Effects of 2＇-O-methyl nucleotide on ligation capability of T4 DNA ligase

To further understand the ligation mechanism, effects of 2＇-O-methyl nucleotide （2＇-OMeN） on the T4 DNA ligation efficiency were investigated. Fluorescence resonance energy transfer assay was used to monitor the nick-joining process by T4 DNA ligase. Results showed that substitutions at 5＇- and 3＇-ends of the nick decreased the ligation efficiency by 48.7% ± 6.7% and 70.6% ±4.0%, respectively. Substitutions at both 5＇- and 3＇-ends decreased the ligation efficiency by 76.6%±1.3%. Corresponding kinetic para- meters, Vmax, Kin, and kcat, have been determined in each case by using the Michaelis-Menten equation. The kinetic data showed that the 2＇-OMeN substitutions reduced the maximal initial velocity and increased the Michaelis constant of T4 DNA ligase. Mismatches at 5＇- and 3＇-ends of the nick have also shown different influences on the ligation. Results here showed that the sugar pucker conformation at 3＇-end impairs the ligation efficiency more profoundly than that at 5＇-end. Different concentrations of Mg2＋, Ca2＋, K＋, Na＋, and ATP were also demonstrated to affect the T4 DNA ligase activ- ity. These results enriched our knowledge about the effects of 2＇-OMEN substitutions on the T4 DNA ligase.     

T4病毒研究进展

T4病毒科由一类单股正链RNA病毒组成,分为松天蛾β样病毒属和松天蛾ω样病毒属。这2个属的病毒具有不同的基因组结构,β样病毒含单组分基因组,其结构蛋白由一亚基因组RNA表达; 而ω样病毒含双组分基因组,2个RNA分子分别编码复制酶蛋白和结构蛋白。在T4病毒基因组RNA 3′端有类似tRNA的二级结构。ω样病毒壳蛋白的氨基酸序列一致性高达66%～86%, 而β样病毒壳蛋白的氨基酸同源性则要低得多。在昆虫细胞中表达壳蛋白基因时都能形成病毒类似粒子。该文还介绍了T4病毒复制机理以及T4病毒与其他病毒的进化关系。     

胸腺肽Tβ4

胸腺肽Tβ4是具有多重生物功能的小肽,是肌动蛋白的结合蛋白之一,该多肽的基因差异性表达与肿瘤恶变和转移、胚胎发育等关系密切,本综述了胸腺肽Tβ4的研究进展。     

Synthesis of (±)-4′-Ethynyl and 4′-Cyano Carbocyclic Analogues of Stavudine (d4T)

The synthesis of (±)-4′-ethynyl (8) and 4′-cyano (9) carbocyclic analogues of the anti-HIV agent stavudine (5, d4T) is reported. The carbocyclic unit (16) was constructed from readily available β-keto ester 10. The ethynyl or cyano group of 8 and 9 were prepared, after the introduction of thymine base to 16, by manipulation of the ester function. Evaluation of the anti-HIV activity of 8 and 9 was also carried out.     

SA-4-1BBL Costimulation Inhibits Conversion of Conventional CD4(+) T Cells into CD4(+)FoxP3(+) T Regulatory Cells by Production of IFN-γ

Tumors convert conventional CD4⁺ T cells into induced CD4⁺CD25⁺FoxP3⁺ T regulatory (iTreg) cells that serve as an effective means of immune evasion. Therefore, the blockade of conventional CD4⁺ T cell conversion into iTreg cells represents an attractive target for improving the efficacy of various immunotherapeutic approaches. Using a novel form of 4-1BBL molecule, SA-4-1BBL, we previously demonstrated that costimulation via 4-1BB receptor renders both CD4⁺and CD8⁺ T effector (Teff) cells refractory to inhibition by Treg cells and increased intratumoral Teff/Treg cell ratio that correlated with therapeutic efficacy in various preclinical tumor models. Building on these studies, we herein show for the first time, to our knowledge, that signaling through 4-1BB inhibits antigen- and TGF-β-driven conversion of naïve CD4⁺FoxP3⁻ T cells into iTreg cells via stimulation of IFN-γ production by CD4⁺FoxP3⁻ T cells. Importantly, treatment with SA-4-1BBL blocked the conversion of CD4⁺FoxP3⁻ T cells into Treg cells by EG.7 tumors. Taken together with our previous studies, these results show that 4-1BB signaling negatively modulate Treg cells by two distinct mechanisms: i) inhibiting the conversion of CD4⁺FoxP3⁻ T cells into iTreg cells and ii) endowing Teff cells refractory to inhibition by Treg cells. Given the dominant role of Treg cells in tumor immune evasion mechanisms, 4-1BB signaling represents an attractive target for favorably tipping the Teff:Treg balance toward Teff cells with important implications for cancer immunotherapy.     

Mutational analysis of the 5′-OH oligonucleotide phosphate acceptor site of T4 polynucleotide kinase

T4 polynucleotide kinase/phosphatase (Pnkp) exemplifies a family of bifunctional enzymes with 5′-kinase and 3′-phosphatase activities that function in nucleic acid repair. The N-terminal kinase domain belongs to the P-loop phosphotransferase superfamily. The kinase is distinguished by a tunnel-like active site with separate entrances on opposite sides of the protein for the NTP phosphate donor and a 5′-OH single-stranded oligonucleotide phosphate acceptor. Here, we probed by mutagenesis the roles of individual amino acids that comprise the acceptor binding site. We thereby identified Glu57 as an important residue, by virtue of its participation in a salt bridge network with two catalytic residues identified previously: Arg38, which binds the 3′-phosphate of the terminal 5′-OH nucleotide, and the putative general base Asp35 that contacts the nucleophilic 5′-OH group. The 5′-OH nucleoside fits into a pocket lined by aliphatic amino acids (Val131, Pro132 and Val135) that make van der Waals contacts to the nucleobase. Whereas subtraction of these contacts by single alanine substitutions for Val131 or Val135 and glycine for Pro132 had modest effects on kinase activity, the introduction of bulkier phenylalanines for Val131 and Val135 were deleterious, especially V131F, which severely impeded both substrate binding (increasing K_m by 15-fold) and catalysis (decreasing k_cat by 300-fold).     

Transformation of a Fragment of β-Structural Bacteriophage T4 Adhesin to Stable α-Helical Trimer

Gene product 12 of bacteriophage T4, adhesin, serves to adhere the virus to host cells. Adhesin is a fibrous homotrimer, and a novel tertiary structure element, a -helix, is supposed to be a major structural feature of this protein. We have constructed two truncated gp12 mutants, 12N1 and 12N2, containing 221 and 135 N-terminal residues, respectively. When expressed in E. coli cells, these gp12 fragments formed labile -structural trimers. Another hybrid protein, 12FN, containing 179 N-terminal amino acid residues of gp12 fused to the C-terminal domain (31 amino acids) of T4 fibritin, was shown to have a trimeric proteolytically resistant a-helical structure. This structure is probably similar to that of fibritin, which has a triple -helical coiled-coil structure. Hence, we have demonstrated the possibility of global transformation of fibrous protein structure using fusion with a C-terminal domain that initiates trimerization.     

Structure–Function Analysis of the DNA Translocating Portal of the Bacteriophage T4 Packaging Machine

Tailed bacteriophages and herpesviruses consist of a structurally well conserved dodecameric portal at a special 5-fold vertex of the capsid. The portal plays critical roles in head assembly, genome packaging, neck/tail attachment, and genome ejection. Although the structures of portals from phages φ29, SPP1, and P22 have been determined, their mechanistic roles have not been well understood. Structural analysis of phage T4 portal (gp20) has been hampered because of its unusual interaction with the Escherichia coli inner membrane. Here, we predict atomic models for the T4 portal monomer and dodecamer, and we fit the dodecamer into the cryo-electron microscopy density of the phage portal vertex. The core structure, like that from other phages, is cone shaped with the wider end containing the “wing” and “crown” domains inside the phage head. A long “stem” encloses a central channel, and a narrow “stalk” protrudes outside the capsid. A biochemical approach was developed to analyze portal function by incorporating plasmid-expressed portal protein into phage heads and determining the effect of mutations on head assembly, DNA translocation, and virion production. We found that the protruding loops of the stalk domain are involved in assembling the DNA packaging motor. A loop that connects the stalk to the channel might be required for communication between the motor and the portal. The “tunnel” loops that project into the channel are essential for sealing the packaged head. These studies established that the portal is required throughout the DNA packaging process, with different domains participating at different stages of genome packaging.     

T cell receptor-mediated activation of CD4+CD44hi T cells bypasses Bcl10: an implication of differential NF-kappaB dependence of naïve and memory T cells during T cell receptor-mediated responses

Previous studies have demonstrated that Bcl10 (B-cell leukemia/lymphoma 10) is essential for T cell receptor-mediated NF-kappaB activation and subsequent proliferation and interleukin 2 (IL2) production. However, here we demonstrate that, contrary to expectations, Bcl10 is differentially required for T cell activation, including for both proliferation and cytokine production. When CD4+ and CD8+ T cells were divided based on expression levels of CD44, which distinguishes na?ve cells (CD44lo) versus those that are antigen-experienced (CD44hi), IL2 production by and proliferation of CD4+CD44lo na?ve cells and both subpopulations of CD8+ T cells were clearly Bcl10-dependent, whereas these same functional properties of CD4+CD44hi T cells occurred largely independent of Bcl10. As with the other subpopulations of T cells, CD4+CD44hi T cells did not activate the NF-kappaB pathway in the absence of Bcl10; nevertheless, these CD4+CD44hi antigen-experienced T cells efficiently secreted IL2 after T cell receptor stimulation. Strikingly, therefore, T cell receptor-mediated IL2 production in these cells is NF-kappaB-independent. Our studies suggest that antigen-experienced CD4+ T cells differ from their na?ve counterparts and from CD8+ T cells in their ability to achieve activation independent of the Bcl10/NF-kappaB pathway.     

Isolation and Th17 Differentiation of Na?ve CD4 T Lymphocytes

Th17 cells are a distinct subset of T cells that have been found to produce interleukin 17 (IL-17), and differ in function from the other T cell subsets including Th1, Th2, and regulatory T cells. Th17 cells have emerged as a central culprit in overzealous inflammatory immune responses associated with many autoimmune disorders. In this method we purify T lymphocytes from the spleen and lymph nodes of C57BL/6 mice, and stimulate purified CD4+ T cells under control and Th17-inducing environments. The Th17-inducing environment includes stimulation in the presence of anti-CD3 and anti-CD28 antibodies, IL-6, and TGF-β. After incubation for at least 72 hours and for up to five days at 37 °C, cells are subsequently analyzed for the capability to produce IL-17 through flow cytometry, qPCR, and ELISAs. Th17 differentiated CD4+CD25- T cells can be utilized to further elucidate the role that Th17 cells play in the onset and progression of autoimmunity and host defense. Moreover, Th17 differentiation of CD4+CD25- lymphocytes from distinct murine knockout/disease models can contribute to our understanding of cell fate plasticity.     

STM of freeze-dried and PtIrC-coated bacteriophage T4 polyheads

STM imaging was carried out on the outer surface of bacteriophage T4 type III polyheads, after freeze-drying and coating with a thin conducting film. By taking advantage of the high resolution power of the STM especially in the z-direction (i.e., perpendicular to the support), an apparent surface topography of only about 1 nm height was resolved. The average capsomere morphology was determined by correlation averaging. Because of the high signal-to-noise ratio of the tunneling data, only a few unit cells were needed to reveal a stable average. Both the topology as well as the absolute height of the polyheads have been reproduced in several experiments. The influence of the tip geometry on the STM topographs is discussed.     

Absence of β2 integrins impairs regulatory T cells and exacerbates CD4+ T cell-dependent autoimmune carditis

The immunopathogenic mechanisms mediating inflammation in multiorgan autoimmune diseases may vary between the different target tissues. We used the K/BxN TCR transgenic mouse model to investigate the contribution of CD4(+) T cells and β(2) integrins in the pathogenesis of autoimmune arthritis and endocarditis. Depletion of CD4(+) T cells following the onset of arthritis specifically prevented the development of cardiac valve inflammation. Genetic absence of β(2) integrins had no effect on the severity of arthritis and unexpectedly increased the extent of cardiovascular pathology. The exaggerated cardiac phenotype of the β(2) integrin-deficient K/BxN mice was accompanied by immune hyperactivation and was linked to a defect in regulatory T cells. These findings are consistent with a model in which the development of arthritis in K/BxN mice relies primarily on autoantibodies, whereas endocarditis depends on an additional contribution of effector T cells. Furthermore, strategies targeting β(2) integrins for the treatment of systemic autoimmune conditions need to consider not only the role of these molecules in leukocyte recruitment to sites of inflammation, but also their impact on the regulation of immunological tolerance.     

Functional analysis of the phage T4 holin in a λ context

Phage lambda hybrids were constructed by inserting the t gene of phage T4 in place of the lambda holin gene, S. Induction of the hybrid phage resulted in lysis that was just as abrupt as, but occurred much earlier in the vegetative cycle than, that obtained with lambda, indicating that t is indeed a holin gene. Moreover, it was possible to impose lysis inhibition (LIN) on induction of the hybrid phage, but not of the parental lambda phage, by superinfection with LIN-competent T4. The imposition of the LIN state was found to depend on the allelic state of the rI and t genes of the superinfecting T4 phage, indicating that the LIN-sensitive state of the T holin is transient. Finally, induction of lysogens carrying both holin genes was shown to result in earlier triggering of lysis than with either holin gene alone. This result suggests that the two very dissimilar holins contribute additively to the physiology of the timing mechanism, or, less likely, that they interact to form one mass-action pool. In either case, these results imply a common pathway for holin timing and function.     

Integrin α4β7 Co-Stimulation of Human Peripheral Blood T Cell Proliferation

The Integrin α4β7 mediates lymphocyte adhesion to VCAM-1 on activated endothelium, fibronectin in the extracellular matrix, and the mucosal vascular addressin MAdCAM-1. It is unclear whether α4β7 performs any function beyond directing specific adhesion reactions. We addressed the possibility that triggering of α4β7 with a specific monoclonal antibody was capable of delivering an accessory stimulus that would coactivate T cells and lead to proliferation. At submitogenic levels of anti-CD3 stimulation, triggering of α4β7 by immobilized mAb ACT-1 resulted in T cell blastogenesis, IL-2 production, expression of the IL-2 receptor α chain CD25, and ultimately DNA synthesis. These results indicate that the integrin α4β7 is involved in more than lymphocyte adhesion and homing but also plays a role in cell signaling.     

Inflammasome-derived IL-1β regulates the production of GM-CSF by CD4(+) T cells and γδ T cells

Recent findings have demonstrated an indispensable role for GM-CSF in the pathogenesis of experimental autoimmune encephalomyelitis. However, the signaling pathways and cell populations that regulate GM-CSF production in vivo remain to be elucidated. Our work demonstrates that IL-1R is required for GM-CSF production after both TCR- and cytokine-induced stimulation of immune cells in vitro. Conventional αβ and γδ T cells were both identified to be potent producers of GM-CSF. Moreover, secretion of GM-CSF was dependent on IL-1R under both IL-12- and IL-23-induced stimulatory conditions. Deficiency in IL-1R conferred significant protection from experimental autoimmune encephalomyelitis, and this correlated with reduced production of GM-CSF and attenuated infiltration of inflammatory cells into the CNS. We also find that GM-CSF production in vivo is not restricted to a defined CD4(+) T cell lineage but is rather heterogeneously expressed in the effector CD4(+) T cell population. In addition, inflammasome-derived IL-1β upstream of IL-1R is a critical regulator of GM-CSF production by T cells during priming, and the adapter protein, MyD88, promotes GM-CSF production in both αβ and γδ T cells. These findings highlight the importance of inflammasome-derived IL-1β and the IL-1R/MyD88 signaling axis in the regulation of GM-CSF production.     

Effect of selenium depletion and supplementation on the kinetics of type 1 5′-iodothyronine deiodinase and T3/T4 in rats

Presently, the effect of selenium (Se) deficiency and excess of Se (1 ppm) on the activity of selenoenzymes type 1 5′-iodothyronine deiodinase (5′-DI), glutathione peroxidase (GSH-Px), and level of thyroid hormones (T₃ and T₄) was studied in rats. Se levels in the serum and liver, T₃ and T₄ in the serum, GSH-Px levels in the liver, and 5′-DI activity in the liver/aorta/thyroid were estimated after 1, 2, and 3 mo of Se-deficient (0.02 ppm), Se-adequate (0.2 ppm), and Se-excess (1 ppm) diet feeding. All of these parameters decreased significantly in the Se-deficient group as compared to the adequate group. Within the deficient group, as the Se deficiency progressed, all of the parameters except 5′-DI decreased after 2 and 3 mo in comparison to 1-mo data. Thyroidal 5′-DI activity in Se deficiency showed the maximum increase. A significant increase was observed in all of the above parameters in the 1 ppm Se-supplemented diet group when compared with the adequate Se group; also, as the Se deposition increased within the Se-excess diet group, a significant increase was observed in all of the above parameters. However, as observed by others, the intake of excess of Se (i.e., 2 ppm in the diet) did not elevate the activities of selenoenzymes and thyroid hormones; rather, it had adverse effects. The present study concludes that Se supplementation at least up to 1 ppm enhances the selenoenzyme activities, and above this level, it may not be considered as an indicator of selenoenzyme activities.