Amplified Rnase H Activity in Escherichia coli B/R Increases Sensitivity to Ultraviolet Radiation

Strains of E. coli B/r transformed with the plasmid pSK760 were found to be sensitized to inactivation by ultraviolet radiation (UV) and to have elevated levels of RNase H activity. Strains transformed with the carrier vector pBR322 or the plasmid pSK762C derived from pSK760 but with an inactivated rnh gene were not sensitized. UV-inactivation data for strains having known defects in DNA repair and transformed with pSK760 suggested an interference by RNase H of postreplication repair: uvrA cells were strongly sensitized, wild-type and uvrA recF cells were moderately sensitized and recA cells were not sensitized; and minimal medium recovery was no longer apparent in sensitized uvrA cells. Biochemical studies showed that post-UV DNA synthesis was sensitized and that the smaller amounts of DNA synthesized after irradiation, while of normal reduced size as indicated by sedimentation position in alkaline sucrose gradients, did not shift to a larger size (more rapidly sedimenting) upon additional incubation. We suggest an excess level of RNase H interferes with reinitiation of DNA synthesis on damaged templates to disturb the normal pattern of daughter strand gaps and thereby to inhibit postreplication repair.     

Genetic differentiation and reduced genetic diversity at the northern range edge of two species with different dispersal modes

Theory predicts that genetic variation should be reduced at range margins, but empirical support is equivocal. Here, we used genotyping‐by‐sequencing technology to investigate genetic variation in central and marginal populations of two species in the marine gastropod genus Crepidula. These two species have different development and dispersal types and might therefore show different spatial patterns of genetic variation. Both allelic richness and the proportion of private alleles were highest in the most central populations of both species, and lower at the margin. The species with low dispersal, Crepidula convexa, showed high degrees of structure throughout the range that conform to the pattern found in previous studies using other molecular markers. The northernmost populations of the high‐dispersing species, Crepidula fornicata, are distinct from more central populations, although this species has been previously observed to have little genetic structure over much of its range. Although genetic diversity was significantly lower at the range margin, the absolute reduction in diversity observed with these genomewide markers was slight, and it is not yet known whether there are functional consequences for the marginal populations.     

The effects of mate removal on pregnancy success in prairie voles (Microtus ochrogaster) and meadow voles (Microtus pennsylvanicus).

The effects of removing the stud male have not been controlled in many studies relating pregnancy block to the presence of an unfamiliar male. We examined the effects of removing the male on pregnancy success in prairie voles and meadow voles, two species that differ in degree of paternal investment. Whereas prairie vole males provide extensive care to offspring and accelerate pup development, meadow vole males display little or no care and delay development of pups. We predicted that removal of the stud male would decrease pregnancy success in prairie voles and either have no effect or increase success in meadow voles. In experiment 1, females were in male-induced estrus, and their mates were either left with them or were removed 4 h, 1 day, 2 days, or 8 days after mating. In experiment 2, females were in postpartum estrus, and their mates were either left with them or were removed 1 day, 2 days, or 8 days after birth of their first litter. Removal of the male soon after mating in postpartum estrus decreased pregnancy success in prairie voles and increased success in meadow voles. Thus, although removal of the stud male influenced litter production, the direction of the effect varied with species.     

Functional significance of active site residues in the enzymatic component of the Clostridium difficile binary toxin

Clostridium difficile binary toxin (CDT) is an ADP-ribosyltransferase which is linked to enhanced pathogenesis of C. difficile strains. CDT has dual function: domain a (CDTa) catalyses the ADP-ribosylation of actin (enzymatic component), whereas domain b (CDTb) transports CDTa into the cytosol (transport component). Understanding the molecular mechanism of CDT is necessary to assess its role in C. difficile infection. Identifying amino acids that are essential to CDTa function may aid drug inhibitor design to control the severity of C. difficile infections. Here we report mutations of key catalytic residues within CDTa and their effect on CDT cytotoxicity. Rather than an all-or-nothing response, activity of CDTa mutants vary with the type of amino acid substitution; S345A retains cytotoxicity whereas S345Y was sufficient to render CDT non-cytotoxic. Thus CDTa cytotoxicity levels are directly linked to ADP-ribosyltransferase activity.     

The Effects of Sub-Regional Climate Velocity on the Distribution and Spatial Extent of Marine Species Assemblages

Many studies illustrate variable patterns in individual species distribution shifts in response to changing temperature. However, an assemblage, a group of species that shares a common environmental niche, will likely exhibit similar responses to climate changes, and these community-level responses may have significant implications for ecosystem function. Therefore, we examine the relationship between observed shifts of species in assemblages and regional climate velocity (i.e., the rate and direction of change of temperature isotherms). The assemblages are defined in two sub-regions of the U.S. Northeast Shelf that have heterogeneous oceanography and bathymetry using four decades of bottom trawl survey data and we explore temporal changes in distribution, spatial range extent, thermal habitat area, and biomass, within assemblages. These sub-regional analyses allow the dissection of the relative roles of regional climate velocity and local physiography in shaping observed distribution shifts. We find that assemblages of species associated with shallower, warmer waters tend to shift west-southwest and to shallower waters over time, possibly towards cooler temperatures in the semi-enclosed Gulf of Maine, while species assemblages associated with relatively cooler and deeper waters shift deeper, but with little latitudinal change. Conversely, species assemblages associated with warmer and shallower water on the broad, shallow continental shelf from the Mid-Atlantic Bight to Georges Bank shift strongly northeast along latitudinal gradients with little change in depth. Shifts in depth among the southern species associated with deeper and cooler waters are more variable, although predominantly shifts are toward deeper waters. In addition, spatial expansion and contraction of species assemblages in each region corresponds to the area of suitable thermal habitat, but is inversely related to assemblage biomass. This suggests that assemblage distribution shifts in conjunction with expansion or contraction of thermal habitat acts to compress or stretch marine species assemblages, which may respectively amplify or dilute species interactions to an extent that is rarely considered. Overall, regional differences in climate change effects on the movement and extent of species assemblages hold important implications for management, mitigation, and adaptation on the U.S. Northeast Shelf.     

Structure, attachment, replacement and growth of teeth in bluefish, Pomatomus saltatrix (Linnaeus, 1776), a teleost with deeply socketed teeth

Tooth replacement poses many questions about development, pattern formation, tooth attachment mechanisms, functional morphology and the evolution of vertebrate dentitions. Although most vertebrate species have polyphyodont dentitions, detailed knowledge of tooth structure and replacement is poor for most groups, particularly actinopterygians. We examined the oral dentition of the bluefish, Pomatomus saltatrix, a pelagic and coastal marine predator, using a sample of 50 individuals. The oral teeth are located on the dentary and premaxillary bones, and we scored each tooth locus in the dentary and premaxillary bones using a four-part functional classification: absent (A), incoming (I), functional (F=fully ankylosed) or eroding (E). The homodont oral teeth of Pomatomus are sharp, deeply socketed and firmly ankylosed to the bone of attachment. Replacement is intraosseus and occurs in alternate tooth loci with long waves of replacement passing from rear to front. The much higher percentage of functional as opposed to eroding teeth suggests that replacement rates are low but that individual teeth are quickly lost once erosion begins. Tooth number increases ontogenetically, ranging from 15–31 dentary teeth and 15–39 premaxillary teeth in the sample studied. Teeth increase in size with every replacement cycle. Remodeling of the attachment bone occurs continuously to accommodate growth. New tooth germs originate from a discontinuous dental lamina and migrate from the lingual (dentary) or labial (premaxillary) epithelium through pores in the bone of attachment into the resorption spaces beneath the existing teeth. Pomatomus shares unique aspects of tooth replacement with barracudas and other scombroids and this supports the interpretation that Pomatomus is more closely related to scombroids than to carangoids.     

Preclinical Assessment of Adjunctive tPA and DNase for Peritoneal Dialysis Associated Peritonitis

A major complication of peritoneal dialysis is the development of peritonitis, which is associated with reduced technique and patient survival. The inflammatory response elicited by infection results in a fibrin and debris-rich environment within the peritoneal cavity, which may reduce the effectiveness of antimicrobial agents and predispose to recurrence or relapse of infection. Strategies to enhance responses to antimicrobial agents therefore have the potential to improve patient outcomes. This study presents pre-clinical data describing the compatibility of tPA and DNase in combination with antimicrobial agents used for the treatment of PD peritonitis. tPA and DNase were stable in standard dialysate solution and in the presence of antimicrobial agents, and were safe when given intraperitoneally in a mouse model with no evidence of local or systemic toxicity. Adjunctive tPA and DNase may have a role in the management of patients presenting with PD peritonitis.     

Properties of r Mutants of Bacteriophage T4 Photodynamically Induced in the Presence of Thiopyronin and Psoralen

About 4 x 10(-4)r mutants were induced per lethal hit, a frequency characteristic of weak mutagens. Collections of mutants produced in the presence of either dye were indistinguishable in most of their properties. The rII mutants differed sharply from spontaneous mutants in their mutational spectra (fine scale map distribution) and their reversion responses to specific mutagens. Few or none of the induced mutants were induced to revert with proflavine (sign mutants; reading frame shift mutants). A majority were induced to revert with base analogues (base pair substitution mutants), and about half of these also responded to the hydroxymethylcytosine-specific agent hydroxylamine. A large minority of the mutants reverted spontaneously but failed to respond either to proflavine or to base analogues. We believe these mutants, as well as some of the mutants which did respond to base analogues, to be transversions (base pair substitutions which reverse the purine-pyrimidine orientation).     

Subcutaneous implantable infusion device for chronic intravenous pulsatile gonadotropin-releasing hormone treatment

Preliminary data indicate the potential utility of an implantable subcutaneous device that facilitates chronic intravenous infusion of pulsatile gonadotropin-releasing hormone (GnRH) for ovulation induction. GnRH distribution curves were congruent in control monkeys and those with implanted devices. Tissue tolerance was good in this brief trial. These findings suggest that use of this or a similar implantable device be considered for chronic GnRH administration in human pulse therapy.     

Automated Assay of Telomere Length Measurement and Informatics for 100,000 Subjects in the Genetic Epidemiology Research on Adult Health and Aging (GERA) Cohort

The Kaiser Permanente Research Program on Genes, Environment, and Health (RPGEH) Genetic Epidemiology Research on Adult Health and Aging (GERA) cohort includes DNA specimens extracted from saliva samples of 110,266 individuals. Because of its relationship to aging, telomere length measurement was considered an important biomarker to develop on these subjects. To assay relative telomere length (TL) on this large cohort over a short time period, we created a novel high throughput robotic system for TL analysis and informatics. Samples were run in triplicate, along with control samples, in a randomized design. As part of quality control, we determined the within-sample variability and employed thresholds for the elimination of outlying measurements. Of 106,902 samples assayed, 105,539 (98.7%) passed all quality control (QC) measures. As expected, TL in general showed a decline with age and a sex difference. While telomeres showed a negative correlation with age up to 75 years, in those older than 75 years, age positively correlated with longer telomeres, indicative of an association of longer telomeres with more years of survival in those older than 75. Furthermore, while females in general had longer telomeres than males, this difference was significant only for those older than age 50. An additional novel finding was that the variance of TL between individuals increased with age. This study establishes reliable assay and analysis methodologies for measurement of TL in large, population-based human studies. The GERA cohort represents the largest currently available such resource, linked to comprehensive electronic health and genotype data for analysis.