Possible plant mitochondria involvement in cell adaptation to drought stress. A case study: durum wheat mitochondria

Although plant cell bioenergetics is strongly affected by abiotic stresses, mitochondrial metabolism under stress is still largely unknown. Interestingly, plant mitochondria may control reactive oxygen species (ROS) generation by means of energy-dissipating systems. Therefore, mitochondria may play a central role in cell adaptation to abiotic stresses, which are known to induce oxidative stress at cellular level. With this in mind, in recent years, studies have been focused on mitochondria from durum wheat, a species well adapted to drought stress. Durum wheat mitochondria possess three energy-dissipating systems: the ATP-sensitive plant mitochondrial potassium channel (PmitoK(ATP)); the plant uncoupling protein (PUCP); and the alternative oxidase (AOX). It has been shown that these systems are able to dampen mitochondrial ROS production; surprisingly, PmitoK(ATP) and PUCP (but not AOX) are activated by ROS. This was found to occur in mitochondria from both control and hyperosmotic-stressed seedlings. Therefore, the hypothesis of a 'feed-back' mechanism operating under hyperosmotic/oxidative stress conditions was validated: stress conditions induce an increase in mitochondrial ROS production; ROS activate PmitoK(ATP) and PUCP that, in turn, dissipate the mitochondrial membrane potential, thus inhibiting further large-scale ROS production. Another important aspect is the chloroplast/cytosol/mitochondrion co-operation in green tissues under stress conditions aimed at modulating cell redox homeostasis. Durum wheat mitochondria may act against chloroplast/cytosol over-reduction: the malate/oxaloacetate antiporter and the rotenone-insensitive external NAD(P)H dehydrogenases allow cytosolic NAD(P)H oxidation; under stress this may occur without high ROS production due to co-operation with AOX, which is activated by intermediates of the photorespiratory cycle.     

An anti-let-7 sponge decoys and decays endogenous let-7 functions

The let-7 family contains 12 members, which share identical seed regions, suggesting that they may target the same mRNAs. It is essential to develop a means that can regulate the functions of all members. Using a DNA synthesis technique, we have generated an anti-let-7 sponge aiming to modulate the function of all members. We found that products of the anti-let-7 construct could bind and inactivate all members of the let-7 family, producing decoy and decay effects. To test the role of the anti-let-7 sponge, we stably expressed the anti-let-7 construct in two types of cells, the breast carcinoma cells MT-1 and the oldest and most commonly used human cervical cancer cell line, HeLa cells. We found that expression of anti-let-7 increased cell survival, invasion and adhesion, which corroborate with known functions of let-7 family members. We further identified a novel target site across all species of the let-7 family in hyaluronan synthase 2 (HAS2). HAS2 overexpression produced similar effects as the anti-let-7 sponge. Silencing HAS2 expression by siRNAs produced opposite effects to anti-let-7 on cell survival and invasion. The ability of anti-let-7 to regulate multiple members of the let-7 family allows us to observe their multiple functions using a single reagent. This approach can be applied to other family members with conserved sequences.     

Human papillomavirus prevalence in lung carcinomas in Bulgaria

A possible association between high‐risk human papillomaviruses (HPV) and lung cancer has been investigated for decades with discrepant results. The aim of this study was to determine the prevalence of HPV16 and 18 in Bulgarian patients with lung cancer. Two hundred and nine biopsy specimens from patients with histologically proven lung cancer and without cancer were analyzed. Each sample was subjected to three parallel PCRs using broad spectrum GP5+/6+ primers and type‐specific (TS) primers for HPV types 16 and 18. Of the 132 lung carcinoma samples, 33 (25%) were positive for HPV16 and/or HPV18 by TS PCR whereas only five (3.8%) samples were HPV positive by consensus PCR. All non‐malignant controls were HPV negative. HPV18 was the more prevalent, being found in 11.4% of samples, followed by HPV16 in 9.1% samples; 4.5% of lesions were positive for both HPV16 and HPV18. HPV16/18 were most prevalent in small cell carcinoma (29.2%) and least prevalent in squamous cell carcinoma (23.3%). HPV was only detected in squamous cell carcinoma and adenosquamous carcinoma by consensus PCR. This study revealed a high HPV16/18 prevalence in lung carcinoma samples from Bulgarian patients when TS PCR was used to detect them. The difference between HPV positivity as detected by consensus and by TS PCR was significant, indicating the importance of methodological issues in explaining the discrepancies between previous studies. HPV18 was more common than HPV16. No association between HPV16/18 status and histopathological diagnosis was identified.

     

In-vitro regeneration of olive tree by somatic embryogenesis

We induced somatic embryogenesis from the cotyledon segments ofOlea europaea (L) cvs. ‘Chetoui’, ‘Chemleli’, and ‘Arbequina’. Calli were established from all three cultvars on OMc media supplemented with IBA and 2i-R The greatest success was obtained with media that contained zero or low concentrations of growth regulators. High levels of hormones (i.e.,>0.5 mgL^-1 IBA and 2i-P) inhibited embryogenesis. Embryos at different maturation stages were observed with continuously proliferating secondary embryogenesis. Abnormally shaped embryos and teratoma were also noted. Four weeks was the optimal incubation period for inducing embryogenesis on the auxin-containing medium. In addition, 30 to 40 gL^-1 sucrose was more effective than glucose in stimulating the growth and maturation of somatic embryos. Embryogeic efficiency was also higher when multivariate combinations of nitrogen sources (inorganic and organic nitrogen forms) were used. The plantlets that were derived from our germinating somatic embryos were similar to those obtained from axillary buds.     

Versican G3 domain modulates breast cancer cell apoptosis: a mechanism for breast cancer cell response to chemotherapy and EGFR therapy

Overexpression of EGFR and versican has been reported in association with breast cancers. Considered oncogenic, these molecules may be attractive therapeutic targets. Possessing anti-apoptotic and drug resistant properties, overexpression of these molecules is accompanied by selective sensitization to the process of apoptosis. In this study, we exogenously expressed a versican G3 construct in breast cancer cell lines and analyzed the effects of G3 on cell viability in fetal bovine serum free conditioned media and evaluated the effects of apoptotic agent C2-ceramide, and chemotherapeutic agents including Docetaxel, Doxorubicin, and Epirubicin. Versican G3 domain enhanced tumor cell resistance to apoptosis when cultured in serum free medium, Doxorubicin, or Epirubicin by up-regulating pERK and GSK-3β (S9P). However, it could be prevented by selective EGFR inhibitor AG 1478 and selective MEK inhibitor PD 98059. Both AG 1478 and PD 98059 enhanced expression of pSAPK/JNK, while selective JNK inhibitor SP 600125 enhanced expression of GSK-3β (S9P). Versican G3 promoted cell apoptosis induced by C2-ceramide or Docetaxel by enhancing expression of pSAPK/JNK and decreasing expression of GSK-3β (S9P), an observation blocked by AG 1478 or SP 6000125. Inhibition of endogenous versican expression by siRNA or reduction of versican G3's expression by linking G3 with 3'UTR prevented G3 modulated cell apoptosis. The dual roles of G3 in modulating breast cancer cell resistance to chemotherapeutic agents may in part explain a potential mechanism for breast cancer cell resistance to chemotherapy and EGFR therapy. The apoptotic effects of chemotherapeutics depend upon the activation and balance of down stream signals in the EGFR pathway. GSK-3β (S9P) appears to function as a key checkpoint in this balance of apoptosis and anti-apoptosis. Investigation and potential consideration of targeting GSK-3β (S9P) merits further study.     

Cytokines Alter IgA1 O-Glycosylation by Dysregulating C1GalT1 and ST6GalNAc-II Enzymes

IgA nephropathy (IgAN), the most common primary glomerulonephritis, is characterized by renal immunodeposits containing IgA1 with galactose-deficient O-glycans (Gd-IgA1). These immunodeposits originate from circulating immune complexes consisting of anti-glycan antibodies bound to Gd-IgA1. As clinical disease onset and activity of IgAN often coincide with mucosal infections and dysregulation of cytokines, we hypothesized that cytokines may affect IgA1 O-glycosylation. We used IgA1-secreting cells derived from the circulation of IgAN patients and healthy controls and assessed whether IgA1 O-glycosylation is altered by cytokines. Of the eight cytokines tested, only IL-6 and, to a lesser degree, IL-4 significantly increased galactose deficiency of IgA1; changes in IgA1 O-glycosylation were robust for the cells from IgAN patients. These cytokines reduced galactosylation of the O-glycan substrate directly via decreased expression of the galactosyltransferase C1GalT1 and, indirectly, via increased expression of the sialyltransferase ST6GalNAc-II, which prevents galactosylation by C1GalT1. These findings were confirmed by siRNA knockdown of the corresponding genes and by in vitro enzyme reactions. In summary, IL-6 and IL-4 accentuated galactose deficiency of IgA1 via coordinated modulation of key glycosyltransferases. These data provide a mechanism explaining increased immune-complex formation and disease exacerbation during mucosal infections in IgAN patients.     

Glycosylation Patterns of HIV-1 gp120 Depend on the Type of Expressing Cells and Affect Antibody Recognition

Human immunodeficiency virus type 1 (HIV-1) entry is mediated by the interaction between a variably glycosylated envelope glycoprotein (gp120) and host-cell receptors. Approximately half of the molecular mass of gp120 is contributed by N-glycans, which serve as potential epitopes and may shield gp120 from immune recognition. The role of gp120 glycans in the host immune response to HIV-1 has not been comprehensively studied at the molecular level. We developed a new approach to characterize cell-specific gp120 glycosylation, the regulation of glycosylation, and the effect of variable glycosylation on antibody reactivity. A model oligomeric gp120 was expressed in different cell types, including cell lines that represent host-infected cells or cells used to produce gp120 for vaccination purposes. N-Glycosylation of gp120 varied, depending on the cell type used for its expression and the metabolic manipulation during expression. The resultant glycosylation included changes in the ratio of high-mannose to complex N-glycans, terminal decoration, and branching. Differential glycosylation of gp120 affected envelope recognition by polyclonal antibodies from the sera of HIV-1-infected subjects. These results indicate that gp120 glycans contribute to antibody reactivity and should be considered in HIV-1 vaccine design.