Differential response of cycling and noncycling cells to inducers of DNA synthesis and mitosis

The objective of this study was to determine whether cells in G(0) phase are functionally distinct from those in G(1) with regard to their ability to respond to the inducers of DNA synthesis and to retard the cell cycle traverse of the G(2) component after fusion. Synchronized populations of HeLa cells in G(1) and human diploid fibroblasts in G(1) and G(0) phases were separately fused using UV-inactivated Sendai virus with HeLa cells prelabeled with [(3)H]ThdR and synchronized in S or G(2) phases. The kinetics of initiation of DNA synthesis in the nuclei of G(0) and G(1) cells residing in G(0)/S and G(1)/S dikaryons, respectively, were studied as a function of time after fusion. In the G(0)/G(2) and G(1)/G(2) fusions, the rate of entry into mitosis of the heterophasic binucleate cells was monitored in the presence of Colcemid. The effects of protein synthesis inhibition in the G(1) cells, and the UV irradiation of G(0) cells before fusion, on the rate of entry of the G(2) component into mitosis were also studied. The results of this study indicate that DNA synthesis can be induced in G(0)nuclei after fusion between G(0)- and S-phase cells, but G(0) nuclei are much slower than G(1) nuclei in responding to the inducers of DNA synthesis because the chromatin of G(0) cells is more condensed than it is in G(1) cells. A more interesting observation resulting from this study is that G(0) cells is more condensed than it is in G(1) cells. A more interesting observation resulting from this study is that G(0) cells differ from G(1) cells with regard to their effects on the cell cycle progression of the G(2) nucleus into mitosis. This difference between G(0) and G(1) cells appears to depend on certain factors, probably nonhistone proteins, present in G(1) cells but absent in G(0) cells. These factors can be induced in G(0) cells by UV irradiation and inhibited in G(1) cells by cycloheximide treatment.     

Biologic properties of transplantation immune sera. IV. Influence of the course of immunization, dilution and complexing to antigen on enhancing activity of Ig classes.

The influence of the course of immunization on the facilitating-enhancing activity of antibody classes has been studied by passive enhancement of growth of A/JAX sarcomas in CBA and IC mice and of C57BL/6 EL 4 leukemia in BALB/c mice. The influence of dilution of antibodies and complexing to antigens was also studied. During immunization (with several boosters), the enhancing capacity of sera increased together with 7S IgG antibody activity, but showed no correlation with 19S IgM antibody activity. It also was mercaptoethanol resistant. IgG1 to be more enhancing than an equal number of hemagglutinating units of IgG2a. When concentrated on a small amount (10(5)) of target sarcoma I cells, complement-fixing IC anti-A antibodies were even inhibitory on Sa I allografted to IC recipients. Progressive dilutions reversed this situation, IgG1 activity disappearing and IgG2 acquiring enhancing activity. After complexing to corresponding antigens IgG2 also (and immune sera with inhibitory properties) acquired enhancing properties. These results may provide a basis for understanding the discrepancies between the results of several groups of authors studying the class(es) of enhancing anibodies.     

The formation of a flexible DNA-binding protein chain is required for efficient DNA unwinding and adenovirus DNA chain elongation

The adenovirus DNA-binding protein (DBP) binds cooperatively to single-stranded DNA (ssDNA) and stimulates both initiation and elongation of DNA replication. DBP consists of a globular core domain and a C-terminal arm that hooks onto a neighboring DBP molecule to form a stable protein chain with the DNA bound to the internal surface of the chain. This multimerization is the driving force for ATP-independent DNA unwinding by DBP during elongation. As shown by x-ray diffraction of different crystal forms of the C-terminal domain, the C-terminal arm can adopt different conformations, leading to flexibility in the protein chain. This flexibility is a function of the hinge region, the part of the protein joining the C-terminal arm to the protein core. To investigate the function of the flexibility, proline residues were introduced in the hinge region, and the proteins were purified to homogeneity after baculovirus expression. The mutant proteins were still able to bind ss- and double-stranded DNA with approximately the same affinity as wild type, and the binding to ssDNA was found to be cooperative. All mutant proteins were able to stimulate the initiation of DNA replication to near wild type levels. However, the proline mutants could not support elongation of DNA replication efficiently. Even the elongation up to 26 nucleotides was severely impaired. This defect was also seen when DNA unwinding was studied. Binding studies of DBP to homo-oligonucleotides showed an inability of the proline mutants to bind to poly(dA)(40), indicating an inability to adapt to specific DNA conformations. Our data suggest that the flexibility of the protein chain formed by DBP is important in binding and unwinding of DNA during adenovirus DNA replication. A model explaining the need for flexibility of the C-terminal arm is proposed.     

V beta T cell repertoire of CD8+ splenocytes selected on nonpolymorphic MHC class I molecules

In this work, we have studied the role of the MHC class Ib molecules in the selection and maintenance of CD8(+) T splenocytes. We have compared the CD8(+) T cell repertoires of wild-type, H-2K-deficient, H-2D-deficient, or double knockout C57BL/6 mice. We show that the different CD8(+) repertoires, selected either by class Ia and class Ib or by class Ib molecules only, use the various V alpha (AV) and V beta (BV) rearrangements in the same proportion and without biases in the CDR3 size distribution. Furthermore, we have estimated the size of the BV repertoire in the four different strains of mice. Interestingly, we have found that the BV repertoire size is proportional to the overall number of CD8(+) splenocytes. This observation implies that BV diversity is positively correlated with the number of CD8(+) cells, even when the number of CD8(+) splenocytes is dramatically reduced (90% in the double knockout mice).     

增强UV-B辐射对蚕豆叶片微粒体膜一些性质的影响

温室条件下,用0（Control）、8．65kJm－2d－1（TI）及11．2KJm－2d－1（t2）不同剂量的UV－B辐射处理蚕豆幼苗。Ca2 ．ATPase及Mg2 －ATPase的活性在辐射处理期间下降。在处理21d,T1和T2微粒体膜的MDA含量明显高于对照,同时IUFA急剧下降,且呈明显的剂量效应。14及21d时,膜磷脂的含量也明显下降。脂氧合酶（Lox）活性在第7及14天与对照相比都显著升高,而21d后迅速下降。结果表明,增强UV－B对微粒体膜的伤害可能是一方面导致正常酶合成与分解之间的平衡失调,另一方面导致了膜脂过氧化作用。     

Comparison of the functions of glutathionylspermidine synthetase/amidase from E. coli and its predicted homologues YgiC and YjfC

Protein function prediction is very important in establishing the roles of various proteins in bacteria; however, some proteins in the E. coli genome have their function assigned based on low percent sequence homology that does not provide reliable assignments. We have made an attempt to verify the prediction that E. coli genes ygiC and yjfC encode proteins with the same function as glutathionylspermidine synthetase/amidase (GspSA). GspSA is a bifunctional enzyme that catalyzes the ATP-dependent formation and hydrolysis of glutathionylspermidine (G-Sp), a conjugate of glutathione (GSH) and spermidine. YgiC and YjfC proteins show 51% identity between themselves and 28% identity to the synthetase domain of the GspSA enzyme. YgiC and YjfC proteins were expressed and purified, and the properties of GspSA, YgiC, and YjfC were compared. In contrast to GspSA, proteins YgiC and YjfC did not bind to G-Sp immobilized on the affinity matrix. We demonstrated that all three proteins (GspSA, YgiC and YjfC) catalyze the hydrolysis of ATP; however, YgiC and YjfC cannot synthesize G-Sp, GSH, or GSH intermediates. gsp, ygiC, and yjfC genes were eliminated from the E. coli genome to test the ability of mutant strains to synthesize G-Sp conjugate. E. coli cells deficient in GspSA do not produce G-Sp while synthesis of the conjugate is not affected in ΔygiC and ΔyjfC mutants. All together our results indicate that YgiC and YjfC are not glutathionylspermidine synthetases as predicted from the amino acid sequence analysis.     

Ezrin/Radixin/Moesin Are Required for the Purinergic P2X7 Receptor (P2X7R)-dependent Processing of the Amyloid Precursor Protein

The amyloid precursor protein (APP) can be cleaved by α-secretases in neural cells to produce the soluble APP ectodomain (sAPPα), which is neuroprotective. We have shown previously that activation of the purinergic P2X7 receptor (P2X7R) triggers sAPPα shedding from neural cells. Here, we demonstrate that the activation of ezrin, radixin, and moesin (ERM) proteins is required for the P2X7R-dependent proteolytic processing of APP leading to sAPPα release. Indeed, the down-regulation of ERM by siRNA blocked the P2X7R-dependent shedding of sAPPα. We also show that P2X7R stimulation triggered the phosphorylation of ERM. Thus, ezrin translocates to the plasma membrane to interact with P2X7R. Using specific pharmacological inhibitors, we established the order in which several enzymes trigger the P2X7R-dependent release of sAPPα. Thus, a Rho kinase and the MAPK modules ERK1/2 and JNK act upstream of ERM, whereas a PI3K activity is triggered downstream. For the first time, this work identifies ERM as major partners in the regulated non-amyloidogenic processing of APP.