REVIEW
Histidine kinase Hik33is an important participant in cold-signal transduction in cyanobacteria
Norio Murata a,*and Dmitry A.Los b
a National Institute for Basic Biology,Myodaiji,Okazaki,444-8585,Japan
b Institute of Plant Physiology,Russian Academy of Sciences,Botanicheskaya Street35,127276Moscow,Russia
Correspondence
*Corresponding author,
e-mail:murata@nibb.ac.jp
Received5September2005;revised17 September2005
doi:10.1111/j.1399-3054.2005.00608.x Acclimation of living organisms to cold stress begins with the perception and transduction of the cold signal.However,traditional methods failed to identify the sensors and transducers of cold stress.Therefore,we combined systematic mutagenesis of potential sensors and transducers with DNA microarray analysis in an attempt to identify these components in the cya-nobacterium Synechocystis sp.PCC6803.We identified histidine kinase Hik33as a potential cold sensor and found that Hik33participates in the regulation of the expression of more than60%of the cold-inducible genes. Further study revealed that Hik33is also involved in the perception of hyperosmotic stress and salt stress and transduction of the signals. Complexity of responses to cold and other environmental stresses is discussed.
Introduction
Decreases in ambient temperature reduce enzymatic activities and ultimately depress various physiological activities.When the temperature changes suddenly and significantly,organisms often fail to survive.When such change is gradual,organisms can acclimate to their environment by sensing the shift in temperature and expressing large numbers of previously unexpressed genes,with resultant synthesis of specific proteins and metabolites that participate in protection against low temperature(Fig.1).Acclimation begins with the per-ception of the shift in temperature and transduction of the resultant https://www.doczj.com/doc/108755380.html,anisms and/or individual cells appear to be equipped with sensors and signal transdu-cers that perceive and transduce cold signals.This review focuses on the initial events in cold-inducible gene expression,describing our analysis of potential sensors and transducers of cold stress in cyanobacteria (also reviews by Los and Murata2002,2004,and by Mikami and Murata2003).
Unicellular cyanobacteria are particularly suitable for studies of stress responses at the molecular level.The general features of their plasma and thylakoid mem-branes resemble those of the chloroplasts of higher plants in terms of both lipid composition and assembly of membranes.Thus,cyanobacteria appear to provide powerful model systems for studies of the molecular mechanisms of acclimation to low temperature (Murata and Wada1995).
Some strains of cyanobacteria,such as Synecho-cystis sp.PCC6803(hereafter,Synechocystis), Synechococcus sp.PCC7942and Synechococcus sp. PCC7002,are naturally competent,incorporating for-eign DNA that is efficiently integrated into their
Abbreviations–Hik,histidine kinase;Rre,response regulator.
This paper is dedicated to Dr Marilyn Griffith,who passed away on February19th,2005,to recognize her outstanding contribution to research on cold stress in plants.
Physiologia Plantarum126:17–27.2006Copyright?Physiologia Plantarum2006,ISSN0031-9317
genomes by homologous recombination (Haselkorn 1991,Williams 1988).Therefore,many researchers have used cyanobacteria for the production of mutants with disrupted genes of interest (for review,see Vermaas 1998).
The Synechocystis genome was sequenced in 1996(Kaneko et al.1996)with subsequent publication of the genome sequences of other cyanobacteria (see Murata and Suzuki 2005for a complete list with references).Genome sequences provide basic information that can be exploited for the genome-wide study of gene expres-sion.A commercially available genome-wide DNA microarray for analysis of gene expression in Synechocystis (Takara Bio Co.,Ohtu,Japan)covers 3079of the 3165(97%)genes on the chromosome of Synechocystis (99genes for transposases are excluded from this calculation).There are also 397genes on plasmids harboured by Synechocystis (Kaneko et al.2003).These genes are not included in the above-mentioned DNA microarray.
Genome-wide analysis of cold-stress-inducible genes in Synechocystis
We used the DNA microarray from Takara to character-ize the genome-wide expression of genes in response
to environmental stresses,starting with cold stress (Suzuki et al.2001).The expression of a large number of genes was enhanced in response to cold stress,while that of another large number of genes was repressed.
Some researchers have inferred that cold stress induces cellular dehydration that is essentially identical to that induced by hyperosmotic stress.To examine whether Synechocystis recognizes these kinds of stress similarly,we compared the effects of cold stress and hyperosmotic stress by DNA microarray analysis (Fig.2;Mikami et al.2002).These two kinds of stress enhanced,in common,the expression of genes for high light-inducible proteins (hliA,hliB,and hliC ),for rare lipoprotein A (rlpA ),for DNA mismatch repair protein (mutS ),for a sigma factor (sigD ),and for proteins with other functions.However,only cold stress enhanced the expression of the rbpA gene for an RNA-binding protein,the ndhD2gene for NADH dehydrogenase subunit 4,the crhR gene for an ATP-dependent RNA helicase,the fus gene for translation elongation factor EF-G,the feoB gene for ferrous iron transport protein,the infB gene for translation initiation factor IF-2,and various genes for proteins of known and unknown function.By contrast,only hyperosmotic stress enhanced the expression of genes for heat-shock proteins (hspA,dnaK2,groEL2,and clpB1),for the synthesis of glucosylglycerol (ggpS and glpD ),for the synthesis of lipids and lipoproteins (fabG and repA ),and for various other proteins (htrA,
Cold stress Sensor
mRNAs Proteins - enzymes
Metabolites
genes Signal transducer
Acclimation to cold conditions
Fig.1.A general scheme for the responses of a cyanobacterial cell to cold stress.
rbpA, ndhD2, ndhF,crhR, rpl3, rpl4,fus, feoB, infB,ycf39, desB, nusG secE, folK, slr0082,slr0616, slr0236,slr1927, sll1611,sll0086
hspA, clpB1, fabG htrA, spkH
sigB, sodB, hik34htpG, dnaK2, dnaJ groES, groEL, groEL2ggpS, glpD gloA, repA
sll0528, sll0846, slr1963slr0959, sll1884slr1603, slr1915slr0967, sll0939……
402051
31
11
hliA, hliB hliC, rlpA,mutS, sigD,slr1544, sll1541sll1862, sll1863sll1483
Hyperosmotic
Cold
Fig.2.Some cold-stress-inducible genes and hyperosmotic stress-inducible genes are the same,and some are different.The diagram includes genes that are induced during incubation of Synechocystis for 20min after a shift in growth temperature from 34 C to 22 C (cold stress)and after addition of sorbitol at 0.5M (hyperosmotic stress).Adapted,with permission,from Mikami et al.(2002)with inclusion of recent results from authors’laboratory.
spkH,sodB,htpG,and gloA).Thus,cold and hypero-smotic stress each induced expression of a number of specific genes,while both stresses induced expression of a relatively small number of common genes(Fig.2), suggesting that Synechocystis recognizes cold stress and hyperosmotic stress as different signals via specific signal-transduction pathways.
Two-component systems:positive regulation and negative regulation
The existence of two-component systems has been well-established in Escherichia coli and Bacillus subtilis (Aguilar et al.2001,Stock et al.2000).Each two-component system consists of a histidine kinase(Hik) and a cognate response regulator(Rre).The Hik per-ceives a change in the environment via its sensor domain,and then a conserved histidine residue within the Hik domain is autophosphorylated,with ATP pro-viding phosphate group(Stock et al.2000)that is trans-ferred from the Hik to a conserved aspartate residue in the receiver domain of the cognate Rre.Upon phos-phorylation,the conformation of the Rre changes, allowing the binding of the Rre to promoter regions of genes that are located downstream in the acclimation pathway(Koretke et al.2000).
In E.coli and B.subtilis,the genes for each Hik and its cognate Rre are generally located close to one another on the chromosome.Moreover,these genes are often located near functionally related genes.
Two-component systems are found also in cyanobac-teria(Mizuno et al.1996).However,genes for Hiks and Rres in Synechocystis are,in most cases,distributed somewhat randomly on the chromosome.Among the 44genes for Hiks,14are located near genes for poten-tially cognate Rres,whereas genes for the other30Hiks are located far from any rre genes.Thus,it is difficult to predict the pairs of Hiks and Rres that might function as individual two-component systems.Therefore,we sys-tematically mutated the genes for all the potential sen-sors and transducers of environmental signals and examined gene expression under various conditions using DNA microarrays.
Synechocystis has3661putative genes,of which47 encode Hiks and45encode Rres(http://www.kazusa. or.jp/cyanobase/Synechocystis/index.html).There are 44genes for Hiks on the chromosome,which we named hik1through hik44.We deduced that three putative Hiks,namely,Hik11,Hik17,and Hik37, might be inactive as Hiks,because they lack the con-served histidine residue in the Hik domain.Hik32might also be inactive as a Hik,because the hik32(sll1473) gene is part of a larger gene,namely,sll1473-sll1475,which is interrupted by a transposon(sll1474)in the strain that was used for genome sequencing and sys-tematic mutagenesis(Okamoto et al.1999).The gene for the Hik encoded by pSYSM,a plasmid harboured by Synechocystis,was designated hik45,and the genes for Hiks encoded by pSYSX,another plasmid,were desig-nated hik46and hik47.There are42chromosomal genes for Rres and two and one on plasmids pSYSX and pSYSM,respectively.We designated the42chro-mosomal genes for rre1through rre42.The rre genes on pSYSM and pSYSX were designated rre43and rre44and 45,respectively.
We inactivated each putative hik gene in Synechocystis by inserting a spectinomycin-resistance gene cassette to create a gene-knockout library(Suzuki et al.2000;CyanoMutants,http://www.kazusa.or.jp/ cyano/Synechocystis/mutants/).This library has proved to be a powerful tool for the identification of sensors of various stimuli and the corresponding signal transducers in Synechocystis(Marin et al.2003,Paithoonrangsarid et al.2004,Shoumskaya et al.2005,Suzuki et al.2000, Suzuki et al.2004,Yamaguchi et al.2002).We exam-ined the genome-wide expression of genes in wild-type cells and in each line of hik mutant cells with DNA microarrays in an attempt to identify the Hiks involved in the regulation of expression of stress-inducible genes. Regulation of gene expression in response to stress can be positive or negative(for details,see Murata and Suzuki2005).In positive regulation,a two-component system is inactive under non-stress conditions.Genes controlled by such systems are silent or expressed. When cells are exposed to the appropriate environmen-tal stress,the two-component system is activated(by phosphorylation in response to the stress),and then the activated system enhances the expression of genes that are silent under non-stress conditions or represses the expression of genes that are expressed under non-stress conditions(Murata and Suzuki2005).Most stress-inducible gene expression in Synechocystis is positively regulated.
In negative regulation,the two-component system is active under non-stress conditions,and the expression of the genes controlled by such a system is either enhanced or repressed.Under appropriate environmen-tal stress,the two-component system becomes inactive. Genes that are expressed or repressed under non-stress conditions become silent or are released from repres-sion,respectively.Levels of expression of genes fall in the former case and rise in the latter.
Changes in phenotype due to mutations in Hiks and Rres,which reflect the effects of such two-component systems on gene expression,differ between positive regulation and negative regulation.A knockout
mutation in either the Hik or the Rre in a two-compo-nent system for negative regulation has marked effects on gene expression.The expression of genes controlled by a negatively regulating two-component system is either enhanced or repressed under non-stress condi-tions.Therefore,a specific signal-transduction pathway with a specific Hik and a specific Rre can be identified with relative ease in cases of negative regulation.
By contrast,a knockout mutation in a Hik or an Rre in a two-component system that operates via positive reg-ulation does not have a significant effect on gene expression under non-stress conditions.In such a sys-tem,the identification of the Hik and the Rre in a specific signal-transduction pathway requires the screening of knockout libraries of hik and rre genes under individual types of stress.
Negative regulation of gene expression Using DNA microarray,we analyzed the effects of muta-tion of each hik gene on gene expression in mutant cells that had been grown under normal conditions.No sig-nificant alterations in gene expression were evident in most of the mutants.However,three mutants,D hik27, D hik34,and D hik20,each with a mutation in the indi-cated Hik,were unique,because,in these mutants,the expression of some genes was enhanced and that of some others was repressed,indicating that Hik27,Hik34,and Hik20might be involved in signal transduction via the negative regulation of gene expression.
We compared gene expression in D hik27cells with that in wild-type cells under normal conditions (Yamaguchi et al.2002).Marked changes,with induc-tion factors higher than10,due to mutation of the hik27 gene(slr0640)were recognized only in the expression of three genes,namely,mntC,mntA,and mntB,which constitute the mntCAB operon that encodes subunits of the ABC-type Mn2ttransporter(Bartsevich and Pakrasi 1995,1996).Under normal growth conditions,Hik27 might transduce a signal that represses the expression of the mntCAB operon.Moreover,disappearance of this signal,due to inactivation of Hik27,might allow the expression of the mntCAB operon.This scenario corre-sponds to the scheme outlined for negative regulation. We examined the effects of mutation of various rre genes on gene expression in mutant cells.The mutation in D rre16cells enhanced the expression of only the mntC,mntA,and mntB genes.This phenomenon was very similar to the change in gene expression detected in the D hik27mutant.In its active form,Rre16might have repressed expression of the mntCAB operon.Then, inactivation of Rre16in D rre16-mutant cells eliminated the repressive effect of Rre16,allowing the expression of the mntCAB operon.These findings suggested that Hik27and Rre16might constitute a two-component system,acting as the sensor and signal transducer of Mn2tdeficiency.Ogawa et al.(2002)identified this two-component system independently by‘traditional’methods.
Mutation of Hik34altered the genome-wide expres-sion of genes under normal conditions,namely,at a growth temperature of34 C(Suzuki et al.2005).In D Hik34cells,levels of transcripts of heat-shock genes, such as htpG,groES,and groEL1,were elevated,sug-gesting that Hik34might act as a negative regulator of the expression of these genes during normal growth. Because Hik34appeared to be a negative regulator of heat-shock-responsive genes,we postulated that its overexpression should result in repressed expression of these genes.We observed that the effect of overexpres-sion of hik34was the opposite of that of inactivation of hik34on the expression of heat-shock genes at the normal growth temperature(Suzuki et al.2005).This observation is consistent with the hypothesis that Hik34 is important in the regulation of expression of heat-shock genes.
Positive regulation by Hik33in response to low temperature
Mutation of the42other hik genes did not induces significant changes in gene expression.Therefore,it is likely that the Hiks encoded by these42genes regulate gene expression in a positive manner.To confirm this hypothesis,we screened our library of hik mutants under specific kinds of stress.
To monitor the inducibility of the cold-inducible desB gene for the o3desaturase,we generated the pdesB::lux strain of Synechocystis,in which the promoter region of the desB gene was ligated to the coding region of the luxAB gene for a bacterial luciferase(Suzuki et al. 2000).Thus,we could use luciferase activity as an indicator of cold-inducible changes in the activity of the desB promoter.Then we inactivated separately the gene for each Hik in pdesB::lux cells by inserting a spectinomycin-resistance cartridge(Sp r),creating a gene-knockout library(Suzuki et al.2000).We screened the members of this library for loss of cold inducibility of gene expression by monitoring luciferase activity at a low temperature.Only pdesB::lux/D Hik33 and pdesB::lux/D Hik19,with disruption of the genes for Hik33and Hik19,respectively,exhibited reduced ability to activate luciferase at low temperature, suggesting that Hik33and Hik19might be involved in the perception and tranduction of cold signals.
DNA microarray analysis of D hik33cells indicated that Hik33regulates the expression of 21of 36cold-inducible genes,with induction factors higher than 3.0(Fig.3;Suzuki et al.2001).These 21genes include ndhD2,hliA,hliB,hliC ,feoB ,crp ,and genes for pro-teins of unknown function.By contrast,15of the 36cold-inducible genes were not regulated by Hik33.
Therefore,we can deduce that Synechocystis might have at least one other pathway for cold-signal trans-duction.DNA microarray analysis of the expression of genes in D hik19cells indicated that Hik19is unlikely to be a cold sensor.
To identify the Rre that is located downstream of Hik33,we screened an Rre-knockout library by RNA slot-blot hybridization using some of the cold-inducible genes controlled by Hik33as probes.We identified Rre26as a candidate for the Rre that,with Hik33,con-stitutes a two-component system for cold-signal trans-duction (our unpublished results;Fig.3A).
Tu et al.(2004)and Hsiao et al.(2004)postulated that Hik33might negatively regulate the expression of a set of photosynthetic and high light-responsive genes.Their hypothesis was based on changes in the global expres-sion of genes under normal conditions and the comple-mentation of the D hik33mutation in Synechocystis by the homologous nblS gene from Synechococcus with respect to the light-induced expression of hli genes,as monitored by Northern blotting.However,because the complementation test did not examine the genome-wide expression of genes,it is possible that their D Hik33mutant cells had,in addition to the mutation in hik33,a further mutation that might have produced changes in gene expression under normal conditions.To confirm our conclusion that Hik33is a positive regulator of signal transduction,we replaced the entire open-reading frame of the hik33gene with a spectino-mycin-resistance gene cassette that contained the O sequence,which is a strong terminator of transcription and inhibits the read-through of inserted genes on both sides of the cassette (our unpublished work).The growth rate of cells with deletion of hik33was similar to that of the wild-type and the insertion mutant of Hik33,contra-dicting the inferences made by Hsiao et al.(2004).DNA microarray analysis of the deletion mutant,comparing the pattern of gene expression with that of the insertion mutant under normal conditions,confirmed the absence of the major changes in gene expression reported by Hsiao et al.(2004).Our results suggest that the Hik33-dependent-signalling pathway involves the positive reg-ulation of gene expression.
Responses to salt stress and hyperosmotic stress
We demonstrated previously that the perception of hyperosmotic stress involves Hik33and other unknown components (Mikami et al.2002).Later when we used the DNA microarray to investigate the impact of muta-tions in each Hik on changes in gene expression under salt stress (Marin et al.2003),we found that the
Cytoplasmic membrane
?
slr1927sll1611slr0955slr0236sll0494slr1436slr1974
Hik33
Rre26
ndhD2hliA hliB hliC fus ycf39sigD
slr1747ssr2016slr0400sll1911slr0401sll0815sll1770
crhR rlpA rbpA cbiO cbiQ mutS desB slr0082
feoB crtP slr1544sll1483sll1541sll0086slr0616
Cold stress
Total: 21Total: 15
A
htrA
Hik10
?
?
Hyperosmotic stress
Hik34
Rre1
sll0939slr0967
Hik41Hik33
fabG hliA hliB hliC gloA sigD sll1483slr1544ssr2016ssl3446sll1541
rlpA repA glpD sll1863sll1862sll1772slr0581hspA clpB1sodB htpG dnak2spkH groES groEL1groEL2dnaJ
Rre3
Rre1
Rre17
Rre31
sigB sll0528slr1119slr0852ssr3188
sll0846slr1963slr0959sll1884slr1603slr1915ssl2971slr1413
ssr1853slr0112sll0294slr0895slr1501sll0293sll0470
Hik2Total: 11Total: 19Total: 5Total: 2Total: 1Total: 14
Cytoplasmic membrane
B
Hik16
Fig.3.Hypothetical schemes showing the two-component systems that are involved in the transduction of cold stress and hyperosmotic stress,as well as the genes that are under the control of the individual two-component systems.Primary signals are represented by open arrows.Hiks are indicated as ellipses,Rres are indicated as hexagons,and selectively regulated genes are shown in boxes.Uncharacterized mechanisms are represented by question marks.Genes with induction factors higher than 3.0are included in these schemes.Minor discrepan-cies with respect to cold-inducible genes between this figure and Fig.2are due to the use of different versions of the DNA microarray in the respective experiments.(A)Cold stress;adapted originally from Suzuki et al.(2001)and Mikami et al.(2002),with inclusion of more recent results from authors’laboratory.(B)Hyperosmotic stress;adapted from Paithoonrangsarid et al.(2004)and Shoumskaya et al.(2005).
inducibility of gene expression by elevated levels of NaCl was significantly affected in D Hik33-,D Hik34-, D Hik16-,and D Hik41-mutant cells.In each of these mutants,several genes were no longer induced by salt, or their inducibility by salt was markedly reduced. Because hyperosmotic stress and salt stress might affect some aspects of the physiology of cyanobacterial cells similarly,we examined whether these Hiks might also regulate gene expression under hyperosmotic stress.DNA microarray analysis demonstrated that they are indeed involved in hyperosmotic-signal trans-duction.Fig.3B shows that they regulate the expression of38of52hyperosmotic stress-inducible genes (Paithoonrangsarid et al.2004).
Screening our Rre-knockout library by RNA slot-blot hybridization and with DNA microarray,we identified four Rres,namely,Rre31,Rre1,Rre3,and Rre17,that are involved in hyperosmotic signal transduction. Further analysis with microarray showed that four Hik-Rre systems,namely,Hik33-Rre31,Hik34-Rre1,Hik10-Rre3,and Hik16-Hik41-Rre17,as well as another sys-tem that included Rre1and possibly Hik2,appeared to be involved in the perception of hyperosmotic stress and transduction of the signal(Paithoonrangsarid et al. 2004).Fig.3B shows a hypothetical model of the hyper-osmotic signal-transducing systems that involve these Hiks and Rres,including the hyperosmotic stress-inducible genes that are controlled by the individual Hik-Rre systems.
The Hik33-Rre31two-component system regulates the expression of11hyperosmotic stress-inducible genes.Inactivation of either Hik33or Rre31resulted in the elimination of or a marked reduction in the hyper-osmotic stress-inducible expression of these genes,indi-cating that Hik33and Rre31are tightly coupled in the signal-transduction pathway.The Hik10-Rre3two-component system regulates the hyperosmotic stress-inducible expression of only htrA,which encodes a serine protease.
The Hik16-Hik41-Rre17system regulates the hyper-osmotic stress-inducible expression of sll0939and slr0967.Inactivation of Hik16,of Hik41,or of Rre17 eliminated the expression of these genes,suggesting that Hik16,Hik41,and Rre17are all essential for the per-ception of hyperosmotic stress and for transduction of the corresponding signal.Hik41probably acts down-stream of Hik16,because Hik41is a hybrid-type Hik that contains both a signal-receiver domain and a Hik domain,whereas Hik16is a typical Hik with a Hik domain and potential sensory domain that,hypotheti-cally,spans the membrane seven times.It is also possi-ble that Hik16and Hik41might perceive hyperosmotic stress as a complex.
The Hik34-Rre1system regulates the expression of19 hyperosmotic stress-inducible genes(for heat-shock pro-teins and for proteins of unknown function).The Hik2-Rre1system regulates the expression of five genes that include the sigB gene for a sigma factor.Rre1might perceive hyperosmotic signals from both Hik34and Hik2.However,the regulated genes are specific to either His34or Hik2(Fig.3B;Paithoonrangsarid et al. 2004).
DNA microarray analysis revealed that expression of 14of the52hyperosmotic stress-inducible genes was not controlled by any of the five Hiks and four Rres discussed above(Fig.3B).The signals,due to hyperos-motic stress,that induce the expression of these genes are probably perceived by unknown mechanisms that differ from typical Hik-Rre two-component systems. Such signals might act directly to regulate either tran-scription or the stability of the transcripts of these indu-cible genes.
Using similar methods,we identified that five two-component systems are involved in the salt-stress signal-transduction pathway.To our surprise,they were iden-tical to those involved in response to hyperosmotic stress.However,the genes controlled by the individual pathways are different(Shoumskaya et al.2005),as discussed below.
His33is a multifunctional regulator
As described above,Hik33is involved in the perception and transduction of the cold signal and the hyperosmotic-stress signal(Fig.3).However,the cold-responsive genes controlled by Hik33are not identical to the hyperosmotic stress-responsive genes controlled by the same system.Fig.4shows that eight genes,including hliA,hliB,hliC,and sigD,are induced by both kinds of stress under the control of Hik33.However,Hik33reg-ulates the expression of eight other genes,including ndhD2,fus,crtP,and feoB,in response to cold stress specifically,and not to hyperosmotic stress,whereas it regulates the expression of three other genes,including fabG and gloA,in response to hyperosmotic stress spe-cifically,and not to cold stress.Thus,cold stress,hyper-osmotic stress,and salt stress are perceived as distinct signals by a sensory system that includes Hik33.In addition,recent studies(Hsiao et al.2004,Tu et al. 2004)suggest that Hik33might be involved in response to light stress.Moreover,a homolog of Hik33in Synechococcus is involved in sensing nutritional deficits (van Waasbergen et al.2002).
Another level of compexity reflects the differential involvement of Rres in signal transduction and gene expression.Microarray analysis indicated that Rre26is
involved in cold-signal tranduction (Fig.3A),whereas Rre31is involved in hyperosmotic-and salt-signal trans-duction (Fig.3B;Paithoonrangsarid et al.2004,Shoumskaya et al.2005).Moreover,eight genes respond,in common,to cold stress and hyperosmotic stress via Rre26and Rre31,respectively.Our observa-tions suggest that the mechanisms for perception of cold stress and hyperosmotic stress by Hik33are complex and that as-yet-unidentified components exist that pro-vide the sensory systems with their respective activities for induction of responses specific to each individual type of stress.
Hik33perceives the cold signal via rigidification of membrane lipids
The Hik33sensory kinase includes a type-P linker,a leucine zipper,and a PAS domain (Fig.5A;Los and Murata 1999,2002,2004,Mikami and Murata 2003).The type-P linker contains two helical regions in tandem that are assumed to transduce stress signals via intramo-lecular structural changes that result from interactions between the two helical regions and lead to intermole-cular dimerization of membrane proteins (Aravind et al.2003,Williams and Stewart 1999).In Hik33,cold stress might promote a conformational change in the type-P linker,with subsequent activation of Hik33via dimeriza-tion of the protein (Fig.5B;Los and Murata 2000,2004).There are two transmembrane domains in the amino-terminal region of Hik33(Los and Murata 2004,Mikami and Murata 2003).Because it has been suggested that
changes in membrane fluidity might be involved in the sensing of temperature (Los and Murata 2000,Murata and Los 1997),it is likely that the transmembrane domains of Hik33can recognize changes in membrane fluidity at low temperatures (Los and Murata 1999,2000,2004).
In 1996,we produced a series of mutants in which the extent of unsaturation of fatty acids is modified in a step-wise manner (Tasaka et al.1996).In one such mutant,the desA and desD genes for the D 12and D 6fatty acid desaturases,respectively,are inactive as a result of targeted mutagenesis.The desA –/desD –-double mutant synthesize only a saturated C16fatty acid and a mono-unsaturated C18fatty acid,regardless of growth temperature,whereas wild-type cells synthesize di-unsaturated and tri-unsaturated C18fatty acids in addi-tion to the mono-unsaturated C18fatty acid (Tasaka et al.1996).FTIR spectrometry revealed that the double mutation of the desA and desD genes rigidified the plasma membrane of Synechocystis at physiological temperatures (Szalontai et al.2000).
ndhD2, fus, crtP, ycf39, feoB, slr1747, sll0086, slr0616
fabG, gloA,ssl3044
8
3
8
11
21
Cold; Hik33-Rre26
Hyperosmotic;Hik33-Rre31hliA, hliB hliC, sigD slr1544ssr2016, sll1483sll1541
Fig.4.A schematic representation of genes that are induced by cold stress and hyperosmotic stress under control of Hik33.The diagram includes genes that are induced during incubation for 20min after a shift in growth temperature from 34 C to 22 C (cold stress)and after addition of sorbitol at 0.5M (hyperosmotic stress).
TM1
TM2
Type-P
PAS His kinase 132
48
201217220270310
350
400
600663
A
B
Type-P PAS
His kinase
H H
H
LZ LZ Fig.5.A hypothetical scheme for the structure of Hik33.(A)Domain structure of Hik33.(B)A putative dimeric form of Hik33and its relation-ship to the cell membrane.A decrease in temperature rigidifies the membrane,leading to compression of the lipid bilayer,which forces the membrane-spanning domains closer changes the linker conforma-tion and finally causes dimerization and autophosphorylation of histi-dine kinase domains.TM1and TM2,Transmembrane domains;LZ,leucine zipper;Type-P,a type-P linker domain;and PAS,PAS domain that contains amino acid motifs P er,Arnt,S im,and phytochrome (Taylor and Zhulin,1999);H in circles,histidine residues that can be phosphorylated in response to cold stress.
Using microarray,we examined the effects of the above-described membrane rigidification on the expres-sion of genes at low temperature in Synechocystis (Inaba et al.2003).We monitored changes in gene expression in wild-type and desA–/desD–cells after growth at34 C and subsequent incubation at22 C for30min.In wild-type cells,cold stress more than doubled the levels of expression of168genes.In desA–/desD–cells,in addition to the enhanced expres-sion of these same168cold-inducible genes,we observed enhanced expression of96additional genes. Thus,rigidification of membrane lipids apparently enhanced the response of gene expression to low tem-perature in Synechocystis.By contrast,under isothermal conditions,the double mutation had no significant effect on gene expression.
We divided cold-inducible genes into three groups according to the effects of the double mutation(Inaba et al.2003).The first group included genes that were not induced by low temperature in wild-type cells but were strongly induced by low temperature in desA–/desD–cells.The second group included genes whose low-temperature inducibility was moderately enhanced by the double mutation.The third group included genes whose inducibility by low temperature was unaffected by the double mutation.The response to low tempera-ture of the expression of the genes in these three groups might be regulated by different mechanisms with respect to membrane rigidity.Induction of expression of the genes in the first group might require greater rigidification of membrane lipids than the low-temperature responses of genes in the second and third groups.The rigidification of membrane lipids did not enhance the cold inducibility of genes in the third group,perhaps because the rigidity of membranes in wild-type cells is sufficient at low temperatures for the maximum induction of expression of these genes.
To examine whether Hik33might regulate the cold-responsive gene expression that depends on membrane rigidity,we examined genome-wide gene expression in desA–/desD–/hik33–cells,in which the hik33gene had been mutated in addition to mutation of the desA and desD genes.Mutation of Hik33abolished or signifi-cantly reduced the inducibility by low temperature of 10of the17genes in the second group and of seven of 25genes in the third group.By contrast,mutation of Hik33had no significant effect on the low-temperature inducibility of genes in the first group.These results indicate that Hik33regulates the expression of many genes in the second and third groups.They also suggest that the activity of Hik33in the sensing of low tempera-ture depends on membrane rigidity and that there are at least two other cold sensors,one of which depends on membrane rigidification,while the other functions inde-pendently of membrane rigidity(Inaba et al.2003). Cold sensors and cold-signal transducers in other organisms
Aguilar et al.(2001)identified DesK of Bacillus subtilis as a cold-sensing Hik and DesR as the cognate Rre that regulate the cold-inducible expression of the des gene for D5desaturase.The desK and desR genes form an operon on the genome of B.subtilis.
DesK is a membrane-bound protein with four trans-membrane domains and a Hik domain.However,in contrast to Hik33,DesK lacks the PAS and leucine zipper domains.DesK is a bifunctional enzyme with kinase and phosphatase activities.It has been suggested that DesK is involved in two signalling reactions:phos-phorylation in response to membrane rigidification and dephosphorylation in response to membrane fluidiza-tion(Mansilla et al.2004).In fact,the carboxy-terminal portion of DesK(DesKC)acts as an autokinase as well as a phosphatase;the phosphoryl group of phosphorylated DesKC is transferred to DesR.The resultant phosphory-lated DesR can be dephosphorylated in the presence of DesKC in vitro.These findings suggest that DesK has the ability to modify DesR through both its kinase and its phosphatase activities,depending on the physical state of the membrane.It is likely but,as yet,unproved that transmembrane segments of DesK sense changes in membrane fluidity due to changes in temperature (Albanesi et al.2004).
DesR binds specifically to the promoter region of the des for the D5desaturase.Induction of expression of des in B.subtilis by the DesK-DesR two-component system is inhibited by exogenous unsaturated fatty acids or isoleucine(Aguilar et al.2001;Cybulski et al.2002), suggesting the presence of a feedback loop between the function of the sensor and the extent of fatty acid unsa-turation.Cybulski et al.(2004)demonstrated that DesK, DesR,and the promoter region of the des gene interact directly with one another.The dephosphorylated form of DesR is unable to bind to a regulatory region of the des gene.DesK phosphorylates dimeric DesR,which becomes a tetramer,and binds upstream of the promo-ter of the des gene in a sequence-specific manner, with activation of des through recruitment of RNA polymerase to the promoter.Thus,the DesK-DesR two-component system regulates the expression of cold-inducible des,allowing the cell to optimize the fluidity of membrane phospholipids(Cybulski et al. 2004,Mansilla and de Mendoza2005).
The DesK-DesR system regulates the cold-inducible expression of the des gene but of no other genes.By
contrast,the cyanobacterial Hik33sensor regulates the expression of more than50cold-inducible genes (Mikami et al.2002,Suzuki et al.2001).
In plants,the discovery of the cold-regulation path-way that involves CBF/DREB led to further progress in the characterization of cold-signal transduction(reviews by Guy1999,Thomashow1999,Yamaguchi-Shinozaki and Shinozaki2005,Xiong et al.2002).Analysis of the transcriptional control of two cold-inducible genes (rd29A and cor15a)in Arabidopsis thaliana led to the identification of a cold-responsive element,the CRT/ DRE[(C-repeat)/(dehydration responsive element)],in their promoters(Shinozaki and Yamaguchi-Shinozaki 2000).Members of a family of AP2-domain-transcription factors,namely,DREB1(DRE-binding protein)and CBF (CRT-binding factor),bind to the CRT/DRE element and activate transcription.Expression of genes for these tran-scription factors is rapidly induced on cold treatment of plants.Moreover,at normal temperatures,overexpres-sion of CBF1,CBF2,or CBF3in transgenic A.thaliana enhanced the expression of41genes,30of which had been identified as cold-inducible genes in wild-type plants(Fowler and Thomashow2003).Thus,CBF tran-scription factors might regulate the expression not only of cold-inducible genes but also of genes whose expres-sion is induced by other signals.By contrast,some cold-inducible genes do not appear to be controlled via the CBF pathway.Thus,other regulatory systems might exist for the cold-inducible regulation of approximately60 genes(Fowler and Thomashow2003).
Despite the accumulation of important results about the cold regulation of gene expression,little is known about the temperature sensors in plants.Sensors and transducers of cold signals in plants remain to be identified.
Conclusion
Genome-based systematic analysis is a powerful techni-que for the identification of Hiks and Rres that are involved in the perception and transduction of cold signals and other kinds of stress https://www.doczj.com/doc/108755380.html,ing this approach,we showed,with relative ease,that Hik33 regulates the expression of most of the cold-inducible genes in Synechocystis,that membrane rigidification is intimately involved in the sensing of low temperature, and that Hik33is also involved in the perception and transduction of other types of stress.We must now determine how the same Hik can perceive and trans-duce more than one kind of environmental signal and regulate the expression of different sets of genes(Los and Murata2002,2004,Mikami et al.2002, Shoumskaya et al.2005).Our findings cannot be explained by the current model of two-component sys-tems,in which a Hik perceives a specific signal and regulates the expression of a particular set of genes via the phosphorylation-dependent activation(or inactiva-tion)of a cognate Rre.It is likely that as-yet-unidentified components are important in determining the specificity of the responses to individual types of stress.It is also possible that the sensors of environmental signals are highly organized protein complexes,in which Hiks, Rres,and various unidentified components are some-how associated.To identify these components,we shall have to develop new techniques,which,most probably,will also exploit the information encoded in the genomes of cyanobacteria and other organisms. Acknowledgements–This work was supported by a Grant-in-Aid for Scientific Research on Priority Areas (no.14086207)from the Ministry of Education, Science,Sports and Culture of Japan to N.M.It was also supported by grants from the Russian Foundation for Basic Research(nos.03-04-48581and05-04-50883) and by a grant from the‘Molecular and Cell Biology Program’of the Russian Academy of Sciences to D.A.L. References
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Edited by C.Guy
白介素IL-6信号转导及其通路研究概述 细胞因子是一类参与免疫系统的细胞之间通信的蛋白质，除此之外，许多细胞因子在免疫系统之外也具有调节功能。1986年白介素IL-6作为B细胞刺激因子被Kishimoto组分子克隆。IL-6在免疫系统外的活性还有肝细胞刺激因子和骨髓细胞分化诱导蛋白。 白介素IL-6含有184个氨基酸，属于糖基化蛋白质。IL-6可以由多种类型细胞合成和分泌，包括单核细胞、T细胞、成纤维细胞和内皮细胞。IL-6结合受体有两种，一种是特异性受体IL-6R（80kDa I型跨膜蛋白），另一种是gp130，是IL-6家族细胞因子的所有成员的常见受体亚单位。gp130可以在所有细胞表达，但IL-6R的表达受到更多的限制，主要发现于肝细胞、嗜中性粒细胞、单核细胞和CD4+ T细胞。 白介素IL-6受体gp130的二聚化会导致两种细胞内信号通路的启动：经典信号通路和反式信号通路（见下文）。白介素IL-6的受体IL-6R可以在细胞膜经过蛋白质水解，形成可溶性的IL-6R（sIL-6R），在人类中，也可以在翻译阶段进行剪接mRNA，进而产生sIL-6R。在经典信号通路中，IL-6与膜上的IL-6R结合，随后与结合在细胞膜上的gp130结合，启动细胞内信号传导。在IL-6反式信号通路中，IL-6与sIL-6R结合，IL-6和sIL-6R的复合物与细胞膜结合的gp130结合，从而引发细胞内信号。 白介素IL-6是最重要的炎症细胞因子之一。IL-6在通过膜结合和可溶性受体的信号传导中是独特的。有趣的是，这两种途径的生物学后果有很大差异，通过膜结合受体的经典IL-6信号通路主要是再生和保护性的，可溶性IL-6R的IL-6反式信号通路是促炎症的。响应于受体激活的IL-6的细胞内信号传导是通过STA T依赖和STAT独立的信号模块，其由复杂的调节网络调节。IL-6的复杂生物学对该细胞因子的治疗靶向具有影响。 白介素IL-6胞内信号通路可以简单的概述为：IL-6与受体复合物结合后，激活JAK1。JAK1磷酸化gp130细胞质部分内的酪氨酸残基，这些磷酸酪氨酸基序是STAT转录因子，SOCS3反馈抑制剂和衔接蛋白和磷酸酶SHP2的募集位点。SHP2连接到MAPK级联，使Gab1磷酸化，磷酸化的Gab1转移到质膜上，协调正在进行的MAPK和PI3K活化。Src家族激酶独立于受体磷酸化并激活Y AP。 白介素IL-6信号转导第一步：激活JAK。 大多数细胞因子受体缺乏胞内激酶活性，生长因子的受体例外。白介素IL-6胞内信号转导首先激活Janus激酶（JAK），开启酶促反应。通过JAK N末端的同源结构域内（JH）
1JAK-STAT信号通路 1)JAK与STAT蛋白 JAK-STAT信号通路是近年来发现的一条由细胞因子刺激的信号转导通路，参与细胞的增殖、分化、凋亡以及免疫调节等许多重要的生物学过程。与其它信号通路相比，这条信号通路的传递过程相对简单，它主要由三个成分组成，即酪氨酸激酶相关受体、酪氨酸激酶JAK和转录因子STAT。(1)酪氨酸激酶相关受体（tyrosinekinaseassociatedreceptor） 许多细胞因子和生长因子通过JAK-STAT信号通路来传导信号，这包括白介素2?7（IL-2?7）、GM-CSF（粒细胞/巨噬细胞集落刺激因子）、GH（生 长激素）、EGF（表皮生长因子）、PDGF（血小板衍生因子）以及IFN（干扰素）等等。这些细胞因子和生长因子在细胞膜上有相应的受体。这些受体的共同特点是受体本身不具有激酶活性，但胞内段具有酪氨酸激酶JAK 的结合位点。受体与配体结合后，通过与之相结合的JAK的活化，来磷酸化各种靶蛋白的酪氨酸残基以实现信号从胞外到胞内的转递。 (2)酪氨酸激酶JAK（Januskinase） 很多酪氨酸激酶都是细胞膜受体，它们统称为酪氨酸激酶受体（receptor tyrosinekinase,RTK），而JAK却是一类非跨膜型的酪氨酸激酶。JAK是英文Januskinase的缩写，Janus在罗马神话中是掌管开始和终结的两面神。之所以称为两面神激酶，是因为JAK既能磷酸化与其相结合的细胞因子受体，又能磷酸、JAK1个成员：4蛋白家族共包括JAK结构域的信号分子。SH2化多个含特定
JAK2、JAK3以及Tyk2，它们在结构上有7个JAK同源结构域（JAKhomologydomain,JH），其中JH1结构域为激酶区、JH2结构域是“假”激酶区、JH6和JH7是受体结合区域。 (3)转录因子STAT（signaltransducerandactivatoroftranscription）STAT被称为“信号转导子和转录激活子”。顾名思义，STAT在信号转导和转录激活上发挥了关键性的作用。目前已发现STAT家族的六个成员，即STAT1-STAT6。STAT蛋白在结构上可分为以下几个功能区段：N-端保守序列、DNA结合区、SH3结构域、SH2结构域及C-端的转录激活区。其中，序列上最保守和功能上最重要的区段是SH2结构域，它具有与酪氨酸激酶Src的SH2结构域完全相同的核心序列“GTFLLRFSS”。 2)JAK-STAT信号通路 与其它信号通路相比，JAK-STAT信号通路的传递过程相对简单。信号传 递过程如下：细胞因子与相应的受体结合后引起受体分子的二聚化，这使得与受体偶联的JAK激酶相互接近并通过交互的酪氨酸磷酸化作用而活化。JAK激活后催化受体上的酪氨酸残基发生磷酸化修饰，继而这些磷酸化的酪氨酸位点与周围的氨基酸序列形成“停泊位点”（dockingsite），同时含有SH2结构域的STAT蛋白被招募到这个“停泊位点”。最后，激酶JAK 催化结合在受体上的STAT蛋白发生磷酸化修饰，活化的STAT蛋白以二 聚体的形式进入细胞核内与靶基因结合，调控基因的转录。值得一提的是，一种JAK激酶可以参与多种细胞因子的信号转导过程，一种细胞因子的信号通路也可以激活多个JAK激酶，但细胞因子对激活的STAT分子却具有一定的选择性。例如IL-4激活STAT6，而IL-12 。STAT4却特异性激活
综述与进展 p38M APK信号转导通路与细胞凋亡研究进展 王誉霖1,张励才2 作者单位：1.安徽省宣城市人民医院麻醉科242000;2江苏徐州医学院作者简介： 王誉霖（1978，女,吉林市人，住院医师，硕士。研究方向：疼痛信号转导及调控。 主题词p38丝裂原活化蛋白激酶类；细胞凋亡;综述 中图分类号R345文献标识码A文章编号1674 8166（201012 1665 03 丝裂原活化蛋白激酶（mitog en2activated pr otein kinase,MA PK级联是细胞内广泛存在的丝/苏氨酸蛋白激酶超家族，是将细胞质的信号传递至细胞核并引起细胞核发生变化的重要物质。目前在人类已鉴定了4条MAPK途径:细胞外信号调节蛋白 激酶（ex tra cellular sig nal regulated protein kinase,ERK途径,C Jun 基末端激酶（c Jun N term inal kinase,JN K/应激活化蛋白（stress activated protein kinase,SAPK途 径,ERK5/大丝裂素活化蛋白激酶1（big MAP MAP kinase,BM K1途径和p38M APK（p38mitogen activated protein kinases,p38MA PK 传导途径［1］。p38 信号途径是 MAPK家族中的重要组成部分，多种炎症因子和生长因子及应激反应可使p38MAPK的酪氨酸和苏氨酸双磷酸化，从而激活p38M APK,使它在炎症、细胞应激、凋亡、细胞周期和生长等多种生理和病理过程中起重要作用。因此,p38MAPK 通路参与了多种刺激引起的信号级联反应，表明它在引起多种细胞反应中起重要作用，并且,p38在细胞凋亡中也有着重要的调节效应。1 p38M APK信号转导通路 丝裂原活化蛋白激酶（m ito gen activated pr otein kinase,MA PK级联是细胞内重 要的信号转导系统之一。在哺乳动物细胞M APK通路主要有:细胞外信号调节激酶（extracellular signal r eg ulated kinase,ERK ffi路、p38MA PK 通路、c jun 氨基末端激酶（c jun N term inal kinase,JNK通路和ERK5 通路［1］。其中,p38MAPK 是M APK 家族中的重要成员。
实验项目名称：基本信号的产生和时频域抽样实验 实验项目性质：普通实验 所属课程名称：信号分析与处理 实验计划学时：2 一、实验目的 1学习使用matlab 产生基本信号波形、实现信号的基本运算 2熟悉连续信号经理想采样前后的频谱变化关系，加深对时域采样定理的理解； 3 加深理解频谱离散化过程中的数学概念和物理概念，掌握频域抽样定理的基本内容。 二、实验内容和要求 1 用Matlab 产生以下序列的样本，并显示其波形： (a): ()(0.9)cos(0.2/3),020n x n n n ππ=+≤≤ (b): )20()5()(---=n u n u n x
(c): )*5.0exp()(n n x -= (d): )1.0sin()(n n x π= (e): ||1000)(t a e t x -=
(f): )()sin()(0t u t Ae t x t a Ω=-α 2 设||1000a )t (x t e -= （a ）：求其傅里叶变换)jw (X a ； （b ）：用频率Hz s 5000F =对)t (x a 进行采样，求出采样所得离散时间信
号]n[ x a1的傅里叶变换) ( X 1 jw a e；再用频率Hz s 1000 F=对)t( x a 进行采样， 求出采样所得离散时间信号]n[ x a2的傅里叶变换) ( X a2 jw e； （c）：分别针对（b）中采样所得离散时间信号]n[ x a1和]n[ x a2 ，重建出 对应的连续时间信号)t( x a1和)t( x a2 ，并分别与原连续时间信号)t( x a 进 行比较；根据抽样定理（即Nyquist定理）的知识，说明采样频率对信号重建的影响。 3 已知序列x[k]={1,1,1;k=0,1,2},对其频谱) (Ωj e X进行抽样，分别取N=2,3,10，观察频域抽样造成的混叠现象。
1 JAK-STAT 信号通路 1) JAK 与STAT 蛋白 JAK-STAT 信号通路是近年来发现的一条由细胞因子刺激的信号转导通路，参与细胞的增殖、分化、凋亡以及免疫调节等许多重要的生物学过程。与其它信号通路相比，这条信号通路的传递过程相对简单，它主要由三个成分组成，即酪氨酸激酶相关受体、酪氨酸激酶JAK和转录因子STAT。 (1) 酪氨酸激酶相关受体( tyrosine kinase associated receptor ) 许多细胞因子和生长因子通过JAK-STAT 信号通路来传导信号，这包括白介素2?7 (IL-2?7 )、GM-CSF (粒细胞/巨噬细胞集落刺激因子)、GH (生长激素)、EGF (表皮生长因子)、PDGF (血小板衍生因子)以及IFN (干扰素)等等。这些细胞 因子和生长因子在细胞膜上有相应的受体。这些受体的共同特点是受体本身不具有激酶活性，但胞内段具有酪氨酸激酶JAK 的结合位点。受体与配体结合后，通过与之相结合的JAK 的活化，来磷酸化各种靶蛋白的酪氨酸残基以实现信号从胞外到胞内的转递。 (2) 酪氨酸激酶JAK ( Janus kinase ) 很多酪氨酸激酶都是细胞膜受体，它们统称为酪氨酸激酶受体( receptor tyrosine kinase, RTK )，而JAK 却是一类非跨膜型的酪氨酸激酶。JAK 是英文Janus kinase 的缩写，Janus 在罗马神话中是掌管开始和终结的两面神。之所以称为两面神激酶，是因为JAK既能磷酸化与其相结合的细胞因子受体，又能磷酸化多个含特定 SH2结构域的信号分子。JAK蛋白家族共包括4个成员：JAK1、JAK2、JAK3以及Tyk2，它们在结构上有7个JAK同源结构域(JAK homology domain, JH )，其中JH1结构域为激酶区、JH2结构域是“假”激酶区、JH6和JH7是受体结合区域。 (3) 转录因子STAT ( signal transducer and activator of transcription ) STAT 被称为“信号转导子和转录激活子”。顾名思义，STAT在信号转导和转录激活上发挥了关键性 的作用。目前已发现STAT家族的六个成员，即STAT1-STAT6。STAT蛋白在结构上可分为以下几个功能区段：N-端保守序列、DNA结合区、SH3结构域、SH2结构域及C-端的转录激活区。其中，序列上最保守和功能上最重要的区段是SH2结构域，它具 有与酪氨酸激酶Src的SH2结构域完全相同的核心序列“ GTFLLRFSS ”。 2) JAK-STAT 信号通路 与其它信号通路相比，JAK-STAT 信号通路的传递过程相对简单。信号传递过程如下：细胞因子与相应的受体结合后引起受体分子的二聚化，这使得与受体偶联的JAK激酶相互接近并通过交互的酪氨酸磷酸化作用而活化。JAK激活后催化受体上的酪氨酸残 基发生磷酸化修饰，继而这些磷酸化的酪氨酸位点与周围的氨基酸序列形成“停泊位
高速信号在提升电子设备性能的的同时，也为检定和调试的设计工程师带来了很多问题。在这些问题中，一类典型的例子是偶发性或间歇性的事件以及一些低占空比的信号，如激光脉冲或亚稳定性，低占空比雷达脉冲等等。这些事件很难识别和检定，要求测试设备同时提供高采样率和超强的数据捕获能力。这对示波器性能提出了极高的要求。在过去，要对这些信号的测试不得不在分辨率和捕获长度之间进行取舍：所有示波器的存储长度都是有限的；在示波器中，采样率×采集时间＝采集内存，以使用示波器的所有采集内存为例，采样率越高，则数据采集的时间窗口越小；另一方面，若需要加长采集时间窗口，则需要以降低水平分辨率(降低采样率)为代价。 当前的高性能示波器提供了高采样率和高带宽，因此现在的关键问题是优化示波器捕获的信号质量，其中包括：怎样以足够高的水平分辨率捕获多个事件，以有效地进行分析；怎样只存储和显示必要的数据，优化存储器的使用。 对于这两个关键问题，泰克的高性能示波器采用FastFrame分段存储技术，改善了存储使用效率和数据采集质量，消除了采集时间窗口和水平分辨率不可兼得的矛盾。 本文将分别介绍传统方法和FastFrame分段存储技术测试偶发性或间歇性的事件以及一些低占空比的信号，从而分析FastFrame分段存储技术在实际测试带来好处。 1. 传统测试方法 传统测试低占空比脉冲等间歇性的信号，通常利用数字示波器。为了提高测试精度，通常使用示波器的最高采样率来采集波形数据。通常在高采样率的支持下，可以看到大部分波形细节，见图1。 但是，如果想查看多个连续脉冲，那么必须提高采集的时间窗口。要让多个脉冲落在示波器提供的有限存储器内，很多时候必须通过降低采样率来达到。显而易见地，降低采样率本身会降低水平分辨率，使得时间测试精度大大下降。当然，用户也可以扩展示波器的存储器的长度，在不降低采样率的情况下提高采集时间窗口。但是，这种方法有其局限性。尽管存储技术不断进步，高速采集存储器仍是一种昂贵的资源，而且很难判断多少存储容量才足够。即使拥有被认为很长的存储器长度，但可能仍不能捕获最后的、可能是最关键的事件。 图2是在长记录长度时以高分辨率捕获的多个脉冲。从图2中可以看出，时间窗口扩展了10倍，可以捕获更多的间歇性脉冲。其实现方式：通常是提高采集数据的时间长度，并提高记录长度，同时保持采样率不变。这种采集方法带来了以下这些缺点： 1.更大的采集数据提高了存储器和硬盘的存储要求。 2.更大的采集数据影响着I/O传送速率。 3.更高的记录长度提高了用户承担的成本。 4.由于示波器要处理更多的信息，因此前后两次采集之间的不活动时间或“死区时间”提高了，导致更新速率下降。 考虑到这些矛盾，必须不断地在高采样率与每条通道提供的存储长度中间做出平衡，并且还是很难达到测试更多个脉冲的需求。
细胞信号转导途径研究方法 一、蛋白质表达水平和细胞内定位研究 1、信号蛋白分子表达水平及分子量检测: Western blot analysis. 蛋白质印迹法是将蛋白质混合样品经SDS-PAGE后,分离为不同条带，其中含有能与特异性抗体(或McAb)相应的待检测的蛋白质(抗原蛋白)，将PAGE胶上的蛋白条带转移到NC膜上此过程称为blotting，以利于随后的检测能够的进行，随后,将NC膜与抗血清一起孵育，使第一抗体与待检的抗原决定簇结合(特异大蛋白条带)，再与酶标的第二抗体反应,即检测样品的待测抗原并可对其定量。 基本流程： 检测示意图：
2、免疫荧光技术 Immunofluorescence （IF） 免疫荧光技术是根据抗原抗体反应的原理，先将已知的抗原或抗体标记上荧光素制成荧光标记物，再用这种荧光抗体（或抗原）作为分子探针检查细胞或组织内的相应抗原（或抗体）。在细胞或组织中形成的抗原抗体复合物上含有荧光素，利用荧光显微镜观察标本，荧光素受激发光的照射而发出明亮的荧光（黄绿色或桔红色），可以看见荧光所在的细胞或组织，从而确定抗原或抗体的性质、定位，以及利用定量技术测定含量。 采用流式细胞免疫荧光技术（FCM）可从单细胞水平检测不同细胞亚群中的蛋白质分子，用两种不同的荧光素分别标记抗不同蛋白质分子的抗体，可在同一细胞内同时检测两种不同的分子（Double IF），也可用多参数流式细胞术对胞内多种分子进行检测。 二、蛋白质与蛋白质相互作用的研究技术 1、免疫共沉淀（Co- Immunoprecipitation, Co-IP）
Co-IP是利用抗原蛋白质和抗体的特异性结合以及细菌蛋白质的“protein A”能特异性地结合到免疫球蛋白的FC片段的现象而开发出来的方法。目前多用精制的protein A预先结合固化在agarose的beads 上，使之与含有抗原的溶液及抗体反应后，beads上的prorein A就能吸附抗原抗体达到沉淀抗原的目的。 当细胞在非变性条件下被裂解时，完整细胞内存在的许多蛋白质－蛋白质间的相互作用被保留了下来。如果用蛋白质X的抗体免疫沉淀X，那么与X在体内结合的蛋白质Y也能沉淀下来。进一步进行Western Blot 和质谱分析。这种方法常用于测定两种目标蛋白质是否在体内结合，也可用于确定一种特定蛋白质的新的作用搭档。缺点：可能检测不到低亲和力和瞬间的蛋白质－蛋白质相互作用。 2、GST pull-down assay GST pull-down assay是将谷胱甘肽巯基转移酶（GST）融合蛋白（标记蛋白或者饵蛋白，GST, His6, Flag, biotin …）作为探针，与溶液中的特异性搭档蛋白(test protein或者prey被扑获蛋白)结合，然后根据谷胱甘肽琼脂糖球珠能够沉淀GST融合蛋白的能力来确定相互作用的蛋白。一般在发现抗体干扰蛋白质－蛋白质之间的相互作用时，可以启用GST沉降技术。该方法只是用于确定体外的相互作用。
实验一 基本信号产生及信号的基本运算 一、实验目的 1.熟悉Matlab 的使用 2.掌握信号处理中基本信号的产生 3.掌握信号处理中信号的基本运算 二、实验内容 1．编程产生以下常用信号并画出波形（n 为信号波形显示区间） （1） )(0n n +δ 0n =-5；0；5 1010≤≤-n （2） )(0n n u + 0n =-5；0；5 3010≤≤-n （3）cos()()8 n u n π 300≤≤n （4）1()()4 n u n 020n ≤≤ 2．编程实现以下基本运算并画出波形 （1）1()()(10)f n u n u n =-- 2020n -≤≤ （2）21()(2)f n f n =- （3）31()()f n f n =- （4）已知(){11021}x n =-，，，，， 求10()2R h n =(n)，求系统的零状态响应。 三、思考题 1．MATLAB 中几种方式可以计算系统的零状态响应？ 四、实验报告要求 1．简述实验目的 2．按实验内容要求编写实验程序，并附上实验结果 3．回答思考题 4．总结实验中的问题
1: n1=-10:10; x1=[(n1-0)==-5];x2=[(n1-0)==0];x3=[(n1-5)==5]; subplot(231) stem(n1,x1,'fill'),grid on xlabel('T1'),title('x(n)=Delta(n-5)') subplot(232) stem(n1,x2,'fill'),grid on xlabel('T2'),title('x(n)=Delta(n+0)') subplot(233) stem(n1,x3,'fill'),grid on xlabel('T3'),title('x(n)=Delta(n+5)') n2=-10:30; u1=[(n2-5)>=0];u2=[(n2-0)>=0];u3=[(n2+5)>=0]; subplot(234) stem(n2,u1,'fill'),grid on xlabel('T1'),title('x(n)=u(n-5）') subplot(235) stem(n2,u2,'fill'),grid on xlabel('T2'),title('x(n)=u(n+0）') subplot(236) stem(n2,u3,'fill'),grid on xlabel('T3'),title('x(n)=u(n+5）') 2: n1=0:30; x1=cos(pi/8*n1); subplot(211); stem(n1,x1,'fill');grid on; title('x(n)=cos(pi/8*n)u(n)'); n2=0:20; x2=(1/4).^n2; subplot(212); stem(n2,x2,'fill');grid on; title('x(n)=(0.25^n)u(n)'); 3: n=-20:20; x1=[(n-0)>=0&(n-10)<=0]; %x1=[(n-0)>=0];x2=[(n-10)>=0];x3=x1-x2; subplot(311); stem(n,x1); title('f1(n)=u(n)-u(n-10)'); subplot(312); stem(n+2,x1)
1 JAK-STAT信号通路 1) JAK与STAT蛋白 JAK-STAT信号通路是近年来发现的一条由细胞因子刺激的信号转导通路，参与细胞的增殖、分化、凋亡以及免疫调节等许多重要的生物学过程。与其它信号通路相比，这条信号通路的传递过程相对简单，它主要由三个成分组成，即酪氨酸激酶相关受体、酪氨酸激酶JAK和转录因子STAT。 (1) 酪氨酸激酶相关受体（tyrosine kinase associated receptor） 许多细胞因子和生长因子通过JAK-STAT信号通路来传导信号，这包括白介素2?7（IL-2?7）、GM-CSF（粒细胞/巨噬细胞集落刺激因子）、GH（生长激素）、EGF （表皮生长因子）、PDGF （血小板衍生因子）以及IFN（干扰素）等等。这些细胞因子和生长因子在细胞膜上有相应的受体。这些受体的共同特点是受体本身不具有激酶活性，但胞内段具有酪氨酸激酶JAK的结合位点。受体与配体结合后，通过与之相结合的JAK的活化，来磷酸化各种靶蛋白的酪氨酸残基以实现信号从胞外到胞内的转递。 (2) 酪氨酸激酶JAK（Janus kinase） 很多酪氨酸激酶都是细胞膜受体，它们统称为酪氨酸激酶受体（receptor tyrosine kinase, RTK），而JAK却是一类非跨膜型的酪氨酸激酶。JAK是英文Janus kinase的缩写，Janus在罗马神话中是掌管开始和终结的两面神。之所以称为两面神激酶，是因为JAK既能磷酸化与其相结合的细胞因子受体，又能磷酸化多个含特定SH2结构域的信号分子。JAK蛋白家族共包括4个成员：JAK1、JAK2、JAK3以及Tyk2，它们在结构上有7个JAK同源结构域（JAK homology domain, JH），其中JH1结构域为激酶区、JH2结构域是“假”激酶区、JH6和JH7是受体结合区域。(3) 转录因子STAT（signal transducer and activator of transcription）STAT被称为“信号转导子和转录激活子”。顾名思义，STAT在信号转导和转录激活上发挥了关键性的作用。目前已发现STAT家族的六个成员，即STAT1-STAT6。STAT蛋白在结构上可分为以下几个功能区段：N-端保守序列、DNA结合区、SH3结构域、SH2结构域及C-端的转录激活区。其中，序列上最保守和功能上最重要的区段是SH2结构域，它具有与酪氨酸激酶Src的SH2结构域完全相同的核心序列“GTFLLRFSS”。 2) JAK-STAT信号通路 与其它信号通路相比，JAK-STAT信号通路的传递过程相对简单。信号传递过程如下：细胞因子与相应的受体结合后引起受体分子的二聚化，这使得与受体偶联的JAK
实验项目一 常见离散信号产生和基本运算 1．实验目的 （1）掌握MATLAB 最基本的矩阵运算语句； （2）掌握对常用离散信号的理解与运算实现。 2．实验内容 （1）熟悉MATLAB 的使用环境和方法； （2）练习使用基本的向量生成、矩阵运算、绘图等语句； 利用冒号（：）生成向量： X1=[1 2 3 4 5] X2=[1.000 1.500 2.000 2.500] X3=[5 4 3 2 1] 分别生成3*3，3*4的全0矩阵，全1矩阵和随机矩阵； 分别输入矩阵： 123 456789A = 1.0 1.1 1.22.0 2.1 2.23.0 3.1 3.2 B = 分别计算A+B ，A-B ，A+3，A-4，A*B ，A.*B ，C=inv （A ）,A/B,A./B ； 分别计算sin(x1),cos(x1),exp(x1),log(x2),sqrt(x2)。 （3）生成以上五种基本离散信号函数； （4）绘出信号zn e n x =)(，当6)12/1(π j z +-=、6)12/1(π j z +=时、 121=z 、62π j z +=、6π j z =时的信号实部和虚部图； （5）绘出信号)1.0*2sin(5.1)(n n x π=的频率是多少？周期是多少？产生一个数字频率为0.9的正弦序列，并显示该信号，说明其周期并绘图。 3．实验技能要求 掌握并能灵活运用MATLAB 语句对离散时间信号进行基本建立和运算。 4．实验操作要求 在实验操作过程中要注意对基本实验仪器的保护。
5．实验场所 魂芯DSP应用实验室 6．实验课后训练 实验课后训练以实验报告为表现形式，在实验报告中要对实验过程中出现的问题进行分析和思考，对所测得的数据进行数据处理，并根据结果进行总结。
东南大学电工电子实验中心 实验报告 课程名称：电子线路实践 第七次实验 实验名称：信号的产生、分解与合成 院（系）：电子科学与工程学院专业： 姓名：姜勖学号：06A11315 实验室:104实验组别：27 同组人员：徐媛媛实验时间：年月日 评定成绩：审阅教师： 实验四信号的产生、分解与合成 一、实验内容及要求 设计并安装一个电路使之能够产生方波，并从方波中分离出主要谐波，再将这些谐波合成为原始信号或其他周期信号。 1.基本要求 （1）设计一个方波发生器，要求其频率为1kHz，幅度为5V； （2）设计合适的滤波器，从方波中提取出基波和3次谐波； （3）设计一个加法器电路，将基波和3次谐波信号按一定规律相加，将合成后的信号与原始信号比较，分析它们的区别及原因。 2.提高要求 设计5次谐波滤波器或设计移相电路，调整各次谐波的幅度和相位，将合成后的信号与原始信号比较，并与基本要求部分作对比，分析它们的区别及原因。 3.创新要求 用类似方式合成其他周期信号，如三角波、锯齿波等。 分析项目的功能与性能指标： 功能：通过振荡电路产生一个方波，并将其通过滤波得到1、3、5次谐波，最后通过加法电路合成新的波形。 性能指标： （1）方波：频率1KHz，幅度5V。 （2）滤波器：基础要求从方波中提取基波和三次谐波，提高要求提取五次谐波。 （3）移相电路：通过移相电路调节滤出来的1、3、5次谐波相位，使得其与原方波相位差近似为0。
（4）加法器电路：将基波和3次谐波和5次谐波信号按一定规律相加。 1、信号的产生 通过震荡电路产生1kHz ，幅度为5V 的方波信号。 2、滤波器的设计 根据方波的傅里叶展开式: 可知原信号分解只包含奇次谐波分量。因此设计不同中心频率的带通滤波器，可将各次谐波滤出。 3、相位校正电路 由于滤波器用到了对不同频率有不同响应的储能元件，对于滤除的波形会产生附加相位。若要让各次谐波叠加出原有信号，必须调节其相位使之同相。用全通滤波器可在不影响相对幅度的前提下改变相位。 4、加法电路 将滤除的基波、3次谐波、5次谐波相加，得到近似的方波信号。对于滤波器对不同频率分量不成比例的衰减，可在加法电路中选择合适的比例给予响应的补偿。 二、电路设计（预习要求） (1) 电路设计思想（请将基本要求、提高要求、创新要求分别表述）： 1、信号发生电路： 利用运放和RC 回路构成振荡电路，通过分别调节正反向RC 回路的时间常数和运放同相输入端的参考电压来调节震荡电路的频率以及占空比。用一对稳压二极管限制输出电压幅度，并对稳压管导通压降进行一定的补偿。 2、有源带通滤波器： 根据实验要求，设计有源带通滤波器，将所需频率的信号以尽量小的衰减输出，同时对其它频率有非常大的衰减。因此需要增加滤波器的阶数。初步选择采用二阶有源带通滤波器，通过理论计算，调节其中一个电阻来改变中心频率。根据实际搭出的电路效果，可尝试使用四阶有源带通滤波器，以求获得更好的滤波效果。 3、相移电路： 由于滤波器难免对滤出的谐波分量产生附加相位，需要在选频电路之后加一全通网络校正相位，抵消相位差。移向电路有两种，分为正向移向和反向移向。 4、加法电路 将所得到的各次谐波分量叠加，得到近似的方波。同时，加法电路可对滤波对原信号分量的衰减进行补偿。 (2) 电路结构框图（请将基本要求、提高要求、创新要求分别画出）： 基础要求：因基础要求与提高要求相比，除缺少5次滤波与移相电路外，其余部分均相同，其结构框图已包含在提高要求的框图中，故不单独列出。 提高要求： （3）电路原理图（各单元电路结构、工作原理、参数计算和元器件选择说明）： 分工：徐媛媛（滤波电路的设计、搭建和调试）；姜勖（方波产生、相移及加法电路设计搭建和调试） 方波振荡及鉴幅电路： 采用迟滞比较及RC 反馈回路以及比较器鉴幅电路，总电路图如下： 设从输出端的对输入端的负反馈电阻分别为1f R 和2f R ，则前部分方波的振荡周期为111222 ln(12)ln(12)f f R R T R C R C R R =+++，通过电位器分别调节1f R 和2f R 的阻值使方波的频率为1kHz ，占空比为50%。
实验二 基本信号的生成 1．实验目的 学会使用MATLAB 产生各种常见的连续时间信号与离散时间信号； 通过MATLAB 中的绘图工具对产生的信号进行观察，加深对常用信号的理解； 熟悉MATLAB 的基本操作，以及一些基本函数的使用，为以后的实验奠定基础。 2．实验内容 (1) 在 k [10:10]=? 范围内产生并画出以下信号： a) 1f [k][k]δ=； b) 2f [k][k+2]δ=； c) 3f [k][k-4]δ=； d) 4f [k]2[k+2][k-4]δδ=?。 (2) 在 k [0:31]=范围内产生并画出以下信号： a) ()()k k 144f [k]sin cos π π=； b) ()2k 24f [k]cos π =； c) ()()k k 348f [k]sin cos π π=。 请问这三个信号的基波周期分别是多少？ 3.实验程序 （1）k=-10:10; flk=[zeros(1,10),1,zeros(1,10)] subplot(2,2,1) stem(k,flk) title('fl[k]')
grid on f2k=[zeros(1,8),1,zeros(1,12)] subplot(2,2,2) stem(k,f2k) title('f2[K]') grid on f3k=[zeros(1,14),1,zeros(1,6)] subplot(2,2,3) stem(k,flk) stem(k,f3k) title('f3[k]') grid on f4k=2*f2k-f3k; subplot(2,2,4) stem(k,f4k) title('f4[k]') grid on （2）k=0:31; f1k=sin(pi/4*k).*cos(pi/4*k); subplot(3,1,1) stem(k,f1k) title('f1[k]') f2k=(cos(pi/4*k)).^2; subplot(3,1,2) stem(k,f2k) title('f2[k]') f3k=sin(pi/4*k).*cos(pi/8*k); subplot(3,1,3) stem(k,f3k) title('f3[k]')
怎样分析喷油脉宽信号? 喷油脉冲宽度是发动机微机控制喷油器每次喷油的时间长度，是喷油器工作是否正常的最主要指标。该参数所显示的喷油脉冲宽度数值单位为ms。该参数显示的数值大，表示喷油器每次打开喷油的时间较长，发动机将获得较浓的混合气；该参数显示的数值小，表示喷油器每次打开喷油的时间较短，发动机将获得较稀的混合气。喷油脉冲宽度没有一个固定的标准，它将随着发动机转速和负荷的不同而变化。 影响喷油脉；中宽度的主要因素如下： (1)λ调节； (2)活性炭罐的混合气浓度； (3)空气温度与密度； (4)蓄电池电压(喷油器打开的快慢)。 喷油量过大常见原因如下： (1)空气流量计损坏； (2)节气门控制单元损坏； (3)有额外负荷； (4)某缸或数缸工作不良。 喷油脉宽在汽车故障诊断中的应用 一、用脉宽诊断一下燃油反馈控制系统 使发动机运转5分钟以上，进入闭环控制状态，氧传感信号参与发动机反馈系统。关掉所有附属用电设备，测量喷油脉宽。 1。取掉油压调节器的真空管，并用软塞堵好，以防进气系统泄漏。此时转速上升，设法堵住回油管，人为使油压增高，如果反馈系统正常，氧传感器正常，可以看出喷油脉宽减少，一般减少0.1--0.2ms，这是电脑对过浓的混合气进行修正的结果。 2。造成真空泄漏，使混合气过稀。如果系统工作正常，脉宽将增加1.01--1.04ms，这是ECU对过稀混合气进行补偿的结果。 老的车型对怠速下氧传感器作用予以忽略，1800r/min转速下进行上述试验。 喷油脉宽在汽车故障诊断中的应用（二） 二、用怠速脉宽诊断油路 1。热车怠速正常运行时，脉宽一般为1.5ms--2.9ms。如果脉宽达到2.9--5.5ms一般是喷嘴有堵的现象。新车运行一段时间后，喷嘴就有不同程度的堵塞，使喷油量减少，电脑认为空燃比增大（即稀），怠速下降，会修正喷油脉宽、修正怠速控制信号，使怠速达到目标转速值。这个循环反复进行，怠速脉宽就越来越大。同时发动机控制电脑就将此时的怠速控制阀位置（步进电机之步数、或脉冲阀的占空比信号）储存下来以备下次起动时参考。由于各缸喷嘴堵塞的程度不一样，而发动机控制电脑向喷嘴提供的喷油脉宽是一致的，导致发动机工作不稳、动力不足、加速性不良、燃油消耗增加等现象产生。此时用一个好的清洗机可基本解决上述问题。 实例：时代超人清洗前脉宽 3.31ms 清洗后脉宽 1.70ms 应该注意，刚清洗好的喷嘴装车后，发动机转速会聚然提高，这是因为ECU长期燃油修正的结果，它记忆着学习以来的数据，以此控制怠速，使混合气过浓，这里有一个重新学习的过程，因车型的不同，学习时间也不尽相同，有些车几秒就可，有些车则需要更长的
细胞凋亡信号转导通路研究 细胞凋亡（apoptosis）是一种正常的生理性细胞死亡，它是在各种细胞因子的参与和严格控制下，有步骤的裂解过程，是为了维持机体内环境的稳定。细胞凋亡不足引起的疾病有肿瘤、自身免疫病等；凋亡过度引起的疾病有心肌缺血、心力衰竭、神经退行性疾病、病毒感染等；动脉粥样硬化是由于细胞凋亡过度与不足并存引起的疾病。由于细胞凋亡和这些重大疾病紧密相连，近些年来被广泛关注。研究最多的主要是细胞凋亡过程中的蛋白因子和通路。目前，凋亡通路一般认为有3条：死亡受体、线粒体和内质网通路。 标签：凋亡；caspases；死亡受体；线粒体；内质网 1死亡受体通路 死亡受体是一类跨膜蛋白，属肿瘤坏死因子受体（TNFR）超家族，该家族也被称为神经生长因子受体（NGFR）超家族。已知的死亡受体有TNFRI、Fas、DR3、DR4和DR5、CAR1。其相应的配体分别为TNF、FasL、Apo-3、Apo-2L、ASLV，这些配体构成了TNF超家族。它们由胞外区，跨膜区和胞内区组成，死亡受体与相应的配体结合后，可以通过一系列的信号转导过程，将凋亡信号传向细胞内部，最终引起caspases级联反应，引起细胞凋亡。 1.1 Fas/FasL介导的細胞凋亡FasL与Fas结合后，诱导Fas分子聚集成三聚体，通过Fas胞浆内死亡结构域DD与适配蛋白FADD结合，FADD的死亡效应结构域DED连接caspase-8的DED部分，形成死亡诱导信号复合体DISC，caspase-8经过加工以活性形式从DISC中释放出来。活化的caspase-8 可以绕开线粒体直接激活caspases家族其他成员caspase-3、6、7等引起细胞凋亡。目前，caspase-8激活下游的caspases诱导凋亡主要存在两种信号通路，这两种途径的激活主要由caspase-8的量决定：当DISC中caspase-8足量时，通过第一条信号途径，激活caspase-3、6、7，引起细胞裂解而凋亡；而当caspase-8少量时，通过第二条信号途径，caspase-8将胞浆中bcl-2家族的促凋亡蛋白分子bid裂解成一个含BH3结构域的tBid和一个小片段jBid[1]。tBid被运送到线粒体，与Bcl-2/Bax 的BH3结构域形成复合物，导致CytC释放，并与Apaf-1结合并活化Apaf-1激活caspase-9，随之激活caspase-3等引起凋亡。最新的研究表明，Fas的DD结构域还可以直接结合DAXX，激活JNK途径引起细胞凋亡。Fas/FasL细胞凋亡最重要的不同就是没有细胞核的参与和基因的活化。 1.2 TNFR1/TNF-α介导的细胞凋亡TNFR1含有3个功能域C端死亡域、ASD 和NSD，前两者在凋亡中起重要作用。 生理条件下，跨膜形式和可溶性TNF-α前体都是以三聚体的形式发挥作用的。TNF-α三聚体与TNFR1相互交联后诱导TNFR1的DD区聚集。TNFR1的DD区与TRADD的DD区相互作用，引起细胞裂解而凋亡或者导致线粒体释放CytC和Smac，活化线粒体凋亡途径。TRADD还可直接与TANK结合，激活JNK
脉冲占空比 占空比 占空比(Duty Ratio)在电信领域中有如下含义： 在一串理想的脉冲序列中（如方波），正脉冲的持续时间与脉冲总周期的比值。 例如：脉冲宽度1μs，信号周期4μs的脉冲序列占空比为0.25。 在一段连续工作时间内脉冲占用的时间与总时间的比值。 在CVSD调制（continuously variable slope delta modulation）中，比特“1”的平均比例（未完成）。 比特 信息量单位（bit） 数码转换器的基本构造，通常分为接收、数码滤波、数/类转换、I/V转换、类比放大等几个部分。以下仅就数码滤波与数/类转换作一浅释。 CD的取样频率为44.1KHz，这个规格的制定是根据Nyquist的取样理论而来，他认为要把类比讯号变成分立的符号（Discrete Time），取样时的频率至少要在原讯号的两倍以上。人耳的听觉极限约在20KHz，所以飞利浦在一九八二年推出CD时就将其制定为44.1KHz。取样是将类比讯号换成数码讯号的第一步，但精密度仍嫌粗糙，所以超取样的技术就出现了。一般八倍超取样就等于将取样频率提高到352.8KHz，一方面提高精度，一方面经过DAC之后产生的类比讯号比较完整，所需的低通滤波器（滤除音取样时产生的超高频）次数与斜率都可大幅降低，相位误差与失真也都会获得巨大改善。不过CD每隔0.00002秒才取样一次，超取样后样本之间就会产生许多空档，这时需要有一些插入的样本来保持讯号完整，而这样的任务就落在数码滤波器身上（Digital Filter）。比较先进的设计是以DSP（Digital Signal Processor）方式计算，以超高取样来求得一个圆滑曲线，例如Krell的64倍超取样，但目前只有Theta、Wadia、Krell、
关于信号转导研究的若干问题 郑仲承 （中国科学院上海生物化学研究所） 目录 第一节信号以及细胞传递信号的主要“设备” 第二节信号转导系统的特征 第三节二聚作用是调节信号转导的一个重要机制 第四节信号转导的生物学效应 第五节以信号转导为靶的疾病治疗 第六节走向未来 打信号（Signalling）是生物结间通消息的一种最基本,最原始和最重要的方式。比如，老虎沿着一个圈撒了一泡尿。这个圈所划定的范围就成为这只老虎的"领地"。别的老虎经过时，闻到这种味道就"识相"地悄悄离去，免遭麻烦。孙悟空用金箍棒在地上划了一个圈，让唐僧、八戒、沙僧和小龙马待在里面。妖怪来了，想抓走唐僧，却被这个圈发出的万道金光所逼退。又如，我国古代的烽火台，在外敌入侵时，狼烟四起，发出警报。交战双方下的战书，包括哀的美登书，都传递了作战的消息。写信、打电话、打手电。发暗示、对口令、对暗号、发SOS求救信号也是发消息，同情报的手段。美好的事情也要用信号来传达。如，蜜蜂告诉伙伴什么地方有美味的花粉时，就在伙伴们面前飞舞。以各种不同的优美舞姿指示食物的方向、方位、品种、数量和距离等等。鸟类在求偶时，相互欢快地仆翼，顶喙；蛇类在交欢时纠缠盘结的双蛇快步舞；昆虫的鸣叫等等。愉悦的信号还有下课的铃声、睡觉的号声、开饭的钟声、空调机的马达声等，当然，还有无线电的歌声，电视机的笑声等等。总之，生物的生命活动离不开信号。 生物的细胞每时每刻都在接触着来自细胞内或者细胞外的各种各样信号。有的信号激奋高昂，促进细胞增殖；有的信号谆谆劝诱，使细胞向一定的方向分化；有的信号如此迷惘，使得细胞误入歧途，无节制地分裂，"疯长"；有的信号哀徊低荡，让细胞心甘情愿地去死亡！ 虽然，我们身居闹市，经常在车辆的轰隆声和不绝耳的喇叭声、小贩的叫卖声、鸟叫蝉鸣、打击碰撞、潺潺流水、电话电视……中煎熬，但是，我们总能我自岿然不动地处变不惊，在这些杂乱无章的信号中找到自己需要的信号，作出正确的反应，安然地生活。即对有些信号置之不理，对有些信号听之任之，对有些信号一关了之，都有些信号则照此办理，作出反应。细胞也有一个接受、归纳、分析、筛选、放大、传达、处理和答复(响应)信号的过程与机制，使得细胞最终决定：是增殖分裂；是分化成熟；是变异追求一时的痛快，求己之生存而不顾其载体的死活，最后落个鸡飞蛋打，统统死光光；还是干干脆脆地自作了断，一死了之。 可见，信号只是个诱因，生理反应是信号作用于细胞的最终结果。相同的信号作用于不同的细胞可以引发完全不同的生理反应；不同的信号作用于同一种细胞却可以引发出相同的生理反应。细胞的一切生命活动都与信号有关，信号是细胞一切活动的始作俑者。因此，对信号转导的研究非常重要，非常有用。无怪乎近几年你也打信号，我也打信号，他、她也打信号，信号转导研究成为一个发烫的热点。 第一节信号以及细胞传递信号的主要“设备” 可以将细胞内的信号转导与电子计算机作比较。那些起着细胞内信号转导通路作用的分子可以视作为细胞内集成电路的分子转换器(开关)，它们放电时就与适当的信号接受器相连接。想象一下吧，尽管有些差异，电子计算机的操作过程与细胞内信号转导事件何其相似乃而！二者都有信息的定向流动；二者都有编纂过的语言，并通过它们将信息加以译释；二者又都有一套套的反应系统，通过这些反应就可以对它们所接受到的输入信号作出响应。当然，有生命的细胞比之电子计算机要高明得多。设想一下，在任何时刻，会有多少不同的细胞外刺激同时施加于细胞之上！它们驱动了多少细胞内信号转导通路！但是，在细胞内，所有这些信号通路都有严密的协调关系。显然，细胞内信号转导是一个有严密组织的，并且是高度网络的过程。 一作用于细胞的信号 生物细胞所接受的信号有多种多样，从这些信号的自然性质来说，可以分为物理信号、化学信号和生物学信号等几大