The ratio of messenger RNA levels of receptor activator of nuclear factor kappa B ligand to osteoprotegerin correlates with bone remodeling indices in normal human cancellous bone but not in osteoarthritis

skeletal disease. Bone remodeling is initiated by osteoclastic resorption followed by osteoblastic formation of new bone. Receptor activator of nuclear factor KB ligand (RANKL) is a newly described regulator of osteoclast formation and function, the activity of which appears to be a balance between interaction with its receptor RANK and with an antagonist binding protein osteoprotegerin (OPG). Therefore, we have examined the relationship between the expression of RANKL, RANK, and OPG and indices of bone structure and turnover in human cancellous bone from the proximal femur. Bone samples were obtained from individuals with osteoarthritis (OA) at joint replacement surgery and from autopsy controls. Histomorphometric analysis of these samples showed that eroded surface (ES/BS) and osteoid surface (OS/BS) were positively associated in both control (p < 0.001) and OA (p < 0.02), indicating that the processes of bone resorption and bone formation remain coupled in OA, as they are in controls. RANKL, OPG, and RANK messenger RNA, (mRNA) were abundant in human cancellous bone, with significant differences between control and OA individuals. In coplotting the molecular and histomorphometric data, strong associations were found between the ratio of RANKL/OPG mRNA and the indices of bone turnover (RANKL/OPG vs. ES/BS: r = 0.93, p < 0.001; RANKL/OPG vs. OS/BS: r = 0.80, p < 0.001). These relationships were not evident in trabecular bone from severe OA, suggesting that bone turnover may be regulated differently in this disease. We propose that the effective concentration of RANKL is related causally to bone turnover.

Interpreting the effects of small uncharged solutes on protein-folding equilibria

Proteins are designed to function in environments crowded by cosolutes, but most studies of protein equilibria are conducted in dilute solution. While there is no doubt that crowding changes protein equilibria, interpretations of the changes remain controversial. This review combines experimental observations on the effect of small uncharged cosolutes (mostly sugars) on protein stability with a discussion of the thermodynamics of cosolute-induced nonideality and critical assessments of the most commonly applied interpretations. Despite the controversy surrounding the most appropriate manner for interpreting these effects of thermodynamic nonideality arising from the presence of small cosolutes, experimental advantage may still be taken of the ability of the cosolute effect to promote not only protein stabilization but also protein self-association and complex formation between dissimilar reactants. This phenomenon clearly has potential ramifications in the cell, where the crowded environment could well induce the same effects.

Activity of recombinant dengue 2 virus NS3 protease in the presence of a truncated NS2B co-factor, small peptide substrates, and inhibitors

Recombinant forms of the dengue 2 virus NS3 protease linked to a 40-residue co-factor, corresponding to part of NS2B, have been expressed in Escherichia coli and shown to be active against para-nitroanilide substrates comprising the P6-P1 residues of four substrate cleavage sequences. The enzyme is inactive alone or after the addition of a putative 13-residue co-factor peptide but is active when fused to the 40-residue co-factor, by either a cleavable or a noncleavable glycine linker. The NS4B/NS5 cleavage site was processed most readily, with optimal processing conditions being pH 9, I = 10 mm, 1 mm CHAPS, 20% glycerol. A longer 10-residue peptide corresponding to the NS2B/NS3 cleavage site (P6-P4') was a poorer substrate than the hexapeptide (P6-P1) para-nitroanilide substrate under these conditions, suggesting that the prime side substrate residues did not contribute significantly to protease binding. We also report the first inhibitors of a co-factor-complexed, catalytically active flavivirus NS3 protease. Aprotinin was the only standard serine protease inhibitor to be active, whereas a number of peptide substrate analogues were found to be competitive inhibitors at micromolar concentrations.

Species dependence for binding of small molecule agonist and antagonists to the C5a receptor on polymorphonuclear leukocytes

This study investigated the receptor binding affinities of a C5a agonist and cyclic antagonists for polymorphonuclear leukocytes (PMNs) isolated from human, sheep, pig, dog, rabbit, guinea pig, rat and mouse. The affinities of the two small molecule antagonists, F-[OPdChaWR] and AcF-[OPdChaWR], and the agonist, YSFKPMPLaR, revealed large differences in C5a receptor (C5aR) affinities between species. The antagonists bound to human, rat and dog PMNs with similar high affinities, but with lower affinities to PMNs from all other species. The C5a agonist also bound with varying affinities between species, but showed a different affinity profile to the antagonists. In contrast, recombinant human C5a had similar affinity for PMNs of all species investigated. The low correlation between the affinities of the antagonists and the agonist between species either suggests that different receptor residues are important for distinguishing between agonist/antagonist binding, or that the agonist and antagonist peptides bind to two distinct sites within the C5aR.

Nuclear PTEN expression and clinicopathologic features in a population-based series of primary cutaneous melanoma

Germline mutations of the PTEN tumor-suppressor gene, on 10q23, cause Cowden syndrome, an inherited hamartoma syndrome with a high risk of breast, thyroid and endometrial carcinomas and, some suggest, melanoma. To date, most studies which strongly implicate PTEN in the etiology of sporadic melanomas have depended on cell lines, short-term tumor cultures and noncultured metastatic melanomas. The only study which reports PTEN protein expression in melanoma focuses on cytoplasmic expression, mainly in metastatic samples. To determine how PTEN contributes to the etiology or the progression of primary cutaneous melanoma, we examined cytoplasmic and nuclear PTEN expression against clinical and pathologic features in a population-based sample of 150 individuals with incident primary cutaneous melanoma. Among 92 evaluable samples, 30 had no or decreased cytoplasmic PTEN protein expression and the remaining 62 had normal PTEN expression. In contrast, 84 tumors had no or decreased nuclear expression and 8 had normal nuclear PTEN expression. None of the clinical features studied, such as Clark's level and Breslow thickness or sun exposure, were associated with cytoplasmic PTEN expressional levels. An association with loss of nuclear PTEN expression was indicated for anatomical site (p = 0.06) and mitotic index (p = 0.02). There was also an association for melanomas to either not express nuclear PTEN or to express p53 alone, rather than both simultaneously (p = 0.02). In contrast with metastatic melanoma, where we have shown previously that almost two-thirds of tumors have some PTEN inactivation, only one-third of primary melanomas had PTEN silencing. This suggests that PTEN inactivation is a late event likely related to melanoma progression rather than initiation. Taken together with our previous observations in thyroid and islet cell tumors, our data suggest that nuclear-cytoplasmic partitioning of PTEN might also play a role in melanoma progression. (C) 2002 Wiley-Liss, Inc.

Nuclear RelB(+) cells are found in normal lymphoid organs and in peripheral tissue in the context of inflammation, but not under normal resting conditions

Differentiated dendritic cells (DC) have been identified by the presence of nuclear RelB (nRelB) and HLA-DR, and the absence of CD20 or high levels of CD68, in lymph nodes and active rheumatoid arthritis synovial tissue. The current studies aimed to identify conditions in which nRelB is expressed in human tissues, by single and double immunohistochemistry of formalin-fixed peripheral and lymphoid tissue. Normal peripheral tissue did not contain nRelB(+) cells. nRelB(+) DC were located only in T- or B-cell areas of lymphoid tissue associated with normal organs or peripheral tissues, including tonsil, colon, spleen and thymus, or in association with T cells in inflamed peripheral tissue. Inflamed sites included skin delayed-type hypersensitivity reaction, and a wide range of tissues affected by autoimmune disease. Nuclear RelB(+) -HLA-DR- follicular DC were located in B-cell follicles in lymphoid organs and in lymphoid-like follicles of some tissues affected by autoimmune disease. Lymphoid tissue T-cell areas also contained nRelB(-) -HLA-DR+ cells, some of which expressed CD123 and/or CD68. Nuclear RelB(+) cells are found in normal lymphoid organs and in peripheral tissue in the context of inflammation, but not under normal resting conditions.

RelB nuclear translocation mediated by C-terminal activator reigons of Epstein Barr virus-encoded latent membrane protein 1 and its effect on antigen-presenting function in B cells

Previous studies have shown that Epstein-Barr virus-encoded latent membrane protein 1 (LMP1) is uniquely able to up-regulate the expression of the peptide transporters (referred to as TAP-1 and TAP-2) and major histocompatibility complex (MHC) class I in Burkitt's lymphoma (BL) cell lines. This up-regulation is often accompanied by a restoration of antigen-presenting function as measured by the ability of these cells to present endogenously expressed viral antigen to cytotoxic T lymphocytes. Here we show that the expression of LMP1 resulted in up-regulation and nuclear translocation of RelB that were coincident with increased expression of MHC class I in BL cells. Deletion of the C-terminal activator regions (CTARs) of LMP1 significantly impaired the abilities of LMP1 to translocate RelB into the nucleus and to up-regulate the expression of antigen-processing genes. Further analysis with single-point mutations within the CTARs confirmed that the residues critical for NF-kappaB activation directly contribute to antigen-processing function regulation in BL cells. This LMP1-mediated effect was blocked following expression of either dominant negative IkappaBalpha S32/36A, an NF-kappaB inhibitor, or antisense RelB. These observations indicate that upregulation of antigen-presenting function in B cells mediated by LMP1 is signaled through the NF-kappaB subunit RelB. The data provide a mechanism by which LMP1 modulates immunogenicity of Epstein-Barr virus-infected normal and malignant cells.

Exact solution at integrable coupling of a model for the Josephson effect between small metallic grains

A model is introduced for two reduced BCS systems which are coupled through the transfer of Cooper pairs between the systems. The model may thus be used in the analysis of the Josephson effect arising from pair tunneling between two strongly coupled small metallic grains. At a particular coupling strength the model is integrable and explicit results are derived for the energy spectrum, conserved operators, integrals of motion, and wave function scalar products. It is also shown that form factors can be obtained for the calculation of correlation functions. Furthermore, a connection with perturbed conformal field theory is made.