Genes required for ionizing radiation resistance in yeast.

The ability of Saccharomyces cerevisiae to tolerate ionizing radiation damage requires many DNA-repair and checkpoint genes, most having human orthologs. A genome-wide screen of diploid mutants homozygous with respect to deletions of 3,670 nonessential genes revealed 107 new loci that influence gamma-ray sensitivity. Many affect replication, recombination and checkpoint functions. Nearly 90% were sensitive to other agents, and most new genes could be assigned to the following functional groups: chromatin remodeling, chromosome segregation, nuclear pore formation, transcription, Golgi/vacuolar activities, ubiquitin-mediated protein degradation, cytokinesis, mitochondrial activity and cell wall maintenance. Over 50% share homology with human genes, including 17 implicated in cancer, indicating that a large set of newly identified human genes may have related roles in the toleration of radiation damage.

Adrenomedullin interacts with VEGF in endometrial cancer and has varied modulation in tumours of different grades.

OBJECTIVE: Endometrial cancer, in developed countries, is the most common malignancy of the female genital tract. Surgery and radiotherapy are successful in many patients but systemic and recurrent diseases have no consistently effective treatments, and for high grade advanced disease the prognosis is poor. The study investigated characteristics of adrenomedullin in endometrial cancer to assist in identifying targets for developing treatments. METHODS: Endometrial samples of women with and without cancer, and the Ishikawa cell line were used to investigate adrenomedullin mRNA regulation, peptide expression, adrenomedullin secretion and effects of adrenomedullin on VEGF secretion. RESULTS: Expression of adrenomedullin mRNA was upregulated compared to that in healthy post-menopausal endometria. Adrenomedullin secretion was increased by cobalt chloride in this study. Secretion was reduced by the naturally-occurring compounds, (-)-epigallocatechin gallate (EGCG) and 3,4',5-trihydroxystilbene (resveratrol), which we have previously demonstrated to also suppress VEGF secretion in endometrial tumour tissue. We noted, for the first time, that adrenomedullin enhanced VEGF secretion from tumour cells. CONCLUSIONS: Increased adrenomedullin expression may result in amplifying both tumorigenic and angiogenic activities. A substantial impact on growth of tumours may result in vivo as a consequence of the synergism between adrenomedullin and VEGF. Adrenomedullin, which has altered cellular characteristics in tumour compared to healthy tissue, offers an understudied target with potential to modify endometrial cancer behaviour, complementing other treatments.

Short communication: Manipulation of neonatal leptin profile via exogenous glucocorticoids in beef calves.

The link between circulating glucocorticoids and leptin in beef calves has not been explored but has been noted in several studies. The aim of this study is to determine the effects of exogenous glucocorticoids given at birth and 1 day of age on serum leptin concentrations in beef calves. Ruminant animals secrete leptin, which is thought to be important for the programming of the hypothalamic appetite centers. Angus crossbred cows (n = 31) bred via natural service were utilized for this experiment. At parturition (day 0), calf BW was recorded and each calf was infused intravenously with either a hydrocortisol sodium succinate solution (HC, 8 males and 8 females) at a dosage of 3.5 µg/kg of BW or a similar volume of saline solution (CONT, 7 males and 8 females). Each calf was given a second infusion of its respective treatment 24 h postpartum at 1.5 µg/kg of BW for HC treatment. Calf treatment was blocked by sex, dam body condition score (BCS), and dam age. Blood samples were taken via jugular venipuncture before infusion, daily from days 0 to 5, then every other day up to day 17. Serum leptin and cortisol concentrations were analyzed via radioimmunoassay. Dam age, dam BCS, calf BW, and serum leptin and cortisol concentrations were analyzed using MIXED procedure of SAS. Dam age was not different (P = 0.81) among HC and CONT calves (4.9±0.5 and 4.7±0.5, respectively). Dam BCS was not different between treatments (5.7±0.2 and 5.6±0.2 HC and CONT, respectively; P = 0.66). There was no difference in calf birth BW between treatments (P = 0.87) and averaged 38.3±1.4 kg. Cortisol concentrations were not different between both treatments (P = 0.23) from birth to day 4 of age. Calves that received the HC treatment showed significantly reduced (P = 0.03) leptin concentrations on days 1 to 13. Calf BW from 60 to 150 days of age was not different between CONT and HC treated calves (P = 0.65). These data indicate that exogenous glucocorticoids can be used to suppress neonatal leptin levels in calves. This could lead to changes in voluntary feed intake of treated calves.

Relationships between older adults' use of time and cardio-respiratory fitness, obesity and cardio-metabolic risk: A compositional isotemporal substitution analysis.

OBJECTIVE: Older adults' health has been linked with time in moderate-to-vigorous physical activity (MVPA), and recent studies suggest time in sedentary behaviour may also be important. Time-use behaviours (MVPA, light physical activity, sedentary time and sleep) are co-dependent, and therefore their associations with health should be examined in an integrated manner. This is the first study to investigate the relationship between older adults' reallocation of time among these time-use behaviours and markers of cardio-respiratory fitness, obesity and cardio-metabolic risk. STUDY DESIGN: Cross-sectional study of 122 Australians (65 ±â€¯3 y, 61% female). MAIN OUTCOME MEASURES: Daily time use: average daily minutes spent in MVPA, light physical activity, sedentary time and sleep derived from 24-h, 7-day accelerometry, were conceptualised as a time-use composition. Cardio-respiratory fitness: graded submaximal cycle ergometer test. Obesity: objectively measured body mass index (BMI) and waist-to-hip ratio (WHR). Cardio-metabolic risk: sphygmomanometer-measured resting blood pressure and fingertip blood sampling for fasting total cholesterol and glucose. RESULTS: Time-use composition was significantly associated with obesity markers (BMI, p = 0.001; WHR, p < 0.001). The reallocation of 15 min to MVPA from any of the other behaviours was associated with approximately +1.1 (95% confidence interval 0.2; 1.9) ml/kg-1 min-1 VO2max, -0.7 (-1.0; -0.3) BMI units and -1.2 (-1.8; -0.7) WHR percentage points, while the opposite reallocation (15 min from MVPA to other behaviours) was associated with larger difference estimates of -1.8 (-3.2; -0.4) ml/kg-1 min-1 VO2max, +1.2 (0.5; 1.9) BMI units and +2.1 (1.2; 3.1) WHR percentage points. CONCLUSION: These findings reinforce the importance of MVPA for health among older adults. Interventions to maintain MVPA, even without increasing it, may be valuable.

The adiposity of children is associated with their lifestyle behaviours: a cluster analysis of school-aged children from 12 nations.

BACKGROUND: The relationship between children's adiposity and lifestyle behaviour patterns is an area of growing interest. OBJECTIVES: The objectives of this study are to identify clusters of children based on lifestyle behaviours and compare children's adiposity among clusters. METHODS: Cross-sectional data from the International Study of Childhood Obesity, Lifestyle and the Environment were used. PARTICIPANTS: the participants were children (9-11 years) from 12 nations (n = 5710). MEASURES: 24-h accelerometry and self-reported diet and screen time were clustering input variables. Objectively measured adiposity indicators were waist-to-height ratio, percent body fat and body mass index z-scores. ANALYSIS: sex-stratified analyses were performed on the global sample and repeated on a site-wise basis. Cluster analysis (using isometric log ratios for compositional data) was used to identify common lifestyle behaviour patterns. Site representation and adiposity were compared across clusters using linear models. RESULTS: Four clusters emerged: (1) Junk Food Screenies, (2) Actives, (3) Sitters and (4) All-Rounders. Countries were represented differently among clusters. Chinese children were over-represented in Sitters and Colombian children in Actives. Adiposity varied across clusters, being highest in Sitters and lowest in Actives. CONCLUSIONS: Children from different sites clustered into groups of similar lifestyle behaviours. Cluster membership was linked with differing adiposity. Findings support the implementation of activity interventions in all countries, targeting both physical activity and sedentary time.

Requirement for end-joining and checkpoint functions, but not RAD52-mediated recombination, after EcoRI endonuclease cleavage of Saccharomyces cerevisiae DNA.

RAD52 and RAD9 are required for the repair of double-strand breaks (DSBs) induced by physical and chemical DNA-damaging agents in Saccharomyces cerevisiae. Analysis of EcoRI endonuclease expression in vivo revealed that, in contrast to DSBs containing damaged or modified termini, chromosomal DSBs retaining complementary ends could be repaired in rad52 mutants and in G1-phase Rad+ cells. Continuous EcoRI-induced scission of chromosomal DNA blocked the growth of rad52 mutants, with most cells arrested in G2 phase. Surprisingly, rad52 mutants were not more sensitive to EcoRI-induced cell killing than wild-type strains. In contrast, endonuclease expression was lethal in cells deficient in Ku-mediated end joining. Checkpoint-defective rad9 mutants did not arrest cell cycling and lost viability rapidly when EcoRI was expressed. Synthesis of the endonuclease produced extensive breakage of nuclear DNA and stimulated interchromosomal recombination. These results and those of additional experiments indicate that cohesive ended DSBs in chromosomal DNA can be accurately repaired by RAD52-mediated recombination and by recombination-independent complementary end joining in yeast cells.

In vivo site-directed mutagenesis using oligonucleotides.

Functional characterization of the genes of higher eukaryotes has been aided by their expression in model organisms and by analyzing site-specific changes in homologous genes in model systems such as the yeast Saccharomyces cerevisiae. Modifying sequences in yeast or other organisms such that no heterologous material is retained requires in vitro mutagenesis together with subcloning. PCR-based procedures that do not involve cloning are inefficient or require multistep reactions that increase the risk of additional mutations. An alternative approach, demonstrated in yeast, relies on transformation with an oligonucleotide, but the method is restricted to the generation of mutants with a selectable phenotype. Oligonucleotides, when combined with gap repair, have also been used to modify plasmids in yeast; however, this approach is limited by restriction-site availability. We have developed a mutagenesis approach in yeast based on transformation by unpurified oligonucleotides that allows the rapid creation of site-specific DNA mutations in vivo. A two-step, cloning-free process, referred to as delitto perfetto, generates products having only the desired mutation, such as a single or multiple base change, an insertion, a small or a large deletion, or even random mutations. The system provides for multiple rounds of mutation in a window up to 200 base pairs. The process is RAD52 dependent, is not constrained by the distribution of naturally occurring restriction sites, and requires minimal DNA sequencing. Because yeast is commonly used for random and selective cloning of genomic DNA from higher eukaryotes such as yeast artificial chromosomes, the delitto perfetto strategy also provides an efficient way to create precise changes in mammalian or other DNA sequences.

Small Steps: Preliminary effectiveness and feasibility of an incremental goal-setting intervention to reduce sitting time in older adults.

OBJECTIVE: This study aimed to evaluate the preliminary effectiveness and feasibility of a theory-informed program to reduce sitting time in older adults. DESIGN: Pre-experimental (pre-post) study. Thirty non-working adult (≥ 60 years) participants attended a one hour face-to-face intervention session and were guided through: a review of their sitting time; normative feedback on sitting time; and setting goals to reduce total sitting time and bouts of prolonged sitting. Participants chose six goals and integrated one per week incrementally for six weeks. Participants received weekly phone calls. OUTCOME MEASURES: Sitting time and bouts of prolonged sitting (≥ 30 min) were measured objectively for seven days (activPAL3c inclinometer) pre- and post-intervention. During these periods, a 24-h time recall instrument was administered by computer-assisted telephone interview. Participants completed a post-intervention project evaluation questionnaire. Paired t tests with sequential Bonferroni corrections and Cohen's d effect sizes were calculated for all outcomes. RESULTS: Twenty-seven participants completed the assessments (71.7 ± 6.5 years). Post-intervention, objectively-measured total sitting time was significantly reduced by 51.5 min per day (p=0.006; d=-0.58) and number of bouts of prolonged sitting by 0.8 per day (p=0.002; d=-0.70). Objectively-measured standing increased by 39 min per day (p=0.006; d=0.58). Participants self-reported spending 96 min less per day sitting (p<0.001; d=-0.77) and 32 min less per day watching television (p=0.005; d=-0.59). Participants were highly satisfied with the program. CONCLUSION: The 'Small Steps' program is a feasible and promising avenue for behavioral modification to reduce sitting time in older adults.

Identification of high affinity binding sites for LexA which define new DNA damage-inducible genes in Escherichia coli.

A multi-step screening procedure was devised to identify new operators for the LexA repressor in the sequenced portions of the genomes of Escherichia coli and its plasmids and bacteriophages. Sequence analysis methods were employed initially to distinguish true LexA operators from "operator-like" sequences stored within the GenBank and EMBL databases. The affinity of purified LexA protein for cloned DNA fragments containing several of the prospective new sites was then assessed using quantitative electrophoretic mobility shift assays and site-directed mutagenesis. Calculated binding affinities were compared directly with values determined for known and mutant LexA operators in concurrent experiments. Three E. coli chromosomal segments (near pyrC, hsdS and ntrla) and two bacteriophage sequences (near the P1 cre and lambda oop genes) bound LexA protein specifically. These sites and most others identified in the screening are located immediately upstream of known genes and/or large open reading frames. These results and additional transcription data demonstrate that several of the sequences define new DNA damage-inducible (din) genes and include the previously uncharacterized dinD locus. Furthermore, the search identified an SOS gene within the genome of P1 which encodes a protein that is homologous to UmuD', the RecA-promoted cleavage product of the umuD gene. The success of the combinatorial approach described here suggests that analogous searches for new regulatory sequences within the E. coli genome and the genomes of other organisms will also yield favorable results.

Repair of endonuclease-induced double-strand breaks in Saccharomyces cerevisiae: essential role for genes associated with nonhomologous end-joining.

Repair of double-strand breaks (DSBs) in chromosomal DNA by nonhomologous end-joining (NHEJ) is not well characterized in the yeast Saccharomyces cerevisiae. Here we demonstrate that several genes associated with NHEJ perform essential functions in the repair of endonuclease-induced DSBs in vivo. Galactose-induced expression of EcoRI endonuclease in rad50, mre11, or xrs2 mutants, which are deficient in plasmid DSB end-joining and some forms of recombination, resulted in G2 arrest and rapid cell killing. Endonuclease synthesis also produced moderate cell killing in sir4 strains. In contrast, EcoRI caused prolonged cell-cycle arrest of recombination-defective rad51, rad52, rad54, rad55, and rad57 mutants, but cells remained viable. Cell-cycle progression was inhibited in excision repair-defective rad1 mutants, but not in rad2 cells, indicating a role for Rad1 processing of the DSB ends. Phenotypic responses of additional mutants, including exo1, srs2, rad5, and rdh54 strains, suggest roles in recombinational repair, but not in NHEJ. Interestingly, the rapid cell killing in haploid rad50 and mre11 strains was largely eliminated in diploids, suggesting that the cohesive-ended DSBs could be efficiently repaired by homologous recombination throughout the cell cycle in the diploid mutants. These results demonstrate essential but separable roles for NHEJ pathway genes in the repair of chromosomal DSBs that are structurally similar to those occurring during cellular development.

Ouabain is not detectable in human plasma.

An enzyme-linked immunosorbent assay is described for the measurement of ouabain in human plasma. This assay is specific for ouabain, strophanthidin, and ouabagenin, with other steroids, including digoxin and vasopressor hormones, exhibiting negligible cross-reactivity. Assay sensitivity was 0.06 nmol/L if 1 mL plasma was extracted and less than 0.005 nmol/L when 20 mL plasma was analyzed. Extracted plasma samples showed ouabainlike immunoreactivity that diluted in parallel with the ouabain standard curve. Repeated extraction and assay of single plasma samples, however, did not produce consistent results in the assay. Increased specificity was obtained by high-performance liquid chromatography of sample extracts before assay. When high-performance liquid chromatographic profiles of plasma spiked with ouabain standard or following bolus intravenous injections of ouabain into normal human volunteers were compared with profiles of unspiked plasma, there was no support for the immunoreactive material in the latter samples being ouabain. We propose that if ouabain is present in the human circulation, its concentration is less than 0.005 nmol/L.

Hemodynamic, hormonal, and renal effects of short-term adrenomedullin infusion in healthy volunteers.

The actions of adrenomedullin (ADM), a 52-amino acid peptide, are not well defined in man. We, therefore, studied eight normal volunteers aged 1832 yr in a placebo-controlled crossover study. On the 2 study days, subjects received, in random order, ADM in "low" and "high" dose (2.9 pmol/kg x min and 5.8 pmol/kg x min for 2 h each) or vehicle (hemaccel) infusion on day 4 of a metabolic diet (Na+ 80 mmol/day, K+ 100 mmol/day). Achieved plasma ADM levels were in the pathophysiological range, and plasma cAMP values rose 5 pmol/L during the higher dose. Compared with time-matched vehicle infusion, high-dose ADM increased peak heart rate by 10 beats per minute (P < 0.05) and lowered diastolic (by 5 mm Hg, P < 0.01) blood pressure. Cardiac output increased in both phases of ADM (low dose, 7.6 L/min; high dose, 10.2 L/min; vehicle, 6 L/min; P < 0.05 for both). Despite a 2-fold rise in PRA during high-dose ADM (P < 0.01), aldosterone levels were unaltered. Norepinephrine levels increased by 50% during high-dose ADM (P < 0.001), but epinephrine levels were unchanged. Plasma PRL levels increased during high-dose ADM (P = 0.014). ADM had no significant effect on urine volume and sodium excretion. Infusion of ADM to achieve pathophysiological plasma levels produced significant hemodynamic effects, stimulated renin but inhibited the aldosterone response to endogenous angiotensin II, and activated the sympathetic system and PRL without altering urine sodium excretion in normal subjects.

Bioactivity and interactions of adrenomedullin and brain natriuretic peptide in patients with heart failure.

Plasma concentrations of the recently discovered hormones adrenomedullin (ADM), from vascular tissue, and brain natriuretic peptide (BNP), secreted by myocardium, are elevated in patients with heart failure. We tested the hypotheses that short-term increments in circulating levels of these hormones, within the pathophysiological range, would have biological effects and that the 2 hormone systems interact. Eight patients with heart failure (left ventricular ejection fractions <35%) received 4-hour infusions of BNP (3.0 pmol. kg(-1). min(-1)) alone, ADM (2.7 pmol. kg(-1). min(-1) and 5.4 pmol. kg(-1). min(-1) for 2 hours each) alone, ADM and BNP combined, and placebo. BNP and ADM infusions raised plasma levels of the respective peptide within the pathophysiological range. Arterial blood pressure fell (P<0.05) with all peptide infusions, but cardiac output was unchanged. Heart rate increased with ADM and combined infusions (P<0.01). Sodium excretion rose (P<0.05), and creatinine clearance was sustained during both BNP and combined infusions. Urine volume increased in response to BNP alone (P=0.02). Despite a >2-fold increase in plasma renin with both ADM and combined infusions (P<0.05), plasma aldosterone remained lower than time-matched placebo levels. Plasma noradrenaline was increased by combined, BNP, and higher dose ADM infusions (P<0.05). ADM suppressed plasma cGMP (P<0.05) and inhibited the plasma cGMP response to BNP (P<0.05). The vascular hormones ADM and BNP, produced by myocardium, at plasma concentrations within the pathophysiological range have hemodynamic, renal, and hormonal effects and measurable interactions in patients with heart failure.

Effects of endothelin-1 on release of adrenomedullin and C-type natriuretic peptide from individual human vascular endothelial cells.

Regulation of cardiovascular system activity involves complex interactions amongst numerous factors. Three of these vasoactive factors are adrenomedullin, C-type natriuretic peptide (CNP) and endothelin-1 (ET-1), each of which is claimed to have important local effects. To investigate paracrine/autocrine regulation of the secretion of these peptides we used a cell immunoblot method. We postulated that basal release of adrenomedullin and CNP by endothelial cells is modulated by ET-1. Dispersed human aortic endothelial cells were attached to a protein binding membrane and incubated for 1 or 4 h with control medium or with ET-1, endothelin receptor antagonists or antibody to ET-1, and then submitted to immunohistochemical staining. Peptides (adrenomedullin, CNP and ET-1) within individual cells were stained, as was peptide secreted and adjacent to the cell. It was demonstrated that adrenomedullin, CNP and ET-1 can be contained within the same cell. In addition, we observed that individual endothelial cells can secrete all three peptides. The endothelin ET-A/ET-B receptor antagonist, bosentan, the ET-B receptor antagonist, BQ-788, and anti-ET-1 serum decreased the percentage of endothelial cells that secreted adrenomedullin and CNP relative to control. Conversely, the addition of ET-1 induced an increase in the number of endothelial cells that secreted adrenomedullin and CNP. These results provide strong evidence that endogenous ET-1, from human vascular endothelial cells, acts in a paracrine/autocrine manner to modulate the basal release of adrenomedullin and CNP. Our observations of this modulation suggest that vascular endothelial cells of humans constitute an important component of a self-responsive vasoregulatory system.

An enzyme linked immunosorbent assay (ELISA) for plasma medroxyprogesterone acetate (MPA).

A single extraction fixed antigen enzyme-linked immunosorbent assay (ELISA) that can be completed in less than 24 h is described for the measurement of medroxyprogesterone acetate (MPA) in plasma. MPA is covalently coupled to bovine thyroglobulin and passively adsorbed in guanidine hydrochloride to a standard 96-well microtitre plate where it competes with MPA in the extracted plasma sample for goat anti-MPA. Antibody binding to the solid phase is determined via binding of a horse-radish peroxidase second antibody which reacts colorimetrically with its substrate. The reaction is stopped by addition of 1.25 M H2SO4 and absorbance read at 492 nm. All steps except for sample addition and extraction can be performed on an automatic ELISA processing machine. The assay is sensitive, specific and precise, with intra- and inter-assay coefficients of variation of less than 10 and 15%, respectively. Assay sensitivity is 0.08 ng/ml. The assay follows established methodology for other assays in this laboratory which assists standardization, cost structure and sample throughput and thus is a useful alternative to radioimmunoassays for the determination of MPA in plasma.

Bioactivity of adrenomedullin and proadrenomedullin N-terminal 20 peptide in man.

Although the biological effects of adrenomedullin (AM) and PAMP have been reported extensively in animal studies and from in-vitro experiments, relatively little information is available on responses to the hormone administered to man. This review summarizes data from the few studies carried out in man. In healthy volunteers, i.v. infusion of AM reduces arterial pressure, probably at a lower rate of administration than is required to elicit other responses. AM stimulates heart rate, cardiac output, plasma levels of cAMP, prolactin, norepinephrine and renin whilst inhibiting any concomitant response in plasma aldosterone. Little or no increase in urine volume or sodium excretion has been observed. Patients with essential hypertension differ only in showing a greater fall in arterial pressure and in the development of facial flushing and headache. In patients with heart failure or chronic renal failure, i.v. AM has similar effects to those seen in normal subjects but also induces a diuresis and natriuresis, depending on the dose administered. Infusion of AM into the brachial artery results in a dose-related increase in forearm and skin blood flow, more prominent and more dependent on endogenous nitric oxide in healthy volunteers than in patients with cardiac failure. When infused into a dorsal hand vein, AM partially reversed the venoconstrictor action of norepinephrine. Although much more information is required to clarify the role of AM under physiological and pathophysiological circumstances, it is clear that it has prominent hemodynamic and neurohormonal effects, though generally lesser urinary effects when administered short-term in doses sufficient to raise its levels in plasma to those seen in a number of clinical disorders. The only study of PAMP in man showed that its skeletal muscle vasodilator potency, when infused into the brachial artery of healthy volunteers, was less than one hundredth that of AM, and it was without effect on skin blood flow.

Degradation of human adrenomedullin(1-52) by plasma membrane enzymes and identification of metabolites.

Very little is known about degradation or metabolism of adrenomedullin. To this end, we incubated adrenomedullin with ovine adrenal, kidney and lung plasma membrane preparations and showed the major degradation products were ADM(2-52) and ADM(8-52). Smaller amounts of ADM(26-52), (27-52), (28-52) and (33-52) were also produced. Degradation was inhibited by EDTA and 1,10 phenanthroline but not by phosphoramidon, leupeptin and pepstatin. The above data are consistent with initial hydrolysis adjacent to hydrophobic residues by a metalloprotease, generating ADM(8-52), (26-52) and (33-52), followed by an aminopeptidase action to produce ADM(2-52), (27-52) and (28-52). Improved understanding of the metabolism of ADM may have therapeutic implications, for example in the treatment of heart failure.

Regional distribution of estradiol receptors in growing antlers.

This study of estrogen receptors (ER) was carried out to confirm their presence and to determine their localisation in antler bones. Partially grown antlers were amputated from red deer (Cervus elaphus) stags, the skin removed, and samples taken of periosteum, cartilaginous tissue including perichondrium, and bone. Capacity and binding of free ER in the samples were calculated by Scatchard analysis of data obtained from a radioreceptor assay which utilised [3H]estradiol as tracer. High affinity ER (ka 1.3-3.4 x 10(10)/M) were detected in all tissues sampled with the exception of bone. Receptor capacity ranged from 12-74 fmol/mg protein, ranking the tissues for capacity in the following descending order: periosteum, cartilage, calcified cartilage. These results demonstrate the presence of ER in growing antlers and indicate regional localization of the receptors within these structures. The absence of ER in bone tissue within the antler suggests that the effect of estradiol on stimulation of mineralization in this tissue is indirect and must occur via its binding to the non-calcified tissues of antlers, e.g., periosteum, perichondrium, and cartilage.

Tying up loose ends: nonhomologous end-joining in Saccharomyces cerevisiae.

The ends of chromosomal DNA double-strand breaks (DSBs) can be accurately rejoined by at least two discrete pathways, homologous recombination and nonhomologous end-joining (NHEJ). The NHEJ pathway is essential for repair of specific classes of DSB termini in cells of the budding yeast Saccharomyces cerevisiae. Endonuclease-induced DSBs retaining complementary single-stranded DNA overhangs are repaired efficiently by end-joining. In contrast, damaged DSB ends (e.g., termini produced by ionizing radiation) are poor substrates for this pathway. NHEJ repair involves the functions of at least 10 genes, including YKU70, YKU80, DNL4, LIF1, SIR2, SIR3, SIR4, RAD50, MRE11, and XRS2. Most or all of these genes are required for efficient recombination-independent recircularization of linearized plasmids and for rejoining of EcoRI endonuclease-induced chromosomal DSBs in vivo. Several NHEJ mutants also display aberrant processing and rejoining of DSBs that are generated by HO endonuclease or formed spontaneously in dicentric plasmids. In addition, all NHEJ genes except DNL4 and LIF1 are required for stabilization of telomeric repeat sequences. Each of the proteins involved in NHEJ appears to bind, directly or through protein associations, with the ends of linear DNA. Enzymatic and/or structural roles in the rejoining of DSB termini have been postulated for several proteins within the group. Most yeast NHEJ genes have homologues in human cells and many biochemical activities and protein:protein interactions have been conserved in higher eucaryotes. Similarities and differences between NHEJ repair in yeast and mammalian cells are discussed.