Oxidative stress and physiological,epigenetic and genetic variability in plant tissue culture: implications for micropropagators and genetic engineers

A number of well defined problems in physiological, epigenetic and genetic quality are associated with the culture of plant cell, tissue and organs in vitro, namely, absence or loss of organogenic potential (recalcitrance), hyperhydricity (`vitrification') and somaclonal variation. These broad terms are used to describe complex phenomena that are known to be genotype and environment dependent. These phenomena affect the practical application of plant tissue culture in plant propagation and in plant genetic manipulation. Here it is hypothesised much of the variability expressed in microplants may be the consequence of, or related to, oxidative stress damage caused to the plant tissues during explant preparation, and in culture, due to media and environmental factors. The characteristics of these phenomena are described and causes discussed in terms of the known effects of oxidative stress on eukaryote genomes. Parameters to characterise the phenomena are described and methods to remediate the causes proposed. This revised version was published online in June 2006 with corrections to the Cover Date.     

Microbiological Study of Lactic Acid Fermentation of Caper Berries by Molecular and Culture-Dependent Methods

Fermentation of capers (the fruits of Capparis sp.) was studied by molecular and culture-independent methods. A lactic acid fermentation occurred following immersion of caper berries in water, resulting in fast acidification and development of the organoleptic properties typical of this fermented food. A collection of 133 isolates obtained at different times of fermentation was reduced to 75 after randomly amplified polymorphic DNA (RAPD)-PCR analysis. Isolates were identified by PCR or 16S rRNA gene sequencing as Lactobacillus plantarum (37 isolates), Lactobacillus paraplantarum (1 isolate), Lactobacillus pentosus (5 isolates), Lactobacillus brevis (9 isolates), Lactobacillus fermentum (6 isolates), Pediococcus pentosaceus (14 isolates), Pediococcus acidilactici (1 isolate), and Enterococcus faecium (2 isolates). Cluster analysis of RAPD-PCR patterns revealed a high degree of diversity among lactobacilli (with four major groups and five subgroups), while pediococci clustered in two closely related groups. A culture-independent analysis of fermentation samples by temporal temperature gradient electrophoresis (TTGE) also indicated that L. plantarum is the predominant species in this fermentation, in agreement with culture-dependent results. The distribution of L. brevis and L. fermentum in samples was also determined by TTGE, but identification of Pediococcus at the species level was not possible. TTGE also allowed a more precise estimation of the distribution of E. faecium, and the detection of Enterococcus casseliflavus (which was not revealed by the culture-dependent analysis). Results from this study indicate that complementary data from molecular and culture-dependent analysis provide a more accurate determination of the microbial community dynamics during caper fermentation.     

Mycobacterial Ser/Thr protein kinases and phosphatases: physiological roles and therapeutic potential

Reversible protein phosphorylation is a major regulation mechanism of fundamental biological processes, not only in eukaryotes but also in bacteria. A growing body of evidence suggests that Ser/Thr phosphorylation play important roles in the physiology and virulence of Mycobacterium tuberculosis, the etiological agent of tuberculosis. This pathogen uses 'eukaryotic-like' Ser/Thr protein kinases and phosphatases not only to regulate many intracellular metabolic processes, but also to interfere with signaling pathways of the infected host cell. Disrupting such processes by means of selective inhibitors may thus provide new pharmaceutical weapons to combat the disease. Here we review the current knowledge on Ser/Thr protein kinases and phosphatases in M. tuberculosis, their regulation mechanisms and putative substrates, and we explore their therapeutic potential as possible targets for the development of new anti-mycobacterial compounds.     

The versatility of mitochondrial calcium signals: from stimulation of cell metabolism to induction of cell death

Both the contribution of mitochondria to intracellular calcium (Ca(2+)) signalling and the role of mitochondrial Ca(2+) uptake in shaping the cytoplasmic response and controlling mitochondrial function are areas of intense investigation. These studies rely on the appropriate use of emerging techniques coupled with judicious data interpretation to a large extent. The development of targeted probes based on the molecular engineering of luminescent proteins has allowed the specific measurement of Ca(2+) concentration ([Ca(2+)]) and adenosine trisphosphate concentration ([ATP]) in intracellular organelles or cytoplasmic subdomains. This approach has given novel information on different aspects of mitochondrial homeostasis.     

Maternal hypoxia activates endovascular trophoblast cell invasion

Oxygen is a critical regulator of placentation. Early placental development occurs in a predominantly low oxygen environment and is, at least partially, under the control of hypoxia signaling pathways. In the present study, in vivo hypobaric hypoxia was used as an experimental tool to delineate hypoxia-sensitive events during placentation. Pregnant rats were exposed to the equivalent of 11% oxygen between days 6.5 and 13.5 of gestation. Pair-fed pregnant animals exposed to ambient conditions were included as a control group. Uterine mesometrial blood vessels in the hypoxia-exposed animals were greatly expanded and some contained large cuboidal cells that were positive for cytokeratin and other markers characteristic of invasive trophoblast cells. Unlike later in gestation, the route of trophoblast cell invasion in the hypoxia-exposed animals was restricted to endovascular, with no interstitial invasion observed. Hypoxia-activated endovascular trophoblast invasion required exposure to hypoxia from gestation day 8.5 to day 9.5. Activation of the invasive trophoblast lineage was also associated with an enlargement of the junctional zone of the chorioallantoic placenta, a source of invasive trophoblast cell progenitors. In summary, maternal hypoxia during early stages of placentation activates the invasive endovascular trophoblast cell lineage and promotes uterine vascular remodeling.     

Shrimp thioredoxin is a potent antioxidant protein

Thioredoxin (TRX) is a main component of the redox homeostasis machinery in the cell and it is required for ribonucleotide reductase function among others. In invertebrates, the redox balance is compromised during disease and changes in the physiological state and it is one of the components of the innate immune response. In this work, the shrimp (Litopenaeus vannamei) LvTRX cDNA was sequenced, cloned and over-expressed in bacteria to further characterize the function of the recombinant protein. LvTRX was able to reduce insulin disulfides and it was a better antioxidant compared to reduced glutathione and ascorbic acid, by means of the Trolox Equivalent Antioxidant Capacity (TEAC) assay. Interestingly, LvTRX contains aside of the canonical active site CXXC disulfide motif, one Cys (C73) residue in the interface of a putative dimer previously reported for human TRX. Using qRT-PCR, we found that shrimp LvTRX is mainly expressed in gills and pleopods; the variation of LvTRX mRNA upon hypoxia and re-oxygenation is not statistically significant. LvTRX stands as an important antioxidant that must be considered in future physiological and immune challenges studies.     

Studies on the reactivity of organometallic Ru-, Rh- and Os-pta complexes with DNA model compounds

The reactions of arene–metal complexes (arene = p-cymene, benzene or pentamethylcyclopentadienyl, metal = Ru, Rh or Os), including 1,3,5-triaza-7-phosphatricyclo-[3.3.1.1]decanephosphine (pta) and chloro co-ligands, with 9-methylguanine, adenine, and a series of nucleosides were studied in water to ascertain the binding modes. The products were characterized by NMR spectroscopy and electrospray ionization mass spectrometry (ESI-MS). Tandem mass spectrometry was found to provide excellent information on preferential binding sites. In general, the N7 position on guanine (the most basic site) was found to be the preferred donor atom for coordination to the metal complexes. The X-ray structures of the precursor complexes, [(η⁵-C₁₀H₁₅)RhCl(pta-Me)₂]Cl₂, [(η⁶-C₁₀H₁₄)OsCl(pta)₂]Cl, and [(η⁶-C₆H₆)OsCl₂(CH₃CN)], are also reported.     

Synthesis, biological evaluation, structural-activity relationship, and docking study for a series of benzoxepin-derived estrogen receptor modulators

The estrogen receptors ERalpha and ERbeta are recognized as important pharmaceutical targets for a variety of diseases including osteoporosis and breast cancer. A series of novel benzoxepin-derived compounds are described as potent selective modulators of the human estrogen receptor modulators (SERMs). We report the antiproliferative effects of these compounds on human MCF-7 breast tumor cells. These heterocyclic compounds contain the triarylethylene arrangement as exemplified by tamoxifen, conformationally restrained through the incorporation of the benzoxepin ring system. The compounds demonstrate potency at nanomolar concentrations in antiproliferative assays against an MCF-7 human breast cancer cell line with low cytotoxicity together with low nanomolar binding affinity for the estrogen receptor. The compounds also demonstrate potent antiestrogenic properties in the human uterine Ishikawa cell line. The effect of a number of functional group substitutions on the ER binding properties of the benzoxepin molecular scaffold is examined through a detailed docking and 2D-QSAR computational investigation. The best QSAR model developed for ERalphabeta selectivity yielded R(2) of 0.84 with an RMSE for the training set of 0.30. The predictive quality of the model was Q(2) of 0.72 and RMSE of 0.18 for the test set. One particular compound bearing a 4-fluoro substituent, exhibits 15-fold selectivity for ERbeta and both our docking and QSAR studies converge on the correlation between enhanced lipophilicity and enhanced ERbeta binding for this benzoxepin ring scaffold.     

Pyrazino[1,2-b]isoquinolines: synthesis and study of their cytostatic and cytotoxic properties

The in vitro antitumor potential of novel pyrazino[1,2-b]-isoquinoline-4-ones that contain a half portion of significant natural products was explored in three cancer cell lines: MDA-MB 231 human breast carcinoma, A-549 human lung carcinoma, and HT-29 human colon carcinoma. In general, these compounds show mid to low muM GI(50)s, but LC(50)s over 100 microM with the exceptions of compounds 3b and 31 that are moderately toxic in all cell lines, while compound 4a is highly toxic and selective for HT-29 cells with LC(50) values in the high nanomolar range. Experiments directed to elucidate possible mechanisms of action with compounds 3a, 29, and 31 showed that compound 3a is able to efficiently induce apoptosis triggered directly from the G2/M phase of cell cycle, while compounds 29 and 31 are potentially cytostatic agents that induce the G1/S arrest of cell cycle. All three compounds do not act through DNA damage, since they do not activate this signaling at the level of sensors, transducers, and executers. Furthermore, the apoptosis induction of 3a is not mediated by activation of pro-apoptotic kinases JNK and p38 or by activation of AKT.