Transformation of Brown Leghorn chicken embryo fibroblasts by avian myeloblastosis virus proviral DNA.

Brown Leghorn chicken embryo fibroblasts were transfected with a mixture of avian myeloblastosis virus (AMV) and myeloblastosis-associated virus type 1 (MAV1) proviral DNA purified from lambda-Charon 4A recombinant clones. A transformed cell line (T1AM) able to grow without anchorage in semisolid medium was obtained. The presence of both proviral AMV and MAV sequences was detected in T1AM DNA by hybridization with v-myb- and MAV1-specific probes. Altered AMV and MAV1 proviral genomes were found in T1AM genome. Characterization of the RNA species expressed in transformed cells showed that in addition to a 2.5-kilobase (kb) putative subgenomic v-myb-specific RNA, three other myb-containing RNAs (9.4, 8.4, and 7.0 kb) were present in T1AM cells. No AMV genomic RNA was detected. Also, a new 5.0-kb MAV1-specific RNA species was expressed in transformed cells in addition to MAV1 genomic RNA species (7.8 kb). No infectious AMV virions are released by T1AM cells. Chicken embryo fibroblasts infected by T1AM-released virions contained and expressed all MAV1 sequences detected in T1AM transformed cells but did not express any transformation parameter. These results indicated that the presence of AMV proviral sequences in T1AM cells is responsible for their transformed phenotype.     

Effects of psoralen on replicon size and mean rate of DNA synthesis in partially synchronized cells of Pisum sativum L.

We have examined by fibre autoradiography the spacing of replicons in pea root meristems during synchronized entry into S phase from arrest at the G1/S boundary. Pretreatment with the DNA cross-linking agent, psoralen, produces a marked shortening of replicon spacing, suggesting that premature arrest of the replication fork results in the recruitment of additional initiation points within a given replicon family. This is discussed in relation to models for the control of DNA replication.     

Evaluation of quenching methods for metabolite recovery in photoautotrophic Synechococcus sp. PCC 7002

The first step of many metabolomics studies is quenching, a technique vital for rapidly halting metabolism and ensuring that the metabolite profile remains unchanging during sample processing. The most widely used approach is to plunge the sample into prechilled cold methanol; however, this led to significant metabolite loss in Synecheococcus sp. PCC 7002. Here we describe our analysis of the impacts of cold methanol quenching on the model marine cyanobacterium Synechococcus sp. PCC 7002, as well as our brief investigation of alternative quenching methods. We tested several methods including cold methanol, cold saline, and two filtration approaches. Targeted central metabolites were extracted and metabolomic profiles were generated using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The results indicate that cold methanol quenching induces dramatic metabolite leakage in Synechococcus, resulting in a majority of central metabolites being lost prior to extraction. Alternatively, usage of a chilled saline quenching solution mitigates metabolite leakage and improves sample recovery without sacrificing rapid quenching of cellular metabolism. Finally, we illustrate that metabolite leakage can be assessed, and subsequently accounted for, in order to determine absolute metabolite pool sizes; however, our results show that metabolite leakage is inconsistent across various metabolite pools and therefore must be determined for each individually measured metabolite.     

The Systemic Immune Network in Recent Onset Type 1 Diabetes: Central Role of Interleukin-1 Receptor Antagonist (DIATOR Trial)

Background

The hypothesis was tested that the systemic immune milieu in recent-onset type 1 diabetes is associated with residual beta cell function and other metabolic patient characteristics.

Methods and Findings

All patients (n = 89, 40% female) of the Diabetes and Atorvastatin (DIATOR) Trial were analyzed at recruitment, i.e. prior to receiving the study medication. Inclusion criteria were insulin dependent diabetes for 2 weeks to 3 months, age range 18–39 years, and islet cell autoantibodies. Blood samples were analyzed for 14 immune mediators by standard methods. Concentrations of all mediators correlated with at least one other mediator (p<0.05, Spearman correlation) giving rise to a network. Interleukin 1 receptor antagonist (IL1-RA) held a central position and was associated with both pro- and anti-inflammatory mediators. Further central elements were the pro-inflammatory mediators CRP and IL-6, the soluble adhesion molecules sICAM-1 and E-selectin, and MCP-4 which held a central position in the chemokine network. The two Th1-associated mediators IFNγ and IP-10 remained outside the network but correlated with each other. All correlations were positive (r = 0.25–0.72), i.e., high levels of pro-inflammatory mediators were accompanied by increased levels of anti-inflammatory mediators. IL-1RA was the only mediator associated with fasting and liquid mixed meal stimulated C-peptide concentrations (r = 0.31 and 0.24, p = 0.003 and 0.025, after adjustment for age, sex, BMI). There were associations between the immune mediator network and BMI (IL-1RA, CRP, IL-6, MCP-4, MIP-1ß) but few or no associations with HbA1c, insulin dose, lipid parameters, age or sex.

Conclusions

In patients with recent onset type 1 diabetes, systemic acute phase proteins, cytokines, chemokines and soluble adhesion molecules form a network. Among the few central elements IL-1RA has a dominant role. IL-1RA is associated with all other groups of mediators and is the only mediator which correlates (positively) with residual beta cell function.

Trial registration

ClinicalTrials.gov registration number: {"type":"clinical-trial","attrs":{"text":"NCT00974740","term_id":"NCT00974740"}}NCT00974740     

Associations of testosterone and sex hormone binding globulin with adipose tissue hormones in midlife women

Objective:

Regulators of adipose tissue hormones remain incompletely understood, but may include sex hormones. As adipose tissue hormones have been shown to contribute to numerous metabolic and cardiovascular disorders, understanding their regulation in midlife women is of clinical importance. Therefore, we assessed the associations between testosterone (T) and sex hormone binding globulin (SHBG) with leptin, high molecular weight (HMW) adiponectin, and the soluble form of the leptin receptor (sOB‐R) in healthy midlife women.

Design and Methods:

Cross‐sectional analyses were performed using data from 1,881 midlife women (average age 52.6 (±2.7) years) attending the sixth Annual follow‐up visit of the multiethnic Study of Women's Health Across the Nation.

Results:

T was weakly negatively associated with both HMW adiponectin and sOB‐R (r = ?0.12 and r = ?0.10, respectively; P < 0.001 for both), and positively associated with leptin (r = 0.17; P < 0.001). SHBG was more strongly and positively associated with both HMW adiponectin and sOB‐R (r = 0.29 and r = 0.24, respectively; P < 0.001 for both), and more strongly and negatively associated with leptin (r = ?0.27; P < 0.001). Adjustment for fat mass, insulin resistance, or waist circumference only partially diminished associations with HMW adiponectin and sOB‐R, but attenuated associations with leptin. In conclusion, in these midlife women, lower SHBG values, and to a lesser extent, higher T levels, were associated with lower, or less favorable, levels of adiponectin and sOB‐R, independent of fat mass.

Conclusions:

These data suggest that variation in these adipose hormones resulting from lower SHBG levels, and possibly, though less likely, greater androgenicity, may contribute to susceptibility for metabolic and cardiovascular outcomes during midlife in women.

     

Maternal condition, yolk androgens and offspring performance: a supplemental feeding experiment in the lesser black-backed gull (Larus fuscus)

It has been proposed that the maternal androgens in avian egg yolk enhance offspring fitness by accelerating growth and improving competitive ability. Because egg quality is strongly influenced by maternal condition, we predicted that females in good condition would produce high-quality eggs with relatively high androgen content. We experimentally enhanced maternal condition by supplementary feeding lesser black-backed gulls (Larus fuscus) during egg formation and compared the concentrations of androstenedione (A4), 5alpha-dihydrotestosterone (DHT) and testosterone (T) in their eggs with those in eggs laid by control females. We also measured circulating levels of T in females immediately after laying. Egg androgens could affect offspring performance directly through chick development and/or indirectly through changes in the competitive ability of a chick relative to its siblings. To avoid confounding these two routes, and to separate effects operating through the egg itself with those operating through experimental changes in parental chick rearing capacity, we fostered eggs from both maternal treatment groups singly into the nests of unmanipulated parents. Contrary to expectation, mothers with experimentally enhanced body condition laid eggs with lower levels of androgens, while exhibiting higher circulating T concentrations post-laying. Despite these lower levels of egg androgen, offspring hatched from eggs laid by mothers in good condition did not show reduced growth or survival when reared in the absence of sibling competition. Our results demonstrate that yolk androgen concentrations vary with the body condition of the female at the time of egg formation and that females in good condition reduced the yolk androgen content of their eggs without altering offspring performance.