CN102631353A - Usage of toosendanin in preparation of targeted drug for resisting tumor - Google Patents
Usage of toosendanin in preparation of targeted drug for resisting tumor Download PDFInfo
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- CN102631353A CN102631353A CN2012101258844A CN201210125884A CN102631353A CN 102631353 A CN102631353 A CN 102631353A CN 2012101258844 A CN2012101258844 A CN 2012101258844A CN 201210125884 A CN201210125884 A CN 201210125884A CN 102631353 A CN102631353 A CN 102631353A
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Abstract
The invention discloses usage of toosendanin in preparation of a targeted drug for resisting tumor, and discloses a human deoxycytidinekinase (dCK) which is taken as a drug target for treating related diseases of the tumor, and the function of mutant of the human dCK in development of the targeted drug for resisting the tumor.
Description
Technical field
The invention belongs to drug world, relate to a kind of antineoplastic target medicine, the especially Toosendanin purposes in preparation antineoplastic target medicine.
Background technology
Targeted therapy is meant unusual molecule and the gene that relates to according in the known cancer generation, and design is to the medicine of these specific moleculars and gene target spot, optionally killing tumor cell.Though and chemotherapy can be played obvious antineoplastic; But it is in killing tumor cell; Also can large-arealy kill and wound the normal cell of body, compare with traditional chemotherapy, targeted therapy is just becoming the focus of oncotherapy research with its low toxicity and high therapeutic effect.
Toosendanin is a kind of a kind of tetracyclic triterpenoid that from traditional bark that drives ascarid Chinese medicine Meliaceae plant Fructus Toosendan Melia Toosendan Sieb.et Zucc or chinaberry Melia azedarach L. or seed, extracts; Have multiple biological activitys such as the ascarid of driving, parasite killing, anti-meat toxic effect, the release of inhibition neurotransmitter, be widely used in industries such as agricultural, medicine.In recent years discover; Toosendanin has apoptotic effect; But the action target of Toosendanin in tumor cell still is not very clear and definite; The signal transduction pathway that target is participated in it be unclear that, and this problem is for the antitumor action of further Toosendanin, and exploitation antineoplastic target medicine has important directive significance.
People source deoxycytidine kinase is one type of key enzyme in the pyrimidine salvage route, and it can its natural substrate of phosphorylation dC, dG, dA.In addition, it can also some nucleoside analog prodrugs of single phosphorylation, make it can continue its activity form of triphosphoric acidization, are incorporated on the DNA chain, suppress the activity of archaeal dna polymerase, stop the extension of DNA chain, cause the DNA chain interruption.Find the earliest and use and clinical nucleoside analog antitumor drug is cytosine arabinoside C (Ara-C), its mechanism be Ara-C by the single phosphorylation of DCK, be Ara-CTP by other enzyme catalysis then, and then disturb that tumor cell DNA's is synthetic.After this, some new nucleoside analogs appear, like his shore, Fuda, gemcitabine etc. again.Research shows that activity that the drug-fast increase of this type of nucleoside analog and DCK genetic flaw or other reason cause lacks relation is arranged, and DCK can these nucleoside analogs of catalysis wears the first step of film phosphorylation.Because it is DCK is bringing into play so important effect, also more and more about the research of its activity and inhibitor thereof.Disturb synthesizing of DNA and at present also only limit to DCK, and then be used for treatment of cancer and antiviral chemotherapy about the report of DCK effect.The Drug resistance that these medicines occur loss of activity or reduction common and DCK have certain relation.Existing discovering, DCK and the generation of immunological diseases also have certain related, and it all has very high expression at thymus and bone marrow, indicates that its effect in the lymphocyte generation can't neglect.Thymus and lymphocytic quantity obviously reduce than wild-type mice in the mice of DCK genetic flaw.And do not have relevant report in the effect aspect the apoptosis about DCK.Therefore, the comprehensive utilization multiple means is illustrated the new in vivo effect of DCK and is had great importance.
At present, the method for searching drug target protein mainly contains affinity chromatography, phage display clone, yeast three-hybrid system, the medicine marking, DNA chip technology, magnetic Nano probe technique etc.Wherein, affinity chromatography is the most classical a kind of method, and common affinity chromatography has solid phase specificity elution method and medicine competition law.But solid phase specificity elution method is vulnerable to " affinity column-target protein " complex velocity effect of dissociating, but also contrast post that must preparation non-activity analog medicine.The medicine competition law is because albumen can be with insoluble drug precipitation, and is very high to the requirement of medicine dissolution property.A kind of new affinity chromatography that can overcome above shortcoming that the series affinity chromatography was just reported in recent years, the target that is not applied to native compound is so far as yet found.
Summary of the invention
Goal of the invention of the present invention provides a kind of preparation of antineoplastic target medicine, that is, and and a kind of new application of Toosendanin.
For reaching the foregoing invention purpose, the technical scheme that the present invention adopts is: the application of Toosendanin in preparation antineoplastic target medicine.
In the technique scheme, with people source deoxycytidine kinase as drug targets.
Toosendanin (Toosendanin, C wherein
30H
38O
11), molecular weight is 574.62.The Toosendanin analog mainly comprises: lanoline alkane type is like pachymic acid, the acid of piece Siberian cocklebur and lanolin alcohol; The ginsenoside of all containing in dammarane type such as rod hammer triterpene A and Radix Ginseng, Radix Notoginseng and the Radix Panacis Quinquefolii; Ah's Fructus Artocarpi Heterophylli type such as Cycloastragenol glycosides; The masticadienonic acid of euphane type such as euphane, Burseraceae Boswellia and isomasticadienonic acid; Cucurbitane type such as blood gallbladder first are plain, blood gallbladder second is plain; Former obedient alkane type such as Rhizoma Alismatis terpene alcohol A and Rhizoma Alismatis terpene alcohol B.
Toosendanin changes the conformation of deoxycytidine kinase and makes its active increasing.Obtain the mutant of a kind of people source deoxycytidine kinase thus.Wherein,
The 35th mutant serine becomes glutamic acid, alanine, glutamine in the aminoacid sequence of people source deoxycytidine kinase;
The 74th amino acids is mutated into glutamic acid, alanine, glutamine in the aminoacid sequence of people source deoxycytidine kinase;
The 128th arginine is mutated into glutamic acid, alanine in the aminoacid sequence of people source deoxycytidine kinase.
The tumor type that above-mentioned targeted drug relates to mainly comprises: breast carcinoma, colorectal carcinoma, nonsmall-cell lung cancer and cancer of pancreas and lymphoma, leukemia and multiple myeloma.Relevant tumor cell mainly comprises adrenal various tumor cell strains of prostate, liver, nervus centralis, blood, lung and rat such as the PC3 that comes from the people, BEL7404, SH-SY5Y, U251, HL-60, U937, A-549, MDA-MB-468, PC12, K562 etc.
The present invention is in the process of research Toosendanin antineoplastic molecule mechanism, and adopting the HL-60 cell is object of study, explores Toosendanin and suppresses HL-60 hyperplasia and the molecular target that promotes its apoptosis.Utilize the Pull-down experimental verification TSN combine for specificity with target protein: use to be connected with TSN and to be molecular probe in post material Sephorase-4B; Adopt the method for serial affinity chromatography; Target protein in specific adsorption and the HL-60 tumor cell; And albumen is carried out simpleness identify that qualification result is that this target protein is deoxycytidine kinase (DCK).DCK is the dimer that to contain two sizes be the same subunit of 30.5kDa; Contain 260 aminoacid; Nucleoside kinase of its folding configuration and Drosophila melanogaster (dNK) and people's dGK is similar; Each monomer contain 5 folding, and around as the center round 10 α spirals, each monomeric the 4th dimer interface with 4 spirals composition of the 7th α spiralization.Owing to the phosphokinase activity to antineoplastic nucleoside analog medicine that DCK has, research active for it in recent years and effect is more and more, and its target spot or first time as the antitumor drug Toosendanin of mentioning among the present invention.
The present invention is after the target protein of having confirmed TSN is DCK, and the Kinase-Glo kinase activity detection kit that adopts Promega company has further been verified the interaction of TSN to DCK external.This test kit is a kind of through kinases and substrate reactions, the method that the homogenizing luciferase detects.Need the participation of ATP in the luciferase reaction, kinase whose phosphorylation also need consume ATP, and kinase reaction finishes, and adds isopyknic Kinase-Glo reagent, measures luminous signal.Residual A TP is directly proportional in luminous signal and the system, is inversely proportional to kinase activity.In this method, kinases is DCK, and substrate is dC.In the reaction system,, also added other a kind of micromolecule, i.e. TSN except adding the kinases and the substrate of reflection.There are many kinds of methods can be used for detecting kinase whose activity; Like bioluminescence; Fluorescence and radioactive label method, then two kinds of methods respectively have limitation when in use; Biloluminescence method is because of its easy operating; The output of low background and luminous value does not receive the interference of fluorescent composition and is employed more and more widely, and great majority reports all be directly in system, add enzyme with and substrate or inhibitor and reaction enzymes is lived, add other micromolecule therein and still monitor it the enzyme whether influential also report not of living.Experimental result of the present invention shows that TSN can play certain activation to DCK, and the activity increase degree of the addition of TSN and DCK has positive correlation (TSN concentration is increased progressively to 100 μ M twice multiple proportions by 12.5 μ M); Experimental result shows, with the increase of Toosendanin concentration, the activity of DCK brings up to original 106.78% respectively; 108.87%, 113.30%, 116.12%; Compare with matched group, significant difference is all arranged.
Bibliographical information is arranged; The autophosphorylation of DCK74 position serine can improve the activity of himself; Therefore the DCK mutant that carries out the aminoacid rite-directed mutagenesis to this site is arranged, in this patent, adopt the method for rite-directed mutagenesis; Made up 8 mutants of DCK, purpose is clear and definite TSN plays activation to DCK a mechanism.Remove 3 existing reports (S74E, S74A, S74Q), in addition 5 all do not have report (S35E, S35A, S35Q, R128E, R128A).Wherein, arginic the choosing of the 35th serine and the 128th carried out interactional molecular simulation with TSN and DCK and is the basis.Through identifying that mutant all makes up success.The mutant of realizing successful abduction delivering have 7 (S35E, S35A, S35Q, S74E, S74Q, 128E, R128A), we adopt wild type DCK as matched group, have detected the activity influence of TSN to 7 mutants of DCK respectively, method therefor is the same.The result does, TSN to 2 mutants (S74E S74Q) still plays activation, and to 5 mutants (S35E, S35A, S35Q, 128E R128A) does not play activation.This shows that TSN is not to carry out through the mechanism of its autophosphorylation to the activation of wild type DCK, but through its conformation change is carried out.
Because the technique scheme utilization, the present invention compared with prior art has advantage:
The invention provides the application of Toosendanin in preparation antineoplastic target medicine; (Human Deoxycytidine kinase is dCK) as drug targets of treating the tumor relevant disease and the effect of mutant in exploitation antineoplastic target medicine thereof to have disclosed people source deoxycytidine kinase.
Description of drawings
Fig. 1 is that embodiment utilizes serial affinity chromatography method to seek the SDS-PAGE analysis of carrying out the bonded target protein of specificity with Toosendanin.
Fig. 2 is the mass spectral analysis of Toosendanin binding proteins specific range of hydrolysed peptides section.
Fig. 3 is that the abduction delivering of wild type people source deoxycytidine kinase among the embodiment two and the SDS-PAGE of purification result analyze.
Fig. 4 detects the reaction principle figure of TSN to the active influence of DCK among the embodiment three.
Fig. 5 be among the embodiment three Toosendanin to the detection of the activity influence of wild type people source deoxycytidine kinase (* P 0.05, * * P 0.01 with matched group relatively).
Fig. 6 is that the mutation induction of people source deoxycytidine kinase among the embodiment four is expressed and the SDS-PAGE of purification result analyzes.
Fig. 7 be among the embodiment five Toosendanin to the detection of mutant protein activity influence (* P 0.05, * * P 0.01 with matched group relatively).
The specific embodiment
Below in conjunction with embodiment the present invention is further described:
Embodiment one: seek and the bonded albumen of Toosendanin specificity with serial affinity chromatography
Utilize Toosendanin and succinic anhydrides to generate the succinate that has carboxyl, the Toosendanin succinate is connected with post material EAH-Sepharose 4B, and the coupling rate is 39.8%, and the coupling amount is 3.22mg/ml.Choose the HL-60 cell and carry out cell culture, cell carries out cracking, gets cell pyrolysis liquid.LH-60 cell pyrolysis liquid 1ml is added in the 100 μ l affinity column materials, mix homogeneously, ice bath jolting reaction 40min, centrifugal, get post material deposition and identify.Supernatant then adds in the 100 μ l affinity column materials once more, mixing, ice bath jolting reaction 40min is centrifugal.Using such method is carried out compatible reaction continuously 3 times, and the post material deposition of 3 secondary response gained is refinement cellular lysate liquid buffer 1ml respectively, shake well, and centrifugal (2000rpm, 1min, 4 ℃) are abandoned supernatant, Using such method continuous washing 5 times.The washing back adds 30 μ l SDS-PAGE sample-loading buffers in deposition, 95 ℃ are heated 5min, fully discharges to be combined in the albumen on the post material, and centrifugal (10000rpm, 2min), supernatant is used for SDS-PAGE and identifies that resolving gel concentration is 13%.With silver staining method dyeing, dye the result behind the electrophoresis, find out the protein band that concentration reduces successively in 3 series and be the bonded destination protein of specificity according to silver.Result such as Fig. 1, albumen is for to carry out the bonded destination protein of specificity with TSN shown in the figure.The destination protein band utilizes efficient liquid-phase chromatography method to separate through the hydrolysis of Trypsin hydrolytic enzyme, the polypeptide fragment of destination protein hydrolysis is carried out the MALDI-TOF structure identify, identifies that this conjugated proteinly is DCK.Result such as Fig. 2.
Embodiment two: the abduction delivering and the purification of people source reorganization deoxycytidine kinase
The pET-28a that will contain people source reorganization deoxycytidine kinase gene transforms and gets into expressive host bacterium BL21, and the positive colony picking is gone into to contain in the LB fluid medium that the kanamycin final concentration is 50 μ g/ml, cultivates 12 ~ 14 hours for 37 ℃, to OD
600nmValue is about at 0.4 ~ 0.6 o'clock, add IPTG to final concentration be 100 μ M, continue at 15 ℃ and cultivate and carried out abduction delivering in 12 ~ 16 hours.The centrifugal collection thalline of 8000rpm/min is with the phosphate buffer washed twice of the 0.05M of PH7.0.The broken appearance smudge cells of high pressure cell, with 12000rpm/min centrifugal 25 minutes, get supernatant after centrifugal, can obtain the extract of people source deoxycytidine kinase.Adopt nickel post (Ni-NTA) affinitive layer purification method that thick enzyme is carried out separation and purification.At first use phosphate buffer (lysis buffer) the balance nickel post of the pH7.0 of 0.05M; Again with supernatant after centrifugal and the abundant mixing absorption of nickel post post material; Take to contain the method for variable concentrations imidazoles gradient elution, gradient is following: phosphate buffer (wash buffer) eluting that 1) contains the 0.05M of 100mM NaCl is adhesion protein not.
2) contain variable concentrations imidazoles (10Mm, 50Mm, 100Mm, 200mM, the phosphate buffer of 0.05M 500Mm) (elution buffer) gradient elution destination protein respectively.
3) use phosphate buffer (lysis buffer) the balance nickel post of the pH7.0 of 0.05M once more, subsequent use.
Carry out SDS-PAGE with the vertical electrophoresis appearance and detect protein sample.Get 20 μ L protein samples, add on the 5 μ L albumen appearance and go up a buffer and handle, sample-loading buffer is 10 μ L, and the protein of standard molecular weight is purchased the Sigma company in the U.S..The gum concentration of SDS-PAGE is 12%, concentrates electrophoresis with 85V voltage earlier, and reuse 130V voltage carries out separation electrophoresis.The result shows, people source deoxycytidine kinase be expressed as solubility expression, single subunit size is about 33kD.Referring to Fig. 3, the SDS-PAGE of people source deoxycytidine kinase purification result analyzes.Swimming lane 1 is 200 mM imidazoles eluting effluent, and swimming lane 2 is the protein standard substance of different molecular weight, and swimming lane 3 is 100 mM imidazoles eluting effluent, and swimming lane 4 is 50 mM imidazoles eluting effluent, and swimming lane 5 is a row cell pyrolysis liquid supernatant.
Embodiment three: TSN is to the detection of DCK activity influence
Adopt Kinase-Glo Plus Luminescent Kinase Assay method to detect TSN to the active influence of DCK.Reaction principle is referring to shown in Figure 4.
Through kinases and substrate reactions, the method that the homogenizing luciferase detects.Need the participation of ATP in the luciferase reaction, kinase whose phosphorylation also need consume ATP, after reaction finishes, detects luminous signal, and residual A TP is directly proportional in luminous signal and the system, is inversely proportional to kinase activity.Reaction system is 100 μ L, and adding final concentration respectively is the pure enzyme of 100nM, the ATP of 1 μ M and the dC of 5 μ M, and TSN is with different final concentrations (0 μ M, 12.5 μ M; 25 μ M, 50 μ M and 100 μ M) be added in the reaction system, with buffer (50mM Tris-HCl, 5mM MgCl; 0.5mM DTT, pH 7.5) complement to 50 μ L, reacted 30 minutes; The Kinase-Glo reagent that adds equal-volume (50 μ L), abundant mixing, room temperature left standstill 10 minutes.Utilize multi-functional ELIASA to detect luminous signal.Experimental result such as Fig. 5 show that TSN can play activation to DCK, and along with the increase of concentration, and the active increase of DCK is significantly (100%, 106.78%, 108.87%, 113.30%, 116.12%) also.
Embodiment four: the construction and expression purification of DCK mutant
(overlap extention PCR, principle OE-PCR) through the two-step pcr reaction, obtain to contain the people source deoxycytidine kinase gene in mutational site to utilize overlapping PCR.At first, design a pair of upstream and downstream primer according to the gene order of DCK, design a pair of mutant primer according to the mutational site to each mutant again, the restriction enzyme site that the design primer uses is NdeI and EcoRI.First step reaction is that template is carried out pcr amplification with the pET-28a that contains the DCK gene, and the genetic fragment that amplification is obtained reclaims, and is that template is carried out pcr amplification with the fragment that is recovered to again, obtains the purpose fragment.Respectively genes of interest that obtains and pET-28a plasmid are carried out enzyme action with NdeI and EcoRI; The enzyme action product connects with the T4 dna ligase; To connect in the competent cell that product is transformed into e. coli bl21 (DE3); Coat in the kanamycin culture dish that contains 50 μ g/ml 37 ℃ of incubated overnight.The monoclonal that sudden change obtains is compared through order-checking, and the aminoacid sequence of mutant protein is compared with wild type and all comprised required mutational site.Adopt the method among the embodiment two that mutant protein is carried out abduction delivering and purification.The result shows that except that mutant S74A, other 7 mutants are all realized successful expression and purification.Result such as Fig. 6 show that swimming lane 1-5 is respectively mutant protein S35E, S35A, and S35Q, S74A, S74E, swimming lane 6 is the protein standard substance of different molecular weight, swimming lane 7-10 is respectively mutant protein S74Q, R128E, R128A, R128Q.
Embodiment five: TSN is to the detection of mutant protein activity influence
Same method among employing and the embodiment three; Detect TSN to the active influence of mutant protein; Result such as Fig. 7 show that TSN still plays activation to the mutain of 74 mutant serines, and 35 serines and 128 mutant proteins for the arginine sudden change are not played activation.
Claims (2)
1. the application of Toosendanin in preparation antineoplastic target medicine.
2. application according to claim 1 is characterized in that: with people source deoxycytidine kinase as drug targets.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104997793A (en) * | 2015-06-24 | 2015-10-28 | 齐齐哈尔大学 | Application of toosendanin in preparation of drugs used for inhibiting lung cancer cell proliferation and inducting lung cancer cell apoptosis |
| CN109316483A (en) * | 2018-10-31 | 2019-02-12 | 上海中医药大学 | The medical usage of isochuanliansu |
-
2012
- 2012-04-26 CN CN2012101258844A patent/CN102631353A/en active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104997793A (en) * | 2015-06-24 | 2015-10-28 | 齐齐哈尔大学 | Application of toosendanin in preparation of drugs used for inhibiting lung cancer cell proliferation and inducting lung cancer cell apoptosis |
| CN109316483A (en) * | 2018-10-31 | 2019-02-12 | 上海中医药大学 | The medical usage of isochuanliansu |
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Application publication date: 20120815 |