Arabidopsis mutants sensitive to gamma radiation include the homologue of the human repair gene ERCC1

Mutants sensitive to ionizing radiation in yeast and mammals include an assortment of DNA repair genes. The majority of these DNA repair genes are involved in the repair of DNA double-strand breaks. In this study a forward genetic screen is used to identify gamma-sensitive mutants of Arabidopsis thaliana. The gamma-plantlet screen used here also reveals two general mutant classes based on size of cotyledons and hypocotyls. One of the mutants discovered is a homologue of the mammalian nucleotide excision repair gene ERCC1.     

Thermodynamic analysis of the nondenaturational conformational change of baker's yeast phosphoglycerate kinase at 24 degrees C

A plot of the Gibbs free energy of unfolding vs. temperature is calculated for baker’s yeast phosphoglycerate kinase in 150 mM sodium phosphate (pH = 7.0) from a combination of reversible differential scanning calorimetry measurements and isothermal guanidine hydrochloride titrations. The stability curve reveals the existence of two stable, folded conformers with an abrupt conformational transition occurring at 24 °C. The transition state thermodynamics for the low- to high-temperature conformational change are calculated from slow-scan-rate differential scanning calorimetry measurements where it is found that the free energy barrier for the conversion is 90 kJ/mol and the transition state possesses a significant unfolding quality. This analysis also confirms a nondenaturational conformational transition at 24 °C. The data therefore suggest that X-ray structures obtained from crystals grown below this temperature may differ considerably from the physiological structure and that the two conformers are not readily interconverted.     

Murine CMV-Induced Hearing Loss Is Associated with Inner Ear Inflammation and Loss of Spiral Ganglia Neurons

Congenital human cytomegalovirus (HCMV) occurs in 0.5–1% of live births and approximately 10% of infected infants develop hearing loss. The mechanism(s) of hearing loss remain unknown. We developed a murine model of CMV induced hearing loss in which murine cytomegalovirus (MCMV) infection of newborn mice leads to hematogenous spread of virus to the inner ear, induction of inflammatory responses, and hearing loss. Characteristics of the hearing loss described in infants with congenital HCMV infection were observed including, delayed onset, progressive hearing loss, and unilateral hearing loss in this model and, these characteristics were viral inoculum dependent. Viral antigens were present in the inner ear as were CD3+ mononuclear cells in the spiral ganglion and stria vascularis. Spiral ganglion neuron density was decreased after infection, thus providing a mechanism for hearing loss. The lack of significant inner ear histopathology and persistence of inflammation in cochlea of mice with hearing loss raised the possibility that inflammation was a major component of the mechanism(s) of hearing loss in MCMV infected mice.     

Risk of suicide,deliberate self‐harm and psychiatric illness after the loss of a close relative: A nationwide cohort study

The loss of a close relative is a common event, yet it is associated with increased risk of serious mental health conditions. No large‐scale study has explored up to now the importance of the bereaved person's relation to the deceased while accounting for gender and age. We performed a nationwide Danish cohort study using register information from 1995 through 2013 on four sub‐cohorts including all persons aged ≥18 years exposed to the loss of a child, spouse, sibling or parent. We identified 1,445,378 bereaved persons, and each was matched by gender, age and family composition to five non‐bereaved persons. Cumulative incidence proportions were calculated to estimate absolute differences in suicide, deliberate self‐harm and psychiatric illness. Cox proportional hazard regression was used to calculate hazard ratios while adjusting for potential confounders. Results revealed that the risk of suicide, deliberate self‐harm and psychiatric illness was increased in the bereaved cohorts for at least 10 years after the loss, particularly during the first year. During that year, the risk difference was 18.9 events in 1,000 persons after loss of a child (95% CI: 17.6‐20.1) and 16.0 events in 1,000 persons after loss of the spouse (95% CI: 15.4‐16.6). Hazard ratios were generally highest after loss of a child, in younger persons, and after sudden loss by suicide, homicide or accident. One in three persons with a previous psychiatric diagnosis experienced suicide, deliberate self‐harm or psychiatric illness within the first year of bereavement. In conclusion, this study shows that the risk of suicide, deliberate self‐harm and psychiatric illness is high after the loss of a close relative, especially in susceptible subgroups. This suggests the need for early identification of high‐risk persons displaying adjustment problems after loss of a close family member, in order to reduce the risk of serious mental health outcomes.     

The evolution of bacterial resistance against bacteriophages in the horse chestnut phyllosphere is general across both space and time

Insight to the spatial and temporal scales of coevolution is key to predicting the outcome of host–parasite interactions and spread of disease. For bacteria infecting long-lived hosts, selection to overcome host defences is just one factor shaping the course of evolution; populations will also be competing with other microbial species and will themselves be facing infection by bacteriophage viruses. Here, we examine the temporal and spatial patterns of bacterial adaptation against natural phage populations from within leaves of horse chestnut trees. Using a time-shift experiment with both sympatric and allopatric phages from either contemporary or earlier points in the season, we demonstrate that bacterial resistance is higher against phages from the past, regardless of spatial sympatry or how much earlier in the season phages were collected. Similarly, we show that future bacterial hosts are more resistant to both sympatric and allopatric phages than contemporary bacterial hosts. Together, our results suggest the evolution of relatively general bacterial resistance against phages in nature and are contrasting to previously observed patterns of phage adaptation to bacteria from the same tree hosts over the same time frame, indicating a potential asymmetry in coevolutionary dynamics.     

Site and sequence specificity of the daunomycin-DNA interaction

The site and sequence specificity of the daunomycin-DNA interaction was examined by equilibrium binding methods, by deoxyribonuclease I footprinting studies, and by examination of the effect of the antibiotic on the cleavage of linearized pBR322 DNA by restriction endonucleases PvuI and EcoRI. These three experimental approaches provide mutually consistent results showing that daunomycin indeed recognizes specific sites along the DNA lattice. The affinity of daunomycin toward natural DNA increases with increasing GC content. The quantitative results are most readily explained by binding models in which daunomycin interacts with sites containing two adjacent GC base pairs, possibly occurring as part of a triplet recognition sequence. Deoxyribonuclease I footprinting studies utilizing the 160 base pair (bp) tyrT DNA fragment and 61 and 53 bp restriction fragments isolated from pBR322 DNA further define the sequence specificity of daunomycin binding. Specific, reproducible protection patterns were obtained for each DNA fragment at 4 degrees C. Seven protected sequences, ranging in size from 4 to 14 bp, were identified within the tyrT fragment. Relative to the overall tyrT sequence, these protected sequences were GC rich and contained a more limited and distinct distribution of di- and trinucleotides. Within all of the protected sequences, a triplet containing adjacent GC base pairs flanked by an AT base pair could be found in one or more copies. Nowhere in the tyrT fragment did that triplet occur outside a protected sequence. The same triplet occurred within seven out of nine protected sequences observed in the fragments isolated from pBR322 DNA. In the two remaining cases, three contiguous GC base pairs were found. We conclude that the preferred daunomycin triplet binding site contains adjacent GC base pairs, of variable sequence, flanked by an AT base pair. This conclusion is consistent with the results of a recent theoretical study of daunomycin sequence specificity [Chen, K.-X., Gresh, N., & Pullman, B. (1985) J. Biomol. Struct. Dyn. 3, 445-466]. Adriamycin and the beta-anomer of adriamycin produce the same qualitative pattern of protection as daunomycin with the tyrT fragment. Daunomycin inhibits the rate of digestion of pBR322 DNA by PvuI (recognition sequence 5'-CGATCG-3') to a greater extent than it does EcoRI (recognition sequence 5'-GAATTC-3'), a finding consistent with the conclusions derived from our footprinting studies. Our results, as a whole, are the clearest indication to date that daunomycin recognizes a specific DNA sequence as a preferred binding site.     

Definition of the MinimalMEN1Candidate Area Based on a 5-Mb Integrated Map of Proximal 11q13: THE EUROPEAN CONSORTIUM ON MEN1

Multiple endocrine neoplasia type 1 (MEN1) is an autosomal dominant disorder with a high penetrance characterized by tumors of the parathyroid glands, the endocrine pancreas, and the anterior pituitary. TheMEN1gene, a putative tumor suppressor gene, has been mapped to a 3- to 8-cM region in chromosome 11q13 but it remains elusive as yet. We have combined the efforts and resources from four laboratories to form the European Consortium on MEN1 with the aims of establishing the genetic and the physical maps of 11q13 and of further narrowing the MEN1 region. A 5-Mb integrated map of the region was established by fluorescencein situhybridization on both metaphase chromosomes and DNA fibers, by hybridization to DNA from somatic cell hybrids containing various parts of human chromosome 11, by long-range restriction mapping, and by characterization of YACs and cosmids. Polymorphic markers were positioned and ordered by physical mapping and genetic linkage in 86 MEN1 families with 452 affected individuals. Two critical recombinants identified in two affected cases placed theMEN1gene in an ≈2-Mb region aroundPYGM,flanked by D11S1883 and D11S449.     

Arrhenius analysis of the electrophorus electricus acetylcholinesterase-catalyzed hydrolysis of acetylthiocholine

Ellman's method was used to determine the Michaelis-Menten parameters for the hydrolysis of acetylthiocholine by Electrophorus electricus acetylcholinesterase from 12 to 37 degrees C. Arrhenius analysis revealed that the activation energy for formation of the enzyme/substrate complex is 22.2 +/- 1.1 kJ/mole. The Arrhenius plot of k(cat) is markedly curved and attributed to comparable rates of acylation and deacylation due to the absence of evidence for a temperature-dependent enzyme conformational change by differential scanning calorimetry.     

Generation of human female reproductive tract epithelium from human embryonic stem cells

Background

Recent studies have identified stem/progenitor cells in human and mouse uterine epithelium, which are postulated to be responsible for tissue regeneration and proliferative disorders of human endometrium. These progenitor cells are thought to be derived from Müllerian duct (MD), the primordial female reproductive tract (FRT).

Methodology/Principal Findings

We have developed a model of human reproductive tract development in which inductive neonatal mouse uterine mesenchyme (nMUM) is recombined with green fluorescent protein (GFP)-tagged human embryonic stem cells (hESCs); GFP-hESC (ENVY). We demonstrate for the first time that hESCs can be differentiated into cells with a human FRT epithelial cell phenotype. hESC derived FRT epithelial cells emerged from cultures containing MIXL1⁺ mesendodermal precursors, paralleling events occurring during normal organogenesis. Following transplantation, nMUM treated embryoid bodies (EBs) generated epithelial structures with a typical MD phenotype that expressed the MD markers PAX2, HOXA10. Functionally, the hESCs derived FRT epithelium responded to exogenous estrogen by proliferating and secreting uterine-specific glycodelin A (GdA).

Conclusions/Significance

These data show nMUM can induce differentiation of hESC to form the FRT epithelium. This may provide a model to study early developmental events of the human FRT.     

Treatment of long-term anestrous sows with estradiol benzoate and GnRH: response of serum LH and occurrence of estrus

Seventeen primiparous sows, anestrous for 41 +/- 4 days after weaning, received i.m. injections of 500 mug estradiol benzoate (EB) or corn oil. At 48 hr after treatment, LH averaged 12.1 +/- 2.6 ng/ml in EB-treated sows and 0.7 +/- 0.1 ng/ml in corn oil-treated sows. At 55 hr after EB or corn oil, each sow was given 50 mug gonadotropin releasing hormone (GnRH). Average LH 1 hr after GnRH was 5.7 +/- 1.1 and 5.1 +/- 0.9 ng/ml in EB- and corn oil-treated sows, respectively. All EB-treated sows exhibited estrus 2.3 +/- 0.2 days after treatment and were mated. None of the corn oil-treated sows exhibited estrus and all were slaughtered two weeks after treatment. Examination of reproductive tracts revealed that the ovaries of corn oil-treated sows were small and did not contain corpora lutea. In mated sows, progesterone concentrations in blood two weeks after mating indicated luteal function in eight of the nine animals. Positive pregnancy diagnoses were made in all eight animals; however, only three sows farrowed, with litter sizes of four, five and seven, respectively. Results of the present experiment indicate that the hypothalamus and anterior pituitary of long-term anestrous sows are capable of responding to endocrine stimuli (i.e. estradiol and GnRH). Moreover, estradiol induced estrus and ovulation, but subsequent farrowing rate was only 33 percent and size of litters was small.