Two binding partners cooperate to activate the molecular motor Kinesin-1

The regulation of molecular motors is an important cellular problem, as motility in the absence of cargo results in futile adenosine triphosphate hydrolysis. When not transporting cargo, the microtubule (MT)-based motor Kinesin-1 is kept inactive as a result of a folded conformation that allows autoinhibition of the N-terminal motor by the C-terminal tail. The simplest model of Kinesin-1 activation posits that cargo binding to nonmotor regions relieves autoinhibition. In this study, we show that binding of the c-Jun N-terminal kinase-interacting protein 1 (JIP1) cargo protein is not sufficient to activate Kinesin-1. Because two regions of the Kinesin-1 tail are required for autoinhibition, we searched for a second molecule that contributes to activation of the motor. We identified fasciculation and elongation protein zeta1 (FEZ1) as a binding partner of kinesin heavy chain. We show that binding of JIP1 and FEZ1 to Kinesin-1 is sufficient to activate the motor for MT binding and motility. These results provide the first demonstration of the activation of a MT-based motor by cellular binding partners.     

Oxidized cholesterol metabolites found in human atherosclerotic lesions promote apolipoprotein C-II amyloid fibril formation

Apolipoprotein amyloid deposits and lipid oxidation products are colocalized in human atherosclerotic tissue. In this study we show that the primary ozonolysis product of cholesterol, 3beta-hydroxy-5-oxo-5,6-secocholestan-6-al (KA), rapidly promotes human apolipoprotein (apo) C-II amyloid fibril formation in vitro. Previous studies show that hydrophobic aldehydes, including KA, modify proteins by the formation of a Schiff base with the lysine epsilon-amino group or N-terminal amino group. High-performance liquid chromatography, mass spectrometry, and proteolysis of KA-modified apoC-II revealed that KA randomly modified six different lysine residues, with primarily one KA attached per apoC-II molecule. Competition experiments showed that an aldehyde scavenging compound partially inhibited the ability of KA to hasten apoC-II fibril formation. Conversely, the acid derivative of KA, lacking the ability to form a Schiff base, accelerated apoC-II fibril formation, albeit to a lesser extent, suggesting that amyloidogenesis triggered by KA involves both covalent and noncovalent mechanisms. The viability of a noncovalent mechanism mediated by KA has been observed previously with alpha-synuclein aggregation, implicated in Parkinson's disease. Electron microscopy demonstrated that fibrils formed in the presence of KA had a similar morphology to native fibrils; however, the isolated KA-apoC-II covalent adducts in the absence of unmodified apoC-II formed fibrillar structures with altered ropelike morphologies. KA-mediated fibril formation by apoC-II was inhibited by the addition of the amine-containing compound hydralazine and the lipid-binding protein apoA-I. These in vitro studies suggest that the oxidized small molecule pool could trigger or hasten the aggregation of apoC-II to form amyloid deposits.     

Identification and synthesis of major metabolites of Vasopressin V2-receptor agonist WAY-151932, and antagonist, Lixivaptan

Small molecule agonists and antagonists of the V(2)-vasopressin receptor have been discovered and have undergone clinical trials. In conjunction with these discovery programs, the synthesis and biological testing of various metabolites associated with these clinical targets were actively pursued. We now report the results of our synthetic efforts and the corresponding biological data generated for several of the metabolites of WAY-151932 and CL-347985 (Lixivaptan).     

Biological solutions to transport network design

Transport networks are vital components of multicellular organisms, distributing nutrients and removing waste products. Animal and plant transport systems are branching trees whose architecture is linked to universal scaling laws in these organisms. In contrast, many fungi form reticulated mycelia via the branching and fusion of thread-like hyphae that continuously adapt to the environment. Fungal networks have evolved to explore and exploit a patchy environment, rather than ramify through a three-dimensional organism. However, there has been no explicit analysis of the network structures formed, their dynamic behaviour nor how either impact on their ecological function. Using the woodland saprotroph Phanerochaete velutina, we show that fungal networks can display both high transport capacity and robustness to damage. These properties are enhanced as the network grows, while the relative cost of building the network decreases. Thus, mycelia achieve the seemingly competing goals of efficient transport and robustness, with decreasing relative investment, by selective reinforcement and recycling of transport pathways. Fungal networks demonstrate that indeterminate, decentralized systems can yield highly adaptive networks. Understanding how these relatively simple organisms have found effective transport networks through a process of natural selection may inform the design of man-made networks.     

Manual curation is not sufficient for annotation of genomic databases

MOTIVATION: Knowledge base construction has been an area of intense activity and great importance in the growth of computational biology. However, there is little or no history of work on the subject of evaluation of knowledge bases, either with respect to their contents or with respect to the processes by which they are constructed. This article proposes the application of a metric from software engineering known as the found/fixed graph to the problem of evaluating the processes by which genomic knowledge bases are built, as well as the completeness of their contents. RESULTS: Well-understood patterns of change in the found/fixed graph are found to occur in two large publicly available knowledge bases. These patterns suggest that the current manual curation processes will take far too long to complete the annotations of even just the most important model organisms, and that at their current rate of production, they will never be sufficient for completing the annotation of all currently available proteomes.     

Rab11-FIP2 regulates differentiable steps in transcytosis

Transcytosis through the apical recycling system of polarized cells is regulated by Rab11a and a series of Rab11a-interacting proteins. We have identified a point mutant in Rab11 family interacting protein 2 (Rab11-FIP2) that alters the function of Rab11a-containing trafficking systems. Rab11-FIP2(S229A/R413G) or Rab11-FIP2(R413G) cause the formation of a tubular cisternal structure containing Rab11a and decrease the rate of polymeric IgA transcytosis. The R413G mutation does not alter Rab11-FIP interactions with any known binding partners. Overexpression of Rab11-FIP2(S229A/R413G) alters the localization of a subpopulation of the apical membrane protein GP135. In contrast, Rab11-FIP2(129-512) alters the localization of early endosome protein EEA1. The distributions of both Rab11-FIP2(S229A/R413G) and Rab11-FIP2(129-512) were not dependent on the integrity of the microtubule cytoskeleton. The results indicate that Rab11-FIP2 regulates trafficking at multiple points within the apical recycling system of polarized cells. Rab11a; immunoglobulin A; trafficking; apical recycling; GP135; early endosome; EEA1; Eps15 homology domain     

Seasonality and coronary heart disease deaths in United States firefighters

United States firefighters have a high on-duty fatality rate, and coronary heart disease is the leading cause. Seasonality affects the incidence of cardiovascular events in the general population, but its effects on firefighters are unknown. This study statistically examined the seasonal and annual variation of all on-duty coronary heart disease deaths among US firefighters between 1994 and 2004 using the chi-square distribution and Poisson regression model of the monthly fatality counts. It also examined the effect of ambient temperature (apparent as well as wind chill temperature) on coronary heart disease fatalities during the study span using a time-stratified, case-crossover study design. When grouped by season, we observed the distribution of the 449 coronary heart disease fatalities to show a relative peak in winter (32%) and relative nadir in spring (21%). This pattern was significantly different (p=0.005) from the expected distribution under the null hypothesis of season having no effect. The pattern persisted in additional analyses, stratifying the deaths by the type of duty in which the firefighters were engaged at the time of their deaths. In the Poisson regression model of the monthly fatality counts, the overall goodness-of-fit between the actual and predicted case counts was excellent (χ₄²=16.63; p=0.002). Two distinct peaks were detected: one in January-February and the other in August-September. Overall temperature was not associated with increased risk of on-duty death. After allowing for different effects of temperature in mild/hot versus cold periods, a 1°C increase was not protective in cold weather; nor did it increase the risk of death in warmer weather. The findings of this study reveal statistical evidence for excess coronary heart disease deaths among firefighters during winter; however, the temporal pattern of coronary heart disease deaths was not linked to temperature variation. The seasonal pattern was also found to be independent of duty-related risks.     

Marsupial oocytes, fertilization and embryonic development can provide useful tools to study developmental mechanisms

An analysis of several features of marsupial oocytes and developmental stages from the zygote to the neonate shows the advantages of studying these to answer questions about mammalian development in general. Marsupials make good model systems providing that species have well established breeding and husbandry protocols, a detailed time table of embryonic development associated with appropriate monitoring to collect desired stages and an induced ovulation protocol to allow collection of normal oocytes and developmental stages. One of the specific features of marsupial development that would provide further insight into problems in mammalian development is distinct oocyte and/or zygote polarity. Additionally, because the conceptus is transparent, cell-cell associations can be studied until well after the hypoblast is formed. These combined features mean that lineage allocation and axis formation can be more easily studied and related to oocyte/zygote polarity and the order of cell division in these conceptuses. Another feature is the ability to collect normal unattached embryonic and fetal stages in some species and to culture conceptuses from the primitive streak stage to within a few hours of birth. This ability means that stages of organogenesis in most organ systems are readily accessible without the complications of implantation.