Epigenetic mechanisms regulate the prostaglandin E receptor 2 in breast cancer

The increase in local oestrogen production seen in oestrogen receptor positive (ER+) breast cancers is driven by increased activity of the aromatase enzyme. CYP19A1, the encoding gene for aromatase, is often overexpressed in the oestrogen-producing cells of the breast adipose fibroblasts (BAFs) surrounding an ER+ tumour, and the molecular processes underlying this upregulation is important in the development of breast-specific aromatase inhibitors for breast cancer therapy. Prostaglandin E2 (PGE2), a factor secreted by tumours, is known to stimulate CYP19A1 expression in human BAFs. The hormonal regulation of this process has been examined; however, what is less well understood is the emerging role of epigenetic mechanisms and how they modulate PGE2 signalling. This present study characterises the epigenetic processes underlying expression of the prostanoid receptor EP2 in the context of ER+ breast cancer. Sodium bisulphite sequencing of CpG methylation within the promoter region of EP2 revealed that an inverse correlation existed between methylation levels and relative EP2 expression in breast cancer cell lines MDA-MB-231, MCF7 and MCF10A but not in HS578t and T47D. Inhibition of DNA methylation with 5-aza-2'-deoxycytidine (5aza) and histone deacetylation with Trichostatin A (TSA) resulted in upregulation of EP2 mRNA in all cell lines with varying influences of each epigenetic process observed. Expression of EP2 was detected in human BAFs despite a natively methylated promoter, and this expression was further increased upon 5aza treatment. An examination of 3 triple negative, 3 ductal carcinoma in situ and 3 invasive ductal carcinoma samples revealed that there was no change in EP2 promoter methylation status between normal and cancer associated stroma, despite observed differences in relative mRNA levels. Although EP2 methylation status is inversely correlated to expression levels in established breast cancer cell lines, we could not identify that such a correlation existed in tumour-associated stroma cells.     

Biosynthesis of a mycobacterial lipopolysaccharide. Incorporation of [14C]acyl groups by whole cells in vivo

1. Mycobacterium phlei (A.T.C.C. 356) cells were incubated with (14)C-labelled short-chain fatty acids and the 6-O-methylglucose-containing lipopolysaccharides that became esterified with radioactive acyl groups were isolated. The pattern of labelling of these lipopolysaccharides with the different acyl groups, the effects of different conditions on labelling patterns, and the kinetics of the turnover of (14)C-labelled acyl groups were studied. 2. The labelling patterns are summarized as follows. [1-(14)C]Acetate was incorporated into all of the acyl groups. [1-(14)C]Propionate led to labelling of propionate and succinate, while [1-(14)C]isobutyrate was incorporated mostly as such, along with a trace amount in iso-octanoate. 3. Under the conditions of the experiments, [1-(14)C]acetate was rapidly incorporated into succinyl (3-carboxypropionyl) and octanoyl groups, whereas the acetyl groups themselves were labelled more slowly. Radioactivity in propionyl and succinyl groups, originating from [1-(14)C]propionate, attained maximum values and then gradually decreased in both. Incorporation of [1-(14)C]isobutyrate proceeded slowly but reached a plateau and remained constant. While n-butyrate is not a normal constituent of methyl-glucose-containing lipopolysaccharides, it was incorporated as such when n-[1-(14)C]-butyrate was supplied in the medium. 4. [1-(14)C]Acetyl groups were readily displaced by unlabelled acetate. On the other hand, the specific radioactivity of the succinyl group continued to increase during a 3h incubation with unlabelled succinate. Propionyl and succinyl groups, labelled by [1-(14)C]propionate, were displaced slowly by unlabelled propionate or succcinate. The isobutyryl group of the lipopolysaccharides did not turn over, in contrast to the results obtained with the other acyl substituents.     

Caenorhabditis elegans reticulon interacts with RME-1 during embryogenesis

Reticulon (RTN) family proteins are localized in the endoplasmic reticulum (ER). At least four different RTN genes have been identified in mammals, but in most cases, the functions of the encoded proteins except mammalian RTN4-A and RTN4-B are unknown. Each RTN gene produces 1-3 proteins by different promoters and alternative splicing. In Caenorhabditis elegans, there is a single gene (rtn gene) encoding three reticulon proteins, nRTN-A, B, and C. mRNA of nRTN-C is expressed in germ cells and embryos. However, nRTN-C protein is only expressed during embryogenesis and rapidly disappears after hatch. By yeast two-hybrid screening, two clones encoding the same C-terminal region of RME-1, a protein functioning in the endocytic recycling, were isolated. These findings suggest that nRTN-C functions in the endocytic pathway during embryogenesis.     

Kinetic studies on redox reactions of hemoproteins. I. Reduction of thermoresistant cytochrome c-552 and horse heart cytochrome c by ferrocyanide

The oxidation-reduction reaction of horse heart cytochrome c and cytochrome c (552, Thermus thermophilus), which is highly thermoresistant, was studied by temperature-jump method. Ferrohexacyanide was used as reductant.

Thermodynamic and activation parameters of the reaction obtained for both cytochromes were compared with each other. The results of this showed that (1) the redox potential of cytochrome c-552,+0.19 V, is markedly less than that of horse heart cytochrome c. (2) ?H_ox³ of cytochrome c-552 is considerably lower than that of horse heart cytochrome c. (3) ?H_ox³ and ?S_red³ of cytoochrome c-552 are more negative than those of horse heart cytochrome c. (4) k_red of cytochrome c-552 is much lower than that of horse heart cytochrome c at room temperature.     

The protective effect of erdosteine on vancomycin-induced pancreatic damage in rats

Objective The goal of this study was to investigate whether vancomycin (VCM) has a negative effect on pancreatic tissue and to elucidate the role of erdosteine (ERD), an expectorant and an antioxidant agent, on possible VCM-induced pancreas impairment in rats. Materials and methods A total of 21 male Wistar albino rats were included in this study. All animals were equally divided into three groups as follows: Controls (n = 7), VCM treated group (200 mg/kg twice daily for 7 days intraperitoneally, n = 7) and VCM (200 mg/kg) + ERD treated group (10 mg/kg day orally ERD, n = 7). The first dose of ERD administration was performed 24 h prior to VCM injection and the study was continued for 7 days. At the end of the study, all animals were sacrificed. Blood and pancreas tissue samples were collected. For biochemical analysis, serum amylase, lipase, alkaline phosphatase (ALP), and gamma glutamyl transferase (GGT) activities were measured. For histopathological examination, pancreas tissue samples were investigated under the light microscope. Results VCM administration has significantly increased the serum amylase, lipase, ALP, and GGT activities, when compared with the controls. VCM + ERD administration significantly decreased the serum lipase, amylase, and GGT activities. There was no statistically significant difference between the VCM + ERD treated group and only VCM treated group by means of serum ALP levels. It has been observed that there was a prominent pancreatic tissue damage in only VCM given group. However, ERD exhibited structural protection against VCM-induced pancreatic damage and this effect was statistically significant. ERD has also obtained a marked reduction in the extent of pancreatic damage. Conclusion Erdosteine may play an important role in the VCM-induced pancreatic damage and may reduce the pancreatic damage both in biochemical and histopathological aspects.     

Non-covalent interactions in blue copper protein probed by Met16 mutation and electronic and resonance Raman spectroscopy of Achromobacter cycloclastes pseudoazurin

We have used low-temperature (77 K) resonance Raman (RR) spectroscopy as a probe of the electronic and molecular structure to investigate weak π-π interactions between the metal ion-coordinated His imidazoles and aromatic side chains in the second coordination sphere of blue copper proteins. For this purpose, the RR spectra of Met16 mutants of Achromobacter cycloclastes pseudoazurin (AcPAz) with aromatic (Met16Tyr, Met16Trp, and Met16Phe) and aliphatic (Met16Ala, Met16Val, Met16Leu, and Met16Ile) amino acid side chains have been obtained and analyzed over the 100-500 cm⁻¹ spectral region. Subtle strengthening of the Cu(II)-S(Cys) interaction on replacing Met16 with Tyr, Trp, and Phe is indicated by the upshifted (0.3-0.8 cm⁻¹) RR bands involving ν(Cu-S)_Cys stretching modes. In contrast, the RR spectra of Met16 mutants with aliphatic amino acids revealed larger (0.2-1.8 cm⁻¹) shifts of the ν(Cu-S)_Cys stretching modes to a lower frequency region, which indicate a weakening of the Cu(II)-S(Cys) bond. Comparisons of the predominantly ν(Cu-S)_Cys stretching RR peaks of the Met16X = Tyr, Trp, and Phe variants, with the molar absorptivity ratio ε₁/ε₂ of σ(∼455 nm)/π(∼595 nm) (Cys)S → Cu(II) charge-transfer bands in the optical spectrum and the axial/rhombic EPR signals, revealed a slightly more trigonal disposition of ligands about the copper(II) ion. In contrast, the RR spectra of Met16Z = Ala, Val, Leu, and Ile variants with aliphatic amino acid side chains show a more tetrahedral perturbation of the copper active site, as judged by the lower frequencies of the ν(Cu-S)_Cys stretching modes, much larger values of the ε₁/ε₂ ratio, and the increased rhombicity of the EPR spectra.     

Artificial hybrids of influenza A virus RNA polymerase reveal PA subunit modulates its thermal sensitivity

Background

Influenza A virus can infect a variety of different hosts and therefore has to adapt to different host temperatures for its efficient viral replication. Influenza virus codes for an RNA polymerase of 3 subunits: PB1, PB2 and PA. It is well known that the PB2 subunit is involved in temperature sensitivity, such as cold adaptation. On the other hand the role of the PA subunit in thermal sensitivity is still poorly understood.

Methodology/Principal Findings

To test which polymerase subunit(s) were involved in thermal stress we reconstituted artificial hybrids of influenza RNA polymerase in ribonucleoprotein (RNP) complexes and measured steady-state levels of mRNA, cRNA and vRNA at different temperatures. The PA subunit was involved in modulating RNP activity under thermal stress. Residue 114 of the PA subunit was an important determinant of this activity.

Conclusions/Significance

These findings suggested that influenza A virus may acquire an RNA polymerase adapted to different body temperatures of the host by reassortment of the RNA polymerase genes.     

CSF-1-dependent red pulp macrophages regulate CD4 T cell responses

The balance between immune activation and suppression must be regulated to maintain immune homeostasis. Tissue macrophages (MΦs) constitute the major cellular subsets of APCs within the body; however, how and what types of resident MΦs are involved in the regulation of immune homeostasis in the peripheral lymphoid tissues are poorly understood. Splenic red pulp MΦ (RPMs) remove self-Ags, such as blood-borne particulates and aged erythrocytes, from the blood. Although many scattered T cells exist in the red pulp of the spleen, little attention has been given to how RPMs prevent harmful T cell immune responses against self-Ags. In this study, we found that murine splenic F4/80(hi)Mac-1(low) MΦs residing in the red pulp showed different expression patterns of surface markers compared with F4/80(+)Mac-1(hi) monocytes/MΦs. Studies with purified cell populations demonstrated that F4/80(hi)Mac-1(low) MΦs regulated CD4(+) T cell responses by producing soluble suppressive factors, including TGF-β and IL-10. Moreover, F4/80(hi)Mac-1(low) MΦs induced the differentiation of naive CD4(+) T cells into functional Foxp3(+) regulatory T cells. Additionally, we found that the differentiation of F4/80(hi)Mac-1(low) MΦs was critically regulated by CSF-1, and in vitro-generated bone marrow-derived MΦs induced by CSF-1 suppressed CD4(+) T cell responses and induced the generation of Foxp3(+) regulatory T cells in vivo. These results suggested that splenic CSF-1-dependent F4/80(hi)Mac-1(low) MΦs are a subpopulation of RPMs and regulate peripheral immune homeostasis.