Molecular and cellular pharmacology
New insights in endogenous modulation of ligand-gated ion channels: Histamine is an inverse agonist at strychnine sensitive
glycine receptors
Olaf Kletke b,Olga A.Sergeeva b,Philipp Lorenz a,Sonja Oberland d,Jochen C.Meier c,
Hanns Hatt a,Günter Gisselmann a,n
a Department of Cell Physiology,Ruhr-University-Bochum,44801Bochum,Germany
b Department of Neurophysiology,Medical Faculty,Heinrich-Heine-University,40225Düsseldorf,Germany
c RNA Editing an
d Hyperexcitability Disorders Helmholtz Group,Max Delbrück Center for Molecular Medicine,13125Berlin,Germany
d FU-Berlin,Fachbereich Biologie,Chemie,Pharmazie,Takustr.3,14195Berlin und Charité—Universit?tsmedizin Berlin,Cluster of Excellenc
e NeuroCure,
Charitéplatz1,10117Berlin,Germany
a r t i c l e i n f o
Article history:
Received9November2012
Received in revised form
28March2013
Accepted3April2013
Available online17April2013
Keywords:
Glycine
Histamine
Modulation
Ligand-gated ion channel
Endogenous modulator
Competitive antagonist
a b s t r a c t
Histamine is involved in many physiological functions in the periphery and is an important neuro-
transmitter in the brain.It acts on metabotropic H1–H4receptors mediating vasodilatation,bronchocon-
striction and stimulation of gastric acid secretion.In the brain histamine is produced by neurons in the
tuberomamillary nucleus(TMN),which controls arousal.Histamine is also a positive modulator of the
inhibitory Cys-loop ligand-gated ion channel GABA A.We investigated now its effect on the second
member of inhibitory Cys-loop ligand-gated ion channels,the strychnine sensitive glycine receptor.We
expressed different human and rat glycine receptor subunits in Xenopus laevis oocytes and characterized
the effect of histamine using the two electrode voltage clamp technique.Furthermore we investigated
native glycine receptors in hypothalamic neurons using the patch-clamp technique.Histamine inhibited
α1βglycine receptors with an IC50of5.270.3mM.In presence of10mM histamine the glycine dose–
response curve was shifted,increasing the EC50for glycine from25.571.4μM to42.472.3μM.In
addition,histamine blocked the spontaneous activity of RNA-editedα3βglycine receptors.Histamine
inhibited glycine receptors expressed in hypothalamic TMN neurons with an IC50of4.670.3mM.Our
results give strong evidence that histamine is acting on the same binding site as glycine,being an inverse
agonist that competitively antagonizes glycine receptors.Thus,we revealed histamine as an endogenous
modulator of glycine receptors.
&2013Elsevier B.V.All rights reserved.
1.Introduction
Histamine is known as a crucial cytokine in the periphery and
an important neurotransmitter in the brain.It is produced and
released by basophiles,mast cells,neurons and enterochromaf?n-
like cells of the stomach.Central histamine regulates multiple
functions including cognition,energy homeostasis,body tempera-
ture and in?ammatory responses(Haas et al.,2008;Schwartz
et al.,1991).In the periphery,histamine causes vasodilatation,
bronchoconstriction(Leurs et al.,1995)and stimulates gastric acid
secretion(Arrang et al.,1983).These physiological functions of
histamine are thought to be mediated via metabotropic histamine
receptors(H1–H4)(Hill et al.,1997),but several evidences point to
a possible involvement of histamine-gated ion channels from
different ligand-gated ion channel species(reviwed by Fleck
et al.,2012).
Glycine released from interneurons controls motor and sensory
pathways(Legendre,2001)by activating strychnine-sensitive
glycine receptors which belong to the‘Cys-loop’ligand-gated ion
channels(Laube et al.,2002;Lynch,2004).Fourαsubunits(α1–α4)
and oneβsubunit have been identi?ed to be expressed as homo-
(α)or heteropentamers(αβ)in the mammalian nervous system
(Bormann et al.,1994),with exception ofα4,which is a pseudo-
gene in humans(Simon et al.,2004)and little evidence for a
functional expression was found in rats(Piechotta et al.,2001).
Several allosteric agents and modulators for glycine receptors
are known to share their pharmacological pro?le with GABA A
receptors(Laube et al.,2002).In contrast to that,the taurine
analog guanidinoethyl sulfonate shows agonistic properties at
striatal GABA A receptors but is a competitive antagonist at native
glycine receptor(Sergeeva et al.,2002).Previously,we showed
that histamine gatedβ2/3homomeric GABA A receptors with a
higher ef?cacy than GABA and that histamine is a positive
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European Journal of Pharmacology
0014-2999/$-see front matter&2013Elsevier B.V.All rights reserved.
https://www.doczj.com/doc/218142906.html,/10.1016/j.ejphar.2013.04.002
n Correspondence to:Ruhr-University Bochum,Department of Cell Physiology,
Building ND4-164,44780Bochum,Germany.Tel.:+492343224106;
fax:+492343214129.
E-mail address:guenter.gisselmann@rub.de(G.Gisselmann).
European Journal of Pharmacology710(2013)59–66
allosteric modulator ofα1β2andα1β2γ2L GABA A receptors(Saras et al.,2008).In two preliminary reports,the fact that histamine is a negative modulator of glycine receptors was mentioned(Kletke et al.,2009,Fleck et al.,2012),however,no detailed experimental data were presented.In this study we analyzed the interaction of histamine with all possible human glycine receptor subunit combinations expressed in Xenopus oocytes and with native hypothalamic glycine receptor.We found that histamine does not gate but competitively antagonizes native and recombinant glycine receptor,being an inverse agonist.
2.Material and methods
2.1.Glycine receptor cDNA and RNAs
Human glycine receptor subunitsα1–2andβcDNAs were courtesy of H.Betz,Frankfurt am Main,Germany(Schmieden et al.,1989;Taleb and Betz,1994).Humanα3cDNA was obtained from RZPD(Berlin,Germany),and ratα3andβcDNAs were generated as described in Meier et al.(2005).All cDNAs were subcloned into pSGEM by PCR-based methods(courtesy of M. Hollmann,Bochum,Germany)using standard molecular biology procedures.
2.2.Expression of receptor cDNA in Xenopus oocytes
Humanα1,α2andβ,as well as ratα3andβglycine receptor subunit cRNAs were generated using the AmpliCap T7high yield message maker kit(Epicenter,Madison,WI)regarding manufac-turer's protocol.Pac I linearized pSGEM plasmids were used as templates.Humanα3cDNA delivered in the pBluescriptR Vector by ImaGenes(Berlin,Germany)was linearized with Sca I and cRNA was synthesized using the mScript mRNA Production System (Epicenter,Madison,WI)https://www.doczj.com/doc/218142906.html,evis oocytes were prepared using standard methods.24h after surgery,stage V–IV oocytes were selected and injected with5–15ng cRNA per oocyte and incubated at171C in ND96(96mM NaCl,2mM KCl,1.8mM CaCl2,1mM MgCl2,5mM HEPES),pH7.2,100U/ml Penicillin,100U/ml strep-tomycin from Antibiotic Antimycotic Solution(100?)(Sigma-Aldrich,St.Louis,MO).After3–7days,oocytes were measured by two-electrode voltage clamp,as described previously(Sergeeva et al.,2010).All tested substances were diluted to the indicated concentration with Frog-Ringer's solution(115mM NaCl,2.5mM KCl, 1.8mM CaCl2,10mM HEPES,pH7.2)and histamine-containing solutions were adjusted to pH7.2if necessary.Mea-surements were done at a membrane potential of?40to?60mV.
2.3.Electrophysiological recordings from mouse TMN neurons
From the brains of three to?ve week old male C57Black6mice, 400μm thick transverse slices containing the TMN region were cut and incubated for1h in a solution containing124mM NaCl, 3.7mM KCl, 1.24mM NaH2PO4, 1.3mM MgSO4, 2.0mM CaCl2, 26mM NaHCO3,20mM glucose,saturated with95%O2and5% CO2prior to transferring to a chamber?lled with the recording solution(150mM NaCl, 3.7mM KCl, 2.0mM CaCl2, 2.0mM MgCl2,10mM HEPES,10mM glucose,pH7.4).Individual neurons were separated by vibrodissociation from the hypothalamic slice (Vorobjev,1991)and recorded with the whole-cell voltage clamp technique as previously described(Sergeeva et al.,2010).The electrode solution was composed of140mM KCl,2mM MgCl2, 0.5mM CaCl2,5mM EGTA,2mM ATP and10mM HEPES (pH7.2adjusted with NaOH).
A fast perfusion technique was used for application of ligands and modulators of fast ionotropic receptors(Vorobjev et al.,1996).TMN neurons were identi?ed according to their size and typical sodium inward current activated by a15ms-long depolarizing step (+40mV from the holding potential?50mV).
2.4.Analysis and statistics
Currents recorded with pCLAMP6and CellWorks were ana-lyzed using pCLAMP10software.Statistical analysis and curve ?tting(3or4-parameter Hill equation)was processed using SigmaPlot V8.0(Systat Software,San Jose,CA).All mean values are7S.E.M.
3.Results
3.1.The action of histamine at recombinant glycine receptors
We investigated the action of histamine on recombinantly expressed human glycine receptors.3–7days after injection of 15ng corresponding cRNA mixtures in https://www.doczj.com/doc/218142906.html,evis oocytes,elicited currents were recorded in the voltage clamp con?guration. All results were summarized in Table1.
Atα1βglycine receptors histamine inhibited glycine elicited currents,whereas histamine alone had no effect(Fig.1A). To characterize the inhibitory effect,we recorded dose–inhibition curves for histamine with glycine concentrations near the EC10, EC50and EC95(Fig.1B and C).Increasing concentrations of glycine decreased the inhibitory effect of histamine.Depending on the glycine concentration,the IC50of histamine varied from2.8to
38.5mM(Table1).Maximal histamine concentrations resulted in
a nearly complete inhibition of the glycine-evoked currents.In addition,the dose–response curve for glycine was shifted right-wards by10mM histamine,increasing the EC50for glycine, changing neither the steepness nor the maximal evoked current (Fig.1D,Table1).
In recombinant expression systems,the agonist EC50on glycine receptors depends on the receptor expression level as described by Taleb&Betz(1994).To investigate whether this also in?uences histamine inhibition,we expressed different amounts of cRNA to
Table1
Histamine inhibition of different glycine receptor subtypes.Statistic p-values are given for the comparison of glycine and glycine plus10mM histamine,n.s.?not signi?cant.Values represent mean7S.E.M.
Glycine
[μM]
Glycine
+10mM
histamine
Statistic
p-value
IC50Histamine
[mM]
hsα1EC5013.6070.14 3.7670.47
(n?3)(n?3;～EC30) hsα1βEC5025.571.842.472.3o0.001 2.7970.60
(n?6)(n?6)(n?6;～EC10) nHill 2.670.2 2.570.1n.s. 5.2370.32
(n?5;～EC50)
I max0.9970.010.9670.01n.s.38.4975.26
(n?5;～EC95)
hsα2EC5030.4470.42 1.7270.27
(n?3)(n?3;～EC50) hsα2βEC5082.470.112.3574.27
(n?3)(n?5;～EC70) EC50454728702737o0.001
(n?7)
hsα3βnHill 2.170.1 2.170.1n.s. 6.4070.5
I max 1.170.02 1.070.03n.s.(n?13;～EC30) rnα3βEC5078.2277.828.4471.16
(n?6)(n?4;～EC30)
rnα3P185LβEC5017.9672.3931.1073.64o0.01 3.9870.59
(n?7)(n?4)(n?4;～EC30)
O.Kletke et al./European Journal of Pharmacology710(2013)59–66 60
generate receptors with a variable EC 50in Xenopus oocytes (Fig.1E).Reducing the injected cRNA amount resulted in an increased glycine EC 50:15ng:25.571.8μM (n ?6,I max ?10.270.8μA)and 5ng:50.472.0μM (n ?4,I max ?7.170.9μA).α1βglycine receptors with lower expression levels (5ng)showed a less pronounced right shift of the glycine dose –response curve by 10mM histamine from 50.4μM to 60.473.7μM (n ?4)compared to oocytes expressing high levels of α1βglycine receptors (25.5μM to 42.472.3μM;p o 0.05).
As the results obtained above are pointing to a competitive mechanism of histaminergic inhibition,we were interested,whether histamine inhibition is general for all human glycine receptors.Thus,we recorded dose –inhibition curves with hista-mine for α2β,α3βand for homopentameric α1and α2glycine receptors (Supplementary Fig.1,Fig.2A)and observed a similar inhibition as described for α1βglycine receptors.With an IC 50of 1.72mM,the homopentameric glycine receptor α2was most sensitive to histamine (Table 1).In the next experiments,we addressed the question if the histamine inhibition is species speci ?c and if RNA editing changes histamine inhibition.We found
that histamine acts as an antagonist at both,human and rat α3βglycine receptors with a comparable potency (Fig.2,Table 1).3.2.Posttranscriptional RNA edited glycine receptors were more sensitive to histamine
Next,we tested if RNA editing changes histamine inhibition.Posttranscriptional RNA editing of the rat α3subunit transcript was described (Meier et al.,2005),which leads to increased agonist potencies.We expressed edited rat α3P185L βglycine receptors in oocytes and saw a signi ?cantly increased glycine potency com-pared to the non-edited receptors (Table 1).Compared to the non-edited α3βglycine receptor,the IC 50for histamine was signi ?cantly lowered at α3P185L βglycine receptors (Fig.2C)and in the presence of 10mM histamine,the EC 50for glycine was signi ?cantly increased (Fig.2D).
Interestingly,when expressed in Xenopus oocytes,α3P185L βchannels show a spontaneous activity in absence of the agonist.Picrotoxin (PTX),an open channel blocker for glycine receptors (Pribilla et al.,1992)inhibited this activity (Fig.2E)whereas
the
Fig.1.The inhibitory effect of histamine on human α1βglycine receptors.(A)Inhibitory effect of histamine on oocytes expressing α1βglycine receptors.Histamine decreased the glycine-evoked current,but histamine alone had no effect.(B)Representative trace of histamine inhibition.Increasing concentrations of histamine inhibited glycine-evoked currents in a dose-dependent manner.(C)Dose –inhibition curve of histamine with various glycine-concentrations near the EC 10[●],EC 50[○]and EC 95[▼]for glycine (10,300100μM).(D)Averaged dose –response curve for glycine [●]and glycine plus 10mM histamine [○].Histamine lowered the EC 50for glycine affecting neither the Hill-coef ?cient nor the maximum evoked current.(E)Variety of EC 50s corresponding to the glycine receptor expression level.The EC 50for glycine increased from high (15ng injected cRNA per oocyte [●])over medium (5ng per oocyte [○])to low expression levels (1ng per oocyte [▼]).(F)Dose –response curve for glycine [●]and glycine plus 10mM histamine [○]at oocytes with a medium expression level (5ng per oocyte)for α1βglycine receptors,with a less pronounced right shift compared to oocytes with high glycine receptor expression levels (D).
O.Kletke et al./European Journal of Pharmacology 710(2013)59–6661
competitive antagonist strychnine (1μM)had no effect (data not shown).With 10mM histamine and low glycine concentrations,the spontaneous activity was decreased about 0.2070.04(n ?4)in relation to the maximum glycine-evoked currents,resulting in a negative starting point of the dose –response curve (Fig.2D and E).Like PTX,histamine closed the constitutive activity in the absence of glycine with an estimated IC 50around 3mM (Fig.2E).
In conclusion,histamine inhibited rat and human α3βglycine receptors in a competitive manner.Since histamine was able to close the constitutively active α3P185L βglycine receptors,histamine is an inverse agonist of glycine receptors.
3.3.α1βglycine receptors expressed in TMN neurons were competitively antagonized by histamine
In accordance with our previous study in rats (Sergeeva et al.,2001)the majority of mouse TMN neurons in the present study (7out of 10)responded to glycine (1mM)with currents of 0.5–2.5nA amplitude,which were abolished by strychnine 100nM (IC 50?10nM).Such high strychnine sensitivity is in line with the dominant role of the α1subunit of glycine receptor.α2homomeric receptors (neonatal type of the spinal glycine recep-tor)were reported to be strychnine-resistant (Kuhse et al.,1990)and recombinant α2receptors are less sensitive to strychnine compared to the α1receptors (Schmieden et al.,1992).
Striatal neurons,lacking α1subunit expression are less sensitive to strychnine (IC 50?74nM,see Sergeeva and Haas,2001)than TMN neurons (IC 50?10nM).Therefore,taking in account previous immunohistochemical studies in the hypothalamus (discussed in (Sergeeva et al.,2001))we conclude that α1βglycine receptor are the dominant subtype in the TMN.The mean EC 50for glycine determined in the present study of 54.870.6μM (n ?7)and the Hill coef ?cient (nHill) 1.470.2(Fig.3)were similar to the previously reported values for rat TMN neurons (Sergeeva et al.,2001).10mM histamine shifted the dose –response curve for glycine rightwards (EC 50?12471.6μM)without affecting the maximal response amplitude or the slope function (nHill ?1.570.02,n ?5),indicating competitive antagonism.Histamine blocked glycine currents with an amplitude below half-maximal but did not affect currents of maximal amplitude.Therefore we used glycine concentrations eliciting responses below 20%of maximal amplitude (on average ～EC 10)to determine histamine
potency.
Fig.2.The effect of histamine on human and rat α3βglycine receptors.(A)Averaged dose –inhibition curve for histamine at glycine concentrations corresponding to the EC 30[●].(B)Averaged dose-response curve for glycine [●]and glycine plus 10mM histamine [○].Histamine shifted the dose –response curve signi ?cantly changing neither the Hill coef ?cient nor the maximum evoked current.(C)Rat α3β[●]and [○]α3P185L βdose –inhibition curve for histamine with a decreased IC 50for histamine compared to non-edited receptors.(D)Averaged dose –response curve for glycine [●]and glycine plus 10mM histamine [○]at α3P185L βglycine receptors.Histamine closed the spontaneously open glycine receptors at low glycine concentrations .(E)Left,direct inhibition of spontaneously open α3P185L βglycine receptors by histamine.Right,glycine elicited responses and (sub)-threshold glycine responses with 10mM histamine.
O.Kletke et al./European Journal of Pharmacology 710(2013)59–66
62
Histamine started to affect glycine responses signi ?cantly at 0.5mM and nearly abolished responses at 30mM (IC 50?4.670.3mM,nHill ?0.9770.07,n ?6)(Fig.3B).
3.4.Histamine related substances were able to inhibit α1βglycine receptors
The competitive antagonism of histamine at glycine receptors raised the question whether ligands of metabotropic histamine receptors (H 1–H 4)or structurally histamine-related substances were able to modulate glycine receptors.EC 50corresponding glycine concentrations were co-applied with 100,300or 1000μM of the tested substances to characterize their effect on α1βglycine receptors (Fig.4A).
SR 95531(Gabazine),a competitive antagonist of GABA A receptors and with lower af ?nity also of glycine receptors,was given as a control and strongly inhibited α1βglycine receptors.Doxylamine (histamine H 1receptor antagonist)and tiotidine (histamine H 2receptor antagonist)were similarly strong inhibitors of α1βglycine receptors and with an estimated IC 50below 100μM more potent than histamine.The application of 100μM was suf ?cient to reduce the glycine-induced currents by about 60%and nearly completely abolished the responses at concentrations
of 1mM.HTMT (histamine H 3receptor agonist),thioperamide (histamine H 3receptor antagonist),pyrilamine (histamine H 1receptor antagonist),famotidine (histamine H 2receptor antago-nist)and cimetidine (histamine H 2receptor antagonist)inhibited the glycine-induced responses about 50%at concentrations of 300μM and about 90%with 1mM.Harmane (imidazoline binding site ligand)and imetit (histamine H 3/H 4receptor agonist)were weaker inhibitors;at concentrations of 1mM they reduced the glycine-elicited currents about 60%.The physiological active amino acid and precursor of histamine,L -histidine,was more potent than the enantiomer D -histidine which is not active in vivo .Carcinine (histamine H 3receptor antagonist)and L -carnosine (dipeptide of β-alanine and histidine)were only weak inhibitors of α1βglycine receptors at all concentrations applied.The chemical structures are displayed in Fig.4B.
4.Discussion
Our study demonstrates that histamine inhibits recombinant and native glycine receptors.Dose –inhibition curves of histamine revealed that glycine receptors were inhibited by histamine in a dose-dependent manner.The IC 50for histamine increased
from
Fig.3.Histamine (HA)competitively antagonizes native glycine receptors.(A)Inhibitory effect of histamine on glycine (Gly)-evoked currents in mouse TMN neurons.Examples of histamine (10mM)block of responses to different glycine concentrations recorded from one cell are shown around an averaged dose-response plot (n ?7)for glycine alone [●]or glycine plus 10mM histamine [○].(B)Increasing concentrations of histamine inhibit glycine-evoked currents in a dose-dependent manner.Representative current traces (left)and an averaged (n ?5)curve for the histamine inhibition of glycine responses (5–20%of maximal glycine (1mM)-response)(right).
O.Kletke et al./European Journal of Pharmacology 710(2013)59–6663
low to high millimolar concentrations while using EC 10,EC 50,and EC 95corresponding glycine concentrations with nearly complete inhibition at high histamine concentrations.In addition,neither the Hill coef ?cient nor the maximal evoked current of the glycine dose –response curve was in ?uenced by a given concentration of histamine;only the potency for glycine was negatively affected.Similar results were obtained at all recombinantly expressed subunit combinations and could also be observed at native α1βglycine receptors expressed in TMN neuron.
As the agonist potency of recombinantly expressed α1βglycine receptors is in ?uenced by the expression level (Taleb and Betz,1994),we used this instance as a tool and could show that a decreased glycine potency resulted in a decreased potency for histamine.The same effect was observed at edited α3P185L βglycine receptors where the potency for histamine and glycine was increased compared to the non-edited form and thus seems to be coupled to the ligand af ?nity.All results point to the fact that the inhibition of histamine underlies a competitive mechanism.Moreover,at constitutively active α3P185L βglycine receptors,histamine could inhibit the spontaneous activity.Taking this behavior in account,histamine is an inverse agonist of glycine receptors.Overall,histamine shows lower potencies compared to glycine but interacts with the same binding site.Grudzinska et al.(2005)have shown that two oppositely charged amino acids of adjacent subunits are relevant for glycine https://www.doczj.com/doc/218142906.html,pared to glycine,histamine is not oppositely charged and larger.GABA with an increased size but similar charge as glycine activates glycine receptors with a reduced potency.The lack of oppositely charged groups and the exceeding size of histamine might account for the reduced potency of inhibition.Histamine seems not to interact with both oppositely charged amino acid residues of the adjacent subunits,but the interaction with one residue seems to be suf ?cient to cover the ligand binding site to stabilize the closed state.
A preferred target for histaminergic inhibition in vivo might be edited α3P185L glycine receptors,as we have shown that these receptors were more sensitive to histamine and glycine compared to the non-edited receptors.In recombinant systems an EC 50for glycine of 17μM (Meier et al.,2005)or 18μM (this study)was reported.However,the edited receptor is more sensitive in native neurons (EC 50of 5μM,(Meier et al.,2005)).As the potency for histamine is coupled to glycine,effective histamine
concentrations
Fig.4.Pharmacological screening of histamine related substances at α1βglycine receptors.(A)Various metabotropic histamine receptor H 1–4agonists/antagonists and substances structurally related to histamine were applied in three concentrations (100μM gray,300μM dark gray,1000μM black bars)at least at three different oocytes with glycine EC 50concentrations.(B)Chemical structure of the above tested substances in order of their inhibitory capabilities.For comparison the structure of histamine and glycine is displayed in the gray box.
O.Kletke et al./European Journal of Pharmacology 710(2013)59–66
64
could be in a micromolar range in vivo.In general,the overall histamine concentration(1.44nmol/g in hypothalamus or 0.30nmol/g in cerebral cortex(Fogel et al.,2001))is too low to modulate glycine receptors.Histamine concentration in vertebrate synaptic vesicles is not known.However,other neurotransmitters can reach concentrations over100mM(e.g.210mM reported for glutamate(Riveros et al.,1986)).In Drosophila,vesicular histamine concentrations can reach up to670mM(Borycz et al.,2005).In a co-culture system,axonal processes of neurons of the tubero-mammillary nucleus form a relatively large number of varicosities. Some of these histaminergic varicosities establish synaptic junc-tions both on spines and on dendritic shafts of hippocampal neurons(Diewald et al.,1997).Histamine levels near to the release area might be suf?cient to modulate edited glycine receptors.Such edited glycine receptors were observed in the spinal cord,cortex, cerebellum and hippocampus(Meier et al.,2005),where they might be associated with histaminergic neurons from the TMN which are projecting to those areas(Haas and Panula,2003).
Furthermore,we observed a constitutive activity atα3P185Lβglycine receptors expressed in Xenopus oocytes.Histamine was able to directly close these receptors showing characteristics of an inverse agonist.The amino acid at position185is located close to the plasma membrane,where the receptors enter the cytosol through the transmembrane domain1,and is distinct from the ligand binding site.The amino acid exchange P185L at the N-terminal domain causes a sterical conformational change which is thought to modify the accessibility of the ligand binding pocket and thus increase the potency for glycine(Legendre et al.,2009). For GABA A receptors spontaneous activity was reported at α1β2E155C receptors,where a structural rearrangement of the ligand binding site causes the constitutive activity(Newell et al.,2004). We propose that the modi?ed accessibility of the ligand binding site results in a structural rearrangement of the binding pocket, which tends to gate the receptor spontaneously.Further investiga-tions of neurons containing edited glycine receptors have to prove the existence of spontaneous activity atα3P185Lβglycine receptors in vivo,but the proposed molecular basis admits the possibility.
The occurrence of RNA-editedα3glycine receptors accompa-nied with spontaneous activity in the spinal cord might lead to tonic,synaptic currents.In patients with temporal lobe epilepsy and hippocampal sclerosis,the amount of editedα3glycine receptors increases.Accompanied with high Cl?equilibrium potentials present under pathophysiological conditions,activation ofα3P185L glycine receptors was proposed to increase the excit-ability of the hippocampal network(Eichler et al.,2009;Eichler et al.,2008).In the presence of histamine,glycine-mediated currents would be antagonized and the tonic,glycine-independent activation would be disrupted.Both effects would act synergistic and would decrease the network activity.Our ?ndings that histamine is an inverse agonist of edited glycine receptors might contribute to new therapeutic approaches in treating temporal lobe epilepsy.However,the physiological role of histaminergic inhibition of glycine receptors and the occurrence of spontaneous activity in vivo remains elusive.
Besides the metabotropic histamine receptors,where hista-mine is the endogenous ligand,it was shown that also ion channels can be gated or modulated by histamine.In acutely-isolated or cultured hippocampal pyramidal cells histamine enhances NMDA receptor-mediated currents with an EC50of 1.7μM(Bekkers,1993;Vorobjev et al.,1993).High histamine concentrations resulted in an inhibition of NMDA-mediated current with an IC50of803μM.At recombinantly expressedα1β2 GABA A receptors histamine was able to potentiate GABA-induced currents with an EC50of965μM(Saras et al.,2008).In insects,a family of Cys-loop ion channels with a homology to vertebrate GABA A and glycine receptors is directly gated by histamine (Gisselmann et al.,2002)or dually gated by GABA and histamine (Gisselmann et al.,2004).Several agonists and antagonists of metabotropic histamine H1–4receptors were also able to inhibit α1βglycine receptors expressed in oocytes.A similar pharmacol-ogy was found for vertebrate GABA A receptors(Saras et al.,2008) and insect HisCl1(Gisselmann et al.,2002),where such substances showed a similar competitive inhibition.In all three cases,the pharmacology of the histamine binding site is clearly distinct from the known H1–H4pharmacology.Histaminergic pharmacology of homo-oligomericβ3GABA A receptors was most recently character-ized by surface plasmon resonance biosensor technology demon-strating that several histaminergic ligands compete with histamine binding(Seeger et al.,2012).
5.Conclusion
In addition to GABA A and NMDA receptors,we identi?ed a further ion channel that is modulated by histamine.We demon-strate that histamine inhibits recombinant and native glycine receptors in a competitive manner and can act as an inverse agonist.
Acknowledgments
Supported by Deutsche Forschungsgemeinschaft SFB575/3and 8and a Heisenberg stipend to OAS and BMBF(ERA-Net Neuron II CIPRESS)to JCM.We are grateful to A.Stoeck and C.Bernert for the excellent molecular biology support.Furthermore we like to thank H.Bartel and W.Grabowsky for their technical assistance. Appendix A.Supporting information
Supplementary data associated with this article can be found in the online version at https://www.doczj.com/doc/218142906.html,/10.1016/j.ejphar.2013.04. 002.
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山西大学计算机与信息技术学院 实验报告 姓名学号专业班级 课程名称 Java实验实验日期成绩指导教师批改日期 实验5 JAVA常用类实验名称 一．实验目的： （1）掌握常用的String，StringBuffer(StringBuilder)类的构造方法的使用；（2）掌握字符串的比较方法，尤其equals方法和==比较的区别； （3）掌握String类常用方法的使用； （4）掌握字符串与字符数组和byte数组之间的转换方法； （5）Date，Math，PrintWriter，Scanner类的常用方法。 二．实验内容 1.二进制数转换为十六进制数（此程序参考例题249页9. 2.13） 程序源代码 import java.util.*; public class BinToHexConversion{ //二进制转化为十六进制的方法 public static String binToHex(String bin){ int temp; //二进制转化为十六进制的位数 if(bin.length()%4==0) temp = bin.length()/4; else temp = bin.length()/4 + 1; char []hex = new char[temp]; //十六进制数的字符形式 int []hexDec = new int[temp];//十六进制数的十进制数形式 int j = 0; for(int i=0;i
工业品营销人员的立体化激励模式 提起营销人员的激励，普遍存在两大认识：一种认为营销人员的激励最简单，因为营销人员的业绩无非就是销售额、回款额，比较容易量化，根据销售额提成或合同毛利提成，就能够直观地反映出营销人员业绩的优良，就能够给予营销人员与贡献相关联的物质回报。目前国内绝大多数的企业都在采用这种激励模式。另一种认为营销人员长期出差在外，其行为难以控制，过程难以管理，尤其是业务费用的控制难度很大，仅用一维的业绩评价难以体现公司的经营导向和对营销人员的行为导向，因此尝试过多种复杂的考核激励办法，比如通过OA 工作日志法监控营销人员的行为等，但实际效果却不甚理想：要么营销人员感觉太繁琐太复杂而不愿配合，认为耗费了本应拜访客户的大量精力；要么投入的管理成本太高，但导致营销效率下降、营销人员积极性受挫，直接危及企业的经济效益。显然这都不是企业想达到的管理效果。以上对营销人员考核激励的两种认识，都存在一个共同的误区：就是把营销人员作为“管理的对象”或“赚钱的工具”，目的之一是希望通过物质刺激让销售人员能够多为企业创造效益；目的之二是提防销售人员不利于企业的利已行为。基于这种出发点的激励设计，其效果是可想而知的。 只有把营销工作看作是企业产生价值的系统工程，把营销人员看做是企业整体价值链中最接近客户、最关键的一个环节，对于营销人员的考核激励才能走出简单的“提成激励”和“管理控制”的误区。因此，在探讨对营销人员的激励模式之前，首先我们先来分析工业品营销的突出特点，其次分析工业品营销对销售人员的要求以及销售人员特点，结合这些特点我们再来思考激励模式的设计要点。 一、工业品营销特点 工业品营销不同于消费品营销，面对的是少数的大客户；单笔合同额一般金额较大；项目的开发周期、合同的执行周期都较长。 1、目标客户集中。 在消费品行业，最常用的武器是利用大众媒体大做广告宣传，而在工业品营销领域，客户或许只有屈指可数的几个大客户，目标客户比较集中。 2、市场区域分散。 目标客户数量少，但地域分布一般较为分散，遍布全国各地。工业品营销一般采用“划界营销”的模式，常常由一个业务经理负责一个省甚至几个省市的客户开发。 3、定制的订单驱动。 消费品定制化通常限于包装、标识或促销上，为广泛的用户提供基础性标准化产品。但工业品客户所采购的产品通常不是单独使用，而是为下一级生产或装配使用的，因此，需要工业品制造商根据客户的需求进行产品定制化设计。 4、购买决策复杂。 因为工业品购买金额较大，不买则已，一买就是“大买卖”，少则几百万，多则数千成，因此客户会相当慎重。在工业品市场上，决策流程通常比较复杂，一套工业设备的购买可能涉及多个部门，如采购部、工程部、财务部、技术部以及高层管理者；从购买角色来看，他们在采购过程中扮演着使用者、影响者、购买者、决定者和把关者等多种不同角色，发挥着不同的作用。因此，工业品营销需要客户经过几轮、多层次的比较和权衡才能最终决策买家。 5、以关系营销为主。 工业品营销的以上的四个特点，决定了“客户关系”成了营销成功的关键因素。有人曾精辟地总结过工业品营销的模式： 工业品营销模式=X%透明营销+Y%灰色营销 其中，Y随着不同的行业数值不同。一般来说，在快消品营销领域，X占到80%以上；而在工业品营销领域，Y或许要占到80%以上也未为可知。随着市场经济的不断深入，Y的比重
工业品销售 工业品销售过程:从粗放到精细管理 目前，工业品销售过程中，因为项目周期长，关系营销比较明显，基础上销售人员掌握大量的客户信息，导致销售过程相对不透明，所以，相对销售过程比较粗放，在实际销售过程就不容易把握销售过程。因此，产生了四大困惑： 困惑之一：20%销售精英带走大客户，怎么办？ 不少企业在销售过程中都曾经遇到过这样的困惑：一个非常重要的营销人员尤其是营销骨干离开公司，跳槽把他所接触的客户和营销网络全部带走。它给企业带来的巨大“阵痛“和“虚空”，让很多企业从此一蹶不振，这种“切肤之痛”，甚至使有些企业从此走上了衰亡的不归路。 几个月前，我遇到一位民营企业家张总。从2002年开始，经过四年把销售额的200万发展到到2500万，但是老板张总向我透露了他所遇到的一件非常烦心的事：属下一位颇有开拓及沟通能力的销售功臣李华突然离职投奔了另一家竞争对手公司，不但让销售工作大受影响，而且还使以前的客户纷纷“倒戈”，不再购买他的产品，为这件事情，张总大伤脑筋。 在实际的市场操作中，类似张总这样的困惑，即营销人员离职带走客户的现象，可以说是比比皆是，屡见不鲜，一个人的出走对一个企业的打击不会太大，但一个团队的出走就会沉重打击企业的经营和商誉。 困惑之二：销售靠艺术还是科学？ 目前，许多营销精英都认为销售是一门艺术，特别是在工业品营销中，
很多人都认为，销售成功归结吃喝营销，就是关系营销，就是拉拢与腐蚀客户的“采购人员”，进行灰色交易，满足他们的吃、拿、卡、要，甚至销售人员应该主动挖掘，进行投怀送抱，产品技术与品牌不是最重要，最关键就是搞定关键人，建立良好的客户关系才是最关键的，这已经成为工业品企业营销过程中的“潜规则”。所以，怎样送、怎样建立关系关键就靠自己的悟性了，悟性好，就有发展，能搞定项目。 所以，长期以来，我们更多地把营销当做一种艺术，经验、悟性、灵感和个人的随机应变占有更重要的地位，甚至有些精英人才不无自豪地说：“营销的奥秘是无法培训传授的。”一些企业也期望靠这些优秀的精英人才打出一片天下。所以项目营销部经理们总是千方百计从各种渠道挖掘精英人才。遗憾的是，“营销精英”们的跳槽频率极高（他们总是竞争对手挖墙脚的对象），管理起来难度也最大。他们既能为企业开发市场，也最容易毁掉企业的市场，甚至将客户带往竞争对手。 5年前，我在一家大型工业机械企业做培训时，老总自豪地向我推荐2名“销售状元”：他们年销售额分别是0.4亿元、0.2亿元，公司73%的销售额都是他们创造出来的。 我马上问了两句话：“公司业务近75%掌握在2个销售精英手中，你没有感觉有风险吗？”、“34个销售人员创造的业绩不好，你该怎办呢？”，那个老总，顿时无语。 在营销工作中，销售人员是非常需要艺术性的灵感和创意，同时，营销更需要科学，艺术的灵感加上科学的分析和决策这双翅膀，才能飞得高远。所以，著名营销专家凯文、克兰斯说：“营销越来越多地成为一门
New York Climate Climate is very important message we can know what kind of clothes we should prepare. The New York climate is Temperate continental. Cold and dry in winter. Dry and hot in summer. In spring and autumn you can feel four season in one day. Building Central Park Central Park plays an essential role for everyday lives of New Yorkers by providing 843 acres of paths. lakes and open spaces for escaping the din and chaos of the surrounding city. Visitors to New York City are often impressed with Central Park's beauty and size, making it a wonderful place for visitors to New York City to enjoy a bit of relaxation and get a better sense of what it's like to live in New York City. Metropolitan Museum of Art One of New York City's most popular tourist attractions, the Metropolitan Museum of Art welcomes over 5 million visitors a year. The Metropolitan Museum of Art's collection and special exhibits offer something for everyone -- from Ancient Egyptian Vases and Roman Statues to Tiffany Stained Glass and Rembrandt Paintings there is something for nearly everyone. If you're overwhelmed by the sheer size and breadth of the Metropolitan Museum of Art's collection, take a Highlights Tour The Statue of Liberty The Statue of Liberty was a gift to the United States in honor of the friendship established during the French Revolution. The Statue of Liberty has become an American symbol of freedom and welcome to the immigrants who come to the USA looking for a better life. Broadway It is a long street which is 25 km long. Many famous theatres has gathered there, so Broadway has become the symbol of the operas of world. Grand Central Terminal Grand Central Terminal is a beautiful Beaux3-arts building in midtown Manhattan. First opened in 1913, Grand Central Terminal underwent a massive restoration to restore it to its former glory, and is now both a transportation hub, as well as a destination in itself for visitors to New York City. The Rockefeller Center The Rockefeller Center complex was conceived by John D. Rockefeller Jr. in early 1929 in the hopes of revitalizing恢复活力the area. Despite the stock market crash, construction continued, providing much needed employment during the Great Depression. This commercial complex of 19 buildings is worth visiting to experience the architecture and art, as well as to enjoy the area's shopping and dining. American Museum of Natural History Since opening to the public in 1869, the American Museum of Natural History has evolved and grown. In addition to the Rose Center planetarium and regular exhibits, the American Museum of Natural History hosts a revolving series of new exhibits, so there is always something new to see
对定量结果进行差异性分析 1.单因素设计一元定量资料差异性分析 1.1.单因素设计一元定量资料t检验与符号秩和检验 T检验前提条件：定量资料满足独立性和正态分布，若不满足则进行单因素设计一元定量资料符号秩和检验。 1.2.配对设计一元定量资料t检验与符号秩和检验 配对设计：整个资料涉及一个试验因素的两个水平，并且在这两个水平作用下获得的相同指标是成对出现的，每一对中的两个数据来自于同一个个体或条件相近的两个个体。 1.3.成组设计一元定量资料t检验 成组设计定义： 设试验因素A有A1，A2个水平，将全部n（n最好是偶数）个受试对象随机地均分成2组，分别接受A1，A2，2种处理。再设每种处理下观测的定量指标数为k，当k=1时，属于一元分析的问题；当k≥2时，属于多元分析的问题。 在成组设计中，因2组受试对象之间未按重要的非处理因素进行两两配对，无法消除个体差异对观测结果的影响，因此，其试验效率低于配对设计。 T检验分析前提条件：
独立性、正态性和方差齐性。 1.4.成组设计一元定量资料Wil coxon秩和检验 不符合参数检验的前提条件，故选用非参数检验法，即秩和检验。1.5.单因素k（k>=3）水平设计定量资料一元方差分析 方差分析是用来研究一个控制变量的不同水平是否对观测变量产生了显著影响。这里，由于仅研究单个因素对观测变量的影响，因此称为单因素方差分析。 方差分析的假定条件为： （1）各处理条件下的样本是随机的。 （2）各处理条件下的样本是相互独立的，否则可能出现无法解析的输出结果。 （3）各处理条件下的样本分别来自正态分布总体，否则使用非参数分析。（4）各处理条件下的样本方差相同，即具有齐效性。 1.6.单因素k（k>=3）水平设计定量资料一元协方差分析 协方差分析(Analysis of Covariance)是将回归分析与方差分析结合起来使用的一种分析方法。在这种分析中，先将定量的影响因素(即难以控制的因素)看作自变量，或称为协变量(Covariate)，建立因变量随自变量变化的回归方程，这样就可以利用回归方程把因变量的变化中受不易控制的定量因素的影响扣除掉，从而，能够较合理地比较定性的影响因素处在不同水平下，经过回归分析手段修正以后的因变量的样本均数之间的差别是否有统计学意义，这就是协方差分析解决问题的基本计算原理。
史上最全Java基础知识点归纳 写这篇文章的目的是想总结一下自己这么多年来使用Java的一些心得体会，主要是和一些Java基础知识点相关的，所以也希望能分享给刚刚入门的Java 程序员和打算入Java开发这个行当的准新手们，希望可以给大家一些经验，能让大家更好学习和使用Java。 这次介绍的主要内容是和J2SE相关的部分，另外，会在以后再介绍些J2EE 相关的、和Java中各个框架相关的内容。 经过这么多年的Java开发，以及结合平时面试Java开发者的一些经验，我觉得对于J2SE方面主要就是要掌握以下的一些内容。 1.JVM相关(包括了各个版本的特性) 对于刚刚接触Java的人来说，JVM相关的知识不一定需要理解很深，对此里面的概念有一些简单的了解即可。不过对于一个有着3年以上Java经验的资
深开发者来说，不会JVM几乎是不可接受的。 JVM作为Java运行的基础，很难相信对于JVM一点都不了解的人可以把Java语言吃得很透。我在面试有超过3年Java经验的开发者的时候，JVM几乎就是一个必问的问题了。当然JVM不是唯一决定技术能力好坏的面试问题，但是可以佐证Java开发能力的高低。 在JVM这个大类中，我认为需要掌握的知识有： JVM内存模型和结构 GC原理，性能调优 调优：Thread Dump，分析内存结构 class二进制字节码结构，class loader体系，class加载过程，实例创建过程 方法执行过程 Java各个大版本更新提供的新特性(需要简单了解) 2.Java的运行（基础必备） 这条可能出看很简单，Java程序的运行谁不会呢？不过很多时候，我们只是单纯通过IDE去执行Java程序，底层IDE又是如何执行Java程序呢？很多人并不了解。
工业产品市场开拓活动方案 为认真做好大宗工业产品市场开发活动，有效帮助企业拓宽销售渠道，促进我市工业经济平稳较快发展，现将我市大宗工业产品市场开发活动工作计划制定如下： 一、指导思想 以党的十七大精神为指导，围绕“保增长、促发展”的要求，以“宣传、促发展、发展”为主题，充分展示我市著名和优秀工业产品的发展成果，积极帮助我市工业企业拓宽销售渠道，渡过当前的经济难关，进一步促进更广泛领域的合作，促进我市工业经济平稳较快发展。 二、活动内容 一是开展城市大型工业产品市场开发活动 XXXX年度市政府集中采购相关产品目录； ⑵市经贸委根据市政府集中采购产品目录对地方企业进行深入调查，初步参与市政府集中采购企业名单； (3)组织地方企业参与市政府集中采购招标； (4)做好中标人产品销售的衔接工作； (5)根据实施情况，市财政局、市政府事务管理办公室将把优质低价的地方产品纳入未来政府集中采购目录。 2、组织轻工(食品)进商场(超市)。由市经贸委牵头，与市供销
协会、农业局、农业局、粮食局等部门合作，组织轻工(食品)进商场(超市)的工贸对接活动，鼓励商场(超市)在同质和价格条件下优先采购本地产品。 (1)市经贸委组织对国贸大厦、苏中大厦、世纪联华超市、农业、工商超市、时代超市等商场(超市)相关采购产品进行调查，并编制进入商场(超市)的当地轻工(食品)产品目录； (二)市经贸委根据采购产品目录对当地企业进行深入调查，初步确定参与工贸对接活动的企业名单； (3)组织当地轻工(食品)进入商场(超市)交易会，实行工贸对接，集中签约； (4)做好承包企业产品与商场(超市)的衔接工作)。 ⑸电视台、广播电台、报纸做好地方名优轻工业(食品)的宣传，积极引导市民购买地方名优轻工业(食品)。 3、积极组织企业参加全国商品交易会。由市政府办公室牵头，与市经贸委、外经贸局、农业局、其他部门合作，利用国内外举办各类商品交易会的机会，组织我市大宗产品生产企业参加国内外大型商品交易会，拓宽产品在国内外的销售渠道。 二认真做好大宗工业产品的市场开发活动 1、积极配合市经贸委牵头组织的大宗工业产品政府集中采购活动，编制全市相关产品目录，组织协调相关企业参加全市产品推广会
介绍美国纽约英语作文 在纽约市和出纽约市要通过两个隧道：林肯隧道和荷兰隧道。林肯隧道是一条位于纽约市的隧道，跨越哈德逊河以连接新泽西州。现在是小编为您整理的介绍美国纽约英语作文，希望对您有所帮助。 介绍美国纽约英语作文1 In 1869, the draft of the statue of Liberty was completed, and Bartholdy began to throw himself into the work of sculpture. He has traveled to the United States to win American support for the statue scheme, but Americans have been slow to realize the value of the gift. Until 1876, Bartholdy held in Philadelphia to celebrate the 100th anniversary independent exposition, in order to attract public attention of , the statue of liberty he held the torch hand on display at the fair, just a sensation. Placed in front of people in this hand only index finger on the length of 2.44 meters, more than 1 meters in diameter, nail thickness of 25 cm, 12 people can stand on the edge of the torch. So this a few days ago also little-known sculpture suddenly worth a hundred times, become the everyone to appreciate art treasures. We boarded the cruise ship and went up to the two floor. The river wind is very cool, put the hair on my forehead to the back, behind the hair along the wind, blown into the "fluttering" hairstyle. I saw a wide river, suffused with blue waves, along the river, or ————来源网络搜集整理，仅供个人学习查参考
第1章 Java语言概述与面向对象思想 1 1.1 Java语言的发展 1 1.1.1 Java语言的产生 1 1.1.2 Java语言的发展 1 1.2 Java语言的特点 2 1.3 面向对象与面向过程的差异 3 1.3.1 面向过程思想回顾 4 1.3.2 面向对象思想介绍 4 1.4 面向对象程序设计中的主要概念和特征 4 1.4.1 主要概念 5 1.4.2 主要特征 5 *1.5 Java与C++的差异 5 1.6本章小结5 习题 5 第2章 Java语言开发环境 6 2.1 JDK 6 2.1.1 JDK的简介6 2.1.2 JDK的构成6 2.1.3 JDK的使用6 2.2 IDE 8 2.2.1 IDE简介8 2.2.2 JBuilder 9 2.2.3 Eclipse 9 2.2.4 相关资源9 2.3 Project管理9 2.3.1 Project的含义9 2.3.2 可行的Project组织模式9 2.3.3 主要开发工具的Project目录10 2.4 本章小结10 习题10 第1章 Java语言概述与面向对象思想 1.1 Java语言的发展 1.1.1 Java语言的产生以介绍面向对象编程的基本概念、基本理论为重点，结合Java语言的语法规则、编程特点和设计思想、强调容易发生错误和编程应注意的地方，使学生能对Java 技术有一个总体了解，通过本课程学习，使学生掌握Java语言的基础知识，理解和掌握面向对象程序设计的基本思想，熟练地使用Java语言进行程序的编写、编译以及调试工作 上世纪90年代初期，Sun公司在研究一种适用于未来的智能设备的编程语言，该语言要具有一些新的特性，以避免C++的一些不足。 该语言起初命名为Oak，来源于语言作者Gosling办公室窗外的一棵橡树（Oak）。后来在注册时候遇到了冲突，于是就从手中的热咖啡联想到了印度尼西亚一个盛产咖啡的岛屿，中文名叫爪哇，Java语言得名于此。 随着Internet的迅速发展，Web应用日益广泛，Java语言也得到了迅速发展。1994年，Gosling
美国纽约市英语简介 纽约(New York)，是纽约都会区的核心，也是美国最大城市，同时也是世界最大的城市之一。纽约位于美国东海岸的东北部，是美国人口最多的城市，也是个多族裔聚居的多元化城市，拥有来自97个国家和地区的移民，在此使用的语言达到800种。截至2014年，纽约大约有849万人，居住在789平方千米的土地上。纽约市是一座世界级国际化大都市，直接影响着全球的经济、金融、媒体、政治、教育、娱乐与时尚界。纽约GDP于2013年超越东京，位居世界第一。下面为大家带来旅游英语美国纽约市英语简介，欢迎大家阅读! 纽约市英语简介： New York City is the most beguiling place there is. You may not think so at first - for the city is admittedly mad, the epitome in many ways of all that is wrong in modern America. But spend even a week here and it happens - the pace, the adrenaline take hold, and the shock gives way to myth. Walking through the city streets is an experience, the buildings like icons to the modern age, and above all to the power of money. Despite all the hype, the movie-image sentimentalism, Manhattan - the central island and the city's real core - has massive romance: whether it's the flickering lights of
一、单选题 1.对类：(B) public class Test( //...do something } 下面那个正确地定义了类Test的构造函数。 A)public void Test() () B)publicTest()(} C ) public static Test() (} D) publicTest(); 2.下面哪个函数是public void example()(...)的重载函数。(A) A)public void example( float f)(...) B)public int example() (...) C)public void example2()(...} D)public int example_overLoad ()(...) 3.下面的代码段中，执行之后i和j的值是_C_。 int i = 1; intj； j = i++； A)1, 1 B) 1,2 C) 2, 1 D) 2,2 4.以下for循环的执行次数是_B o for(int x=0,y=0;(y !=0)&&(x<4) ;x++); A)无限次B) 一次也不执行 C)执行4次D)执行3次 5.下面程序的输出结果是—C o public class People( String name; int id; public People( String str, int n )( name = str; id = n; } public String toString(){ return id + " ：” + name; } public String print()(
JAVA中常用类的常用方法 一、https://www.doczj.com/doc/218142906.html,ng.Object类 1、clone()方法 创建并返回此对象的一个副本。要进行“克隆”的对象所属的类必须实现https://www.doczj.com/doc/218142906.html,ng. Cloneable接口。 2、equals(Object obj)方法 ?功能：比较引用类型数据的等价性。 ?等价标准：引用类型比较引用，基本类型比较值。 ?存在特例：对、Date及封装类等类型来说，是比较类型及对象的内容而不考虑引用的是否为同一实例。 3、finalize()方法 当垃圾回收器确定不存在对该对象的更多引用时，由对象的垃圾回收器调用此方法。 4、hashCode()方法返回该对象的哈希码值。 5、notify()方法唤醒在此对象监视器上等待的单个线程。 6、notifyAll()方法唤醒在此对象监视器上等待的所有线程。 7、toString()方法 返回该对象的字符串表示。在进行String与其它类型数据的连接操作时，自动调用toString()方法。可以根据需要重写toString()方法。 8、wait()方法 在其他线程调用此对象的notify() 方法或notifyAll() 方法前，导致当前线程等待。 二、字符串相关类 l String类 charAt(int index) 返回指定索引处的char 值。 compareTo(String anotherString) 按字典顺序比较两个字符串。 compareToIgnoreCase(String str) 按字典顺序比较两个字符串，不考虑大小写。 concat(String str) 将指定字符串连接到此字符串的结尾。 endsWith(String suffix) 测试此字符串是否以指定的后缀结束。 equals(Object anObject) 将此字符串与指定的对象比较。 equalsIgnoreCase(String anotherString) 将此String 与另一个String 比较，不考虑大小写。indexOf(int ch) 返回指定字符在此字符串中第一次出现处的索引。 indexOf(String str) 返回第一次出现的指定子字符串在此字符串中的索引。 lastIndexOf(int ch) 返回指定字符在此字符串中最后一次出现处的索引。 length() 返回此字符串的长度。 replace(char oldChar, char newChar) 返回一个新的字符串，它是通过用newChar 替换此字符串中出现的所有oldChar 得到的。split(String regex) 根据给定正则表达式的匹配拆分此字符串。 startsWith(String prefix) 测试此字符串是否以指定的前缀开始。 substring(int beginIndex) 返回一个新的字符串，它是此字符串的一个子字符串。该子字符串始于指定索引处的字符，一直到此字符串末尾。 substring(int beginIndex, int endIndex) 返回一个新字符串，它是此字符串的一个子字符串。该子字符串从指定的beginIndex 处开
工业品营销-区域市场开拓与经销商管理7 区域市场开拓与经销商管理 一、区域经理的角色定位 了解自己的筹码 SWOT分析 做最有把握的事 有所为有所不为 选择自己最有利的战场 用适当的方式营销——自营.代理.批发.特许.加盟.直销整合——定位竞争对手，制定攻守方略 案例讨论：区域经理及经销商经理工作职责 （分享）自我负责任的态度 二、区域市场的有效拓展 市场开拓不良造成的影响 影响市场开拓的主要因素 制定市场开拓目标与计划 了解区域市场的方法
经销商选择与评估要素 准确部署及有效启动市场 产品铺货与管理方向 解决经销商的困难问题 实施有效的市场支持 促进经销商业绩提高的办法 三、区域市场的品牌建设 “需要、需求、欲望”实战品牌营销法则；品牌营销的时机把握与阶段性策略； 符合市场的品牌表现及推广策略； 产品品牌与企业品牌的组织管理； 品牌运营的组织架构与管理方式； 四、区域市场的项目管理 评价地区市场管理的好坏 评估地区市场三大能力 地区市场渠道维护与管理 改善与提升市场绩效
区域市场的扩张攻略 区域市场上精耕细作 帮助经销商获得成功 做经销商的教练 有效培训经销商 提高地区市场核心竞争力 提高区域市场总体执行力 五、区域经理的团队沟通 亲和力的塑造---模仿 同理心是建立双方沟通的平台 同理心的建立---站在对方的立场 建立与销售渠道的沟通渠道 如何为渠道的业务增值 改善渠道关系的有效途径 六、如何与分销商建立长期的合作伙伴关系管理分销商的五条原则 供应商的销售人员所需的技能
分销商的销售人员所需的技能如何为分销商的业务增值 改善分销商关系的有效途径
工业品营销渠道建设 在工业品市场，营销渠道仍是产品流向最终用户的主要通道。但工业品市场不同于消费品市场，它有其自身的特征，因此，工业品营销渠道在渠道类型、渠道策略、渠道成员等方面与消费品都有所区别。 1、工业品市场的渠道成员 在工业品市场，营销渠道的主要成员有：经销商、代理商和其他一些中间商。 1）经销商 经销商是指，拿着钱，从生产者那里买来产品或服务，通过提供各种相关的服务，再把产品或服务卖给其他用户。他们购买产品或服务不是自己用，而是转手卖出去，对于他们只是经过手，再销售而已。他们关注的利差，而不是实际的价格。企业对他们不是赊销，而是收到了钱的。因此经销商对他们经营的产品拥有所有权，可以自主经营。所以“经销商”，一般是企业用来说从企业拿钱进货的营销渠道机构。 经销商是提供服务的中间人，它在生产企业和工业品最终消费者之间的很大范围内发挥着市场销售渠道的功能，包括：提供市场信息；提供区域性市场覆盖；进行市场销售；仓储管理；处理订单；为顾客提供咨询和技术帮助。 经销商接待顾客，通过提供必要的援助服务，如送货、信用保证、技术咨询、维修服务、装配和推销，最终使顾客获得商品。一般来说，经销商经营的是牵涉面小、潜在顾客多的商品。这些商品通常可以储藏，以小数量销售，而且需要快捷的送货和服务。这就意味着这些中间商最适合做需购零配件和需要二次购买商品的那些用户(而不是第一次购买者)的生意。 然而，并不是所有的经销商都经营老一套的商品。有时，经销商还主动为新产品提供特别的服务，为此，他们为新顾客提供重要的技术咨询。当新产品在市场上逐渐成熟，更加标准化和广为人知之后，经销商所充当的教育角色就不那么重要了，而考虑送货和价格上的服务则变得更为关键。与该发展过程相联系的是经销商从事商业活动所采用的方式也将随之改变。新的业务会要求经销商在利用训练有素的外部销售人员的同时，还要利用内部销售人员以及远方的用户，通过增强顾客的信心进行重复销售。
第一章SAS系统概况 SAS（Statistic Analysis System）系统是世界领先的信息系统，它由最初的用于统计分析经不断发展和完善而成为大型集成应用软件系统;具有完备的数据存取、管理、分析和显示功能。在数据处理和统计分析领域，SAS系统被誉为国际上的标准软件系统。 SAS系统是一个模块化的集成软件系统。SAS系统提供的二十多个模块（产品）可完成各方面的实际问题，功能非常齐全，用户根据需要可灵活的选择使用。 ●Base SAS Base SAS软件是SAS系统的核心。主要功能是数据管理和数据加工处理，并有报表生成和描述统计的功能。Base SAS软件可以单独使用，也可以同其他软件产品一起组成一个用户化的SAS系统。 ●SAS/AF 这是一个应用开发工具。利用SAS/AF的屏幕设计能力及SCL语言的处理能力可快速开发各种功能强大的应用系统。SAS/AF采用先进的OOP（面向对象编程）的技术，是用户可方便快速的实现各类具有图形用户界面（GUI）的应用系统。 ●SAS/EIS 该软件是SAS系统种采用OOP（面向对象编程）技术的又一个开发工具。该产品也称为行政信息系统或每个人的信息系统。利用该软件可以创建多维数据库（MDDB），并能生成多维报表和图形。 ●SAS/INTRNET ●SAS/ACCESS 该软件是对目前许多流行数据库的接口组成的接口集，它提供的与外部数据库的接口是透明和动态的。 第二章Base SAS软件 第一节SAS编程基础 SAS语言的编程规则与其它过程语言基本相同。 SAS语句 一个SAS语句是有SAS关键词、SAS名字、特殊字符和运算符组成的字符串，并以分号（；）结尾。 注释语句的形式为：/*注释内容*/ 或*注释内容。 二、SAS程序 一序列SAS语句组成一个SAS程序。SAS程序中的语句可分为两类步骤：DA TA步和
联纵智达咨询集团事业部 总经 理：李天，高级咨询师、马松林 该文 刊登在《现代营销 —学 苑版》 2007年第十二期 提起营销工作，很多企业都抱怨， 现在的市场竞争越来越激烈，客户越来越挑剔了，生意 也越来越难做了。事实上，市场营销过程就是企业追求 利益最大化、客户追求价值最大化的博弈过程，能够寻 找到企业利益和客户价值之间的利益平衡点也就是市场 营销活动的魅力所在。目前的市场属于买方市场，客户 处于主导地位，企业更多地是处于被动地位，所以企业 必须以市场营销观念为核心，以客户价值观为导向，才 能做好市场工作，在提高客户价值的同时达到企业销售 业绩提升的同步提升 必须认识什么是客户价值。客户价值指客户购买商品 （包括服务）所获得的价值或利益总和，既包括商品本 身有形价值，也包括服务价值、心理满足感、额外便利 性、身份形象等无形价值。客户价值观，就是围绕提高 客户价值，实现客户价值增值为目标来开展市场营销工 作的一种营销观念。这种观念贯穿于各个行业各个阶段 的营销工作 要做到以客户价值观为导向， 作为众多工业品行 之一，客户价值观却显得更为重要。这里所提及的工 品产品，指工业用工业品产品（下同），客户价值观之 所以对其更为重要，更多是由于工业品产品的采购特点 工业品采购特点决定客户价值 通常情况 所决定的。 中，而工业品行 工业品营销营销的特点与策略
下，工业品产品的采购具有以下特点： 一、采购决策 的主体一般是一个小组或委员会，而不是具体的消费者 般情况下，购买工业品都有特定的用途，而且 都是用于工程 项目的居多，比如工业厂房建设、办公楼 宇的建设和装修、市政工程施工、文体设施建设等。项 目采购内容多而繁杂，采购金额大而敏感，而且采购事 项涉及关联方多（比如设计单位、建设单位、施工单 位），采购是一项庞大而系统的工作，无法由个人来完 成， 性能参数、包装、保质期等质量因素，追求产品 的使用价值最大化。 二、采购决策过程复杂，时间 长。工业品产品的采购有两种形式：一种是公开招标采 购，通过向符合条件的供应商发放招标书，然后供应商 递交投标书，采购单位评标选择最终中标人；另外一种 是普通的洽谈采购，采购方经过考察确定几家供应商进 行报价，采购方进行对比后选择供应商。不论哪种方式 由于所采购材料涉及金额较大，事关应用对象的长期质 量和影响，因此在采购流程上都要经过前期考察、报 价、洽谈、形成初步意见、报批、签订合同、执行合同 等过个人。 为便于管理，通常都是纳入到项目建设小组或委员会 来主管，下面再设立不同的岗位来具体执行，因此所有 大宗材料的采购都是通过项目建设小组或委员会来集体 决策的，决策过程更为理性，更注重产品的原料、生产 工艺、
java常用类知识点总结 Java常用类 要求: 1、掌握String和StringBuffer的区别，可以熟练使用String和StringBuffer的各 种方法进行相关操作。 2、能够自己编写一个得到日期的操作类，并将日期进行格式化操作。 3、掌握比较器及其基本原理，并可以通过比较器进行对象数组的比较操作。 4、掌握对象克隆技术及其实现 5、能够灵活应用正则表达式对字符串的组成进行判断 6、掌握Math、Random、基本数据类型的包装类的使用 7、描述出Object System对垃圾收集的支持 8、使用NumberFormat、DecimalFormat、BigInteger、BigDecimal进行数字的操 作 String和StringBuffer String的内容一旦声明不可改变，如果要改变，改变的是String的引用地址，如果一个字符串要经常改变，必须使用StringBuffer。 在一个字符串内容需要频繁修改时，使用StringBuffer可以提升操作性能，因为StringBuffer内容可以改变，而String内容不可改变。StringBuffer支持的方法大部分与String类似。 StringBuffer常见用法: (1) 字符串的连接操作
String类可以通过“+“进行字符串的连接，而StringBuffer中却只能使用append方法进行字符串的连接，而且此方法返回一个StringBuffer类的实例，这样就可以采用代码链的形式一直调用append方法。 (2) 在任意位置处为StringBuffer添加内容 可以使用insert方法在指定位置上为StringBuffer添加内容 字符串的反转操作(较为常见的操作，使用reverse方法) (3) 替换指定范围的内容 replace方法可对指定范围的内容进行替换。在String中如果要替换，使用的是replaceAll (4) 字符串截取(使用subString方法从指定范围中截取内容) (5) 删除指定范围的字符串(使用delete方法删除指定范围内容) (6) 查找指定内容是否存在(indexOf查找指定内容，查找到返回内容的位置， 没查到返回-1) 问题:(1)String s = "Hello";s = s + " world!";这两行代码执行后，原始的String对象中的内容到底变了没有, 没有。因为String被设计成不可变(immutable)类，所以它的所有对象都是不可变对象。在这段代码中，s原先指向一个String对象，内容是 "Hello"，然后我们对s进行了+操作，那么s所指向的那个对象是否发生了改变呢,答案是没有。这时，s不指向原来那个对象了，而指向了另一个 String对象，内容为"Hello world!"，原来那个对象还存在于内存之中，只是s这个引用变量不再指向它了。通过上面的说明，我们很容易导出另一个结论，如果经常对字符串进行各种各样的修改， 或者说，不可预见的修改，那么使用String来代表字符串的话会引起很大的内存开销。因为 String对象建立之后不能再改变，所以对于每一个不同的字符