Abietane diterpenes from the cones of Cedrus atlantica

Five new abietanes, three of them isolated as the corresponding acetate derivatives, i.e., 9alpha,13alpha-epidioxiabiet-8(14)-en-18-ol, 7alpha,18-diacetoxy, 9beta,13beta-epidioxiabiet-8(14)-ene, 7alpha,18-diacetoxyabiet-8(14)-en-13beta-ol, 7alpha,18-diacetoxy-13beta-methoxyabiet-8(14)-ene, and 13beta-hydroxyabiet-8(14)-en-7-one, were isolated from the neutral part of the hexane extract of the cones of Cedrus atlantica collected in Middle Atlas, Morocco. The structures of these compounds were established by spectroscopic techniques, including 2D NMR spectra, and in the case of 1, by chemical correlation. The cytotoxicity of these abietane diterpenoids was tested against five cell lines.     

Characterization of the Mechanism and Magnitude of Cytoglobin-mediated Nitrite Reduction and Nitric Oxide Generation under Anaerobic Conditions

Cytoglobin (Cygb) is a recently discovered cytoplasmic heme-binding globin. Although multiple hemeproteins have been reported to function as nitrite reductases in mammalian cells, it is unknown whether Cygb can also reduce nitrite to nitric oxide (NO). The mechanism, magnitude, and quantitative importance of Cygb-mediated nitrite reduction in tissues have not been reported. To investigate this pathway and its quantitative importance, EPR spectroscopy, spectrophotometric measurements, and chemiluminescence NO analyzer studies were performed. Under anaerobic conditions, mixing nitrite with ferrous-Cygb triggered NO formation that was trapped and detected using EPR spin trapping. Spectrophotometric studies revealed that nitrite binding to ferrous-Cygb is followed by formation of ferric-Cygb and NO. The kinetics and magnitude of Cygb-mediated NO formation were characterized. It was observed that Cygb-mediated NO generation increased linearly with the increase of nitrite concentration under anaerobic conditions. This Cygb-mediated NO production greatly increased with acidosis and near-anoxia as occur in ischemic conditions. With the addition of nitrite, soluble guanylyl cyclase activation was significantly higher in normal smooth muscle cells compared with Cygb knocked down cells with Cygb accounting for ∼40% of the activation in control cells and ∼60% in cells subjected to hypoxia for 48 h. Overall, these studies show that Cygb-mediated nitrite reduction can play an important role in NO generation and soluble guanylyl cyclase activation under hypoxic conditions, with this process regulated by pH, oxygen tension, nitrite concentration, and the redox state of the cells.     

Role of Mechanical Cues in Cell Differentiation and Proliferation: A 3D Numerical Model

Cell differentiation, proliferation and migration are essential processes in tissue regeneration. Experimental evidence confirms that cell differentiation or proliferation can be regulated according to the extracellular matrix stiffness. For instance, mesenchymal stem cells (MSCs) can differentiate to neuroblast, chondrocyte or osteoblast within matrices mimicking the stiffness of their native substrate. However, the precise mechanisms by which the substrate stiffness governs cell differentiation or proliferation are not well known. Therefore, a mechano-sensing computational model is here developed to elucidate how substrate stiffness regulates cell differentiation and/or proliferation during cell migration. In agreement with experimental observations, it is assumed that internal deformation of the cell (a mechanical signal) together with the cell maturation state directly coordinates cell differentiation and/or proliferation. Our findings indicate that MSC differentiation to neurogenic, chondrogenic or osteogenic lineage specifications occurs within soft (0.1-1 kPa), intermediate (20-25 kPa) or hard (30-45 kPa) substrates, respectively. These results are consistent with well-known experimental observations. Remarkably, when a MSC differentiate to a compatible phenotype, the average net traction force depends on the substrate stiffness in such a way that it might increase in intermediate and hard substrates but it would reduce in a soft matrix. However, in all cases the average net traction force considerably increases at the instant of cell proliferation because of cell-cell interaction. Moreover cell differentiation and proliferation accelerate with increasing substrate stiffness due to the decrease in the cell maturation time. Thus, the model provides insights to explain the hypothesis that substrate stiffness plays a key role in regulating cell fate during mechanotaxis.     

Deficiency of functional mannose-binding lectin is not associated with infections in patients with systemic lupus erythematosus

Infection imposes a serious burden on patients with systemic lupus erythematosus (SLE). The increased infection rate in SLE patients has been attributed in part to defects of immune defence. Recently, the lectin pathway of complement activation has also been suggested to play a role in the occurrence of infections in SLE. In previous studies, SLE patients homozygous for mannose-binding lectin (MBL) variant alleles were at an increased risk of acquiring serious infections in comparison with patients who were heterozygous or homozygous for the normal allele. This association suggests a correlation between functional MBL level and occurrence of infections in SLE patients. We therefore investigated the biological activity of MBL and its relationship with the occurrence of infections in patients with SLE. Demographic and clinical data were collected in 103 patients with SLE. Functional MBL serum levels and MBL-induced C4 deposition were measured by enzyme-linked immunosorbent assay using mannan as coat and an MBL- or C4b-specific monoclonal antibody. The complete MBL-dependent pathway activity was determined by using an assay that measures the complete MBL pathway activity in serum, starting with binding of MBL to mannan, and was detected with a specific monoclonal antibody against C5b-9. Charts were systematically reviewed to obtain information on documented infections since diagnosis of SLE. Major infections were defined as infections requiring hospital admission and intravenous administration of antibiotics. In total, 115 infections since diagnosis of lupus, including 42 major infections, were documented in the 103 SLE patients (mean age 41 +/- 13 years, mean disease duration 7 +/- 4 years). The percentage of SLE patients with severe MBL deficiency was similar to that in 100 healthy controls: 13% versus 14%, respectively. Although deposition of C4 to mannan and MBL pathway activity were reduced in 21% and 43% of 103 SLE patients, respectively, neither functional MBL serum levels nor MBL pathway activity was associated with infections or major infections in regression analyses. In conclusion, SLE patients frequently suffer from infections, but deficiency of functional MBL does not confer additional risk.     

Pathogenicity of Bacillus Strains to Cotton Seedlings and Their Effects on Some Biochemical Components of the Infected Seedlings

Pathogenicity of eight Bacillus strains to seedlings of four cotton cultivars was evaluated under greenhouse conditions. Each of the tested cultivars was individually treated with powdered inoculum of each bacterial strain. Untreated seeds were planted as control treatments in autoclaved soil. Effects of the tested strains on levels and activities of some biochemical components of the infected seedlings were also assayed. The biochemical components included total soluble sugars, total soluble proteins, total free amino acids, peroxidase, polyphenol oxidase, phenols, and lipid peroxidation. ANOVA showed that Bacillus strain (B) was a very highly significant source of variation in damping-off and dry weight. Cotton cultivar (V) was a nonsignificant source of variation in damping-off while it was a significant source of variation in dry weight. B × V interaction was a significant source of variation in damping- off and a nonsignificant source of variation in dry weight. Bacillus strain was the most important source of variation as it accounted for 59.36 and 64.99% of the explained (model) variation in damping-off and dry weight, respectively. The lack of significant correlation between levels and activities of the assayed biochemical components and incidence of damping-off clearly demonstrated that these biochemical components were not involved in the pathogenicity of the tested strains. Therefore, it was hypothesized that the pathogenicity of the tested strains could be due to the effect of cell wall degrading enzymes of pathogenic toxins. Based on the results of the present study, Bacillus strains should be considered in studying the etiology of cotton seedling damping-off.     

Selective inhibition of toxic cyanobacteria by β-carboline-containing bacterium Bacillus flexus isolated from Saudi freshwaters

A bacterial strain SSZ01 isolated from a eutrophic lake in Saudi Arabia dominated by cyanobacterial blooms, showed an antialgal activity against cyanobacteria species. Based on the analysis of the 16S rDNA gene sequence, the isolated strain (SSZ01) most likely belonged to the genus Bacillus with a 99% similarity to Bacillus flexus strain EMGA5. The thin layer chromatography (TLC) analysis of the ethyl acetate extract of this bacterium revealed that this strain can produce harmine and norharmane compared to different β-carboline analog standards. Harmine and norharmane were also detected in considerable amounts in bacterial growth medium, indicating a potential excretion of these compounds into the aquatic environment. The crude extract of Bacillus flexus as well as pure materials of harmine and norharmane inhibited the growth of tested species of cyanobacteria. However, the bacterial crude extract has a higher toxicity against tested species of cyanobacteria than harmine and norharmane. In addition, harmine was more toxic to cyanobacteria than norharmane. On the other hand, neither pure compounds of harmine and norharmane nor crude bacterial extract showed any antialgal activity against tested species of green algae. The results of the present study suggest that B. flexus SSZ01 or its crude extract containing harmine and norharmane could be a candidate for the selective control of cyanobacterial blooms without affecting other algal species.     

Impact of Donation Mode on the Proportion and Function of T Lymphocytes in the Liver

Background

Liver T-cells respond to the inflammatory insult generated during organ procurement and contribute to the injury following reperfusion. The mode of liver donation alters various metabolic and inflammatory pathways but the way it affects intrahepatic T-cells is still unclear.

Methods

We investigated the modifications occurring in the proportion and function of T-cells during liver procurement for transplantation. We isolated hepatic mononuclear cells (HMC) from liver perfusate of living donors (LD) and donors after brain death (DBD) or cardiac death (DCD) and assessed the frequency of T-cell subsets, their cytokine secretion profile and CD8 T-cell cytotoxicity function, responsiveness to a danger associated molecular pattern (High Mobility Group Box1, HMGB1) and association with donor and recipient clinical parameters and immediate graft outcome.

Results

We found that T-cells in healthy human livers were enriched in memory CD8 T-cells exhibiting a phenotype of non-circulating tissue-associated lymphocytes, functionally dominated by more cytotoxicity and IFN-γ-production in DBD donors, including upon activation by HMGB1 and correlating with peak of post-transplant AST. This liver-specific pattern of CD8 T-cell was prominent in DBD livers compared to DCD and LD livers suggesting that it was influenced by events surrounding brain death, prior to retrieval.

Conclusion

Mode of liver donation can affect liver T-cells with increased liver damage in DBD donors. These findings may be relevant in designing therapeutic strategies aimed at organ optimization prior to transplantation.     

Prostatic Cell-Specific Regulation of the Synthesis of MUC1-Associated Sialyl Lewis a

Sialyl Lewis antigens are selectin ligands involved in leukocyte trafficking and cancer metastasis. Biosynthesis of these selectin ligands occurs by the sequential actions of several glycosyltransferases in the Golgi apparatus following synthesis of the protein backbone in the endoplasmic reticulum. In this study, we examine how the synthesis of sialyl Lewis a (sLe^a) is regulated in prostatic cells and identify a mucin that carries this glycotope. We treat human prostatic cells including one normal and three cancerous cells with histone deacetylase inhibitors, valproic acid, tricostatin A (TSA), and suberoylanilide hydroxamic acid (SAHA), and then monitor the expression of sLe^a. We have found that SAHA enhances the production of sLe^a in normal prostatic RWPE-1 cells but not prostatic cancer cells. Employing siRNA technology and co-immunoprecipitation, we show that the sLe^a is associated with MUC1, which is confirmed by confocal immunofluorescence microscopy and proximity ligation assay. The SAHA-induced production of sLe^a in RWPE-1 cells is resulted from upregulation of B3GALT1 gene via enhancement of acetylated histone-3 and histone-4. Interestingly, PC3 and LNCaP C-81 cells do not produce detectable amounts of sLe^a despite expressing high levels of B3GALT1. However, the MUC1-associated sLe^a is generated in these cells after introduction of MUC1 cDNA. We conclude that the synthesis of sLe^a is controlled by not only peptide backbone of the glycoprotein but also glycoprotein-specific glycosyltransferases involved in the synthesis of sLe^a. Further, the SAHA induction of this selectin ligand in normal prostatic cells may pose a potentially serious side effect of this drug recently approved by the US Food and Drug Administration.