Acute responses to phytoestrogens in small arteries from men with coronary heart disease

The aim of this study was to investigate acute vasodilator responses to phytoestrogens and selective estrogen receptor-alpha (ERalpha) agonist in isolated small arteries from men with established coronary heart disease (CHD) and with a history of myocardial infarction versus healthy male control subjects. As to methodology, small arteries obtained from subcutaneous fat biopsies and mounted on a wire myograph were preconstricted with norepinephrine, and dilator responses to increasing nanomolar-micromolar concentrations of the phytoestrogens resveratrol and genistein (predominantly ERbeta agonists) and to propyl-[1H]-pyrazole-1,3,5-triyl-trisplenol (PPT, a selective ERalpha agonist) were determined. These were compared with responses to reference compound 17beta-estradiol (17beta-E2). Concentration-response curves were constructed before and after nitric oxide (NO) synthase inhibition with Nomega-nitro-L-arginine methyl ester. As a result, relaxation induced by the investigated compounds was similar in men with CHD and control men, but in both groups PPT and genistein-induced relaxation was greater than that of resveratrol and 17beta-E2. NO contributed to both phytoestrogens and PPT-induced relaxation but not to 17beta-E2 responses in arteries from control men. This NO-mediated component of relaxation was absent in arteries from men with established CHD. In conclusion, phytoestrogens, at concentrations achievable by ingestion of phytoestrogen-rich food products, evoke dilatation ex vivo of small peripheral arteries from normal men and those with established CHD. The contribution of NO to dilatory responses by these compounds is pertinent to arteries from control males, whereas other NO-independent dilatory mechanism(s) are involved in arteries from CHD.     

The mechanism of autocatalytic activation of plant-type L-asparaginases

Plant l-asparaginases and their bacterial homologs, such as EcAIII found in Escherichia coli, form a subgroup of the N-terminal nucleophile (Ntn)-hydrolase family. In common with all Ntn-hydrolases, they are expressed as inactive precursors that undergo activation in an autocatalytic manner. The maturation process involves intramolecular hydrolysis of a single peptide bond, leading to the formation of two subunits (alpha and beta) folded as one structural domain, with the nucleophilic Thr residue located at the freed N terminus of subunit beta. The mechanism of the autocleavage reaction remains obscure. We have determined the crystal structure of an active site mutant of EcAIII, with the catalytic Thr residue substituted by Ala (T179A). The modification has led to a correctly folded but unprocessed molecule, revealing the geometry and molecular environment of the scissile peptide bond. The autocatalytic reaction is analyzed from the point of view of the Thr(179) side chain rotation, identification of a potential general base residue, and the architecture of the oxyanion hole.     

Induction and Role of Indoleamine 2,3 Dioxygenase in Mouse Models of Influenza A Virus Infection

Influenza infection stimulates protective host immune responses but paradoxically enhances lung indoleamine 2,3 dioxygenase (IDO) activity, an enzyme that suppresses helper/effector T cells and activates Foxp3-lineage regulatory CD4 T cells (Tregs). Influenza A/PR/8/34 (PR8) infection stimulated rapid elevation of IDO activity in lungs and lung-draining mediastinal lymph nodes (msLNs). Mice lacking intact IDO1 genes (IDO1-KO mice) exhibited significantly lower morbidity after sub-lethal PR8 infection, and genetic or pharmacologic IDO ablation led to much faster recovery after virus clearance. More robust influenza-specific effector CD8 T cell responses manifested in lungs of PR8-infected IDO1-KO mice, though virus clearance rates were unaffected by IDO ablation. Similar outcomes manifested in mice infected with a less virulent influenza A strain (X31). IDO induction in X31-infected lungs was dependent on IFN type II (IFNγ) signaling and was restricted to non-hematopoietic cells, while redundant IFN type 1 or type II signaling induced IDO exclusively in hematopoietic cells from msLNs. Memory T cells generated in X31-primed IDO1-KO mice protected mice from subsequent challenge with lethal doses of PR8 (100×LD₅₀). However recall T cell responses were less robust in lung interstitial tissues, and classic dominance of TCR Vβ8.3 chain usage amongst memory CD8⁺ T cells specific for influenza nucleoprotein (NP₃₆₆) did not manifest in IDO1-KO mice. Thus, influenza induced IDO activity in lungs enhanced morbidity, slowed recovery, restrained effector T cell responses in lungs and shaped memory T cell repertoire generation, but did not attenuate virus clearance during primary influenza A infection.     

Nested PCR for the detection of Aspergillus species in maxillary sinus samples of patients with chronic sinusitis

Background

Fungal rhinosinusitis has become an increasingly recognized disease, being Aspergillus species responsible for most of the cases. Its diagnosis is quite difficult because of the non-specific symptoms and low sensitivity of the current diagnostic methods.

Diverse mechanisms of metaeffector activity in an intracellular bacterial pathogen,Legionella pneumophila

Pathogens deliver complex arsenals of translocated effector proteins to host cells during infection, but the extent to which these proteins are regulated once inside the eukaryotic cell remains poorly defined. Among all bacterial pathogens, Legionella pneumophila maintains the largest known set of translocated substrates, delivering over 300 proteins to the host cell via its Type IVB, Icm/Dot translocation system. Backed by a few notable examples of effector–effector regulation in L. pneumophila, we sought to define the extent of this phenomenon through a systematic analysis of effector–effector functional interaction. We used Saccharomyces cerevisiae, an established proxy for the eukaryotic host, to query > 108,000 pairwise genetic interactions between two compatible expression libraries of ~330 L. pneumophila‐translocated substrates. While capturing all known examples of effector–effector suppression, we identify fourteen novel translocated substrates that suppress the activity of other bacterial effectors and one pair with synergistic activities. In at least nine instances, this regulation is direct—a hallmark of an emerging class of proteins called metaeffectors, or “effectors of effectors”. Through detailed structural and functional analysis, we show that metaeffector activity derives from a diverse range of mechanisms, shapes evolution, and can be used to reveal important aspects of each cognate effector's function. Metaeffectors, along with other, indirect, forms of effector–effector modulation, may be a common feature of many intracellular pathogens—with unrealized potential to inform our understanding of how pathogens regulate their interactions with the host cell.     

The relationship between chronotype and personality among patients with alcohol dependence syndrome: Pilot study

The study investigates the distribution of chronotypes among alcohol-dependent subjects and the relationship between personality and chronotype. Fifty-eight alcohol-dependent patients and 29 age-matched healthy controls were studied using Ogińska’s Chronotype Questionnaire (ChQ), Eysenck’s Personality Questionnaire – Revised (EPQ-R), Selzer’s Michigan Alcoholism Screening Test (MAST) and a sociodemographic status questionnaire designed by the authors. The alcohol-dependent patients tended to be morning type, based on the morningness–eveningness ChQ scale, with a weakly marked rhythm, based on the distinctness ChQ scale. Preference towards morningness was associated with older age, but no relation between chronotype and severity of alcohol dependence was found. A high amplitude of the rhythm was associated with higher neuroticism. Therefore, despite being in the minority, patients with a distinct circadian rhythm (i.e. with a high amplitude) are at greater risk of mood and anxiety disorders and hence should be given special consideration.     

The Impact of Short-Term,Intensive Antifolate Treatment (with Pyrimethamine and Sulfadoxine) and Antibiotics Followed by Long-Term,Secondary Antifolate Prophylaxis on the Rate of Toxoplasmic Retinochoroiditis Recurrence

PurposeTo assess the impact of intensive antifolate treatment, followed by secondary antifolate prophylaxis (A-SP) on the recurrence rate of toxoplasmic retinochoroiditis (TRC). To investigate whether there are any other factors potentially predisposing for recurrence.ResultsWhen secondary antifolate prophylaxis (A-SP) was instituted immediately after the treatment for TRC, the probability of 3-year recurrence–free survival after the first course of A-SP was 90.9%. A recurrence was most likely approximately 3.5 years after the first treatment. A univariate Cox regression model demonstrated that a risk for recurrence was 2.82 times higher (p = 0.02) in patients with retinal scars. In the multivariate analysis, the risk for recurrence was 2.41 higher (p = 0.06). In patients with haemorrhagic lesions the risk for recurrence was lower, aRR = 0.17 (approaching borderline statistical significance p = 0.08).ConclusionsWith the institution of A-SP of immediately after the intensive treatment for TRC, i.e. when a reactivation was most likely, there was no recurrence during A-SP. Following A-SP the recurrence rates were low and recurrence-free periods tended to be longer. The treatment regimen employed had a beneficial effect on the recurrence interval as it reduced and delayed the highest probability of recurrence.     

On the role of steric clashes in methylation control of restriction endonuclease activity

Restriction-modification systems digest non-methylated invading DNA, while protecting host DNA against the endonuclease activity by methylation. It is widely believed that the methylated DNA would not ‘fit’ into the binding site of the endonuclease in the productive orientation, and thus steric clashes should account for most of the protection. We test this concept statistically by grafting methyl groups in silico onto non-methylated DNA in co-crystal structures with restriction endonucleases. Clash scores are significantly higher for protective than non-protective methylation (P < 0.05% according to the Wilcoxon rank sum test). Structural data alone are sufficient to distinguish between protective and non-protective DNA methylation with 90% confidence and decision thresholds of 1.1 Å and 48 ų for the most severe distance-based and cumulative volume-based clash with the protein, respectively (0.1 Å was deducted from each interatomic distance to allow for coordinate errors). The most severe clashes are more pronounced for protective methyl groups attached to the nitrogen atoms (N6-methyladenines and N4-methylcytosines) than for C5-methyl groups on cytosines. Cumulative clashes are comparable for all three types of protective methylation.     

In vitro metabolism of 17beta-estradiol by human liver tissue.

17beta-[6,7- 3H]Estradiol was incubated with adult human liver slices in Krebs-Ringer phosphate buffer containing glucose. Of the identified 3H recovered, 51-76 percent consisted of estrone-3-sulfate (E13S) and 17 beta-estradiol-3-sulfate (E23S). E13S was the main metabolite and was found in both tissue and medium. E23S was present only in the medium. Minor amounts of estrogen glucuronides were formed. When a human liver homogenate was incubated with [3H]E2 in a medium fortified with excess uridine diphosphate glucuronic acid only some 4 percent of conjugation with glucuronic acid was observed. It is suggested that human liver favors sulfurylation as the conjugating mechanism for E2 and E1.     

Suppression of the E. coli SOS response by dNTP pool changes

The Escherichia coli SOS system is a well-established model for the cellular response to DNA damage. Control of SOS depends largely on the RecA protein. When RecA is activated by single-stranded DNA in the presence of a nucleotide triphosphate cofactor, it mediates cleavage of the LexA repressor, leading to expression of the 30⁺-member SOS regulon. RecA activation generally requires the introduction of DNA damage. However, certain recA mutants, like recA730, bypass this requirement and display constitutive SOS expression as well as a spontaneous (SOS) mutator effect. Presently, we investigated the possible interaction between SOS and the cellular deoxynucleoside triphosphate (dNTP) pools. We found that dNTP pool changes caused by deficiencies in the ndk or dcd genes, encoding nucleoside diphosphate kinase and dCTP deaminase, respectively, had a strongly suppressive effect on constitutive SOS expression in recA730 strains. The suppression of the recA730 mutator effect was alleviated in a lexA-deficient background. Overall, the findings suggest a model in which the dNTP alterations in the ndk and dcd strains interfere with the activation of RecA, thereby preventing LexA cleavage and SOS induction.