Antimicrobial agents from higher plants: two dimethylbenzisochromans from Karwinskia humboldtiana

Two new antimicrobial dimethylbenzisochromans were isolated from the roots of Karwinskia humboldtiana together with the known 7-acetyl-6,8-dimethoxy-3-methyl-l-naphthol. The structures and absolute configurations were determined by spectroscopic examination and by chemical transformation to the known quinones eleutherin and 7-methoxyeleutherin.     

The N-Terminal Extension of Cardiac Troponin T Stabilizes the Blocked State of Cardiac Thin Filament

Cardiac troponin T (cTnT) is a key component of contractile regulatory proteins. cTnT is characterized by a ～32 amino acid N-terminal extension (NTE), the function of which remains unknown. To understand its function, we generated a transgenic (TG) mouse line that expressed a recombinant chimeric cTnT in which the NTE of mouse cTnT was removed by replacing its 1–73 residues with the corresponding 1–41 residues of mouse fast skeletal TnT. Detergent-skinned papillary muscle fibers from non-TG (NTG) and TG mouse hearts were used to measure tension, ATPase activity, Ca²⁺ sensitivity (pCa₅₀) of tension, rate of tension redevelopment, dynamic muscle fiber stiffness, and maximal fiber shortening velocity at sarcomere lengths (SLs) of 1.9 and 2.3 μm. Ca²⁺ sensitivity increased significantly in TG fibers at both short SL (pCa₅₀ of 5.96 vs. 5.62 in NTG fibers) and long SL (pCa₅₀ of 6.10 vs. 5.76 in NTG fibers). Maximal cross-bridge turnover and detachment kinetics were unaltered in TG fibers. Our data suggest that the NTE constrains cardiac thin filament activation such that the transition of the thin filament from the blocked to the closed state becomes less responsive to Ca²⁺. Our finding has implications regarding the effect of tissue- and disease-related changes in cTnT isoforms on cardiac muscle function.     

Adiponectin blocks interleukin-18-mediated endothelial cell death via APPL1-dependent AMP-activated protein kinase (AMPK) activation and IKK/NF-kappaB/PTEN suppression

The adipocyte-derived cytokine adiponectin is known to exert anti-inflammatory and anti-apoptotic effects. In patients with atherosclerotic cardiovascular disease, circulating levels of adiponectin correlate inversely with those of the proinflammatory, proapoptotic cytokine interleukin (IL)-18. The opposing actions of IL-18 and adiponectin on both cell survival and inflammation led us to investigate whether adiponectin signaling antagonizes IL-18-mediated endothelial cell death and to identify the underlying molecular mechanisms. Treatment with IL-18 suppressed Akt phosphorylation and its associated kinase activity, induced IkappaB kinase (IKK)-NF-kappaB-dependent PTEN activation, and promoted endothelial cell death. Pretreatment with adiponectin stimulated APPL1-dependent AMPK activation, reversed Akt inhibition in a phosphatidylinositol 3-kinase-dependent manner, blocked IKK-NF-kappaB-PTEN signaling, reduced caspase-3 activity, blocked Bax translocation, and inhibited endothelial cell death. The cytoprotective effect of adiponectin signaling was recapitulated by treatment with the pharmacological AMPK activator 5-aminoimidazole-4-carboxamide-1-beta-riboside. Collectively, these results demonstrated that adiponectin reverses IL-18-mediated endothelial cell death through an AMPK-associated mechanism, which may thus have therapeutic potential for diminishing IL-18-dependent vascular injury and inflammation.     

Mechanism for signaling initiation and termination of B lymphocyte proliferation induced by anti-immunoglobulin

Summary Several lines of evidence were explored which taken together indicate that both the initiation and the termination signal for activation of rabbit lymphocytes to synthesize DNA in response to anti-rabbit immunoglobulin occurs at an immunoglobulin receptor on the surface membrane of B cells. Thus, the premature removal of anti-rabbit immunoglobulin by simply washing the cells at the 31st hour of a 48-h incubation period caused a 60% decrease in the induction of DNA synthesis. The addition of rabbit immunoglobulin to compete with B cell surface immunoglobulin for the combining sites on anti-rabbit immunoglobulin yielded a markedly diminished activation. Addition of rabbit immunoglobulin even during the latter part of a pulse label period with [³H]-thymidine was sufficient to result in reduced activation. Finally, insoluble anti-rabbit immunoglobulin at the same nominal concentration as soluble anti-rabbit immunoglobulin also was effective in inducing cells to DNA synthesis. However, it is noteworthy that under the incubation conditions used it was not possible to derive a soluble component from insoluble anti-rabbit immunoglobulin which stimulated DNA synthesis. These data have been interpreted to indicate a need for a continuous surface presence of anti-rabbit immunoglobulin to stimulate activation in a process that is not dependent upon internalization of anti-rabbit immunoglobulin.     

Relevance and follow-up of positive results in in vitro genetic toxicity assays: an ILSI-HESI initiative

In vitro genotoxicity assays are often used to screen and predict whether chemicals might represent mutagenic and carcinogenic risks for humans. Recent discussions have focused on the high rate of positive results in in vitro tests, especially in those assays performed in mammalian cells that are not confirmed in vivo. Currently, there is no general consensus in the scientific community on the interpretation of the significance of positive results from the in vitro genotoxicity assays. To address this issue, the Health and Environmental Sciences Institute (HESI), held an international workshop in June 2006 to discuss the relevance and follow-up of positive results in in vitro genetic toxicity assays. The goals of the meeting were to examine ways to advance the scientific basis for the interpretation of positive findings in in vitro assays, to facilitate the development of follow-up testing strategies and to define criteria for determining the relevance to human health. The workshop identified specific needs in two general categories, i.e., improved testing and improved data interpretation and risk assessment. Recommendations to improve testing included: (1) re-examine the maximum level of cytotoxicity currently required for in vitro tests; (2) re-examine the upper limit concentration for in vitro mammalian studies; (3) develop improved testing strategies using current in vitro assays; (4) define criteria to guide selection of the appropriate follow-up in vivo studies; (5) develop new and more predictive in vitro and in vivo tests. Recommendations for improving interpretation and assessment included: (1) examine the suitability of applying the threshold of toxicological concern concepts to genotoxicity data; (2) develop a structured weight of evidence approach for assessing genotoxic/carcinogenic hazard; and (3) re-examine in vitro and in vivo correlations qualitatively and quantitatively. Conclusions from the workshop highlighted a willingness of scientists from various sectors to change and improve the current paradigm and move from a hazard identification approach to a "realistic" risk-based approach that incorporates information on mechanism of action, kinetics, and human exposure..     

Poster Sessions BP02: Oligodendrocytes and Schwann Cells

Neurofibromatosis Type 1 tumors are highly vascularized and contain Schwann cells with hyperactivated Ras. In vitro , the NF1-derived neurofibromin deficient Schwann cells have an angiogenic profile, which favors angiogenesis and sustains the growth of the NF1-derived tumors. This study examined the relationship of the activation state of Ras as it related to the expression of angiogenic and antiangiogenic factors in both cultured NF1-derived Schwann cells and normal human Schwann cells. Western blot analysis of normal human Schwann cells revealed low expression of angiogenic vascular endothelial growth factor (VEGF) as well as low expression of the antiangiogenic pigment epithelium derived factor (PEDF). Relative to normal human Schwann cells, NF1-derived Schwann cells have increased RAS activity and a three-fold increase in VEGF expression. Surprisingly, PEDF was also expressed in the NF1-derived Schwann cells at approximately the same level as VEGF expression. Using a retroviral construct, we introduced the GAP-related domain of neurofibromin into the NF1-derived Schwann cells to reduce the level of activated Ras. Relative to the untreated NF1-derived Schwann cells the Schwann cells expressing the GAP-related domain expressed about one-half the VEGF but twice the PEDF. We conclude that decreasing the Ras activity in NF1-drived Schwann cells will not only decrease proliferation, but also slow tumor angiogenesis due to the decreased expression of angiogenic and increased expression of antiangiogenic factors.