Regulation of basal and induced levels of hsp70 is critical for cellular homeostasis. Ataxin-3 is a deubiquitinase with several cellular functions including transcriptional regulation and maintenance of protein homeostasis. While investigating potential roles of ataxin-3 in response to cellular stress, it appeared that ataxin-3 regulated hsp70. Basal levels of hsp70 were lower in ataxin-3 knockout (KO) mouse brain from 2 to 63 weeks of age and hsp70 was also lower in fibroblasts from ataxin-3 KO mice. Transfecting KO cells with ataxin-3 rescued basal levels of hsp70 protein. Western blots of representative chaperones including hsp110, hsp90, hsp70, hsc70, hsp60, hsp40/hdj2, and hsp25 indicated that only hsp70 was appreciably altered in KO fibroblasts and KO mouse brain. Turnover of hsp70 protein was similar in wild-type (WT) and KO cells; however, basal hsp70 promoter reporter activity was decreased in ataxin-3 KO cells. Transfecting ataxin-3 restored hsp70 basal promoter activity in KO fibroblasts to levels of promoter activity in WT cells; however, mutations that inactivated deubiquitinase activity or the ubiquitin interacting motifs did not restore full activity to hsp70 basal promoter activity. Hsp70 protein and promoter activity were higher in WT compared to KO cells exposed to heat shock and azetidine-2-carboxylic acid, but WT and KO cells had similar levels in response to cadmium. Heat shock factor-1 had decreased levels and increased turnover in ataxin-3 KO fibroblasts. Data in this study are consistent with ataxin-3 regulating basal level of hsp70 as well as modulating hsp70 in response to a subset of cellular stresses.
Summary Reversion of the malignant phenotype of erbB2-transformed cells can be driven by anti-erbB2/neu monoclonal antibodies (mAb), which disrupt the receptor's kinase activity. We examined the biologic effects of IFN-γ alone or after anti-erbB2/neu mAb treatment of erbB2-positive cells. IFN-γ had no effect on its own. Treatment of the tumors with anti-erbB2/neu mAb followed by IFN-γ led to dramatic inhibition of tumor growth in vitro and in vivo with minimal mAb dosing. Sequential therapy enhanced the effects of chemotherapy. Moreover, IFN-γ with mAb treatment of mice with IFNγR knock down tumors did not demonstrate marked synergistic eradication effects, indicating an unexpected role of IFN-γ on the tumor itself. Additionally, mAb and IFN-γ treatment also in duced immune host responses that enhanced tumor eradication. Biochemical analyses identified loss of Snail expression in tumor cells, reflecting diminution of tumor stem cell-like properties as a consequence of altered activity of GSK3-β and KLF molecules.
To address the growing need for new antimicrobial agents, we explored whether inhibition of bacterial signaling machinery could inhibit bacterial growth. Because bacteria rely on two-component signaling systems to respond to environmental changes, and because these systems are both highly conserved and mediated by histidine kinases, inhibiting histidine kinases may provide broad spectrum antimicrobial activity. The histidine kinase ATP binding domain is conserved with the ATPase domain of eukaryotic Hsp90 molecular chaperones. To find a chemical scaffold for compounds that target histidine kinases, we leveraged this conservation. We screened ATP competitive Hsp90 inhibitors against CckA, an essential histidine kinase in Caulobacter crescentus that controls cell growth, and showed that the diaryl pyrazole is a promising scaffold for histidine kinase inhibition. We synthesized a panel of derivatives and found that they inhibit the histidine kinases C. crescentus CckA and Salmonella PhoQ but not C. crescentus DivJ; and they inhibit bacterial growth in both Gram-negative and Gram-positive bacterial strains.
The role of macrophages in medulloblastoma, the most common malignant pediatric brain tumor, is 3 unclear. Using single-cell RNA sequencing in a mouse model of sonic hedgehog medulloblastoma 4 and analysis of bulk RNA sequencing of human medulloblastoma, we investigated macrophage 5 heterogeneity. Our findings reveal differential recruitment of macrophages with molecular-targeted 6 versus radiation therapy and identify an immunosuppressive monocyte-derived macrophages 7 following radiation treatment of mouse medulloblastoma, uncovering potential strategies for 8 immunomodulation as adjunctive therapy. 9 10 11 Main Text 12 13Macrophages in the brain tumor microenvironment are emerging as a predictor of clinical outcome 1,2 . 14 However, targeting macrophages for immunotherapy, or indeed any immunotherapy, has yet to be 15 proven effective for brain tumor treatment. A major barrier is our incomplete understanding of the 16 heterogeneity of tumor-associated macrophages (TAMs) and how they respond to treatment. Within 17 tumors, anti-inflammatory (M2-polarized) macrophages drive immunosuppression while pro-18 inflammatory (M1-polarized) macrophages support anti-tumor immunity. Macrophages in normal 19 tissue are phenotypically, functionally, and ontologically heterogeneous. It is currently unclear 20 whether tumor-associated macrophages display similar heterogeneity and if insights into the nature of 21 their heterogeneity can inform their variable functions. 22
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