Multiple distinct assemblies reveal conformational flexibility in the small heat shock protein Hsp26

Small heat shock proteins are a superfamily of molecular chaperones that suppress protein aggregation and provide protection from cell stress. A key issue for understanding their action is to define the interactions of subunit domains in these oligomeric assemblies. Cryo-electron microscopy of yeast Hsp26 reveals two distinct forms, each comprising 24 subunits arranged in a porous shell with tetrahedral symmetry. The subunits form elongated, asymmetric dimers that assemble via trimeric contacts. Modifications of both termini cause rearrangements that yield a further four assemblies. Each subunit contains an N-terminal region, a globular middle domain, the alpha-crystallin domain, and a C-terminal tail. Twelve of the C termini form 3-fold assembly contacts which are inserted into the interior of the shell, while the other 12 C termini form contacts on the surface. Hinge points between the domains allow a variety of assembly contacts, providing the flexibility required for formation of supercomplexes with non-native proteins.     

Applications of systems biology towards microbial fuel production

Harnessing the immense natural diversity of biological functions for economical production of fuel has enormous potential benefits. Inevitably, however, the native capabilities for any given organism must be modified to increase the productivity or efficiency of a biofuel bioprocess. From a broad perspective, the challenge is to sufficiently understand the details of cellular functionality to be able to prospectively predict and modify the cellular function of a microorganism. Recent advances in experimental and computational systems biology approaches can be used to better understand cellular level function and guide future experiments. With pressure to quickly develop viable, renewable biofuel processes a balance must be maintained between obtaining depth of biological knowledge and applying that knowledge.     

Use of recombinant adenovirus vectored consensus IFN-α to avert severe arenavirus infection

Several arenaviruses can cause viral hemorrhagic fever, a severe disease with case-fatality rates in hospitalized individuals ranging from 15-30%. Because of limited prophylaxis and treatment options, new medical countermeasures are needed for these viruses classified by the National Institutes of Allergy and Infectious Diseases (NIAID) as top priority biodefense Category A pathogens. Recombinant consensus interferon alpha (cIFN-α) is a licensed protein with broad clinical appeal. However, while cIFN-α has great therapeutic value, its utility for biodefense applications is hindered by its short in vivo half-life, mode and frequency of administration, and costly production. To address these limitations, we describe the use of DEF201, a replication-deficient adenovirus vector that drives the expression of cIFN-α, for pre- and post-exposure prophylaxis of acute arenaviral infection modeled in hamsters. Intranasal administration of DEF201 24 h prior to challenge with Pichindé virus (PICV) was highly effective at protecting animals from mortality and preventing viral replication and liver-associated disease. A significant protective effect was still observed with a single dosing of DEF201 given two weeks prior to PICV challenge. DEF201 was also efficacious when administered as a treatment 24 to 48 h post-virus exposure. The protective effect of DEF201 was largely attributed to the expression of cIFN-α, as dosing with a control empty vector adenovirus did not protect hamsters from lethal PICV challenge. Effective countermeasures that are highly stable, easily administered, and elicit long lasting protective immunity are much needed for arena and other viral infections. The DEF201 technology has the potential to address all of these issues and may serve as a broad-spectrum antiviral to enhance host defense against a number of viral pathogens.     

Three-Dimensional Reconstruction of the Mammalian Centriole from Cryoelectron Micrographs: The Use of Common Lines for Orientation and Alignment

The microtubule organizing center of the animal cell (S. D. Fulleret al.,1992,Curr. Opin. Struct. Biol.2,264–274; D. M. Gloveret al.,1993,Sci. Am.268,62–68; E. B. Wilson, 1925), (The Cell in Development and Heredity) comprises two centrioles and the pericentriolar material. We have completed several three-dimensional reconstructions of individual centrioles from tilt series of cryoelectron micrographs. The reconstruction procedure uses minimization of the common lines residual to define the orientation of the centriolar ninefold symmetry axis and then uses this symmetry to generate a structure by weighted backprojection to 28-nm resolution. Many of the features of these reconstructions agree with previous, conventional transmission electron microscopy studies (M. Paintrandet al.,1992,J. Struct. Biol.108,107–128). The microtubule barrel of the centriole is roughly 500 nm long and 300 nm in diameter and the microtubule bundles appear to taper toward the distal end. In addition, we see a handedness to the pericentriolar material at the base (distal end) of the centriole which is opposite to the skew of the microtubule triplets. The region at which the microtubule barrel joins this base is intriguingly complex and includes an internal cylindrical feature which is a site of γ tubulin localization.     

Inherent optical properties of the Irish Sea and their effect on satellite primary production algorithms

Three cruises were conducted in the Irish Sea during May, Juneand July 2001 to determine the variability in inherent opticalproperties (IOP), photo physiological parameters and primaryproduction (PP) and to assess the effect of IOP on satellitePP algorithms. The absorption coefficients of phytoplankton(a_ph), coloured dissolved organic material (a_CDOM) and nonalgalparticles (a_NAP) were higher during May than June and July.A radiative transfer model was used to model the in-water lightfield based on a_ph (case 1) and a_ph, a_CDOM and a_NAP (case 2).When PP was compared using these light fields, there was a 46%difference in estimates. The case 2 in-water light field wascoupled to a wavelength resolving satellite model of PP (PP_case2)and had a low root mean square error (RMS) (0.27 log₁₀PP) comparedwith in situ PP_case2. IOP absorption, especially a_CDOM, hada significant effect on the performance of this algorithm, butscattering of light by suspended particulate material had asmall effect. A look-up table was generated from the in situa_ph, a_CDOM and a_NAP measurements, which can be used in conjunctionwith satellite products to produce satellite maps of PP. Therewas <25% difference between in situ PP_case2 and the satellitePP maps, which suggests that they could be produced routinelyand accurately to monitor PP in the Irish Sea and other coastaland estuarine areas.     

Hemorrhagic fever of bunyavirus etiology: disease models and progress towards new therapies

A growing number of bunyaviruses are known to cause viral hemorrhagic fever (VHF), a severe febrile illness which can progress to hypovolemic shock and multi-organ failure and is characterized by hematologic abnormalities and vascular leak. At present, there are no approved vaccines or antiviral therapies to effectively prevent or treat VHF caused by pathogenic bunyaviruses. Advances in the modeling of bunyaviral infections have facilitated efforts towards the development of novel post-exposure prophylactic and therapeutic countermeasures, several of which may some day be approved for human use. Here, we review recent progress in animal models of severe bunyaviral infections essential to this mission, as well as promising antivirals and biologicals that are at various stages of the development process.     

Cdc6 stability is regulated by the Huwe1 ubiquitin ligase after DNA damage

The Cdc6 protein is an essential component of pre-replication complexes (preRCs), which assemble at origins of DNA replication during the G1 phase of the cell cycle. Previous studies have demonstrated that, in response to ionizing radiation, Cdc6 is ubiquitinated by the anaphase promoting complex (APC(Cdh1)) in a p53-dependent manner. We find, however, that DNA damage caused by UV irradiation or DNA alkylation by methyl methane sulfonate (MMS) induces Cdc6 degradation independently of p53. We further demonstrate that Cdc6 degradation after these forms of DNA damage is also independent of cell cycle phase, Cdc6 phosphorylation of the known Cdk target residues, or the Cul4/DDB1 and APC(Cdh1) ubiquitin E3 ligases. Instead Cdc6 directly binds a HECT-family ubiquitin E3 ligase, Huwe1 (also known as Mule, UreB1, ARF-BP1, Lasu1, and HectH9), and Huwe1 polyubiquitinates Cdc6 in vitro. Degradation of Cdc6 in UV-irradiated cells or in cells treated with MMS requires Huwe1 and is associated with release of Cdc6 from chromatin. Furthermore, yeast cells lacking the Huwe1 ortholog, Tom1, have a similar defect in Cdc6 degradation. Together, these findings demonstrate an important and conserved role for Huwe1 in regulating Cdc6 abundance after DNA damage.     

Observation and simulation of the spring bloom in the northwestern Irish Sea

A two-layer, coupled physical-biological model was used to investigatephytoplankton dynamics in the north-western Irish Sea. The modelwas parameterized with values obtained from the published literature,and predicted the concentration of chlorophyll and dissolvedinorganic nitrogen in an upper and lower layer. Simulationswere driven with measured hourly values of meteorological data.The timings of the spring bloom predicted by the model at asummer thermally stratified site and a vertically mixed sitewere compared with observations made during 1992. At the mixedsite, good agreement was found between the model and observations.At the stratified site, the results are less satisfactory andshow that the model predicted a spring bloom 13 days later thanobserved. The difference was probably due to overestimationof the surface mixed-layer depth leading to underestimationof upper-layer irradiance. A simple numerical experiment demonstratedthat the timing of the spring bloom was best simulated usingthe maximum theoretically justifiable value for photosyntheticefficiency.