Elucidating the ticking of an in vitro circadian clockwork

A biochemical oscillator can be reconstituted in vitro with three purified proteins, that displays the salient properties of circadian (daily) rhythms, including self-sustained 24-h periodicity that is temperature compensated. We analyze the biochemical basis of this oscillator by quantifying the time-dependent interactions of the three proteins (KaiA, KaiB, and KaiC) by electron microscopy and native gel electrophoresis to elucidate the timing of the formation of complexes among the Kai proteins. The data are used to derive a dynamic model for the in vitro oscillator that accurately reproduces the rhythms of KaiABC complexes and of KaiC phosphorylation, and is consistent with biophysical observations of individual Kai protein interactions. We use fluorescence resonance energy transfer (FRET) to confirm that monomer exchange among KaiC hexamers occurs. The model demonstrates that the function of this monomer exchange may be to maintain synchrony among the KaiC hexamers in the reaction, thereby sustaining a high-amplitude oscillation. Finally, we apply the first perturbation analyses of an in vitro oscillator by using temperature pulses to reset the phase of the KaiABC oscillator, thereby testing the resetting characteristics of this unique circadian oscillator. This study analyzes a circadian clockwork to an unprecedented level of molecular detail.     

A Biomechanical Model of Tumor-Induced Intracranial Pressure and Edema in Brain Tissue

Brain tumor growth and tumor-induced edema result in increased intracranial pressure (ICP), which, in turn, is responsible for conditions as benign as headaches and vomiting or as severe as seizures, neurological damage, or even death. Therefore, it has been hypothesized that tracking ICP dynamics may offer improved prognostic potential in terms of early detection of brain cancer and better delimitation of the tumor boundary. However, translating such theory into clinical practice remains a challenge, in part because of an incomplete understanding of how ICP correlates with tumor grade. Here, we propose a multiphase mixture model that describes the biomechanical response of healthy brain tissue—in terms of changes in ICP and edema—to a growing tumor. The model captures ICP dynamics within the diseased brain and accounts for the ability/inability of healthy tissue to compensate for this pressure. We propose parameter regimes that distinguish brain tumors by grade, thereby providing critical insight into how ICP dynamics vary by severity of disease. In particular, we offer an explanation for clinically observed phenomena, such as a lack of symptoms in low-grade glioma patients versus a rapid onset of symptoms in those with malignant tumors. Our model also takes into account the effects tumor-derived proteases may have on ICP levels and the extent of tumor invasion. This work represents an important first step toward understanding the mechanisms that underlie the onset of edema and ICP in cancer-afflicted brains. Continued modeling effort in this direction has the potential to make an impact in the field of brain cancer diagnostics.     

Structure of cytochrome c oxidase: a comparison of the bacterial and mitochondrial enzymes

It has been almost 5 years since the first structures of cytochrome c oxidase, from Paracoccus denitrificans and bovine heart mitochondria, were revealed. Since then many different proton pumping mechanisms have been proposed for the enzyme; however, no definitive conclusion has been achieved. In this article, we revisit the original structures of bacterial and mitochondrial oxidases and try to clarify similarities as well as differences between the two structures.     

An insensitive acetylcholinesterase confers resistance to methomyl in the beet armyworm Spodoptera exigua (Lepidoptera: Noctuidae)

Two forms of acetylcholinesterase were identified in field populations of the beet armyworm, Spodoptera exigua (Hübner), collected from cotton in San Joaquin Valley, CA. Strains (BESS and BKRR) homogeneous for each variant were isolated and their relative susceptibilities to methomyl, chlorpyrifos, and chlorpyrifos-oxon assessed by topical application bioassay. In comparisons with a laboratory susceptible strain (DOW), BKRR and BESS expressed 68-fold and sevenfold resistance, respectively, to the carbamate methomyl. Neither strain was cross-resistant to chlorpyrifos or its oxygen analog (chlorpyrifos-oxon). In biochemical studies, the BKRR AChE enzyme was approximately 30-fold and sixfold more insensitive to methomyl and chlorpyrifos-oxon, respectively, compared with the DOW enzyme. The correlation between the toxicological and biochemical studies provides strong evidence that target-site insensitivity is an important mechanism of resistance to methomyl. The lack of significant cross-resistance to chlorpyrifos suggests that the insensitive AChE in these field populations was selected by methomyl alone and not by the organophosphate.     

Il-17 mobilizes peripheral blood stem cells with short- and long-term repopulating ability in mice

Autologous and allogeneic bone marrow transplantations have evolved as important cancer therapy modalities. For both indications, peripheral blood has been shown to have distinct advantages over bone marrow as the stem cell source. Cytokine combinations for mobilization have enhanced stem cell yield and accelerated engraftment. However, novel mobilizing agents and strategies are needed to further improve clinical outcomes. Within the donor graft, the dynamic equilibrium between T cells and stem cells critically influences engraftment and transplantation results. IL-17 is a cytokine produced almost exclusively from activated T cells. IL-17 was expressed in vivo with adenovirus technology. Here, proof-of-principle studies demonstrate that IL-17 effectively mobilizes hemopoietic precursor cells (CFU-granulocyte-erythrocyte-macrophage-monocyte, CFU-high proliferative potential) and primitive hemopoietic stem cells (Lin(-/low)c-kit(+)Sca1(+)). Moreover, mouse IL-17 adenovirus-mobilized peripheral blood stem cells rescued lethally irradiated mice. Bone marrow was found to be 45-75% of donor origin at 1 year. In secondary recipients, donor-derived bone marrow cells ranged from 45 to 95%. These data show that IL-17 mobilizes stem cells in mice with short- and long-term reconstituting capacity. Additional comparative studies are needed as well as studies in tumor models to refine distinct potential clinical applications for IL-17-mobilized peripheral blood stem cells.