Why have organelles retained genomes?

The observation that chloroplasts and mitochondria have retained relics of eubacterial genomes and a protein-synthesizing machinery has long puzzled biologists. If most genes have been transferred from organelles to the nucleus during evolution, why not all? What selective pressure maintains genomes in organelles? Electron transport through the photosynthetic and respiratory membranes is a powerful - but dangerous - source of energy. Recent evidence suggests that organelle genomes have persisted because structural proteins that maintain redox balance within bioenergetic membranes must be synthesized when and where they are needed, to counteract the potentially deadly side effects of ATP-generating electron transport.     

Inactivation of Prions and Amyloid Seeds with Hypochlorous Acid

Hypochlorous acid (HOCl) is produced naturally by neutrophils and other cells to kill conventional microbes in vivo. Synthetic preparations containing HOCl can also be effective as microbial disinfectants. Here we have tested whether HOCl can also inactivate prions and other self-propagating protein amyloid seeds. Prions are deadly pathogens that are notoriously difficult to inactivate, and standard microbial disinfection protocols are often inadequate. Recommended treatments for prion decontamination include strongly basic (pH ≥~12) sodium hypochlorite bleach, ≥1 N sodium hydroxide, and/or prolonged autoclaving. These treatments are damaging and/or unsuitable for many clinical, agricultural and environmental applications. We have tested the anti-prion activity of a weakly acidic aqueous formulation of HOCl (BrioHOCl) that poses no apparent hazard to either users or many surfaces. For example, BrioHOCl can be applied directly to skin and mucous membranes and has been aerosolized to treat entire rooms without apparent deleterious effects. Here, we demonstrate that immersion in BrioHOCl can inactivate not only a range of target microbes, including spores of Bacillus subtilis, but also prions in tissue suspensions and on stainless steel. Real-time quaking-induced conversion (RT-QuIC) assays showed that BrioHOCl treatments eliminated all detectable prion seeding activity of human Creutzfeldt-Jakob disease, bovine spongiform encephalopathy, cervine chronic wasting disease, sheep scrapie and hamster scrapie; these findings indicated reductions of ≥10³- to 10⁶-fold. Transgenic mouse bioassays showed that all detectable hamster-adapted scrapie infectivity in brain homogenates or on steel wires was eliminated, representing reductions of ≥~10^5.75-fold and >10⁴-fold, respectively. Inactivation of RT-QuIC seeding activity correlated with free chlorine concentration and higher order aggregation or destruction of proteins generally, including prion protein. BrioHOCl treatments had similar effects on amyloids composed of human α-synuclein and a fragment of human tau. These results indicate that HOCl can block the self-propagating activity of prions and other amyloids.     

Crystal Structures of Cif from Bacterial Pathogens Photorhabdus luminescens and Burkholderia pseudomallei

A pre-requisite for bacterial pathogenesis is the successful interaction of a pathogen with a host. One mechanism used by a broad range of Gram negative bacterial pathogens is to deliver effector proteins directly into host cells through a dedicated type III secretion system where they modulate host cell function. The cycle inhibiting factor (Cif) family of effector proteins, identified in a growing number of pathogens that harbour functional type III secretion systems and have a wide host range, arrest the eukaryotic cell cycle. Here, the crystal structures of Cifs from the insect pathogen/nematode symbiont Photorhabdus luminescens (a γ-proteobacterium) and human pathogen Burkholderia pseudomallei (a β-proteobacterium) are presented. Both of these proteins adopt an overall fold similar to the papain sub-family of cysteine proteases, as originally identified in the structure of a truncated form of Cif from Enteropathogenic E. coli (EPEC), despite sharing only limited sequence identity. The structure of an N-terminal region, referred to here as the ‘tail-domain’ (absent in the EPEC Cif structure), suggests a surface likely to be involved in host-cell substrate recognition. The conformation of the Cys-His-Gln catalytic triad is retained, and the essential cysteine is exposed to solvent and addressable by small molecule reagents. These structures and biochemical work contribute to the rapidly expanding literature on Cifs, and direct further studies to better understand the molecular details of the activity of these proteins.     

Framing Responsibility

How can we register the participation of a range of elements, extending beyond the human subject, in the production of HIV events? In the context of proposals around biomedical prevention, there is a growing awareness of the need to find ways of responding to complexity, as everywhere new combinations of treatment, behavior, drugs, norms, meanings and devices are coming into encounter with one another, or are set to come into encounter with one another, with a range of unpredictable effects. In this paper I consider the operation of various framing devices that attribute responsibility and causation with regard to HIV events. I propose that we need to sharpen our analytic focus on what these devices do, their performativity??that is, their full range of worldly implications and effects. My primary examples are the criminal law and the randomized control trial. I argue that these institutions operate as framing devices: They attribute responsibility for HIV events and externalize other elements and effects in the process. Drawing on recent work in science and technology studies as well as queer theory, I set out an analytic frame that marks out a new role for HIV social research. Attentiveness to the performative effects of these devices is crucial, I suggest, if we want better to address the global HIV epidemic.     

Local and Regional Applications of Hydrogen Peroxide in the Control of Clostridial Myositis in Rabbits

The intra-arterial infusion of hydrogen peroxide has been used as a method for producing a hyperoxic environment in experimental animals for the treatment of experimentally induced clostridial myositis. Eighty-five rabbits were employed in this study; 43 were controls and 42 were experimental animals. In the experimental study, 21 animals were treated with hydrogen peroxide by each route of administration. In this group, 52.4% of the animals receiving the intra-arterial infusion and 66.6% receiving intramuscular clysis survived. There were no survivors past 72 hr in the control group.     

Insertion of the enteropathogenic Escherichia coli Tir virulence protein into membranes in vitro

Insertion of the enteropathogenic Escherichia coli Tir protein into the plasma membrane of intestinal epithelial cells is a crucial event in infection because it provides a receptor for intimate bacterial adherence. This interaction with the bacterial outer membrane protein intimin is also essential in generating a number of signaling activities associated with virulence. Tir can be modified at various sites by phosphorylation and functionally interacts with multiple host proteins. To investigate the mechanism of membrane insertion and to establish a model system in which the multiple interactions/functions of Tir can be uncoupled and independently characterized, we used intrinsic tryptophan fluorescence, surface plasmon resonance, and protease digestion assays to show that Tir can insert directly into phospholipid vesicles in a composition-dependent manner to generate the topology reported in vivo. This is the first time that Tir has been shown to insert into membranes in a simple model system in the absence of chemical modification or other factors. These data are consistent with the protein interacting with lipids through two sites. The major site is localized to the transmembrane/intimin-binding domain region and includes Trp235, which is shown to be an effective reporter of interaction. The minor site is located within the C-terminal domain. Together, these data support a model in which Tir is released into the cytoplasm by the type III translocon and then independently inserts into the plasma membrane from a cytoplasmic location. A thorough understanding of this mechanism will be crucial to understand the subtleties of enteropathogenic E. coli pathogenesis.