Changes in dry weight and mineral composition of some oil producing plants over a range of salinity stresses

Using water culture technique, some experiments have been performed to investigate the effect of 60 days salinization treatments (0.0–100 meq 1⁻¹ NaCl) on dry weight and on the content of some nutrient elements (Na, K, Ca, Mg, P, N) in castor bean, sunflower and flax plants. In general the content of sodium increased progressively with the rise of salinity level. The relatively low and moderate salinization levels (20 and 40 meq I⁻¹ NaCl) resulted in a promotion rather than inhibition of the dry weight and in the content of most of the investigated elements in the different organs of the test plants. However with the rise of salinization level from 60 to 100 meq l⁻¹, the dry weight and the content of these nutrient elements were mostly reduced.     

Two-photon fluorescence spectroscopy and microscopy of NAD(P)H and flavoprotein

Two-photon (2P) ratiometric redox fluorometry and microscopy of pyridine nucleotide (NAD(P)H) and flavoprotein (FP) fluorescence, at 800-nm excitation, has been demonstrated as a function of mitochondrial metabolic states in isolated adult dog cardiomyocytes. We have measured the 2P-excitation spectra of NAD(P)H, flavin adenine dinucleotide (FAD), and lipoamide dehydrogenase (LipDH) over the wavelength range of 720-1000 nm. The 2P-excitation action cross sections (sigma2P) increase rapidly at wavelengths below 800 nm, and the maximum sigma2P of LipDH is approximately 5 and 12 times larger than those of FAD and NAD(P)H, respectively. Only FAD and LipDH can be efficiently excited at wavelengths above 800 nm with a broad 2P-excitation band around 900 nm. Two autofluorescence spectral regions (i.e., approximately 410-490 nm and approximately 510-650 nm) of isolated cardiomyocytes were imaged using 2P-laser scanning microscopy. At 750-nm excitation, fluorescence of both regions is dominated by NAD(P)H emission, as indicated by fluorescence intensity changes induced by mitochondrial inhibitor NaCN and mitochondria uncoupler carbonyl cyanide p-(trifluoromethoxy) phenyl hydrazone (FCCP). In contrast, 2P-FP fluorescence dominates at 900-nm excitation, which is in agreement with the sigma2P measurements. Finally, 2P-autofluorescence emission spectra of single cardiac cells have been obtained, with results suggesting potential for substantial improvement of the proposed 2P-ratiometric technique.     

‘Tethering’ fragment-based drug discovery to identify inhibitors of the essential respiratory membrane protein type II NADH dehydrogenase

Energy generation is a promising area of drug discovery for both bacterial pathogens and parasites. Type II NADH dehydrogenase (NDH-2), a vital respiratory membrane protein, has attracted attention as a target for the development of new antitubercular and antimalarial agents. To date, however, no potent, specific inhibitors have been identified. Here, we performed a site-directed screening technique, tethering-fragment based drug discovery, against wild-type and mutant forms of NDH-2 containing engineered active-site cysteines. Inhibitory fragments displayed IC₅₀ values between 3 and 110?μM against NDH-2 mutants. Possible binding poses were investigated by in silico modelling, providing a basis for optimisation of fragment binding and improved potency against NDH-2.     

Inflammation-induced reactive nitrogen species cause proteasomal degradation of dimeric peroxiredoxin-1 in a mouse macrophage cell line

Peroxiredoxin 1 (PRDX1) is an antioxidant enzyme that, when secreted, can act as a proinflammatory signal. Here we studied the regulation of intracellular PRDX1 by lipopolysaccharide (LPS) and interferon-gamma (IFN-γ) in the RAW 264.7 mouse macrophage cell line. While LPS or IFN-γ alone did not affect PRDX1 protein levels, their combination led to an almost complete loss of the PRDX1 dimer. This was likely mediated by the increased production of nitric oxide (NO) as it was reversed by the NO synthase inhibitor L-N-methylarginine (L-NMMA), while a NO-releasing agent decreased PRDX1 levels. Inhibition of the proteasome with MG132 also prevented the loss of the PRDX1 dimer, suggesting that the decrease is due to a NO-activated proteasomal degradation pathway. By contrast with the decrease in protein levels, LPS increased PRDX1 mRNA and this effect was amplified by IFN-γ. Two other Nrf2 target genes, thioredoxin reductase (TXNRD1) and haem oxygenase (HMOX1), were also induced by LPS but IFN-γ did not increase their expression further. This study shows that inflammation differentially regulates PRDX1 at the levels of protein stability and gene expression, and that NO plays a key role in this mechanism.     

Germline Mutations in NFKB2 Implicate the Noncanonical NF-κB Pathway in the Pathogenesis of Common Variable Immunodeficiency

Common variable immunodeficiency (CVID) is a heterogeneous disorder characterized by antibody deficiency, poor humoral response to antigens, and recurrent infections. To investigate the molecular cause of CVID, we carried out exome sequence analysis of a family diagnosed with CVID and identified a heterozygous frameshift mutation, c.2564delA (p.Lys855Serfs^∗7), in NFKB2 affecting the C terminus of NF-κB2 (also known as p100/p52 or p100/p49). Subsequent screening of NFKB2 in 33 unrelated CVID-affected individuals uncovered a second heterozygous nonsense mutation, c.2557C>T (p.Arg853^∗), in one simplex case. Affected individuals in both families presented with an unusual combination of childhood-onset hypogammaglobulinemia with recurrent infections, autoimmune features, and adrenal insufficiency. NF-κB2 is the principal protein involved in the noncanonical NF-κB pathway, is evolutionarily conserved, and functions in peripheral lymphoid organ development, B cell development, and antibody production. In addition, Nfkb2 mouse models demonstrate a CVID-like phenotype with hypogammaglobulinemia and poor humoral response to antigens. Immunoblot analysis and immunofluorescence microscopy of transformed B cells from affected individuals show that the NFKB2 mutations affect phosphorylation and proteasomal processing of p100 and, ultimately, p52 nuclear translocation. These findings describe germline mutations in NFKB2 and establish the noncanonical NF-κB signaling pathway as a genetic etiology for this primary immunodeficiency syndrome.     

Bacillus cereus biofilm formation on central venous catheters of hospitalised cardiac patients

Formation of bacterial biofilms is a risk with many in situ medical devices. Biofilm-forming Bacillus species are associated with potentially life-threatening catheter-related blood stream infections in immunocompromised patients. Here, bacteria were isolated from biofilm-like structures within the lumen of central venous catheters (CVCs) from two patients admitted to cardiac hospital wards. Isolates belonged to the Bacillus cereus group, exhibited strong biofilm formation propensity, and mapped phylogenetically close to the B. cereus emetic cluster. Together, whole genome sequencing and quantitative PCR confirmed that the isolates constituted the same strain and possessed a range of genes important for and up-regulated during biofilm formation. Antimicrobial susceptibility testing demonstrated resistance to trimethoprim-sulphamethoxazole, clindamycin, penicillin and ampicillin. Inspection of the genome revealed several chromosomal β-lactamase genes and a sulphonamide resistant variant of folP. This study clearly shows that B. cereus persisting in hospital ward environments may constitute a risk factor from repeated contamination of CVCs.     

Quantitative analysis of the fluorescence properties of intrinsically fluorescent proteins in living cells

The main potential of intrinsically fluorescent proteins (IFPs), as noninvasive and site-specific markers, lies in biological applications such as intracellular visualization and molecular genetics. However, photophysical studies of IFPs have been carried out mainly in aqueous solution. Here, we provide a comprehensive analysis of the intracellular environmental effects on the steady-state spectroscopy and excited-state dynamics of green (EGFP) and red (DsRed) fluorescent proteins, using both one- and two-photon excitation. EGFP and DsRed are expressed either in the cytoplasm of rat basophilic leukemia (RBL-2H3) mucosal mast cells or anchored (via LynB protein) to the inner leaflet of the plasma membrane. The fluorescence lifetimes (within approximately 10%) and spectra in live cells are basically the same as in aqueous solution, which indicate the absence of both IFP aggregation and cellular environmental effects on the protein folding under our experimental conditions. However, comparative time-resolved anisotropy measurements of EGFP reveal a cytoplasmic viscosity 2.5 +/- 0.3 times larger than that of aqueous solution at room temperature, and also provide some insights into the LynB-EGFP structure and the heterogeneity of the cytoplasmic viscosity. Further, the oligomer configuration and internal depolarization of DsRed, previously observed in solution, persists upon expression in these cells. DsRed also undergoes an instantaneous three-photon induced color change under 740-nm excitation, with efficiently nonradiative green species. These results confirm the implicit assumption that in vitro fluorescence properties of IFPs are essentially valid for in vivo applications, presumably due to the beta-barrel protection of the embodied chromophore. We also discuss the relevance of LynB-EGFP anisotropy for specialized domains studies in plasma membranes.     

Conformational dependence of intracellular NADH on metabolic state revealed by associated fluorescence anisotropy

Global analysis of fluorescence and associated anisotropy decays of intrinsic tissue fluorescence offers a sensitive and non-invasive probe of the metabolically critical free/enzyme-bound states of intracellular NADH in neural tissue. Using this technique, we demonstrate that the response of NADH to the metabolic transition from normoxia to hypoxia is more complex than a simple increase in NADH concentration. The concentration of free NADH, and that of an enzyme bound form with a relatively low lifetime, increases preferentially over that of other enzyme bound NADH species. Concomitantly, the intracellular viscosity is reduced, likely due to the osmotic swelling of mitochondria. These conformation and environmental changes effectively decrease the tissue fluorescence average lifetime, causing the usual total fluorescence increase measurements to significantly underestimate the calculated concentration increase. This new discrimination of changes in NADH concentration, conformation, and environment provides the foundation for quantitative functional imaging of neural energy metabolism.     

Molecular perspective of antigen-mediated mast cell signaling

Antigen-mediated cross-linking of the high affinity receptor for IgE (Fc epsilon RI), in the plasma membrane of mast cells, is the first step in the allergic immune response. This event triggers the phosphorylation of specific tyrosines in the cytoplasmic segments of the beta and gamma subunits of Fc epsilon RI by the Src tyrosine kinase Lyn, which is anchored to the inner leaflet of the plasma membrane. Lyn-induced phosphorylation of Fc epsilon RI occurs in a cholesterol-dependent manner, leading to the hypothesis that cholesterol-rich domains, or "lipid rafts," may act as functional platforms for IgE receptor signaling. Testing this hypothesis under physiological conditions remains challenging because of the notion that these functional domains are likely transient and much smaller than the diffraction limit of optical microscopy. Here we use ultrafast fluorescence dynamics to investigate the correlation between nanostructural changes in the plasma membrane (labeled with 1,1'-dioctadecyl-3,3,3',3'-tetramethyl-indocarbocyanine (diI-C18)) and IgE-Fc epsilon RI cross-linking in adherent RBL mast cells stimulated with multivalent antigen. Time-dependent two-photon fluorescence lifetime imaging microscopy of diI-C18 shows changes in lifetime that agree with the kinetics of stimulated tyrosine phosphorylation of Fc epsilon RI, the first identifiable biochemical step of the allergic response, under the same conditions. In addition, two-photon fluorescence lifetime imaging microscopy of Alexa Fluor 488-labeled IgE indicates that F?rster resonance energy transfer occurs with diI-C18 in the plasma membrane. Our live cell studies provide direct evidence for the association of IgE-Fc epsilon RI with specialized cholesterol-rich domains within approximately 4-nm proximity and with an energy transfer efficiency of 0.22 +/- 0.01 at maximal association during IgE receptor signaling.     

The stabilisation of purified, reconstituted P-glycoprotein by freeze drying with disaccharides

The drug efflux pump P-glycoprotein (P-gp) (ABCB1) confers multidrug resistance, a major cause of failure in the chemotherapy of tumours, exacerbated by a shortage of potent and selective inhibitors. A high throughput assay using purified P-gp to screen and characterise potential inhibitors would greatly accelerate their development. However, long-term stability of purified reconstituted ABCB1 can only be reliably achieved with storage at −80 °C. For example, at 20 °C, the activity of ABCB1 was abrogated with a half-life of <1 day. The aim of this investigation was to stabilise purified, reconstituted ABCB1 to enable storage at higher temperatures and thereby enable design of a high throughput assay system. The ABCB1 purification procedure was optimised to allow successful freeze drying by substitution of glycerol with the disaccharides trehalose or maltose. Addition of disaccharides resulted in ATPase activity being retained immediately following lyophilisation with no significant difference between the two disaccharides. However, during storage trehalose preserved ATPase activity for several months regardless of the temperature (e.g. 60% retention at 150 days), whereas ATPase activity in maltose purified P-gp was affected by both storage time and temperature. The data provide an effective mechanism for the production of resilient purified, reconstituted ABCB1.