Synthesis of nucleoside libraries on solid support. II. 2,6,8-Trisubstituted purine nucleosides using 8-bromoguanosine as precursor

A series of 2,6,8-trisubstituted purine nucleoside libraries was prepared by parallel solid-phase synthesis using 8-bromoguanosine as a common synthetic precursor. Polystyrene-methoxytrityl chloride resin was linked to the N2 or O5' position of the guanosine analogues. 8-Bromoguanosine was derivatized at the C8 position via carbon-carbon bond formation. Nucleophilic aromatic substitution at C2 and/or C6 positions with various amines produced two series of purine nucleoside libraries with very diverse substitution.     

Anti-malarial activity of N(6)-substituted adenosine derivatives. Part I

The synthesis and biological evaluation of novel N(6)-substituted adenosine derivatives is reported. The first series of compounds was obtained using an established procedure for the nucleophilic substitution of a 1-(6-chloro-purin-9-yl)-beta-D-1-deoxy-ribofuranose with various amines. In addition, attachment of two different amino-functionalised spacer arms at the N(6)-position of adenosine enabled derivatisation by an innovative polymer-assisted protocol. Thus, we were able to prepare three series of substituted derivatives that displayed activity versus the multiresistant Plasmodium falciparum strain Dd2 in cell culture experiments.     

Body nitrosation potential measured by a novel 15N breath test

Oxygenated nitrogen species, for example, the protonated form of nitrous acid (H2ONO+), dinitrogentrioxide (N2O3), dinitrogentetroxide (N2O4), or peroxynitrite (ONOO-), can react with amines to form molecular nitrogen. These reactions can occur spontaneously with primary aliphatic amines or via cytochrome P450 catalysed reactions with secondary amines. In principle measurements of the excretion of the molecular nitrogen generated by these reactions could be used as an index of the levels of oxygenated nitrogen compounds acting as nitrosating agents. To test this idea, [15N2]urea (3 mmol) was administered orally to five patients infected with Helicobacter pylori (as diagnosed by the [13C]urea breath test) and to four healthy volunteers. All participants ingested 3-mmol sodium nitrate as a precursor for NA 5 min before the ingestion of the nitrogen tracer. During the test the participants breathed 100% oxygen to increase the sensitivity of detection of endogenous molecular nitrogen. After the administration of [15N2]urea, the patients with H. pylori showed significantly increased 15N enrichments of exhaled N2, expressed as delta value (per 1000), compared with healthy volunteers (patients: 3.5 +/- 0.9 vs. volunteers: 1.3 +/- 0.4; p < .05). We speculate that the endogenous production of molecular nitrogen is a protective process controlling the body NO and nitrite levels. The 15N breath technique allows the noninvasive estimation of the body nitrosation and could indicate the health risk, possibly the oxidative stress status, caused by highly reactive oxygenated nitrogen species and carbenium ion intermediates.     

Analysis of ifosfamide, 4-hydroxyifosfamide, N2-dechloroethylifosfamide, N3-dechloroethylifosfamide and iphosphoramide mustard in plasma by gas chromatography-mass spectrometry

A sensitive and specific method for the simultaneous quantitation of ifosfamide (IF), 4-hydroxylifosfamide (4-OHIF), N2-dechloroethylifosfamide (N2D), N3-dechloroethylifosfamide (N3D) and iphosphoramide mustard (IPM) has been developed using gas chromatography-mass spectrometry (GC-MS) with an ion-trap mass spectrometer. Deuterium labeled analogues for each of these analytes were synthesized as the internal standards. The labile 4-OHIF in plasma was first converted to the more stable cyanohydrin adducts before dichloromethane extraction. IPM was extracted by C₁₈ reversed-phase resin. All analytes were converted to their silyl derivatives before GC-MS analysis. The sensitivity limits ranged from 0.1 to 0.5 μg/ml when 100 μl of plasma was used. This method was validated with within-run coefficients of variation less than 5% (n=8) and between-run coefficients of variation less than 12% (n=6). The method was applied to the determination of plasma levels of IF and metabolites in the rat.     

Chemical modification of N6-(N-threonylcarbonyl) adenosine. Part II. Condensation of the carboxyl group with amines.

Carboxyl group of N6-/N-threonylcarbonyl/adenosine was quantitatively modified with amines/aniline, glycine ethyl ester and ethylenediamine/in the presence of a water-soluble carbodiimide, yielding the respective amides. The reaction was carried out in a water solution of pH about 4 at 20 degrees C and was finished within minutes. The structure of the products was confirmed by UV and PMR spectra, and by chemical reactivity. Under conditions applied for modification of T6A, four common nucleosides and internucleotide linkage of UpA were unreactive, while 5'-AMP was transformed to the respective phosphoramides. At pH 4, the rate of 5'-AMP modification was over 100 times lower than the rate of t6A reaction.     

Simultaneous determination of histamine and N tau-methylhistamine with high-performance liquid chromatography using electrochemical detection

We have developed a liquid chromatographic method which uses electrochemical detection for the simultaneous quantitation of histamine and N tau-methylhistamine in rat brain. The amines are derivatized with the water-soluble Bolton-Hunter reagent (sulfo B-H). Perchloric acid extracts of rat brains are chromatographed on a strong cation-exchange resin. The eluate is evaporated and allowed to react with sulfo B-H at pH 9.8 at room temperature. The derivatization is complete after 30 s vortexing. The derivatives are purified using a cellulose-phosphate fibrous cation exchanger. They are quantified with an electrochemical detector at a potential of 0.56 V after preoxidizing the sample at 0.47 V. The derivatives of histamine, N tau-methylhistamine, and N alpha-methylhistamine are completely separated without interfering peaks. Since no N alpha-methylhistamine was detected in rat brain it was used as an internal standard. The detection limits are 0.1 pmol of histamine and 0.2 pmol of N tau-methylhistamine. The precision of this method is high, with within-run and between-run coefficients of variation of 2-7% and linearity of 0.999. Both histamine and N tau-methylhistamine peak heights increased significantly and selectively after treatment with pargyline. Because of the high sensitivity, accuracy, and precision, the histamine and N tau-methylhistamine contents of single nuclei of the rat hypothalamus can be routinely quantified.     

An efficient gel-phase synthesis of peptides on a high capacity flexible crosslinked polystyrene support: comparison with Merrifield resin.

A highly solvating copolymer was prepared in high yield by introducing a flexible crosslinker, 1,4-butanedioldimethacrylate, into the polystyrene matrix by a free radical aqueous suspension polymerization. A 2 mol% crosslinked resin showed rigidity and mechanical characteristics comparable to those of divinylbenzene-crosslinked polystyrene (Merrifield resin, DVB-PS) support. Swelling and solvation characteristics of the new resin, BDDMA-PS, were much higher than DVB-PS support in all solvents used for solid phase peptide synthesis. The diacrylate crosslinks in the resin network were found to be highly stable even after 48 h treatment with neat TFA, 6 N HCl and 6 N KOH at 110 degrees C. To demonstrate the usefulness of the new resin in high capacity peptide synthesis, a typical difficult peptide, acyl carrier protein (ACP) fragment (65-74), was synthesized on commercially available 1 mol% crosslinked DVB-PS and 2 mol% crosslinked BDDMA-PS resins under identical conditions. A protocol using NMP/DMSO mediated coupling was employed for chain assembly. The yield and purity of the product from BDDMA-PS resin was higher than when the DVB-PS resin was used. The mechanistic reason behind the synthetic efficiency of the new resin was found to be its ability to induce random coil conformation to the growing peptide chains.     

Sensitization of mice with wild-type and cold-adapted influenza virus variants: immune response to two H1N1 and H3N2 viruses

Two A strain influenza viruses, A/Hong Kong/123/77 (A/HK/123/77) (H1N1) and A/Queensland/6/72 (A/Qld/6/72) (H3N2), and the two cold-adapted reassortants which possess the surface antigens of these strains (CR35 and CR6, respectively) were tested for their ability both to induce primary cytotoxic T-cell (Tc cell) responses in mice and to sensitize mice for a second Tc cell response when challenged with a distantly related A strain virus, A/Shearwater/72 (H6N5). After intranasal inoculation, A/Qld/6/72 replicated to higher titers in the lung (1 to 2 log10 50% egg infective doses) than did A/HK/123/77 or either of the reassortants. A/Qld/6/72 induced higher Tc cell responses in the lung than did CR6, and both were more effective than either A/HK/123/77 or CR35 in this respect. When similar doses (10 or 10(3) hemagglutinin units) of each virus were injected intravenously into mice and the spleens were tested for Tc cell activity 6 days later, both A/Qld/6/72 and CR6 were ca. 100-fold better at inducing a primary Tc cell response than A/HK/123/77 or CR35. In contrast, the H1N1 and H3N2 viruses gave rather similar anti-hemagglutinin antibody titers (after intravenous injection) and delayed-type hypersensitivity reactions (after subcutaneous injection). If mice were primed with a low dose of these viruses (10(4) 50% egg infective doses intranasally), A/Qld/6/72 and CR6 were more effective than A/HK/123/77 or CR35 at sensitizing for a secondary Tc cell response when challenged with A/Shearwater/72, but if larger doses were given either intranasally (10(6) 50% egg infective doses) or intravenously (10 to 10(3) hemagglutinin units), all viruses sensitized the mice equally well, despite the fact the A/Shearwater/72 gives a poor primary Tc cell response in mice. Thus, the viral glycoprotein antigens can be important in determining the immunogenicity of the virus and, particularly, the class I antigen-restricted Tc cell response of the host.     

[15N]aspartate metabolism in cultured astrocytes. Studies with gas chromatography-mass spectrometry.

The metabolism of 2.5 mM-[15N]aspartate in cultured astrocytes was studied with gas chromatography-mass spectrometry. Three primary metabolic pathways of aspartate nitrogen disposition were identified: transamination with 2-oxoglutarate to form [15N]glutamate, the nitrogen of which subsequently was transferred to glutamine, alanine, serine and ornithine; condensation with IMP in the first step of the purine nucleotide cycle, the aspartate nitrogen appearing as [6-amino-15N]adenine nucleotides; condensation with citrulline to form argininosuccinate, which is cleaved to yield [15N]arginine. Of these three pathways, the formation of arginine was quantitatively the most important, and net nitrogen flux to arginine was greater than flux to other amino acids, including glutamine. Notwithstanding the large amount of [15N]arginine produced, essentially no [15N]urea was measured. Addition of NaH13CO3 to the astrocyte culture medium was associated with the formation of [13C]citrulline, thus confirming that these cells are capable of citrulline synthesis de novo. When astrocytes were incubated with a lower (0.05 mM) concentration of [15N]aspartate, most 15N was recovered in alanine, glutamine and arginine. Formation of [6-amino-15N]adenine nucleotides was diminished markedly compared with results obtained in the presence of 2.5 mM-[15N]aspartate.     

Pathogenicity of Nocardia caviae,N. asteroides and N. brasiliensis

The pathogenicity ofNocardia caviae, N. asteroides andN. brasiliensis has been tested for white mice, guinea pigs and rabbits and chorio-allantoic membrane of the developing chick embryo. Altogether, 14 strains belonging to the 3Nocardia species originating from soil, human and animal sources in India or abroad were tested. All of them proved pathogenic though the degree of virulence varied from strain to strain. Incorporation of hog gastric mucin in the inoculum enhanced the virulence of all the 3Nocardia species for white mice.N. caviae strains were uniformly more virulent than those ofN. asteroides andN. brasiliensis.In the white mice inoculated intraperitoneally, a greater dissemination of the disease was apparent withN. caviae than withN. asteroides. Of the 6 strains ofN. caviae tested, 5 disseminated to the lung, 3 to the heart and 2 to the brain. InN. asteroides dissemination of the disease to the brain was observed with 2 of its 3 strains.N. brasiliensis showed no dissemination.N. caviae was found to be equally virulent for white mice, guinea pigs and rabbits. On the other hand,N. asteroides andN. brasiliensis were more virulent for white mice than for guinea pigs and rabbits. The lesions caused byN. caviae in mice, guinea pigs and rabbits persisted up to 4 weeks. In strong contrast to this the lesions due toN. asteroides andN. brasiliensis found in the guinea pigs and rabbits showed a strong tendency towards spontaneous clearance.Histologically, the lesions caused byN. caviae, N. asteroides andN. brasiliensis in mice, guinea pigs and rabbits were in the form of abscesses which showed an acute or chronic reaction. In the case ofN. caviae these abscesses showed both granules and freely dispersed cocco-bacillary bodies or filaments. As forN. asteroides it occurred in the form of cocco-bacillary bodies or filaments whereasN. brasiliensis consistently produced granules in the lesions.The lesions caused by the 3Nocardia species on the chorio-allantoic membrane of the developing chick embryo were in the form of abscesses which contained cocco-bacillary bodies and branching filaments but no granules.This forms a part of the thesis submitted by P.V.K. for Ph. D. degree, of the University of Delhi.     

Isosafrole-induced cytochrome P2-450 in DBA/2N mouse liver. Characterization and genetic control of induction

Mouse "cytochrome P2-450" is defined as that form of isosafrole-induced P-450 in DBA/2N liver most specifically correlated with isosafrole metabolism. Isosafrole pretreatment does not induce aryl hydrocarbon hydroxylase activity ("cytochrome P1-450") in C57BL/6N or DBA/2N mice, induces acetanilide 4-hydroxylase activity ("cytochrome P3-450") more than 3-fold in C57BL/6N but not in DBA/2N mice, and induces isosafrole metabolite formation more than 3-fold in both C57BL/6N and DBA/2N mice. P2-450 was, therefore, purified from isosafrole-treated DBA/2N liver microsomes having negligible amounts of contaminating P1-450 and P3-450. The apparent molecular weight of P2-450 is 55,000, and the protein appears homogeneous on sodium dodecyl sulfate-polyacrylamide gels. The Soret peak of the reduced purified cytochrome X CO complex is 448 nm. Purified P2-450, reconstituted in vitro, metabolizes acetanilide poorly and benzo[a]pyrene hardly at all. Anti-(P2-450) inhibits (90 to 100%) liver microsomal isosafrole metabolite formation, yet has no effect on aryl hydrocarbon hydroxylase, acetanilide 4-hydroxylase, biphenyl 2- or 4-hydroxylase, or 7-ethoxycoumarin O-de-ethylase activities. 3-Methylcholanthrene induces anti-(P2-450)-precipitable protein about 12-fold in C57BL/6N and 2-fold in DBA/2N liver; 2,3,7,8-tetrachlorodibenzo-p-dioxin (10 micrograms/kg), about 12-fold in both C57BL/6N and DBA/2N liver; isosafrole, more than 3-fold in both C57BL/6N and DBA/2N. Benzo[a]anthracene at maximal doses induces anti-(P2-450)-precipitable protein in C57BL/6N liver no more than 2-fold, yet is known to be a highly potent inducer of P1-450 mRNA in C57BL/6N liver. The sensitivity of the P2-450 induction process to isosafrole is inherited as an autosomal additive trait; studies of offspring from the C57BL/6N(DBA/N)F1 X DBA/2N backcross confirm involvement of the Ah locus or s closely segregating gene. In contrast, among crosses between C57BL/6N and DBA/2N, sensitivity of the P1-450 and P3-450 induction process to 3-methylcholanthrene or 2,3,7,8-tetrachlorodibenzo-p-dioxin is inherited as an autosomal dominant trait. These data suggest that, although P1-450, P2-450, and P3-450 proteins are controlled by the Ah locus, either a P-450 protein polymorphism exists between C57BL/6N and DBA/2N mice or subtle differences may exist in the interaction of various inducers with Ah receptor.     

Parallel synthesis of DAPT derivatives and their gamma-secretase-inhibitory activity

Parallel synthesis of the C-terminal-modified DAPT (1) derivatives was accomplished utilizing our novel resin 7. Condensation reaction of the N-acylamino acid 10 with the amines 11a-o proceeded smoothly to give the corresponding amides 6a-o without any epimerization. Among the analogues, the benzophenonemethyl amide derivative 6o showed 30 times more potent activity than the original DAPT (1).     

A new arylating agent, 2-carboxy-4,6-dinitrochlorobenzene. Reaction with model compounds and bovine pancreatic ribonuclease.

The reagent 2-carboxy-4,6-dinitrochlorobenzene (CDNCB) reacts with the imino, amino and sulfhydryl groups of model compounds. At pH 8.2, sulfhydryl groups react much faster than do amines. N alpha-Acetylhistidine, N alpha-acetyltyrosine and N alpha-acetyltryptophan do not react. Poly(L-Lysine) and poly(DL-lysine) react about 50 times as fast as does N alpha-acetyllysine. A dichloroanalog, 6-carboxy-2,4-dinitro-1,3-dichlorobenzene, shows stepwise reactivity with amines. With bovine pancreatic ribonuclease, which contains no sulfhydryl, CDNCB reacts preferentially with the epsilon-amino of Lys-41 at 450 times the rate with the epsilon-amino of N alpha-acetyllysine. The preferential reactivity at Lys-41 is discussed in relation to the pK of Ly-41, the cationic character of the active site cleft, and the mechanism of RNAase action on substrates.     

Synthesis and antifilarial evaluation of N1,Nn- xylofuranosylated diaminoalkanes

A series of N(1),N(n)-xylofuranosylated diaminoalkanes (3-9 and 11-18) has been synthesized either by reductive amination of deoxy xylouloses (2a, 2b) with amines followed by one pot reduction with NaBH(4) or NaCNBH(3); or by 1,4-conjugate addition of amines to glycosyl olefinic esters (10a, 10b). The compounds were screened for their interference with filarial worms' glutathione metabolism, a potential target for chemotherapeutic attack. Interestingly, these compounds affected intracellular glutathione, gamma-glutamyl cysteine synthetase, glutathione reductase and glutathione-S-transferase(s) of bovine filarial worms to varying degrees. Some of the compounds though effected the motility and MTT reduction potential of filarial worms Brugia malayi, however, little microfilaricidal and macrofilaricidal were noted with compounds at 50mg/kg oral dose. Compounds 6, 16 and 17 were evaluated also for in vivo activity.     

The N domain of Smad7 is essential for specific inhibition of transforming growth factor-beta signaling.

Inhibitory Smads (I-Smads) repress signaling by cytokines of the transforming growth factor-beta (TGF-beta) superfamily. I-Smads have conserved carboxy-terminal Mad homology 2 (MH2) domains, whereas the amino acid sequences of their amino-terminal regions (N domains) are highly divergent from those of other Smads. Of the two different I-Smads in mammals, Smad7 inhibited signaling by both TGF-beta and bone morphogenetic proteins (BMPs), whereas Smad6 was less effective in inhibiting TGF-beta signaling. Analyses using deletion mutants and chimeras of Smad6 and Smad7 revealed that the MH2 domains were responsible for the inhibition of both TGF-beta and BMP signaling by I-Smads, but the isolated MH2 domains of Smad6 and Smad7 were less potent than the full-length Smad7 in inhibiting TGF-beta signaling. The N domains of I-Smads determined the subcellular localization of these molecules. Chimeras containing the N domain of Smad7 interacted with the TGF-beta type I receptor (TbetaR-I) more efficiently, and were more potent in repressing TGF-beta signaling, than those containing the N domain of Smad6. The isolated N domain of Smad7 physically interacted with the MH2 domain of Smad7, and enhanced the inhibitory activity of the latter through facilitating interaction with TGF-beta receptors. The N domain of Smad7 thus plays an important role in the specific inhibition of TGF-beta signaling.     

A substrate-cofactor free radical intermediate in the reaction mechanism of copper amine oxidase.

Reduction of copper amine oxidase with substrate led to the appearance of a free radical which can be detected in anaerobiosis by ESR and optical spectroscopy. The origin of this radical was examined through studies of the semiquinones of 6-hydroxydopamine, an analogue of the recently identified cofactor 6-hydroxydopa. The ESR spectrum of the 6-hydroxydopamine radical was too narrow to account for the enzyme radical signal; however, after spontaneous reaction with primary amines the hyperfine splittings and spectral width obtained by modulation broadening became very similar to those observed for the oxidase radical species. This effect was ascribed to covalent binding of a nitrogen atom directly to the aromatic ring structure, suggesting that the amine oxidase radical is an amino-6-hydroxydopa semiquinone. Identical ESR spectra were obtained using the amines putrescine, cadaverine, p-[(dimethylamino)methyl]benzylamine, and ethylenediamine; these oxidase substrates gave identical enzyme radical spectra as well. The interaction between cofactor and substrate was proved unambiguously by the technique of isotopic labeling: addition of [15N2]ethylenediamine instead of the normal 14N-labeled compound changed the ESR spectra of both the enzyme radical and its 6-hydroxydopamine counterpart. The results were confirmed by optical spectroscopy measurements; 6-hydroxydopamine and oxidized 6-hydroxydopamine gave spectra identical to those of reduced and oxidized amine oxidase, respectively. The 6-hydroxydopamine radical showed a sharp peak at 440 nm; upon addition of amines the maximum shifted to 460 nm, as found for the enzyme. It is proposed that copper amine oxidase represents the first example of a mixed substrate-cofactor radical within the family of tyrosine radical enzymes.     

Influence of N2 or O2 on two alkylating agents in Neurospora crassa.

Previous studies have indicated that the alkylating agent, 2-methoxy-6-chloro-9-(3-[ethyl-2-chloroethyl]aminopropylamino)acridine dihydrochloride (ICR-170), induces much more killing and mutation in conidia of Neurospora crassa treated in an atmosphere of N2 than in an atmosphere of O2. It was desirable to determine if a similar effect--more killing and mutation in N2 than in O2--could be observed with two other known alkylating agents, beta-propiolactone (BPL) and ethyl methanesulfonate (EMS), in the same test system. Conidia of a heterokaryotic strain of N. crassa were bubbled with N2 or O2 during treatment with BPL or EMS. Forward-mutation was measured in the ad-3 region by a direct method. The results indicate that N2 or O2 do not influence the lethal and mutagenic activities of BPL or EMS during treatment of conidia. Hence the influence of N2 or O2 on the lethal and mutagenic activites of ICR-170 is different from the influence of these gases on BPL or EMS using the ad-3 test system in N. crassa.     

Reactions of plant copper/topaquinone amine oxidases with N6-aminoalkyl derivatives of adenine

Plant copper/topaquinone-containing amine oxidases (CAOs, EC 1.4.3.6) are enzymes oxidising various amines. Here we report a study on the reactions of CAOs from grass pea (Lathyrus sativus), lentil (Lens esculenta) and Euphorbia characias, a Mediterranean shrub, with N6-aminoalkyl adenines representing combined analogues of cytokinins and polyamines. The following compounds were synthesised: N6-(3-aminopropyl)adenine, N6-(4-aminobutyl)adenine, N6-(4-amino-trans-but-2-enyl) adenine, N6-(4-amino-cis-but-2-enyl) adenine and N6-(4-aminobut-2-ynyl) adenine. From these, N6-(4-aminobutyl) adenine and N6-(4-amino-trans-but-2-enyl)adenine were found to be substrates for all three enzymes (Km approximately 10(-4)M). Absorption spectroscopy demonstrated such an interaction with the cofactor topaquinone, which is typical for common diamine substrates. However, only the former compound provided a regular reaction stoichiometry. Anaerobic absorption spectra of N6-(3-aminopropyl)adenine, N6-(4-amino-cis-but-2-enyl)adenine and N6-(4-aminobut-2-ynyl)adenine reactions revealed a similar kind of initial interaction, although the compounds finally inhibited the enzymes. Kinetic measurements allowed the determination of both inhibition type and strength; N6-(3-aminopropyl)adenine and N6-(4-amino-cis-but-2-enyl)adenine produced reversible inhibition (Ki approximately 10(-5) - 10(-4) M) whereas, N6-(4-aminobut-2-ynyl)adenine could be considered a powerful inactivator.     

Liquid phase synthesis of arylamines and its application to the benzimidazolone via nucleophilic aryl substitution.

A method for soluble, inexpensive polymer-supported synthesis of aryl amines and benzimidazolone on the basis of nucleophilic aryl substitution (S(N)Ar) is described. This method involves a direct coupling reaction between resin bound aryl fluoride and amines at ambient temperature. The products are isolated in quantitative yields and excellent purity by simple precipitation and washing. This liquid phase method proves to be a useful tool for constructing combinatorial arylamine and benzimidazolone libraries.     

甲1(H1N1)和甲3(H3N2)型流感病毒在小鼠体内诱导细胞免疫反应的研究

比较了甲型流感病毒A/HK/123/77(H1N1)和A/Qld/6/72(H3N2)及其具有相应表面抗原的两个冷适应基因重分配株(Cold-Adapted Reassortant)的子代毒株CR35和CR6,在小鼠体内诱导初次细胞毒性T细胞(简称Tc)反应的能力。静脉注射相同剂量病毒后第6天,甲3型A/QLd和CR6所诱导的脾细胞Tc活性均比甲1型A/HK和CR35者高100倍。编码内部蛋白的基因相同而表面抗原的基因不同的CR6与CR35所诱导的Tc活性也不同,说明了表面抗原决定着初次Tc反应的强度。用无关的流感病毒A/SW/72(H6N5)攻击已用低剂量(10~4EID_(?))病毒致敏的小鼠表明,对A/Qld和CR6致敏者所诱导的第二次Tc活性,比A/HK和CR3S致敏者的稍高;但对用大剂量病毒致敏者,则均能产生同样好的第二次Tc活性。对Tc识别感染靶细胞上的病毒成份进行了讨论。