Patterns of mitochondrial DNA instability in Brassica campestris cultured cells

We previously showed that the mitochondrial DNA (mtDNA) of a Brassica campestris callus culture had undergone extensive rearrangements (i.e. large inversions and a duplication) relative to DNA of the control plant [54]. In this study we observed that after continued growth, the mtDNA of this culture continues to change, with rearranged forms amplifying and diminishing to varying proportions. Strikingly similar changes were detected in the mtDNA profiles of a variety of other long- and short-term callus and cell suspension lines. However, the proportions of parental (unrearranged) and novel (rearranged) forms varied in different cultured cell mtDNAs. To address the source of this heterogeneity, we compared the mtDNA organization of 28 individual plants from the parental seed stock. With the exception of one plant containing high levels of a novel plasmid-like mtDNA molecule, no significant variation was detected among individual plants and therefore source plant variation is unlikely to have contributed to the diversity of mitochondrial genomes observed in cultured cells. The source of this culture-induced heterogeneity was also investigated in 16 clones derived from single protoplasts. A mixed population of unrearranged and rearranged mtDNA molecules was apprent in each protoclone, suggesting that the observed heterogeneity in various cultures might reflect the genomic composition of each individual cell; however, the induction of an intercellular heterogeneity subsequent to the protoplast isolation was not tested and therefore cannot be ruled out. The results of this study support our earlier model that the rapid structural alteration of B. campestris mtDNA in vitro results from preferential amplification and reassortment of minor pre-existing forms of the genome rather than de novo rearrangement. Infrequent recombination between short dispersed repeated elements is proposed as the underlying mechanism for the formation of these minor mtDNA molecules.     

Effect of self-association on the structural organization of partially folded proteins: inactivated actin

The propensity to associate or aggregate is one of the characteristic properties of many nonnative proteins. The aggregation of proteins is responsible for a number of human diseases and is a significant problem in biotechnology. Despite this, little is currently known about the effect of self-association on the structural properties and conformational stability of partially folded protein molecules. G-actin is shown to form equilibrium unfolding intermediate in the vicinity of 1.5 M guanidinium chloride (GdmCl). Refolding from the GdmCl unfolded state is terminated at the stage of formation of the same intermediate state. An analogous form, known as inactivated actin, can be obtained by heat treatment, or at moderate urea concentration, or by the release of Ca(2+). In all cases actin forms specific associates comprising partially folded protein molecules. The structural properties and conformational stability of inactivated actin were studied over a wide range of protein concentrations, and it was established that the process of self-association is rather specific. We have also shown that inactivated actin, being denatured, is characterized by a relatively rigid microenvironment of aromatic residues and exhibits a considerable limitation in the internal mobility of tryptophans. This means that specific self-association can play an important structure-forming role for the partially folded protein molecules.     

Antagonist-mediated down-regulation of toll-like receptors increases the prevalence of human papillomavirus infection in systemic lupus erythematosus

IntroductionPrevalence of an abnormal Papanicolaou smear was significantly increased in lupus patients in cross-sectional studies, associated with a higher prevalence of high-risk human papillomavirus (HPV) infection. The nucleic acid-specific Toll-like receptors (TLRs) locate at the endolysosomal compartments and trigger the induction of cytokines for the innate immune response. This study evaluated whether abnormal host innate immune response in lupus patients may enhance HPV persistence.MethodsProtein levels of TLRs 3, 7, 8 and 9 in cervical epithelial cells of lupus patients and controls with or without HPV infection were assessed using flow cytometry. Characteristics associated with the differential expression of TLRs in systemic lupus erythematosus (SLE) were elucidated. The effect and interferon-stimulated genes (ISGs) (ISG15 and Mx-1) gene expressions were then measured in oncogenic HeLa (HPV18), CaSki (HPV) and C33A (HPV negative) cell lines using flow cytometry and quantitative real-time PCR. Ex vivo productions of cytokines and interferon-gamma (IFN-γ) upon TLR ligands stimulations were subsequently measured using cytometric bead array and ELISA.ResultsFor subjects with HPV infection, levels of TLR3 and TLR7 were significantly lower in lupus patients compared with controls. Significantly decreased TLRs 7, 8 and 9 levels were observed in HPV-negative SLE compared to healthy controls. For SLE with and without HPV infection, TLR7 and 9 levels were significantly lower in infected SLE than those in HPV-negative patients. Independent explanatory variables associated with down-regulation of TLR7 level included HPV infection and a higher cumulative dose of prednisolone; while a higher cumulative dose of hydroxychloroquine and HPV infection were associated with down-regulation of TLR9 level. In cervical cell lines, TLRs 3, 7, 8, 9 protein levels and antiviral ISG15 and Mx-1 gene expressions were inhibited in two oncogenic HPV types. Functional data showed that the induction of pro-inflammatory cytokines by TLR ligands (R837, ssRNA and ODN2395) was greatly impaired in CaSki and HeLa than C33A cells.ConclusionsIn conclusion, prednisolone and TLR antagonist (hydroxychloroquine) may down-regulate protein levels of TLR7 and TLR9 in lupus patients, thereby decreasing the innate immune response against HPV infection. Upon infection, HPV further down-regulate TLR7 and 9 levels for viral persistence. Furthermore, reduction of nucleic acid-sensing TLRs 7, 8 and 9 in carcinogenic HPVs ensures that the expression of inducible pro-inflammatory cytokines is minimized to prevent the expression of antiviral ISGs (ISG15 and Mx-1) on a biologically relevant antiviral response.     

Interactions between charged, uncharged, and zwitterionic bilayers containing phosphatidylglycerol.

Pressure vs. distance relationships have been obtained for phosphatidylglycerol bilayers, in both charged and uncharged states. Water was removed from the lipid multilayers by the application of osmotic pressures in the range of 0-2.7 x 10(9) dyn/cm2, and the distance between adjacent bilayers was obtained from Fourier analysis of lamellar x-ray diffraction data. For phosphatidylglycerol bilayers made electrically neutral either by lowering the pH or by adding equimolar concentrations of the positively charged lipid stearylamine, the pressure-distance data could be fit with a single exponential. The measured decay lengths were 1.1 A at low pH and 1.5 A with stearylamine, which are similar to decay lengths of the hydration pressure found for gel phases of other neutral bilayers. In addition, the magnitude of this repulsive pressure was proportional to the square of the Volta potential (equivalent to the dipole potential for electrically neutral bilayers) measured in monolayers in equilibrium with bilayers, in agreement with results previously found for the hydration pressure between phosphatidylcholine bilayers. For charged phosphatidylglycerol bilayers, the pressure-distance relation had two distinct regions. For bilayer separations greater than 10 A, the pressure-distance data had an exponential decay length (11 A) and a magnitude consistent with that expected for electrostatic repulsion from double-layer theory. For bilayer separations of 2-10 A, the pressure decayed much more rapidly with increasing bilayer separation (decay length less than 1 A). We interpret these data at low bilayer separations in terms of a combination of hydration repulsion and steric hindrance between the lipid head groups and the sodium ions trapped between apposing bilayers.     

The impact of black wattle encroachment of indigenous grasslands on soil carbon,Eastern Cape,South Africa

Black wattle (Acacia mearnsii, De Wild.) is a fast growing tree species introduced into South Africa in the nineteenth century for commercial purposes. While being an important source of timber and firewood for local communities, black wattle is an aggressive invasive species and has pervasive adverse environmental impacts in South Africa. Little is known about the effects of black wattle encroachment on soil carbon, therefore the aim of this study was to investigate the impact of black wattle encroachment of natural grassland on soil carbon stocks and dynamics. Focussing on two sites in the Eastern Cape, South Africa, the study analysed carbon stocks in soil and litter on a chronosequence of black wattle stands of varying ages (up to >50 years) and compared these with adjacent native grassland. The study found that woody encroachment of grassland at one site had an insignificant effect on soil and litter carbon stocks. The second site showed a clear decline in combined soil and litter carbon stocks following wattle encroachment. The lowest stock was in the oldest wattle stand, meaning that carbon stocks are still declining after 50 years of encroachment. The results from the two sites demonstrate the importance of considering changes in soil carbon when evaluating ecosystem effects of invasive species.     

Long-term fertilisation strategies and form affect nutrient budgets and soil test values,soil carbon retention and crop yield resilience

Plant and Soil - The aim of this study was to evaluate the effects of long-term mineral and organic fertilisation on crop performance and soil fertility. The Long-Term Nutrient Depletion Trial...