Combined effects of NaCl and Cd2+ stress on the photosynthetic apparatus of Thellungiella salsuginea

Plants show complex responses to abiotic stress while, the effect of the stress combinations can be different to those seen when each stress is applied individually. Here, we report on the effects of salt and/or cadmium on photosynthetic apparatus of Thellungiella salsuginea. Our results showed a considerable reduction of plant growth with some symptoms of toxicity, especially with cadmium treatment. The structural integrity of both photosystems (PSI and PSII) was mostly maintained under salt stress. Cadmium induced a considerable decrease of both PSI and PSII quantum yields and the electron transport rate ETR(I) and ETR(II) paralleled by an increase of non-photochemical quenching (NPQ). In addition, cadmium alone affects the rate of primary photochemistry by an increase of fluorescence at O-J phase and also the photo-electrochemical quenching at J-I phase. A positive L-band appeared with (Cd) treatment as an indicator of lower PSII connectivity, and a positive K-band reflecting the imbalance in number of electrons at donor and acceptor side. In continuity to our previous studies which showed that NaCl supply reduced Cd²⁺ uptake and limited its accumulation in shoot of divers halophyte species, here as a consequence, we demonstrated the NaCl-induced enhancement effect of Cd²⁺ toxicity on the PSII activity by maintaining the photosynthetic electron transport chain as evidenced by the differences in ψ_O, φ_Eo, ABS/RC and TR₀/RC and by improvement of performance index PI_(ABS), especially after short time of treatment. A significant decrease of LHCII, D1 and CP47 amounts was detected under (Cd) treatment. However, NaCl supply alleviates the Cd²⁺ effect on protein abundance including LHCII and PSII core complex (D1 and CP47).     

Overexpression of the triose phosphate translocator (TPT) complements the abnormal metabolism and development of plastidial glycolytic glyceraldehyde‐3‐phosphate dehydrogenase mutants

The presence of two glycolytic pathways working in parallel in plastids and cytosol has complicated the understanding of this essential process in plant cells, especially the integration of the plastidial pathway into the metabolism of heterotrophic and autotrophic organs. It is assumed that this integration is achieved by transport systems, which exchange glycolytic intermediates across plastidial membranes. However, it is unknown whether plastidial and cytosolic pools of 3‐phosphoglycerate (3‐PGA) can equilibrate in non‐photosynthetic tissues. To resolve this question, we employed Arabidopsis mutants of the plastidial glycolytic isoforms of glyceraldehyde‐3‐phosphate dehydrogenase (GAPCp) that express the triose phosphate translocator (TPT) under the control of the 35S (35S:TPT) or the native GAPCp1 (GAPCp1:TPT) promoters. TPT expression under the control of both promoters complemented the vegetative developmental defects and metabolic disorders of the GAPCp double mutants (gapcp1gapcp2). However, as the 35S is poorly expressed in the tapetum, full vegetative and reproductive complementation of gapcp1gapcp2 was achieved only by transforming this mutant with the GAPCp1:TPT construct. Our results indicate that the main function of GAPCp is to supply 3‐PGA for anabolic pathways in plastids of heterotrophic cells and suggest that the plastidial glycolysis may contribute to fatty acid biosynthesis in seeds. They also suggest a 3‐PGA deficiency in the plastids of gapcp1gapcp2, and that 3‐PGA pools between cytosol and plastid do not equilibrate in heterotrophic cells.     

Identification of C-terminal Phosphorylation Sites of N-Formyl Peptide Receptor-1 (FPR1) in Human Blood Neutrophils

Accumulation, activation, and control of neutrophils at inflammation sites is partly driven by N-formyl peptide chemoattractant receptors (FPRs). Occupancy of these G-protein-coupled receptors by formyl peptides has been shown to induce regulatory phosphorylation of cytoplasmic serine/threonine amino acid residues in heterologously expressed recombinant receptors, but the biochemistry of these modifications in primary human neutrophils remains relatively unstudied. FPR1 and FPR2 were partially immunopurified using antibodies that recognize both receptors (NFPRa) or unphosphorylated FPR1 (NFPRb) in dodecylmaltoside extracts of unstimulated and N-formyl-Met-Leu-Phe (fMLF) + cytochalasin B-stimulated neutrophils or their membrane fractions. After deglycosylation and separation by SDS-PAGE, excised Coomassie Blue-staining bands (∼34,000 M_r) were tryptically digested, and FPR1, phospho-FPR1, and FPR2 content was confirmed by peptide mass spectrometry. C-terminal FPR1 peptides (Leu³¹²–Arg³²² and Arg³²³–Lys³⁵⁰) and extracellular FPR1 peptide (Ile¹⁹¹–Arg²⁰¹) as well as three similarly placed FPR2 peptides were identified in unstimulated and fMLF + cytochalasin B-stimulated samples. LC/MS/MS identified seven isoforms of Ala³²³–Lys³⁵⁰ only in the fMLF + cytochalasin B-stimulated sample. These were individually phosphorylated at Thr³²⁵, Ser³²⁸, Thr³²⁹, Thr³³¹, Ser³³², Thr³³⁴, and Thr³³⁹. No phospho-FPR2 peptides were detected. Cytochalasin B treatment of neutrophils decreased the sensitivity of fMLF-dependent NFPRb recognition 2-fold, from EC₅₀ = 33 ± 8 to 74 ± 21 nm. Our results suggest that 1) partial immunopurification, deglycosylation, and SDS-PAGE separation of FPRs is sufficient to identify C-terminal FPR1 Ser/Thr phosphorylations by LC/MS/MS; 2) kinases/phosphatases activated in fMLF/cytochalasin B-stimulated neutrophils produce multiple C-terminal tail FPR1 Ser/Thr phosphorylations but have little effect on corresponding FPR2 sites; and 3) the extent of FPR1 phosphorylation can be monitored with C-terminal tail FPR1-phosphospecific antibodies.     

Essential role of protein kinase C delta in platelet signaling, alpha IIb beta 3 activation, and thromboxane A2 release

The protein kinase C (PKC) family is an essential signaling mediator in platelet activation and aggregation. However, the relative importance of the major platelet PKC isoforms and their downstream effectors in platelet signaling and function remain unclear. Using isolated human platelets, we report that PKCdelta, but not PKCalpha or PKCbeta, is required for collagen-induced phospholipase C-dependent signaling, activation of alpha(IIb)beta(3), and platelet aggregation. Analysis of PKCdelta phosphorylation and translocation to the membrane following activation by both collagen and thrombin indicates that it is positively regulated by alpha(IIb)beta(3) outside-in signaling. Moreover, PKCdelta triggers activation of the mitogen-activated protein kinase-kinase (MEK)/extracellular-signal regulated kinase (ERK) and the p38 MAPK signaling. This leads to the subsequent release of thromboxane A(2), which is essential for collagen-induced but not thrombin-induced platelet activation and aggregation. This study adds new insight to the role of PKCs in platelet function, where PKCdelta signaling, via the MEK/ERK and p38 MAPK pathways, is required for the secretion of thromboxane A(2).     

Mutations in CNNM4 Cause Jalili Syndrome, Consisting of Autosomal-Recessive Cone-Rod Dystrophy and Amelogenesis Imperfecta

The combination of recessively inherited cone-rod dystrophy (CRD) and amelogenesis imperfecta (AI) was first reported by Jalili and Smith in 1988 in a family subsequently linked to a locus on chromosome 2q11, and it has since been reported in a second small family. We have identified five further ethnically diverse families cosegregating CRD and AI. Phenotypic characterization of teeth and visual function in the published and new families reveals a consistent syndrome in all seven families, and all link or are consistent with linkage to 2q11, confirming the existence of a genetically homogenous condition that we now propose to call Jalili syndrome. Using a positional-candidate approach, we have identified mutations in the CNNM4 gene, encoding a putative metal transporter, accounting for the condition in all seven families. Nine mutations are described in all, three missense, three terminations, two large deletions, and a single base insertion. We confirmed expression of Cnnm4 in the neural retina and in ameloblasts in the developing tooth, suggesting a hitherto unknown connection between tooth biomineralization and retinal function. The identification of CNNM4 as the causative gene for Jalili syndrome, characterized by syndromic CRD with AI, has the potential to provide new insights into the roles of metal transport in visual function and biomineralization.     

An Assay for Measuring the Activity of Escherichia coli Inducible Lysine Decarboxyase

Escherichia coli is an enteric bacterium that is capable of growing over a wide range of pH values (pH 5 - 9)¹ and, incredibly, is able to survive extreme acid stresses including passage through the mammalian stomach where the pH can fall to as low as pH 1 - 2². To enable such a broad range of acidic pH survival, E. coli possesses four different inducible amino acid decarboxylases that decarboxylate their substrate amino acids in a proton-dependent manner thus raising the internal pH. The decarboxylases include the glutamic acid decarboxylases GadA and GadB³, the arginine decarboxylase AdiA⁴, the lysine decarboxylase LdcI^{5, 6} and the ornithine decarboxylase SpeF⁷. All of these enzymes utilize pyridoxal-5''-phospate as a co-factor⁸ and function together with inner-membrane substrate-product antiporters that remove decarboxylation products to the external medium in exchange for fresh substrate². In the case of LdcI, the lysine-cadaverine antiporter is called CadB. Recently, we determined the X-ray crystal structure of LdcI to 2.0 Å, and we discovered a novel small-molecule bound to LdcI the stringent response regulator guanosine 5''-diphosphate,3''-diphosphate (ppGpp) ¹⁴. The stringent response occurs when exponentially growing cells experience nutrient deprivation or one of a number of other stresses⁹. As a result, cells produce ppGpp which leads to a signaling cascade culminating in the shift from exponential growth to stationary phase growth¹⁰. We have demonstrated that ppGpp is a specific inhibitor of LdcI ¹⁴. Here we describe the lysine decarboxylase assay, modified from the assay developed by Phan et al.¹¹, that we have used to determine the activity of LdcI and the effect of pppGpp/ppGpp on that activity. The LdcI decarboxylation reaction removes the α-carboxy group of L-lysine and produces carbon dioxide and the polyamine cadaverine (1,5-diaminopentane)⁵. L-lysine and cadaverine can be reacted with 2,4,6-trinitrobenzensulfonic acid (TNBS) at high pH to generate N,N''-bistrinitrophenylcadaverine (TNP-cadaverine) and N,N′-bistrinitrophenyllysine (TNP-lysine), respectively¹¹. The TNP-cadaverine can be separated from the TNP-lysine as the former is soluble in organic solvents such as toluene while the latter is not (See Figure 1). The linear range of the assay was determined empirically using purified cadaverine.Download video file.^{(50M, mov)}     

Screening of natural products for therapeutic activity against solid tumors

Most of the currently used cancer therapeutics are natural products. These agents were generally discovered based on their toxicity to tumour cells using various bioassays. Although the exact mechanisms of action of the most commonly used cancer therapeutics such as anthracyclins, podophyllotoxins and camptothecin are incompletely understood, it is becoming increasingly clear that these agents often show complex modes of action at the cellular level, interacting with numerous targets. Such complex modes of action may be the very reason for clinical efficacy. For discovering new cytotoxic anticancer drugs sophisticated screening methods were used. The principles of such screening projects conducted, using collections of purified natural products or extracts from plants have been described. By performing simple but robust prescreening tests such as the brine shrimp assay, bioactive extracts can be identified. Extracts (65) prepared from a collection of Egyptian plants were identified that showed cytotoxity on HepG2 cells. Interestingly, 22 (33%) of these raw extracts, induced > 2-fold induction of caspase-cleavage activity in a colon carcinoma cell line, consistent with induction of apoptosis. Only a fraction of the diversity of the biosphere has been tested for biological activity and novel cancer therapeutics remains to be discovered.     

Intron 1 mediated regulation of bovine prion protein gene expression: Role of donor splicing sites, sequences with potential enhancer and suppressor activities

Prion protein plays a key role in the pathogenesis of transmissible spongiform encephalopathies. Because changes in expression of the prion protein gene (PRNP) alter the incubation time and severity of prion diseases. Our previous work revealed a strong association between the promoter (spanning base pairs (bp) −88 to −30) and intron 1 (spanning bp +114 to +892) that leads to optimum expression of the bovine PRNP. Here, we employed two mutation analysis strategies (deletion and insertion) and two reporter assay systems (luciferase and GFP expression) to define the regulatory domains within intron 1 and further elucidate its role in regulating the promoter activity of the bovine prion protein gene. We identified DNA sequences with potential suppressor and enhancer activities within the 5′ end of intron 1. Moreover stability analyses for PRNP mRNAs demonstrated that splicing sites and mechanism are critical for bovine PRNP expression.     

Vaccination with attenuated Salmonella enterica Dublin expressing E coli O157:H7 outer membrane protein Intimin induces transient reduction of fecal shedding of E coli O157:H7 in cattle

Background  

Escherichia coli serogroup O157:H7 has emerged as an important zoonotic bacterial pathogen, causing a range of symptoms from self-limiting bloody diarrhea to severe hemorrhagic colitis and hemolytic-uremic syndrome in humans. Beef and dairy cattle are considered the most important animal reservoirs for this pathogen. One of the important virulence characteristics of E. coli O157:H7 is the eaeA gene encoding the 97 kDa surface protein intimin. Intimin is required for attachment and effacement during the interaction of enterohemorrhagic E. coli with human and bovine neonatal enterocytes. The present study was undertaken to test the hypothesis that an adaptive mucosal immune response directed against intimin will reduce or prevent enteric colonization and fecal shedding of E. coli O157:H7 in cattle.     

Preferential selection of titanium-bearing minerals in agglutinated Foraminifera: Ilmenite (FeTiO3) in Textularia hauerii d’Orbigny from the Bazaruto Archipelago, Mozambique

The biserial agglutinated foraminifer Textularia hauerii d’Orbigny frequently occurs at shallow depths in reefal habitats off Bazaruto Island, Mozambique. X-ray diffraction, scanning electron- and light microscopy analyses show the enhanced concentration of the titanium-bearing mineral ilmenite (FeTiO₃) in the agglutinated tests of this species. The mineral constitutes an insignificant component in the surrounding sediment (< 1%), but accounts for more than 26% of the foraminiferal test content. The accumulation of ilmenite in T. hauerii and its absence in co-occurring agglutinated Foraminifera suggest the preferential selection for ilmenite. The tendency for ilmenite particles to be arranged along sutures and on the apertural face (1) provides further evidence that certain Foraminifera can specifically select grains, and (2) implies that there exists a selective mechanism to control the timing and placement during test and chamber construction. The preferential uptake represents the first record of ilmenite selection in Foraminifera. Potential driving forces controlling the selective agglutination of Ti minerals, the preferential adsorption to Ti surfaces, and interactions between cellular compounds of the foraminiferal reticulopods and the mineral surfaces are explored.