Reactivity of anti-proliferating cell nuclear antigen (PCNA) murine monoclonal antibodies and human autoantibodies to the PCNA multiprotein complexes involved in cell proliferation

Proliferating cell nuclear Ag (PCNA) occurs as a component of multiprotein complexes during cell proliferation. We found the complexes to react with murine anti-PCNA mAbs, but not with anti-PCNA Abs in lupus sera. The complexes were purified from rabbit thymus extract by affinity chromatography using anti-PCNA mAbs (TOB7, TO17, and TO30) and analyzed by ELISA, immunoprecipitation, immunoblotting, and HPLC gel filtration. That PCNA was complexed with other proteins was demonstrated by its copurification with a group of proteins excluded by an HPLC G3000 SW column. Although immunoblot analysis showed the mAbs to react exclusively with the 34-kDa PCNA polypeptide, they nonetheless immunoprecipitated the same group of proteins, confirming the interaction of the isolated PCNA with other proteins. Anti-PCNA sera, including AK, which reacts with biologically functional sites on PCNA, did not react with complexed PCNA, but did react with it once it was dissociated from the complexes. PCNA complexes in turn reacted with murine anti-DNA mAbs, as well as with Abs against p21, replication protein A, DNA helicase II, cyclin-dependent kinases 4 and 5, and topoisomerase I. These findings suggest that the PCNA complexes purified using anti-PCNA mAbs comprise the "protein machinery" for DNA replication and cell cycle regulation. They also suggest that anti-PCNA mAbs are useful tools with which to characterize the protein-protein interactions within PCNA complexes, as well as the autoimmune responses to proteins interacting with PCNA, which may shed light on the mechanisms of autoantibody production in lupus patients.     

Crystal structure of alkaline cellulase K: insight into the alkaline adaptation of an industrial enzyme

The crystal structure of the catalytic domain of alkaline cellulase K was determined at 1.9 A resolution. Because of the most alkaliphilic nature and it's highest activity at pH 9.5, it is used commercially in laundry detergents. An analysis of the structural bases of the alkaliphilic character of the enzyme suggested a mechanism similar to that previously proposed for alkaline proteases, that is, an increase in the number of Arg, His, and Gln residues, and a decrease in Asp and Lys residues. Some ion pairs were formed by the gained Arg residues, which is similar to what has been found in the alkaline proteases. Lys-Asp ion pairs are disfavored and partly replaced with Arg-Asp ion pairs. The alkaline adaptation appeared to be a remodeling of ion pairs so that the charge balance is kept in the high pH range.     

Immune response induced by airway sensitization after influenza A virus infection depends on timing of antigen exposure in mice

To study which phase of viral infection promotes antigen sensitization via the airway and which type of antigen-presenting cells contributes to antigen sensitization, BALB/c mice were sensitized by inhalation of ovalbumin (OA) during the acute phase or the recovery phase of influenza A virus infection, and then 3 weeks later animals were challenged with OA. The numbers of eosinophils and lymphocytes, the amounts of interleukin-4 (IL-4) and IL-5 in the bronchoalveolar lavage fluid, and the serum levels of OA-specific immunoglobulin G1 (IgG1) and IgE increased in mice sensitized during the acute phase (acute phase group), while a high level of gamma interferon production was detected in those sensitized during the recovery phase (recovery phase group). In the acute phase group, both major histocompatibility complex class II molecules and CD11c were strongly stained on the bronchial epithelium; in the recovery phase group, however, neither molecule was detected. OA-capturing dendritic cells (DCs) migrated to the regional lymph nodes, and a small number of OA-capturing macrophages were also observed in the lymph nodes of the acute phase group. In the recovery group, however, no OA-capturing DCs were detected in either the lungs or the lymph nodes, while OA-capturing macrophages were observed in the lymph nodes. These results indicate that the timing of antigen sensitization after viral infection determines the type of immune response.     

Enhanced conformational diversity search of CDR-H3 in antibodies: role of the first CDR-H3 residue

Through a conformation search by a simulation calculation, the relationships between the amino acid sequences and the conformations of the third complementarity-determining region of the antibody heavy chain (CDR-H3) were investigated to characterize the large conformational varieties of antibodies. Here, we focused on the structural role of the first CDR-H3 residue, and we selected two antibodies, 28B4 and PLG, whose CDR-H3 conformations are significantly different, having Trp and Gly at the first position, respectively. Multicanonical molecular dynamics simulations, with the advantage of enhanced sampling efficiency, were performed for the CDR-H3 fragments of 28B4 and PLG, and a modified CDR-H3 model of 28B4, where the first Trp residue was substituted with Gly. When the first CDR-H3 residue is Trp, almost all of the observed CDR-H3 loops were bent at the first residue. In contrast, when the first residue is Gly, large varieties of loop conformations were observed. The structural role of this Gly residue is discussed from the perspective of the other antibody structures in the database. When the surrounding residues were included in the calculations, CDR-H3 loop structures similar to those in the crystal structures were reproduced as the major conformations for both the 28B4 and PLG antibodies.     

Identification of three clones which commonly interact with the kinase domains of highly homologous two receptor-like kinases, RLK902 and RKL1

We have previously reported the characterization of highly homologous two leucine-rich repeat (LRR)-receptor-like kinase (RLK) genes, RLK902 and RKL1, which showed 75% identity at the amino acid sequence level. To investigate the RLK902 and RKL1 mediated signal transduction pathways, we performed yeast two-hybrid screening using the kinase domains of RLK902 and RKL1 as baits. Three clones, Y-1, 2 and 3, were found to interact commonly with the kinase domain of RLK902 and RKL1 and not to interact with the kinase domain of BRI1, a member of LRR-RLKs. This result suggests that RLK902 and RKL1 may have common biochemical functions, especially in their downstream signal transduction. Furthermore, the detail analysis of their responsiveness to various conditions suggests their involvement in such stress conditions as mechanical wounding, treatment with salicylic acid, and pathogen infection.     

Identification of peptidyl mimics of bioactive gibberellin recognized by an antibody

We screened a phage display peptide library for peptidyl mimotopes of gibberellin against anti-bioactive gibberellin antibody. The peptides obtained were grouped into two homologous sequences and their binding to the antibody was put in competition with free GA(4) but not with GA(4) methylester, suggesting that the peptides behave as mimics of GA(4). As an application, the phage display peptide was shown to work as a tracer for enzyme-linked immunosorbent assay (ELISA) analysis of GA(4).     

Metabolic regulation of leptin production in adipocytes: a role of fatty acid synthesis intermediates

In addition to a signal arising from the physical "stretching" of the adipocytes, metabolic and endocrine regulation of leptin production seems to operate in adipocytes. There is however a paucity of literature examining direct role of fatty acid synthesis in regulating adipocytes leptin production. To clarify the relation between fatty acid synthesis and leptin release in adipocytes, we examined leptin release from primary cultured rat epididymal adipocytes with several substances relevance to de novo fatty acid syntyhesis. Bezafibrate (0.5 or 1.0 mM), known to inhibit acetyl-CoA carboxylase, decreased leptin release to 60.3 +/- 7.2 or 47.3 +/- 11.9%, while cerulenin (15, 30, or 75 mM), an inhibitor of fatty acid synthase, increased it by 20.5 +/- 7.7, 58.5 +/- 12.1 or 105.0 +/- 35.0% of the control. Exogenous pyruvate (2.5, 5.0, or 10.0 mM) and malonyl-CoA (10, 20, or 40 mM), substrates and intermediate of fatty acid synthesis, increased leptin release by 11.0 +/- 3.3, 21.5 +/- 5.4, or 61.0 +/- 10.7%, and 11.1 +/- 3.0, 41.1 +/- 9.7 or 56.7 +/- 7.9% of the control, respectively. Considering difference in the site of action of bezafibrate and cerulenin along fatty acid synthesis pathway, one plausible explanation is that malonyl-CoA levels act as a signal of fuel availability to trigger leptin synthesis and/or secretion in adipocytes. Keywords: Leptin secretion; Fatty acid synthesis; Malonyl-CoA; Rat adipocytes.     

Design and synthesis of dysidiolide analogs from vitamin D3: novel class of Cdc25A inhibitors

Potent dysidiolide analogs were synthesized by structural hybridization of dysidiolide and vitamin D(3). These analogs exhibited strong inhibitory activity toward dual-specificity phosphatase Cdc25A (IC(50)=0.44-0.89 microM).