Mms2-Ubc13-dependent and -independent roles of Rad5 ubiquitin ligase in postreplication repair and translesion DNA synthesis in Saccharomyces cerevisiae

The Rad6-Rad18 ubiquitin-conjugating enzyme complex of Saccharomyces cerevisiae promotes replication through DNA lesions via three separate pathways that include translesion synthesis (TLS) by DNA polymerases eta and zeta and postreplicational repair (PRR) of discontinuities that form in the newly synthesized DNA opposite from DNA lesions, mediated by the Mms2-Ubc13 ubiquitin-conjugating enzyme and Rad5. Rad5 is an SWI/SNF family ATPase, and additionally, it functions as a ubiquitin ligase in the ubiquitin conjugation reaction. To decipher the roles of these Rad5 activities in lesion bypass, here we examine the effects of mutations in the Rad5 ATPase and ubiquitin ligase domains on the PRR of UV-damaged DNA and on UV-induced mutagenesis. Even though the ATPase-defective mutation confers only a modest degree of UV sensitivity whereas the ubiquitin ligase mutation causes a high degree of UV sensitivity, we find that both of these mutations produce the same high level of PRR defect as that conferred by the highly UV-sensitive rad5Delta mutation. From these studies, we infer a requirement of the Rad5 ATPase and ubiquitin ligase activities in PRR, and based upon the effects of different rad5 mutations on UV mutagenesis, we suggest a role for Rad5 in affecting the efficiency of lesion bypass by the TLS polymerases. In contrast to the role of Rad5 in PRR, however, where its function is coupled with that of Mms2-Ubc13, Rad5 function in TLS would be largely independent of this ubiquitin-conjugating enzyme complex.     

Whole genome comparison of six Crocosphaera watsonii strains with differing phenotypes

Crocosphaera watsonii, a unicellular nitrogen‐fixing cyanobacterium found in oligotrophic oceans, is important in marine carbon and nitrogen cycles. Isolates of C. watsonii can be separated into at least two phenotypes with environmentally important differences, indicating possibly distinct ecological roles and niches. To better understand the evolutionary history and variation in metabolic capabilities among strains and phenotypes, this study compared the genomes of six C. watsonii strains, three from each phenotypic group, which had been isolated over several decades from multiple ocean basins. While a substantial portion of each genome was nearly identical to sequences in the other strains, a few regions were identified as specific to each strain and phenotype, some of which help explain observed phenotypic features. Overall, the small‐cell type strains had smaller genomes and a relative loss of genetic capabilities, while the large‐cell type strains were characterized by larger genomes, some genetic redundancy, and potentially increased adaptations to iron and phosphorus limitation. As such, strains with shared phenotypes were evolutionarily more closely related than those with the opposite phenotype, regardless of isolation location or date. Unexpectedly, the genome of the type‐strain for the species, C. watsonii WH8501, was quite unusual even among strains with a shared phenotype, indicating it may not be an ideal representative of the species. The genome sequences and analyses reported in this study will be important for future investigations of the proposed differences in adaptation of the two phenotypes to nutrient limitation, and to identify phenotype‐specific distributions in natural Crocosphaera populations.     

Decreased Catalase Activity but Unchanged Superoxide Dismutase Activity in Brains of Patients with Dementia of Alzheimer Type

Abstract: "Oxidative stress" may be of significance in the etiopathogenesis of dementia of Alzheimer type (DAT). Therefore, we measured activities of the enzymes superoxide dismutase (SOD) and catalase (CAT), which detoxicate reactive oxygen species. Enzyme activities were measured postmortem in basal ganglia, cortical, and limbic brain regions of patients with DAT and age-matched controls. SOD activity increased with age in basal nucleus of Meynert. However, there was no significant difference in SOD activity between DAT and controls. CAT activity was independent of age and postmortem time. There were significant reductions in CAT activity in parietotemporal cortex, basal ganglia, and amygdala in DAT compared with controls ( p < 0.05 to 0.01). Our findings are in line with the assumption that reactive oxygen species could contribute to the pathogenesis of DAT. Absence of these changes in basal nucleus of Meynert might reflect retrograde degeneration of cholinergic fibers.     

The Association between Regional Environmental Factors and Road Trauma Rates: A Geospatial Analysis of 10 Years of Road Traffic Crashes in British Columbia,Canada

BackgroundBritish Columbia, Canada is a geographically large jurisdiction with varied environmental and socio-cultural contexts. This cross-sectional study examined variation in motor vehicle crash rates across 100 police patrols to investigate the association of crashes with key explanatory factors.MethodsEleven crash outcomes (total crashes, injury crashes, fatal crashes, speed related fatal crashes, total fatalities, single-vehicle night-time crashes, rear-end collisions, and collisions involving heavy vehicles, pedestrians, cyclists, or motorcyclists) were identified from police collision reports and insurance claims and mapped to police patrols. Six potential explanatory factors (intensity of traffic law enforcement, speed limits, climate, remoteness, socio-economic factors, and alcohol consumption) were also mapped to police patrols. We then studied the association between crashes and explanatory factors using negative binomial models with crash count per patrol as the response variable and explanatory factors as covariates.ResultsBetween 2003 and 2012 there were 1,434,239 insurance claim collisions, 386,326 police reported crashes, and 3,404 fatal crashes. Across police patrols, there was marked variation in per capita crash rate and in potential explanatory factors. Several factors were associated with crash rates. Percent roads with speed limits ≤ 60 km/hr was positively associated with total crashes, injury crashes, rear end collisions, and collisions involving pedestrians, cyclists, and heavy vehicles; and negatively associated with single vehicle night-time crashes, fatal crashes, fatal speeding crashes, and total fatalities. Higher winter temperature was associated with lower rates of overall collisions, single vehicle night-time collisions, collisions involving heavy vehicles, and total fatalities. Lower socio-economic status was associated with higher rates of injury collisions, pedestrian collisions, fatal speeding collisions, and fatal collisions. Regions with dedicated traffic officers had fewer fatal crashes and fewer fatal speed related crashes but more rear end crashes and more crashes involving cyclists or pedestrians. The number of traffic citations per 1000 drivers was positively associated with total crashes, fatal crashes, total fatalities, fatal speeding crashes, injury crashes, single vehicle night-time crashes, and heavy vehicle crashes. Possible explanations for these associations are discussed.ConclusionsThere is wide variation in per capita rates of motor vehicle crashes across BC police patrols. Some variation is explained by factors such as climate, road type, remoteness, socioeconomic variables, and enforcement intensity. The ability of explanatory factors to predict crash rates would be improved if considered with local traffic volume by all travel modes.     

Locating the rate-determining step(s) for three-step hydrolase-catalyzed reactions with DYNAFIT

Hydrolytic reactions of oligopeptide 4-nitroanilides catalyzed by human-alpha-thrombin, human activated protein C and human factor Xa were studied at pH 8.0-8.4 and 25.0+/-0.1 degrees C by the progress curve method and individual rate constants were calculated mostly within 10% internal error using DYNAFITV. A systematic strategy has been developed for fitting a three-step consecutive mechanism to eighteen hundred to six thousand time-course data points polled from two to four independent kinetic experiments. Enzyme and substrate concentrations were also calculated. Individual rate constants well reproduce published values obtained under comparable conditions and the Michaelis-Menten kinetic parameters calculated from these elementary rate constants are also within reasonable limits of published values. For comparison, the integrated Michaelis-Menten equation was also fitted to data from twelve sets. Both the k(cat) and k(cat)/K(m) values are within 15% agreement with those calculated using the elementary rate constants obtained with DYNAFITV. Rate constants for the second and third consecutive steps are within 3-4 fold indicating that both determine the overall rate. The Factor Xa-catalyzed hydrolysis of N-alpha-Z-D-Arg-Gly-Arg-pNA.2HCl at pH 8.4 in a series of buffers containing increasing fractions of deuterium at 25.0+/-0.1 degrees C shows a very strong dependence of k(3) and a moderate dependence of k(2) on D content in the buffer: the fractionation factors are: 0.49+/-0.03 for K(1,) 0.70+/-0.05 for k(2), and (0.32+/-0.03)(2) for k(3).     

Construction of a Global Pain Systems Network Highlights Phospholipid Signaling as a Regulator of Heat Nociception

The ability to perceive noxious stimuli is critical for an animal''s survival in the face of environmental danger, and thus pain perception is likely to be under stringent evolutionary pressure. Using a neuronal-specific RNAi knock-down strategy in adult Drosophila, we recently completed a genome-wide functional annotation of heat nociception that allowed us to identify α2δ3 as a novel pain gene. Here we report construction of an evolutionary-conserved, system-level, global molecular pain network map. Our systems map is markedly enriched for multiple genes associated with human pain and predicts a plethora of novel candidate pain pathways. One central node of this pain network is phospholipid signaling, which has been implicated before in pain processing. To further investigate the role of phospholipid signaling in mammalian heat pain perception, we analysed the phenotype of PIP5Kα and PI3Kγ mutant mice. Intriguingly, both of these mice exhibit pronounced hypersensitivity to noxious heat and capsaicin-induced pain, which directly mapped through PI3Kγ kinase-dead knock-in mice to PI3Kγ lipid kinase activity. Using single primary sensory neuron recording, PI3Kγ function was mechanistically linked to a negative regulation of TRPV1 channel transduction. Our data provide a systems map for heat nociception and reinforces the extraordinary conservation of molecular mechanisms of nociception across different species.     

ELECTROPHORETIC STUDY OF 5-HYDROXYTRYPTOPHAN DECARBOXYLASE FROM BRAIN AND LIVER IN SEVERAL SPECIES

—An electrophoretic investigation in acrylamide gels of 5-hydroxytryptophan decarboxylase, obtained mostly from mouse, rat, and beef brain and also from beef and human liver, showed electrophoretic differences between species. With the exception of the rat, only one molecular species was found (the same in beef brain and liver). In the rat, polymers form spontaneously and are, at least in part, disaggregated by urea and by triton. Mouse-rat or beef-rat molecular hybrids form in the admixtures. No electrophoretic differences were found in five mice strains that were investigated. Techniques of electrophoretic analysis and of assay of 5-hydroxytryptophan decarboxylase are described, which can be easily applied to other enzymes, provided a substrate is available in radioactive form.     

Spacer length-dependent protection of specific activity of pollen and/or embryo promoters from influence of CaMV 35S promoter/enhancer in transgenic plants

The influence of the CaMV 35S promoter/enhancer on expression profiles of four Arabidopsis thaliana pollen- and/or embryo-specific promoters, APRS, ESL, MXL, and DLL, was tested in transgenic tobacco plants. Individual promoters were fused to the gus reporter gene and cloned in head-to-head orientation with the CaMV 35S:hpt expression unit within the same T-DNA. With the exception of the TATA-less promoter DLL, all other combinations generated interactions between the promoter under investigation and 35S promoter/enhancer resulting in ectopic β-glucuronidase (GUS) expression in vegetative organs and tissues, the most susceptible being the stem, followed by callus, leaf, and root. To eliminate this crosstalk, DNA spacers of length 1, 2 and 5 kb were cloned between the interacting sequences. Ectopic GUS staining was dependent on the affected promoter as well as the distance between the 5′-end of the CaMV 35S promoter and the reporter gene translation start site. When this distance was less than 1 kb strong ectopic GUS staining was observed in all vegetative tissues, similar to the CaMV35S:gus expression profile in transgenic tobacco plants. Insertion of spacer DNA sequences of increasing length resulted in gradual reduction of ectopic GUS staining in tested plants. Of the tissues and organs related to plant reproduction, only anthers and seed coats in the early stages of seed development showed ectopic GUS staining. Developing pollen and embryos showed a pattern of GUS activity consistent with the predicted role of a developmental stage-specific promoter in transgenic tobacco plants.     

Phenotypic and Gene Expression Changes among Clonal Type I Strains of Toxoplasma gondii

Toxoplasma gondii has an unusual population structure consisting of three clonal lineages that predominate in North America and Europe. This simple pattern has encouraged the use of only a few laboratory isolates that are representative of each lineage. Principle among these is the type I RH strain, originally isolated from a child with encephalitis some 70 years ago. Comparison of different passages of the RH strain that have been propagated differently over the intervening time period revealed that the commonly used clonal line called RH-ERP was not representative of natural isolates of the type I lineage. Notably, RH-ERP formed much larger plaques than other type 1 strains, including a separate, earlier derived isolate of the RH strain. The RH-ERP variant also showed enhanced extracellular survival, faster growth, and decreased differentiation compared to the prototype type I strain GT1. Comparison of gene expression differences in the RH-ERP line revealed that several ABC transporters were upregulated, which may provide a growth advantage in vitro. These findings illustrate that dramatic phenotypic changes can arise in laboratory strains, emphasizing the need for comparison with recent clinical isolates.Toxoplasma gondii is a widespread protozoan parasite in the phylum Apicomplexa, an ancient group of protozoan parasites containing more than 5,000 species (27). T. gondii infects a wide range of warm-blooded animals and, while not part of the natural transmission, human infections can lead to severe disease in immunocompromised individuals (19). Using a unique form of actin-based motility, T. gondii penetrates host cells, where it rapidly multiplies within a nonfusigenic vacuole and eventually causes lysis of the host cell (44). During the acute phase of the infection, repeated cycles of cell invasion, replication, and lysis contribute to rapid spread and tissue damage (2, 28). In addition to this rapid lytic form of growth, asexual replication by slow-growing bradyzoites contributes to long-term chronic infection in a variety of hosts (52). In contrast, sexual development occurs only in the intestinal epithelial cells of cats (11). Despite this sexual phase in the life cycle, T. gondii maintains a highly clonal population structure that consists of three lineages type I, II and III, which predominate in North America and Europe (1, 17). These three clonal lineages are the result of recent expansion from either a genetic bottleneck or selective sweep that occurred within the last 10,000 years (48). Within each lineage, all strains are thought to be genetically identical except for random mutations that have occurred since their recent origin. Differences between the three clonal lineages of T. gondii are also limited, and they display only 1 to 2% divergence at the nucleotide level for most loci (6). Despite this similarity, the different clonal lineages differ dramatically in biological traits such as growth (37), migration (2), and pathogenesis in laboratory mice (28). The type I lineage has been shown to exclusively contain those strains of T. gondii that are acutely virulent in laboratory mice (43, 49). The lethal dose of type I strains in outbred mice is estimated to be a single organism based on limiting dilution studies, whereas types II and III have 50% lethal dose that are ∼4 logs higher than this. It has been suggested that the virulence of type I strains may be due to continuous laboratory passage (12). However, more than 20 natural isolates that share this clonal genotype and acute virulence phenotype have been described (43, 49), indicating that acute virulence is genetically encoded and not simply a consequence of passage history. Consistent with this, genetic mapping has recently revealed that the acute virulence of type I strains in the mouse model is controlled by the rhoptry protein ROP18, which encodes a secretory serine threonine kinase (50). ROP18 shows dramatic levels of polymorphism and, although highly expressed in the type I and II lineages, it is expressed at very low levels in the type III lineage owing to the presence of an ancestral upstream region that influences expression (23).The commonly used type I strain known as RH was originally isolated by Albert Sabin from a child who died with toxoplasmic encephalitis in 1939 (40). The RH strain was reported to be virulent on primary inoculation in laboratory mice (40). Since then, it has been serially passaged in mice by many laboratories. The RH strain was adapted for in vitro culture ca. 1977 by Elmer Pfefferkorn, who generated a clonal line that has also been widely distributed (36). This RH-ERP line was used to develop procedures for plaque formation on monolayers of host cells, providing a very useful in vitro assay that captures a combination of invasion, replication, egress, and spread. The ability of T. gondii to form such plaques has been used to generate temperature sensitive mutants (36), define the molecular basis of various chemically derived mutants (33-35), test drug sensitivity of parasite strains (29, 30), develop genetic transformation (45), and to test the phenotype of gene knockouts (20).In the 70 plus years since its isolation, the RH strain has been widely distributed and become the most commonly used laboratory strain. Previous genetic analysis has shown that, whereas type I strains are highly similar, isolates of RH obtained from different laboratories show minor genetic differences in some polymorphic DNA markers (18). This likely reflects minor genetic drift; however, the significance of these relatively small differences is unclear. To determine whether long-term in vitro passage may have affected growth, we compared a number of type I strains by using a plaque formation assay. Surprisingly, this comparison revealed that the clonal isolate RH-ERP differed substantially in plaque size from other type I strains. Additional differences were apparent in extracellular survival after egress, growth rate, differentiation, and changes in gene expression. Our findings indicate that the isolation of cloned lines can give rise to variants that are no longer representative of natural isolates, demonstrating the plasticity of phenotypic traits and limiting the utility of laboratory isolates.