Structure-activity studies of a series of dipyrazolo[3,4-b:3',4'-d]pyridin-3-ones binding to the immune regulatory protein B7.1

The interaction of co-stimulatory molecules on T cells with B7 molecules on antigen presenting cells plays an important role in the activation of naive T cells. Consequently, agents that disrupt these interactions should have applications in treatment of transplant rejection as well as autoimmune diseases. To this end, specific small molecule inhibitors of human B7.1 were identified and characterized. Herein, we report the identification of potent small molecule inhibitors of the B7.1-CD28 interaction. In a high-throughput screen we identified several leads that prevented the interaction of B7.1 with CD28 with activities in the nanomolar to low micromolar range. One of these, the dihydrodipyrazolopyridinone 1, was subsequently shown to bind the V-like domain of human B7.1 at equimolar stoichiometry. With this as a starting point, we report here the synthesis and initial in vitro structure-activity relationships of a series of these compounds.     

Measuring mast seeding behavior: relationships among population variation,individual variation and synchrony

Mast seeding, or masting, is the variable production of flowers, seeds, or fruit across years more or less synchronously by individuals within a population. A critical issue is the extent to which temporal variation in seed production over a collection of individuals can be viewed as arising from a combination of individual variation and synchrony among individuals. Studies of masting typically quantify such variation in terms of the coefficient of variation (CV). In this paper we examine mathematically how the population CV relates to the mean individual CV and synchrony, concluding that the relationship is a complex one which cannot isolate an overall measure of synchrony, and involves additional factors, principally the number of plants sampled and the mean productivity per plant. Our development suggests some simple approximate relationships of population CV to individual variability, synchrony and the number of individuals. These were found to fit quite well when applied to data from 59 studies which included seed production at the individual level.     

Ca2+-independent protein kinase Cs mediate heterologous desensitization of leukocyte chemokine receptors by opioid receptors

Heterologous desensitization of chemokine receptors by opioids has been considered to contribute to their immunosuppressive effects. Previous studies show that Met-enkephalin, an endogenous opioid, down-regulates chemotaxis of selected chemokine receptors via phosphorylation. In the present study, we further investigated the molecular mechanism of such cross-regulation. Our data showed that preincubation with Met-enkephalin inhibited both MIP-1 alpha-mediated chemotaxis and Ca(2+) flux of monocytes in a dose-dependent manner. The inhibitory effects were maximal using nanomolar concentrations of activating chemokines, a concentration found in physiological conditions. A decrease both in chemokine receptor affinity and in coupling efficiency between receptors and G protein were observed, which directly contributed to the desensitization effects. However, comparing with chemokines such as MIP-1 alpha and MCP-1, opioids did not elicit a calcium flux, failed to induce MIP-1 alpha receptors internalization, and mediated a less potent heterologous desensitization. We hypothesized that these differences might originate from the involvement of different protein kinase C (PKC) isotypes. In our studies, opioid-mediated down-regulation of MIP-1 alpha receptors could be blocked by the general PKC inhibitor calphostin C, but not by the calcium-dependent classic PKC inhibitor Go6976. Western blotting analysis and immunofluorescent staining further showed that only calcium-independent PKCs were activated upon opioid stimulation. Thus, opioids achieve desensitization of chemokine receptors via a unique pathway, involving only calcium-independent PKC isotypes.     

Control of the estrogen-like actions of the tamoxifen-estrogen receptor complex by the surface amino acid at position 351

Tamoxifen is a valuable therapeutic agent with applications in the treatment and prevention of breast cancer. However, the development of drug resistance limits the usefulness of tamoxifen therapy. One form of drug resistance in breast cancer is tamoxifen-stimulated growth. We have addressed a mechanism how the tamoxifen–estrogen receptor (ER) complex can convert from being a blocking to stimulatory signal in breast cancer. We have described an effective assay system to study the action of antiestrogen–ER complex through the activation of transforming growth factor alpha gene in situ. The MDA-MB-231 breast cancer cells were stably transfected with cDNAs for wtER (D351), mutant Asp351Tyr ER (D351Y) and mutant Asp351Gly ER (D351G). The D351Y ER can enhance the estrogenic properties of 4OHT and change the pharmacology of raloxifene by converting it from antiestrogen to estrogen. We hypothesized that alterations in the charge of amino acid (aa) 351, and changes in the interaction with the side chain of an antiestrogen, are critical for the subsequent estrogenicity of the complex. Our goal was (1) to modulate the estrogenicity of the antiestrogen–ER complex by different aa substitutions at position 351 and (2) to examine the role of alterations in the side chain of antiestrogens on the estrogenicity of the complex. Substitution of tyrosine for aspartate at aa351 results in increased estrogenicity for a series of tamoxifen derivatives–ER complexes and the conversion of EM 652-ER and GW 7604-ER complexes from antiestrogenic to estrogen-like. Substitution of glycine for aspartate at aa 351 results in the conversion of 4OHT-ER complex from estrogen-like to antiestrogenic. We propose that the side chain of antiestrogens either neutralizes or displaces the charge at aspartate 351 thereby removing a charged site for the opportunistic binding of a novel coactivator. If no charge is present (D351G) then no coactivator can bind and the complex with any antiestrogen is not estrogen-like. However, if the charge is extended beyond the reach of an antiestrogen side chain (D351Y), then the coactivators bind and compounds are estrogen-like. The establishment of a relationship between the structure of the antiestrogen–ER complex and its function will enhance the development of novel compounds with unique biological activities and potentially avoid premature drug resistance.     

Transgenic expression of Theiler's murine encephalomyelitis virus genes in H-2(b) mice inhibits resistance to virus-induced demyelination

We investigated the role of the immune system in protecting against virus-induced demyelination by generating lines of transgenic B10 (H-2(b)) congenic mice expressing three independent contiguous coding regions of the Theiler's murine encephalomyelitis virus (TMEV) under the control of a class I major histocompatibility complex (MHC) promoter. TMEV infection of normally resistant B10 mice results in virus clearance and development of inflammatory demyelination in the spinal cord. Transgenic expression of the viral capsid genes resulted in inactivation of virus-specific CD8(+) T lymphocytes (class I MHC immune function) directed against the relevant peptides, but it did not affect production of virus capsid-specific antibodies or lymphocyte proliferation to the virus antigen (class II MHC immune functions). Following intracerebral infection with TMEV, all three lines of mice survived the acute encephalitis but transgenic mice expressing VP1 (or the cluster of virus capsid proteins [VP4, VP2, and VP3] mapping to the left of VP1 in the TMEV genome) developed virus persistence and subsequent demyelination in spinal cord white matter. Transgenic mice expressing noncapsid proteins mapping to the right of VP1 (2A, 2B, 2C, 3A, 3B, 3C, and 3D) cleared the virus and did not develop demyelination. These results are consistent with the hypothesis that virus capsid gene products of TMEV stimulate class I-restricted CD8(+) T-cell immune responses, which are important for virus clearance and for protection against myelin destruction. Presented within the context of self-antigens, inactivation of these cells by ubiquitous expression of relevant virus capsid peptides partially inhibited resistance to virus-induced demyelination.     

Snow tussocks, chaos, and the evolution of mast seeding

One hitherto intractable problem in studying mast seeding (synchronous intermittent heavy flowering by a population of perennial plants) is determining the relative roles of weather, plant reserves, and evolutionary selective pressures such as predator satiation. We parameterize a mechanistic resource-based model for mast seeding in Chionochloa pallens (Poaceae) using a long-term individually structured data set. Each plant's energy reserves were reconstructed using annual inputs (growing degree days), outputs (flowering), and a novel regression technique. This allowed the estimation of the parameters that control internal plant resource dynamics, and thereby allowed different models for masting to be tested against each other. Models based only on plant size, season degree days, and/or climatic cues (warm January temperatures) fail to reproduce the pattern of autocovariation in individual flowering and the high levels of flowering synchrony seen in the field. This shows that resource-matching or simple cue-based models cannot account for this example of mast seeding. In contrast, the resource-based model pulsed by a simple climate cue accurately describes both individual-level and population-level aspects of the data. The fitted resource-based model, in the absence of environmental forcing, has chaotic (but often statistically periodic) dynamics. Environmental forcing synchronizes individual reproduction, and the models predict highly variable seed production in close agreement with the data. An evolutionary model shows that the chaotic internal resource dynamics, as predicted by the fitted model, is selectively advantageous provided that adult mortality is low and seeds survive for more than 1 yr, both of which are true for C. pallens. Highly variable masting and chaotic dynamics appear to be advantageous in this case because they reduce seed losses to specialist seed predators, while balancing the costs of missed reproductive events.     

High mutation rates in human and ape pseudoautosomal genes

It has been suggested that recombination may be mutagenic, which, if true, would inflate intraspecies diversity and interspecies silent divergence in regions of high recombination. Here, we test this hypothesis comparing human/orangutan genome-wide non-coding divergence (K) to that in the pseudoautosomal genes which were reported to recombine much more frequently than the rest of the genome. We demonstrate that, compared to the average human/orangutan non-coding divergence (K=3%), the substitution rate is significantly elevated in the introns of SHOX (K=5.7%), PPP2R3L (K=8.7%) and ASMT (K=6.5%) genes located in the human and orangutan Xp/Yp pseudoautosomal region (p-PAR), where recombination is over 20-fold higher than the genomic average. On the other hand, human/orangutan non-coding divergence at the Xp/Yp pseudoautosomal boundary (K=3.5%) and in the SYBL1 gene (K=2.7%), located in the human Xq/Yq pseudoautosomal region (q-PAR), where recombination is known to be less frequent than in p-PAR, was not significantly higher than the genome average. The data are consistent with the hypothesis that recombination may be mutagenic.     

Mast cells: an unexpected finding in the modulation of cutaneous wound repair by charged beads

Increased numbers of mast cells are affiliated with a broad spectrum of pathologic skin conditions, including ulcers, atopic dermatitis, neurofibromatosis, hemangiomas, keloids, and hypertrophic scars. It has been proposed that mast cells play a primary pathophysiologic role in these disorders and that their presence represents not merely a secondary event. While investigating their recent hypothesis that positively charged cross-linked diethylaminoethyl dextran (CLDD) beads potentiate cutaneous wound healing, the authors serendipitously observed increased numbers of mast cells in the deep dermis of wounds treated with CLDD beads. The authors propose that mast cells may play an important role in the modulation of healing seen with CLDD beads. Incisional wounds were studied in 30 Sprague-Dawley rats partitioned into two groups that were killed 7 or 14 days after wounding. The wounds were treated with positively, negatively, or neutrally charged CLDD beads. Physiologic saline served as a control. At the designated times after incisional wounding, biopsy specimens were tested for wound breaking strength or processed for histologic testing, fixed in 4% paraformaldehyde, and stained with Giemsa and Goldner-Masson trichrome. Mast cells were counted under light microscopy in a blinded fashion and were expressed as the number of cells per millimeter squared. Significant increases in the number of mast cells were observed in the deep dermis of incisional wounds after implantation with positively or negatively charged CLDD beads. In contrast, neutrally charged beads had no effect on mast cell numbers. At 7 days, the incisions treated with positively charged beads averaged 2.1 times more mast cells compared with those treated with physiologic saline or neutrally charged beads, whereas the incisions treated with negatively charged beads displayed 3.2 times more mast cells. By day 14, the incisions treated with positively charged beads averaged 2.5 times more mast cells than those wounds treated with saline or neutrally charged beads; the incisions treated with negatively charged CLDD beads had 3.4 times more mast cells. The 7-day tensiometric data indicated that wounds treated with negatively charged CLDD beads had increased breaking strength compared with wounds treated with neutrally charged beads or saline (1.8 and 1.7 times, respectively; p = 0.01 and p = 0.02). Wounds treated with positively charged beads also showed increased breaking strength compared with wounds treated with neutrally charged beads or saline (1.5 and 1.4 times greater); however, this did not reach statistical significance. There was no apparent difference in breaking strength when neutrally charged beads were compared with those treated with saline. At 14 days, there was no statistically significant difference in wound breaking strength between different treatments. These findings are clinically germane to the assessment of proposed therapeutic applications of CLDD beads for a variety of impaired wound-healing states. Furthermore, if increased mast cell populations are intimately linked to hypertrophic scar and keloid formation, the results of the authors' study suggest that CLDD bead therapy of cutaneous wounds may lead to pathologic wound healing in humans.