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.     

Modelling the internalization of labelled cells in tumour spheroids

In this paper a mathematical model is developed to describe the migration of labelled particles within a multicell spheroid. In the model, spatial variations in cell proliferation and death create an internal velocity field which leads to redistribution of the labelled and unlabelled cells. By applying a range of numerical and analytical techniques to the model equations, it is possible to show that, whilst the speed with which the labelled cells migrate through the tumour is independent of the type of cells that are labelled, their limiting distribution depends crucially on whether inert polystyrene microspheres or live tumour cells are labelled. These predictions are shown to be in good qualitative agreement with independent experimental results.     

The complete genomic organization of the human MUC6 and MUC2 mucin genes

The complete genomic organization of the two mucin genes MUC2 and MUC6 was obtained by comparison of new and published mRNA sequences with newly available human genomic sequence. The two genes are located 38.5 kb apart in a head-to-head orientation within a gene complex on chromosome 11p15.5. The N-terminal organization of MUC6 is highly similar to that of MUC2, containing the D1, D2, D', and D3 Von Willebrand factor domains followed by the large tandem repeat domains located in exons 31 and 30, respectively. MUC6 has a much smaller C-terminal domain (101 amino acids) encoded by 2 exons containing only the CK domain, compared with MUC2, which has a C-terminal domain of 859 amino acids containing the D4, C, D, and CK domains, encoded by 19 exons. The gene structures agreed partially but not completely with predictions from gene prediction programs.     

Responses of mycorrhizal and non-mycorrhizal<Emphasis Type="Italic"> Erica cinerea</Emphasis> and<Emphasis Type="Italic"> Vaccinium macrocarpon</Emphasis> to<Emphasis Type="Italic"> Glomus mosseae</Emphasis>

An investigation was carried out on the mycorrhizal colonisation, growth and nutrition of two members of the Ericaceae in close proximity to an arbuscular mycorrhizal (AM) association. This was undertaken by separating mycorrhizal (EM) and non-mycorrhizal (NEM) Erica cinerea and Vaccinium macrocarpon from AM (inoculated by Glomus mosseae) and non-mycorrhizal (NAM) Plantago lanceolata using a 30 µm nylon mesh in a sand culture/pot system. Ericoid mycorrhizal colonisation by Hymenoscyphus ericae on root systems of E. cinerea and V. macrocarpon was in the range 14–22% and 58–69%, respectively. The presence of AM P. lanceolata had no effect on the ericoid mycorrhizal colonisation of E. cinerea and V. macrocarpon. NEM E. cinerea showed reductions in shoot biomass and shoot nitrogen concentrations after exposure to AM P. lanceolata after incubations of 6 and 9 weeks but there were no differences in dry mass, length, and nitrogen and phosphorus concentrations of the root systems between the treatment combinations. Reductions were also found, after incubations of 6 and 9 weeks, in shoot dry mass, leaf area and shoot nitrogen concentrations of NEM V. macrocarpon in the presence of AM P. lanceolata but no changes occurred in the length and dry mass of the root systems. There were no differences in maximum photosynthesis in V. macrocarpon between treatment combinations but NEM V. macrocarpon in the presence of AM P. lanceolata had the lowest transpiration rates and stomatal conductance and the highest nitrogen- and phosphorus-use efficiencies compared with the other treatment combinations. These results are discussed in relation to the type of interaction found in these compatible and incompatible mycorrhizal associations.