Regulation of aromatase activity by cytokines, PGE2 and 2-methoxyoestrone-3-O-sulphamate in fibroblasts derived from normal and malignant breast tissues

Synthesis of oestrone from androstenedione within tumours, by the aromatase enzyme complex, is an important source of oestrogen that is available to support the growth of hormone-dependent breast tumours. In view of the central role that the aromatase enzyme has in oestrogen synthesis there has been considerable interest in understanding its regulation and developing inhibitors to block its action. In the present study we have derived fibroblasts from breast tumours (TFs), tissue proximal to tumours (PFs) and reduction mammoplasty tissue (RMFs) and used them to investigate the regulation of aromatase activity by PGE(2), IL-6 plus its soluble receptor (SR) or TNFalpha. In addition we have examined the ability of 2-methoxyoestrone sulphamate (2-MeOEMATE), a compound which alters microtubule stability, to block the stimulation of aromatase activity by these factors. Basal aromatase activity in PFs was significantly higher (p<0.001) than in TFs or RMFs. The combination of IL-6 plus SR or TNFalpha produced the greatest stimulation of aromatase activity in TFs (up to 61-fold) while having a much lower stimulatory effects on aromatase activity in PFs (up to 60% increase) or RMFs (up to 192% increase). 2-MeOEMATE reduced basal aromatase activity in TFs by 87% and completely abrogated the ability of PGE(2), IL-6 plus SR or TNFalpha to stimulate aromatase activity in these fibroblasts. Results from these studies indicate that while PFs have the highest level of non-stimulated aromatase activity, aromatase activity in TFs shows the greatest response to cytokines. These findings suggest that intrinsic difference may exist for the different types of fibroblasts in the way in which they respond to regulatory factors. The ability of 2-MeOEMATE to block cytokine stimulated aromatase activity suggests that, in addition to its other anti-cancer properties, this compound may also act to inhibit cytokine-stimulated aromatase activity in breast tumours.     

Anti-cancer activities of novel D-ring modified 2-substituted estrogen-3-O-sulfamates

Sulfamoylated derivatives of the endogenous estrogen metabolite 2-methoxyestradiol (2-MeOE2 (7)), such as 2-methoxy-3-O-sulfamoyl estrone (2-MeOEMATE (1)), display greatly enhanced activity against the proliferation of human cancer cells and inhibit steroid sulphatase (STS), another current oncology target. We explore here the effects of steroidal D-ring modification on the activity of such 2-substituted estrogen-3-O-sulfamates in respect of inhibition of tumour cell proliferation and steroid sulphatase. The novel 17-deoxy analogues of 2-MeOEMATE and the related 2-ethyl and 2-methylsulfanyl compounds showed greatly reduced inhibition of MCF-7 proliferation. Introduction of a 17alpha-benzyl substituent to such 2-substituted estrogen sulfamates also proved deleterious to anti-proliferative activity but could, in one case, enhance STS inhibition with respect to the parent substituted estrone sulfamate. In contrast, selected 17-oxime derivatives of 2-MeOEMATE displayed an enhanced anti-proliferative activity. These results illustrate that enhanced in vitro anti-cancer activity can be achieved in the 2-substituted estrogen sulfamate series and highlight, in particular, the importance of potential hydrogen bonding effects around the steroidal D-ring in the activity of these molecules. The SAR parameters established herein will assist the future design of anti-proliferative and anti-endocrine agents as potential therapeutics for both hormone dependent and independent cancers.     

Forces during bacteriophage DNA packaging and ejection

The conjunction of insights from structural biology, solution biochemistry, genetics, and single-molecule biophysics has provided a renewed impetus for the construction of quantitative models of biological processes. One area that has been a beneficiary of these experimental techniques is the study of viruses. In this article we describe how the insights obtained from such experiments can be utilized to construct physical models of processes in the viral life cycle. We focus on dsDNA bacteriophages and show that the bending elasticity of DNA and its electrostatics in solution can be combined to determine the forces experienced during packaging and ejection of the viral genome. Furthermore, we quantitatively analyze the effect of fluid viscosity and capsid expansion on the forces experienced during packaging. Finally, we present a model for DNA ejection from bacteriophages based on the hypothesis that the energy stored in the tightly packed genome within the capsid leads to its forceful ejection. The predictions of our model can be tested through experiments in vitro where DNA ejection is inhibited by the application of external osmotic pressure.     

In vitro Growth and Shoot Multiplication of Achras zapota in a Controlled Carbon Dioxide Environment

The culture vessels with multiplying shoots of Achras zapota L. on Schenk and Hildebrandt (SH) medium containing 8.88 M 6-benzylaminopurine (BAP) with or without sucrose were kept under varied CO₂ concentrations ranging from 0.6 to 40.0 g m^–3 using different concentrations of sodium bicarbonate (NaHCO₃), sodium carbonate (Na₂CO₃), potassium bicarbonate (KHCO₃), and potassium carbonate (K₂CO₃) in small acrylic chambers. Complete absence of carbon source caused death of shoots within 20 d. Under elevated concentrations of CO₂ (10.0 and 40.0 g m^–3) the shoots grew photoautotrophically on sucrose-free medium. The growth of cultures was better at 40.0 g (CO₂) m^–3 than on 3.0 % sucrose under ambient air of growth room. However, the best response was obtained at 10.0 g (CO₂) m^–3 and 3.0 % sucrose where maximum number of shoots, shoot length, fresh and dry mass, total number of leaves and leaf area was observed.     

Impaired E-cadherin expression in human spermatozoa in a male factor infertility subset signifies E-cadherin-mediated adhesion mechanisms operative in sperm-oolemma interactions

Cadherins comprise a family of calcium-dependent glycoproteins that function in mediating cell-cell adhesion in virtually all solid tissues of multicellular organisms. We have examined the presence of a cadherin on spermatozoon and its possible involvement in sperm-oocyte interaction. Spermatozoa from fertile human subjects showed the presence of E-cadherin on its head domains, detectable only after permeabilization of the surface membranes. On the contrary, spermatozoa from oligozoospermic subjects did not possess E-cadherin on their principal acrosomal and equatorial domains. Immunoprecipitation and Western blot studies also showed the presence of E-cadherin in spermatozoa from fertile males and its absence in oligozoospermic males. Using RT-PCR, we detected E-cadherin message in the round cells of fertile males, which was absent in the cells from oligozoospermic males. The presence of anti-E-cadherin antibody brought about quantitative reduction in the sperm-oocyte binding in vitro. These observations indicate the possibility of the interplay of a cadherin-dependent homophilic and/or heterophilic adhesion interaction between spermatozoa and oocyte during fertilization. The absence of a key adhesion molecule in a human male infertility disorder points towards genetic defects causing failure in gamete interactions.     

Regulation of immunity by a novel population of Qa-1-restricted CD8alphaalpha+TCRalphabeta+ T cells

Regulatory mechanisms involving CD8+ T cells (CD8 regulatory T cells (Tregs)) are important in the maintenance of immune homeostasis. However, the inability to generate functional CD8 Treg clones with defined Ag specificity has precluded a direct demonstration of CD8 Treg-mediated regulation. In the present study, we describe the isolation of functional lines and clones representing a novel population of TCRalphabeta+ Tregs that control activated Vbeta8.2+ CD4 T cells mediating experimental autoimmune encephalomyelitis. They express exclusively the CD8alphaalpha homodimer and recognize a peptide from a conserved region of the TCR Vbeta8.2 chain in the context of the Qa-1a (CD8alphaalpha Tregs). They secrete type 1 cytokines but not IL-2. CD8alphaalpha Tregs kill activated Vbeta8.2+ but not Vbeta8.2- or naive T cells. The CD8alphaalpha Tregs prevent autoimmunity upon adoptive transfer or following in vivo activation. These findings reveal an important negative feedback regulatory mechanism targeting activated T cells and have implications in the development of therapeutic strategies for autoimmune diseases and transplantation.     

Effect of controlled carbon dioxide on in vitro shoot multiplication in Feronia limonia (L.) Swingle

The effect of controlled carbon dioxide environment on in vitro shoot growth and multiplication in Feronia limonia (a tropical fruit plant, Family- Rutaceae) was studied. Carbon dioxide available in the ambient air of the growth room was insufficient for in vitro growth of the shoots alone. Also, the presence of sucrose only as the C-source in the medium (without CO₂), was found to be inadequate for sustainable growth and multiplication of shoots. The carbon dioxide enrichment promoted shoot multiplication and overall growth. The promotory effect of CO₂ was independent of the presence of sucrose in the medium. In the presence of both CO₂ and sucrose, an additive effect was observed producing maximum shoot growth. In the absence of sucrose a higher concentration of CO₂ (10.0)g m⁻³ was required to achieve photoautotrophic shoot multiplication comparable to ambient air controls. Highest leaf area per shoot cluster promoting shoot growth and multiplication was recorded under this treatment. Shoots growing on sucrose containing medium under controlled CO₂ environment of 0.6 g m⁻³ concentration evoked better response than ambient air controls (shoots growing on sucrose containing medium) in growth room. This treatment produced the overall best response. The present study highlighted the possibility of photoautotrophic multiplication which might prove useful for successful hardening and acclimatization in tissue culture plants.     

Secretory factors of human neuroblastoma (IMR-32) and human glioblastoma (U87MG) cell lines induce neurite outgrowths in PC12 cells

Neurite outgrowth is essential for the communication of the nervous system. The rat Pheochromocytoma (PC12) cells are commonly used in the neuronal cell study. It is well known that exogenous stimuli such as Nerve Growth Factor (NGF) induce neurite outgrowth. In the present study it has been investigated whether or not the conditioned medium from human neuroblastoma cell line (IMR-32) and human glioblastoma cell line (U87MG) may augment neurite outgrowth in PC12 cells. PC12 were cultured with and without conditioned media of IMR-32 and U87MG. The result showed that both the conditioned media induce neurite outgrowth within 48 hr and stops further proliferation of PC12 cells. However no outgrowth was noted in PC12 cells incubated without conditioned medium. In conclusion, it is shown that both the conditioned media (IMR-32 and U87MG) have the potential to induce the neurite outgrowth in the PC12 cells.     

Detection of 65 kD heat shock protein in cerebrospinal fluid of tuberculous meningitis patients

Background  

Diagnosis of tuberculous meningitis (TBM) is difficult. Rapid confirmatory diagnosis is essential to initiate required therapy. There are very few published reports about the diagnostic significance of 65 kD heat shock protein (hsp) in TBM patients, which is present in a wide range of Mycobacterium tuberculosis species and elicits a cellular and humoral immune response. In the present study we have conducted a prospective evaluation for the demonstration of 65 kD hsp antigen in cerebrospinal fluid (CSF) of TBM patients, by indirect ELISA method using monoclonal antibodies (mAb) against the 65 kD hsp antigen, for the diagnosis of TBM.     

Metastases suppressor NME2 associates with telomere ends and telomerase and reduces telomerase activity within cells

Analysis of chromatin-immunoprecipitation followed by sequencing (ChIP-seq) usually disregards sequence reads that do not map within binding positions (peaks). Using an unbiased approach, we analysed all reads, both that mapped and ones that were not included as part of peaks. ChIP-seq experiments were performed in human lung adenocarcinoma and fibrosarcoma cells for the metastasis suppressor non-metastatic 2 (NME2). Surprisingly, we identified sequence reads that uniquely represented human telomere ends in both cases. In vivo presence of NME2 at telomere ends was validated using independent methods and as further evidence we found intranuclear association of NME2 and the telomere repeat binding factor 2. Most remarkably, results demonstrate that NME2 associates with telomerase and reduces telomerase activity in vitro and in vivo, and sustained NME2 expression resulted in reduced telomere length in aggressive human cancer cells. Anti-metastatic function of NME2 has been demonstrated in human cancers, however, mechanisms are poorly understood. Together, findings reported here suggest a novel role for NME2 as a telomere binding protein that can alter telomerase function and telomere length. This presents an opportunity to investigate telomere-related interactions in metastasis suppression.