lost their transport activity. These results indicate that the amino acid residues of V70 and F72 3 and the last seven amino acids of C-terminal are essential to the transport activity of NET.
文摘Venlafaxine, an SNRI （serotonin-norepinephrine reuptake inhibitor） drug, is commonly used to treat depression. Although it is very rare, venlafaxine may lead to addiction or abuse in some patients. To date, there are very limited data available on venlafaxine addiction. The aim of this article is to draw an attention to dependence of venlafaxine in clinical practice. Significant point of this article is： Our patient was addicted by ＂high＂ doses of venlafaxine for getting high effects. Overdose of venlafaxine produces an amphethamine-like ＂high＂ with experiences of euphoria by its ability to increase neurotransmitter levels in addiction literature [1-3]. We present a man aged 42 years, who was referred to our clinic for detoxification from venlafaxine addiction. He had been taking venlafaxine upto 1,950-2,100 mg/day to get high for the past 10 years. Physicians must be careful when prescribing antidepressants to patients, especially those with a history of alcohol and/or drug addictions or abuse.
基金supported by the grants from National Marine Public Welfare Research Program (201205023)the Scientific and Technical Supporting Program (2011 BAD13B03)Shandong Seed Project
文摘The effects of four ions and eight neuroactive compounds on inducing larval settlement ofA. japonicus were assessed in the present study. All bioassays were conducted in 60 × 9mm Petri dishes, each contained 10mL of the test solution and 10 doliolaria larvae. There were significant inductive effects of K＋ （10 mmol L-l）, NH4＋ （0.1 mmol L-l）, GABA （10-3 tool L-l）, acetylcholine （10-5 molL-l）, L-DOPA （10-SmolL-1）, norepinephrine （10-SmolL-1） and dopamine （10-TmolL-1 and 10-5 molL-1） on the settlement of sea cucumber larvae. L-DOPA and dopamine are the most efficient chemical cues to induce A.japonicus larvae to settle. The highest percentage of larval settlement was induced by 10-5 tool L-1 L-DOPA and dopamine （33% and 40%） compared to the control （7%）. However, Ca2＋, Mg2＋, choline, serotonin, and epinephrine were less effective on larval settlement at all tested concentrations. This study evaluated the stability and feasibility of chemical cues for larval settlement in different culture systems, which can be applied to improve the hatchery production of this valuable species.
文摘A cDNA molecule encoding a major part of the hu-man Norepinephrine transporter(hNET) was synthesized by means of Polymerase Chain Reaction(PCR) technique and used as a probe for selecting the human genomic NET gene. A positive clone harbouring the whole gene was ob-tained from a human lymphocyte genomic library through utilizing the "genomic walking" technique. The clone, des-ignated as phNET, harbours a DNA fragment of about 59 kb in length inserted into BamH Ⅰ site in cosmid pWE15.The genomic clone contains 14 exons encoding all amino acid residues in the protein. A single exon encodes a dis-tinct transmembrane domaill, except for transmembrane domain 10 and 11, which are encoded by part of two ex-ons respectively, and exon 12, which encodes part of do-main 11 and all of domain 12. These results imply that there is a close relationship between exon splicing of a gene and structural domains of the protein, as is the case for the human γ-aminobutyric acid transporter(hGAT) and a number of other membrane proteins.
