DNA Sequence Analysis of Mutagenicity and Site Specificity of Ethyl Methanesulfonate in Uvr+ and UvrB - Strains of ESCHERICHIA COLI

EMS-induced mutations within a 180 base pair region of the lacI gene of E. coli were cloned and sequenced. In total, 105 and 79 EMS-induced mutations from a Uvr+ and a UvrB- strain, respectively, were sequenced. The specificity of EMS-induced mutagenesis was very similar in the two strains; G:C----A:T transitions accounted for all but three of the mutants. The overall frequency of induced mutation was fivefold higher in the UvrB- strain compared to the Uvr+ strain. This demonstrates, at the DNA sequence level, that the presumed premutagenic lesion, O6-ethylguanine, is subject to repair by the uvrABC excision repair system of E. coli. An analysis of mutation frequencies with respect to neighboring base sequence, in the two strains, shows that O6-ethylguanine lesions adjacent to A:T base pairs present better targets for the excision repair machinery than those not adjacent to A:T base pairs.     

Biosynthesis of human preapolipoprotein A-IV

The primary translation product of human intestinal apolipoprotein A-IV mRNA was purified from ascites and wheat germ cell-free systems. Comparison of its NH2-terminal sequence with mature, chylomicron-associated apo-A-IV revealed that apo-A-IV was initially synthesized with a 20-amino acid long NH2-terminal extension: Met-X-Leu-X-Ala-Val-Val-Leu-X-Leu-Ala-Leu-Val-Ala-Val-Ala-Leu-X-X-Ala. Co-translational cleavage of the cell-free product as well as Edman degradation of the stable intracellular form of the protein recovered from Hep G2 cells indicated that this entire 20-amino acid sequence behaved as a signal peptide. There is at least 55% sequence homology between the rat and human apo-A-IV signal peptides and 33% homology between the human A-I and A-IV presegments. Agarose gel chromatography of Hep G2 culture media indicated that neither apo-A-IV nor -A-I is associated with particles that have physical properties resembling any of the plasma lipoprotein density classes. Incubation of plasma with Hep G2 media resulted in transfer of A-I but not A-IV to lipoproteins. Since the NH2 termini of co-translationally cleaved and chylomicron-associated apo-A-IV are identical, it is apparent that 1) this polypeptide does not undergo NH2-terminal post-translational proteolysis like proapo-A-II or proapo-A-I, and 2) regulation of A-IV-lipoprotein interaction is not dependent on any NH2-terminal proteolytic processing event.     

A molecular analysis of the RK mutatest

We have shown in our earlier reports (Arimoto et al., 1980a, b) that hemin and some other porphyrins can inhibit the mutagenicity in Salmonella of heterocyclic amines derived from cooking of proteins. A direct interaction between hemin and the mutagens was implicated on the basis of the observation that some of the mutagens were inactivated when they had been metabolically converted into direct-acting mutagens before the treatment with hemin. Hemin is a bound constituent of various proteins including hemoglobin and myoglobin, which are abundantly present in the blood and muscles, respectively. An interesting question is whether or not hemoglobin and myoglobin can inhibit the activities of the mutagenic heterocyclic amines.     

Increased photic sensitivity for phase resetting but not melatonin suppression in Siberian hamsters under short photoperiods

Light regulates a variety of behavioral and physiological processes, including activity rhythms and hormone secretory patterns. Seasonal changes in the proportion of light in a day (photoperiod) further modulate those functions. Recently, short (SP) versus long days (LP) were found to markedly increase light sensitivity for phase shifting in Syrian hamsters. To our knowledge, photoperiod effects on light sensitivity have not been studied in other rodents, nor is it known if they generalize to other circadian responses. We tested whether photic phase shifting and melatonin suppression vary in Siberian hamsters maintained under LP or SP. Select irradiances of light were administered, and shifts in activity were determined. Photic sensitivity for melatonin suppression was examined in a separate group of animals via pulses of light across a 4 log-unit photon density range, with post-pulse plasma melatonin levels determined via RIA. Phase shifting and melatonin suppression were greater at higher irradiances for both LP and SP. The lower irradiance condition was below threshold for phase shifts in LP but not SP. Melatonin suppression did not vary by photoperiod, and the half saturation constant for fitted sigmoid curves was similar under LP and SP. Thus, the photoperiodic modulation of light sensitivity for phase shifting is conserved across two hamster genera. The dissociation of photoperiod effects on photic phase shifting and melatonin suppression suggests that the modulation of sensitivity occurs downstream of the common retinal input pathway. Understanding the mechanistic basis for this plasticity may yield therapeutic targets for optimizing light therapy practices.     

Intranasal vaccine trial for canine infectious tracheobronchitis (kennel cough)

Two field trials were conducted during periods of endemic (summer) and epizootic (winter) canine infectious tracheobronchitis activity to evaluate the efficacy of three intranasal vaccines in a closed commercial beagle breeding kennel. A trivalent vaccine containing Bordetella bronchiseptica, canine parainfluenza, and canine adenovirus-2 was administered at 3 weeks of age. The vaccine was 71.2% and 81.8% effective in decreasing the incidence of coughing during the winter and summer trials, respectively. The number of deaths was lower in each of the vaccine groups than in the placebo groups. No adverse reactions were observed with any of the intranasal vaccines.     

Simulating Henipavirus Multicycle Replication in a Screening Assay Leads to Identification of a Promising Candidate for Therapy

Nipah (NiV) and Hendra (HeV) viruses are emerging zoonotic paramyxoviruses that cause encephalitis in humans, with fatality rates of up to 75%. We designed a new high-throughput screening (HTS) assay for inhibitors of infection based on envelope glycoprotein pseudotypes. The assay simulates multicycle replication and thus identifies inhibitors that target several stages of the viral life cycle, but it still can be carried out under biosafety level 2 (BSL-2) conditions. These features permit a screen for antivirals for emerging viruses and select agents that otherwise would require BSL-4 HTS facilities. The screening of a small compound library identified several effective molecules, including the well-known compound chloroquine, as highly active inhibitors of pseudotyped virus infection. Chloroquine inhibited infection with live HeV and NiV at a concentration of 1 μM in vitro (50% inhibitory concentration, 2 μM), which is less than the plasma concentrations present in humans receiving chloroquine treatment for malaria. The mechanism for chloroquine''s antiviral action likely is the inhibition of cathepsin L, a cellular enzyme that is essential for the processing of the viral fusion glycoprotein and the maturation of newly budding virions. Without this processing step, virions are not infectious. The identification of a compound that inhibits a known cellular target that is important for viral maturation but that had not previously been shown to have antiviral activity for henipaviruses highlights the validity of this new screening assay. Given the established safety profile and broad experience with chloroquine in humans, the results described here provide an option for treating individuals infected by these deadly viruses.Nipah (NiV) and Hendra (HeV) viruses are newly emerging zoonoses that cause encephalitis in humans, with fatality rates of up to 75% (3, 7, 8, 12, 13, 30). NiV has caused at least nine significant outbreaks in Bangladesh and India since its emergence in Malaysia in 1998 (3, 7, 8, 12, 13, 30). The virus emerged from the fruit bat (flying fox) mammalian reservoir, via the pig, into the human population. However, direct transmission from bats to humans can bypass the pig host, and person-to-person transmission also has now become a primary mode of NiV spread (2, 5). HeV, via the same bat host, has caused disease in horses, with transmission to horse-handlers and veterinarians, and since 1995 has caused sporadic illness in Australia (12). Both viruses, in addition to acute disease, may cause asymptomatic infection in up to 60% of exposed people and may lead to late-onset disease or the relapse of encephalitis years after initial infection (25), as well as persistent or delayed neurological sequelae (11). The vast geographic range of the fruit bat mammalian reservoir raises the possibility of a wide spread of these human diseases, which presently have no clinical treatment or vaccine.The first step in infection with HeV or NiV is binding to the target cells, via the interaction of the viral envelope protein (G) with specific receptor molecules on the cell surface. The receptor for HeV is Ephrin B2 (EFNB2) and for NiV is either EFNB2 or EFNB3 (11). The fusion of the viral envelope with the plasma membrane of the cell is then mediated by the viral fusion protein (F). The F protein is synthesized as a precursor protein (F0) that is proteolytically processed posttranslationally to form a trimer of disulfide-linked heterodimers (F1 + F2). This cleavage event places the fusion peptide at the F1 terminus in the mature F protein and is essential for membrane fusion activity. During viral entry, the fusion peptides, which are buried in the F trimer, must be exposed transiently so that they can insert into the target cell membrane. The conformational change that leads to the exposure of the fusion peptides requires an activation step (22), which is initiated by the interaction of G with its receptor. Only virions bearing the mature, cleaved F can undergo activation and thus are infectious (4, 14, 15).We introduce here a biosafety level 2 (BSL-2)-amenable high-throughput screening (HTS) assay (9) for inhibitors that target several stages of the henipavirus viral cycle, based on envelope glycoprotein pseudotypes. The cell-based assay allows for the simultaneous evaluation of antiviral activity and the cytotoxicity of compounds. We have validated the method with several different classes of henipavirus entry inhibitors as well as protease inhibitors. For this assay, HeV envelope glycoproteins were pseudotyped onto a recombinant vesicular stomatitis virus (VSV) that expresses red fluorescent protein (RFP) but lacks its attachment protein, G (19, 20). The resulting pseudotyped virus bears the HeV binding and fusion proteins. The infection of target cells by pseudotyped virus in the absence and presence of compounds is quantified by assessing the production of red fluorescence. This pseudotyped viral entry assay, unlike previous ones (31), simulates multicycle replication because the monolayer cells, which express viral glycoproteins, will generate more pseudotyped particles when infected. Compounds found to be active in this assay may be those that either block binding, interfere with F activation or fusion, or interfere with the protease processing of F. However, the assay is safe, because these particles can only produce infectious progeny in cells expressing HeV G/F. These features allow experimentation and antiviral assessment for emerging viruses and select agents that otherwise would require BSL-4 HTS facilities. We report the use of this screen to discover effective inhibitors of henipavirus replication and the evaluation of a well-known compound with previously unidentified properties that may allow its immediate use for henipaviruses.     

Rpn1 and Rpn2 coordinate ubiquitin processing factors at proteasome

Substrates tagged with (poly)ubiquitin for degradation can be targeted directly to the 26 S proteasome where they are proteolyzed. Independently, ubiquitin conjugates may also be delivered by bivalent shuttles. The majority of shuttles attach to the proteasome through a ubiquitin-like domain (UBL) while anchoring cargo at a C-terminal polyubiquitin-binding domain(s). We found that two shuttles of this class, Rad23 and Dsk2, dock at two different receptor sites embedded within a single subunit of the 19 S proteasome regulatory particle, Rpn1. Their association/dissociation constants and affinities for Rpn1 are similar. In contrast, another UBL-containing protein, the deubiquitinase Ubp6, is also anchored by Rpn1, yet it dissociates slower, thus behaving as an occasional proteasome subunit that is distinct from the transiently associated shuttles. Two neighboring subunits, Rpn10 and Rpn13, show a marked preference for polyubiquitin over UBLs. Rpn10 attaches to the central solenoid portion of Rpn1, although this association is stabilized by the presence of a third subunit, Rpn2. Rpn13 binds directly to Rpn2. These intrinsic polyubiquitin receptors may compete with substrate shuttles for their polyubiquitin-conjugate cargos, thereby aiding release of the emptied shuttles. By binding multiple ubiquitin-processing factors simultaneously, Rpn1 is uniquely suited to coordinate substrate recruitment, deubiquitination, and movement toward the catalytic core. The broad range of affinities for ubiquitin, ubiquitin-like, and non-ubiquitin signals by adjacent yet nonoverlapping sites all within the base represents a hub of activity that coordinates the intricate relay of substrates within the proteasome, and consequently it influences substrate residency time and commitment to degradation.     

Changing Patterns in Place of Cancer Death in England: A Population-Based Study

Background

Most patients with cancer prefer to die at home or in a hospice, but hospitals remain the most common place of death (PoD).This study aims to explore the changing time trends of PoD and the associated factors, which are essential for end-of-life care improvement.

Methods and Findings

The study analysed all cancer deaths in England collected by the Office for National Statistics during 1993–2010 (n = 2,281,223). Time trends of age- and gender-standardised proportion of deaths in individual PoDs were evaluated using weighted piecewise linear regression. Variables associated with PoD (home or hospice versus hospital) were determined using proportion ratio (PR) derived from the log-binomial regression, adjusting for clustering effects. Hospital remained the most common PoD throughout the study period (48.0%; 95% CI 47.9%–48.0%), followed by home (24.5%; 95% CI 24.4%–24.5%), and hospice (16.4%; 95% CI 16.3%–16.4%). Home and hospice deaths increased since 2005 (0.87%; 95% CI 0.74%–0.99%/year, 0.24%; 95% CI 0.17%–0.32%/year, respectively, p<0.001), while hospital deaths declined (−1.20%; 95% CI −1.41 to −0.99/year, p<0.001). Patients who died from haematological cancer (PRs 0.46–0.52), who were single, widowed, or divorced (PRs 0.75–0.88), and aged over 75 (PRs 0.81–0.84 for 75–84; 0.66–0.72 for 85+) were less likely to die in home or hospice (p<0.001; reference groups: colorectal cancer, married, age 25–54). There was little improvement in patients with lung cancer of dying in home or hospice (PRs 0.87–0.88). Marital status became the second most important factor associated with PoD, after cancer type. Patients from less deprived areas (higher quintile of the deprivation index) were more likely to die at home or in a hospice than those from more deprived areas (lower quintile of the deprivation index; PRs 1.02–1.12). The analysis is limited by a lack of data on individual patients'' preferences for PoD or a clinical indication of the most appropriate PoD.

Conclusions

More efforts are needed to reduce hospital deaths. Health care facilities should be improved and enhanced to support the increased home and hospice deaths. People who are single, widowed, or divorced should be a focus for end-of-life care improvement, along with known at risk groups such as haematological cancer, lung cancer, older age, and deprivation. Please see later in the article for the Editors'' Summary     

A dual role for mycobacterial RecO in RecA-dependent homologous recombination and RecA-independent single-strand annealing

Mycobacteria have two genetically distinct pathways for the homology-directed repair of DNA double-strand breaks: homologous recombination (HR) and single-strand annealing (SSA). HR is abolished by deletion of RecA and reduced in the absence of the AdnAB helicase/nuclease. By contrast, SSA is RecA-independent and requires RecBCD. Here we examine the function of RecO in mycobacterial DNA recombination and repair. Loss of RecO elicits hypersensitivity to DNA damaging agents similar to that caused by deletion of RecA. We show that RecO participates in RecA-dependent HR in a pathway parallel to the AdnAB pathway. We also find that RecO plays a role in the RecA-independent SSA pathway. The mycobacterial RecO protein displays a zinc-dependent DNA binding activity in vitro and accelerates the annealing of SSB-coated single-stranded DNA. These findings establish a role for RecO in two pathways of mycobacterial DNA double-strand break repair and suggest an in vivo function for the DNA annealing activity of RecO proteins, thereby underscoring their similarity to eukaryal Rad52.