3-D modelling of chloroplast structure under (Mg) magnesium ion treatment. Relationship between thylakoid membrane arrangement and stacking

We performed for the first time three-dimensional (3D) modelling of the entire chloroplast structure. Stacks of optical slices obtained by confocal laser scanning microscope (CLSM) provided a basis for construction of 3D images of individual chloroplasts. We selected pea (Pisum sativum) and bean (Phaseolus vulgaris) chloroplasts since we found that they differ in thylakoid organization. Pea chloroplasts contain large distinctly separated appressed domains while less distinguished appressed regions are present in bean chloroplasts. Different magnesium ion treatments were used to study thylakoid membrane stacking and arrangement. In pea chloroplasts, as demonstrated by 3D modelling, the increase of magnesium ion concentration changed the degree of membrane appression from wrinkled continuous surface to many distinguished stacked areas and significant increase of the inter-grana area. On the other hand 3D models of bean chloroplasts exhibited similar but less pronounced tendencies towards formation of appressed regions. Additionally, we studied arrangements of thylakoid membranes and chlorophyll-protein complexes by various spectroscopic methods, Fourier-transform infrared spectroscopy (FTIR) among others. Based on microscopic and spectroscopic data we suggested that the range of chloroplast structure alterations under magnesium ions treatment is a consequence of the arrangement of supercomplexes. Moreover, we showed that stacking processes always affect the structural changes of chloroplast as a whole.     

Does the KIR2DS5 Gene Protect from Some Human Diseases?

Background

KIR2DS5 gene encodes an activating natural killer cell receptor whose ligand is not known. It was recently reported to affect the outcome of hematopoietic stem cell transplantation.

Methodology/Principal Findings

In our studies on KIR2DS5 gene associations with human diseases, we compared the frequencies of this gene in patients and relevant controls. Typing for KIR2DS5 gene was performed by either individual or multiplex polymerase chain reactions which, when compared in the same samples, gave concordant results. We noted an apparently protective effect of KIR2DS5 gene presence in several clinical conditions, but not in others. Namely, this effect was observed in ankylosing spondylitis (p = 0.003, odds ratio [OR] = 0.47, confidence interval [CI] = 0.28–0.79), endometriosis (p = 0.03, OR = 0.25, CI = 0.07–0.82) and acute rejection of kidney graft (p = 0.0056, OR = 0.44, CI = 0.24–0.80), but not in non-small-cell lung carcinoma, rheumatoid arthritis, spontaneous abortion, or leukemia (all p>0.05). In addition, the simultaneous presence of KIR2DS5 gene and HLA-C C1 allotype exhibited an even stronger protective effect on ankylosing spondylitis (p = 0.0003, OR = 0.35, CI = 0.19–0.65), whereas a lack of KIR2DS5 and the presence of the HLA-C C2 allotype was associated with ankylosing spondylitis (p = 0.0017, OR = 1.92, CI = 1.28–2.89), whereas a lack of KIR2DS5 and presence of C1 allotype was associated with rheumatoid arthritis (p = 0.005, OR = 1.47, CI = 1.13–1.92). The presence of both KIR2DS5 and C1 seemed to protect from acute kidney graft rejection (p = 0.017, OR = 0.47, CI = 0.25–0.89), whereas lack of KIR2DS5 and presence of C2 seemed to favor rejection (p = 0.0015, OR = 2.13, CI = 1.34–3.37).

Conclusions/Significance

Our results suggest that KIR2DS5 may protect from endometriosis, ankylosing spondylitis, and acute rejection of kidney graft.     

Role of Mast Cells in Inflammatory Bowel Disease and Inflammation-Associated Colorectal Neoplasia in IL-10-Deficient Mice

Background

Inflammatory bowel disease (IBD) is hypothesized to result from stimulation of immune responses against resident intestinal bacteria within a genetically susceptible host. Mast cells may play a critical role in IBD pathogenesis, since they are typically located just beneath the intestinal mucosal barrier and can be activated by bacterial antigens.

Methodology/Principal Findings

This study investigated effects of mast cells on inflammation and associated neoplasia in IBD-susceptible interleukin (IL)-10-deficient mice with and without mast cells. IL-10-deficient mast cells produced more pro-inflammatory cytokines in vitro both constitutively and when triggered, compared with wild type mast cells. However despite this enhanced in vitro response, mast cell-sufficient Il10 ^−/− mice actually had decreased cecal expression of tumor necrosis factor (TNF) and interferon (IFN)-γ mRNA, suggesting that mast cells regulate inflammation in vivo. Mast cell deficiency predisposed Il10 ^−/− mice to the development of spontaneous colitis and resulted in increased intestinal permeability in vivo that preceded the development of colon inflammation. However, mast cell deficiency did not affect the severity of IBD triggered by non-steroidal anti-inflammatory agents (NSAID) exposure or helicobacter infection that also affect intestinal permeability.

Conclusions/Significance

Mast cells thus appear to have a primarily protective role within the colonic microenvironment by enhancing the efficacy of the mucosal barrier. In addition, although mast cells were previously implicated in progression of sporadic colon cancers, mast cells did not affect the incidence or severity of colonic neoplasia in this inflammation-associated model.     

Multiplexed glycoproteomic analysis of glycosylation disorders by sequential yolk immunoglobulins immunoseparation and MALDI-TOF MS

This study applied yolk immunoglobulins immunoaffinity separation and MALDI-TOF MS for clinical proteomics of congenital disorders of glycosylation (CDG) and secondary glycosylation disorders [galactosemia and hereditary fructose intolerance (HFI)]. Serum transferrin (Tf) and alpha1-antitrypsin (AAT) that are markers for CDG, were purified sequentially to obtain high-quality MALDI mass spectra to differentiate single glycoforms of the native intact glycoproteins. The procedure was found feasible for the investigation of protein macroheterogeneity due to glycosylation site underoccupancy then ensuing the characterization of patients with CDG group I (N-glycan assembly disorders). Following PNGase F digestion of the purified glycoprotein, the characterization of protein microheterogeneity by N-glycan MS analysis was performed in a patient with CDG group II (processing disorders). CDG-Ia patients showed a typical profile of underglycosylation where the fully glycosylated glycoforms are always the most abundant present in plasma with lesser amounts of partially and unglycosylated glycoforms in this order. Galactosemia and HFI are potentially fatal diseases, which benefit from early diagnosis and prompt therapeutic intervention. In symptomatic patients with galactosemia and in those with HFI, MALDI MS of Tf and AAT depicts a hypoglycosylation profile with a significant increase of underglycosylated glycoforms that reverses by dietary treatment, representing a clue for diagnosis and treatment monitoring.     

Sulphated flavonoid glycosides from leaves of Atriplex hortensis

Two flavonoid sulphates, i.e. quercetin 3-O-sulphate-7-O-α-arabinopyranoside and kaempferol 3-O-sulphate-7-O-α-arabinopyranoside, were isolated from leaves of Atriplex hortensis L. The structures of these compounds were established by UV, ¹H and ¹³C NMR, 2D NMR and MS spectra. The compounds were isolated for the first time from plant material.     

5S Ribosomal RNA Database

Ribosomal 5S RNA (5S rRNA) is an integral component of the large ribosomal subunit in all known organisms with the exception only of mitochondrial ribosomes of fungi and animals. It is thought to enhance protein synthesis by stabilization of a ribosome structure. This paper presents the updated database of 5S rRNA and their genes (5S rDNA). Its short characteristics are presented in the Introduction. The database contains 2280 primary structures of 5S rRNA and 5S rRNA genes. These include 536 eubacterial, 61 archaebacterial, 1611 eukaryotic and 72 organelle sequences. The database is available on line through the World Wide Web at http://biobases.ibch.poznan.pl/5SData/.     

On the different nature of top-down and bottom-up effects in pelagic food webs

SUMMARY 1. Each individual planktonic plant or animal is exposed to the hazards of starvation and risk of predation, and each planktonic population is under the control of resource limitation from the bottom up (growth and reproduction) and by predation from the top down (mortality). While the bottom-up and top-down impacts are traditionally conceived as compatible with each other, field population-density data on two coexisting Daphnia species suggest that the nature of the two impacts is different. Rates of change, such as the rate of individual body growth, rate of reproduction, and each species' population growth rate, are controlled from the bottom up. State variables, such as biomass, individual body size and population density, are controlled from the top down and are fixed at a specific level regardless of the rate at which they are produced.
2. According to the theory of functional responses, carnivorous and herbivorous predators react to prey density rather than to the rate at which prey are produced or reproduced. The predator's feeding rate (and thus the magnitude of its effect on prey density) should hence be regarded as a functional response to increasing resource concentration.
3. The disparity between the bottom-up and top-down effects is also apparent in individual decision making, where a choice must be made between accepting the hazards of hunger and the risks of predation (lost calories versus loss of life).
4. As long as top-down forces are effective, the disparity with bottom-up effects seems evident. In the absence of predation, however, all efforts of an individual become subordinate to the competition for resources. Biomass becomes limited from the bottom up as soon as the density of a superior competitor has increased to the carrying capacity of a given habitat. Such a shift in the importance of bottom-up control can be seen in zooplankton in habitats from which fish have been excluded.     

The impact of different soil moisture and soil compaction on the growth of triticale root system

Effects of different soil moisture (soil drought and waterlogging) and soil compaction (1.33 and 1.50 g·cm⁻³) on the growth and morphological traits of the root system were studied in four breeding forms and seven cultivars of triticale. Morphological changes, including the restriction of root extension, expansion and proliferation of laterals roots, occur in plants grown in different soil moisture and in compact soil. The investigations comprised quantitative and qualitative analyses of a developed plant root system through determining the number, length and dry matter of the particular components of the root system. Obtained results have demonstrated a relatively broad variation in the habit of the triticale root system. Plants grown under compact soil and low or high soil water content showed a smaller number and less dry matter of lateral branching than plants grown in control conditions. The harmful effects of compact soil and drought conditions on the growth of roots was greater when compared with that of plants exposed to waterlogging. The observed effects of all treatments were more distinct in a drought sensitive strains. The drought resistant forms were a more characterize with extensive rooting and by smaller alterations in the root morphology under the stress conditions compared with drought sensitive one. Results confirm that the breeding forms (CHD-12 and CHD-173) of a high drought susceptibility was found to be also more sensitive to periodical soil water excess. A more efficient water use and a lower shoot to root (S/R) ratio were found to be major reasons for a higher stress resistance of the breeding forms (CHD-220 and CHD-247). The reasons for a different response of the examined breeding forms and cultivars to the conditions of drought or waterlogging may be a more economical water balance and more favourable relations between the shoot and root dimensions in the drought resistant forms and cultivars. The results suggest that the morphological traits of the triticale root system may be used in practice as direct or indirect selection criteria in maize breeding.     

Light-dependent reversal of dark-chilling induced changes in chloroplast structure and arrangement of chlorophyll-protein complexes in bean thylakoid membranes

Changes in chloroplast structure and rearrangement of chlorophyll-protein (CP) complexes were investigated in detached leaves of bean (Phaseolus vulgaris L. cv. Eureka), a chilling-sensitive plant, during 5-day dark-chilling at 1 °C and subsequent 3-h photoactivation under white light (200 μmol photons m⁻² s⁻¹) at 22 °C. Although, no change in chlorophyll (Chl) content and Chl a/b ratio in all samples was observed, overall fluorescence intensity of fluorescence emission and excitation spectra of thylakoid membranes isolated from dark-chilled leaves decreased to about 50%, and remained after photoactivation at 70% of that of the control sample. Concomitantly, the ratio between fluorescence intensities of PSI and PSII (F736/F681) at 120 K increased 1.5-fold upon chilling, and was fully reversed after photoactivation. Moreover, chilling stress seems to induce a decrease of the relative contribution of LHCII fluorescence to the thylakoid emission spectra at 120 K, and an increase of that from LHCI and PSI, correlated with a decrease of stability of LHCI-PSI and LHCII trimers, shown by mild-denaturing electrophoresis. These effects were reversed to a large extent after photoactivation, with the exception of LHCII, which remained partly in the aggregated form. In view of these data, it is likely that dark-chilling stress induces partial disassembly of CP complexes, not completely restorable upon photoactivation. These data are further supported by confocal laser scanning fluorescence microscopy, which showed that regular grana arrangement observed in chloroplasts isolated from control leaves was destroyed by dark-chilling stress, and was partially reconstructed after photoactivation. In line with this, Chl a fluorescence spectra of leaf discs demonstrated that dark-chilling caused a decrease of the quantum yield PSII photochemistry (F_v/F_m) by almost 40% in 5 days. Complete restoration of the photochemical activity of PSII required 9 h post-chilling photoactivation, while only 3 h were needed to reconstruct thylakoid membrane organization and chloroplast structure. The latter demonstrated that the long-term dark-chilled bean leaves started to suffer from photoinhibition after transfer to moderate irradiance and temperature conditions, delaying the recovery of PSII photochemistry, independently of photo-induced reconstruction of PSII complexes.     

Mycobacterium avium Subspecies paratuberculosis Cultured from Locally and Commercially Pasteurized Cow's Milk in the Czech Republic

Between November 2002 and April 2003, 244 bottles and cartons of commercially pasteurized cow's milk were obtained at random from retail outlets throughout the Czech Republic. During the same period, samples of raw milk and of milk that was subsequently subjected to a minimum of 71.7°C for 15 s in a local pasteurization unit were also obtained from two dairy herds, designated herds A and B, with low and high levels, respectively, of subclinical Mycobacterium avium subsp. paratuberculosis infection, and from one herd, herd C, without infection. Infection in individual cows in each herd was tested by fecal culturing. Milk samples were brought to the Veterinary Research Institute in Brno, Czech Republic, processed, inoculated onto Herrold's egg yolk slants, and incubated for 32 weeks. Colonies were characterized by morphology, Ziehl-Neelsen staining, mycobactin J dependency, and IS900 PCR results. M. avium subsp. paratuberculosis was cultured from 4 of 244 units (1.6%) of commercially pasteurized retail milk. M. avium subsp. paratuberculosis was also cultured from 2 of 100 (2%) cartons of locally pasteurized milk derived from infected herds A and B and from 0 of 100 cartons of milk from uninfected herd C. Raw milk from 1 of 10 (10%) fecal culture-positive cows in herd A and from 13 of 66 (19.7%) fecal culture-positive cows in herd B was culture positive for M. avium subsp. paratuberculosis. These findings confirm that M. avium subsp. paratuberculosis is present in raw milk from subclinically infected dairy cows. The culture of M. avium subsp. paratuberculosis in the Czech Republic from retail milk that had been pasteurized locally or commercially to the required national and European Union standards is in agreement with similar research on milk destined for consumers in the United Kingdom and the United States and shows that humans are being exposed to this chronic enteric pathogen by this route.