Cholesterol affects spectrin-phospholipid interactions in a manner different from changes resulting from alterations in membrane fluidity due to fatty acyl chain composition

We previously showed that erythrocyte and brain spectrins bind phospholipid vesicles and monolayers prepared from phosphatidylethanolamine and phosphatidylserine and their mixtures with phosphatidylcholine (Review: A.F. Sikorski, B. Hanus-Lorenz, A. Jezierski, A. R. Dluzewski, Interaction of membrane skeletal proteins with membrane lipid domain, Acta Biochim. Polon. 47 (2000) 565). Here, we show how changes in the fluidity of the phospholipid monolayer affect spectrin-phospholipid interaction. The presence of up to 10%-20% cholesterol in the PE/PC monolayer facilitates the penetration of the monolayer by both types of spectrin. For monolayers constructed from mixtures of PI/PC and cholesterol, the effect of spectrins was characterised by the presence of two maxima (at 5 and 30% cholesterol) of surface pressure for erythroid spectrin, and a single maximum (at 20% cholesterol) for brain spectrin. The binding assay results indicated a small but easily detectable decrease in the affinity of erythrocyte spectrin for FAT-liposomes prepared from a PE/PC mixture containing cholesterol, and a 2- to 5-fold increase in maximal binding capacity (B_max) depending on the cholesterol content. On the other hand, the results from experiments with a monolayer constructed from homogenous synthetic phospholipids indicated an increase in Δπ change with the increase in the fatty acyl chain length of the phospholipids used to prepare the monolayer. This was confirmed by the results of a pelleting experiment. Adding spectrins into the subphase of raft-like monolayers constructed from DOPC, SM and cholesterol (1/1/1) induced an increase in surface pressure. The Δπ change values were, however, much smaller than those observed in the case of a natural PE/PC (6/4) monolayer. An increased binding capacity for spectrins of liposomes prepared from a “raft-like” mixture of lipids could also be concluded from the pelleting assay. In conclusion, we suggest that the effect of membrane lipid fluidity on spectrin-phospholipid interactions is not simple but depends on how it is regulated, i.e., by cholesterol content or by the chemical structure of the membrane lipids.     

Extracellular phospholipases in atherosclerosis

Phospholipases A2 (PLA2) are a family of enzymes that catalyze the hydrolysis of the sn-2 ester bond of glycerophospholipids liberating lysophospholipids and free fatty acids; important second messengers involved in atherogenesis. Plasma PAF-acetylhydrolase (PAF-AH) or Lp-PLA2 is a Ca²⁺-independent PLA2 which is produced by monocyte-derived macrophages and by activated platelets, and circulates in plasma associated with lipoproteins. PAF-AH catalyzes the removal of the acetyl/short acyl group at the sn-2 position of PAF and oxidized phospholipids produced during inflammation and oxidative stress. In humans, PAF-AH is mainly associated with small dense LDL and to a lesser extent with HDL and with lipoprotein(a). PAF-AH is N-glycosylated prior to secretion which diminishes its association with HDL raising the question of its distribution between the proatherogenic LDL vs the antiatherogenic HDL. Hypercholesterolemic patients have higher plasma PAF-AH activity which is reduced upon hypolipidemic therapy. PAF-AH specific inhibitor darapladib stabilizes human and swine plaques, therefore challenging the antiatherogenic potential of PAF-AH shown in small animal models.     

Bifidobacteria-mediated immune system imprinting early in life

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Movement of fluorescent dyes Lucifer Yellow (LYCH) and carboxyfluorescein (CF) in Medicago truncatula Gaertn. roots and root nodules

Lucifer Yellow (LYCH) and carboxyfluorescein (CF) served in Medicago truncatula roots and root nodules as the markers of apoplastic and symplastic transport, respectively. The aim of this study was to understand better the water and photoassimilate translocation pathways to and within nodules. The present study shows that in damaged roots LYCH moves apoplastically through the vascular elements but it was not detected within the nodule vascular bundles. In intact roots, the outer cortex was strongly labeled but the dye was not present in the interior of intact root nodules. The inwards movement of LYCH was halted in the endodermis. When the dye was introduced into a damaged nodule by infiltration, it spread only in the cell walls and the intercellular spaces up to the inner cortex. Our research showed that in addition to the outer cortex, the inner tissue containing bacteroid-infected cells is also an apoplastic domain. Our results are consistent with the hypothesis that nodules do not receive water from the xylem but get it and photoassimilates from phloem. A comparison between using LYCH and LYCH followed by glutaraldehyde fixation indicates that glutaraldehyde is responsible for fluorescence of some organelles within root nodule cells. The influence of the fixation on nodule fluorescence has not been reported before but must be taken into consideration to avoid errors. An attempt was made to follow carboxyfluorescein (6(5) CF) translocation from leaflets into roots and root nodules. In root nodules, CF was present in all or a couple of vascular bundles (VB), vascular endodermis and some adjacent cells. The leakage of CF from the VBs was observed, which suggests symplastic continuity between the VBs and the nodule parenchyma. The lack of CF in inner tissue was observed. Therefore, photoassimilate entry to the infected region of nodule must involve an apoplastic pathway.     

Biological control of Radopholus similis in banana by combined application of the mutualistic endophyte Fusarium oxysporum strain 162, the egg pathogen Paecilomyces lilacinus strain 251 and the antagonistic bacteria Bacillus firmus

The biological control efficacy of single or multiple applications of the mutualistic endophyte Fusarium oxysporum strain 162, the egg pathogen Paecilomyces lilacinus strain 251 and the antagonistic bacteria Bacillus firmus toward Radopholus similis was investigated in pot trials with banana under glasshouse conditions. R. similis was controlled substantially in single and combined applications of F. oxysporum with P. lilacinus or B. firmus. The combination of F. oxysporum and P. lilacinus caused a 68.5% reduction in nematode density whereas the individual applications reduced the density by 27.8% and 54.8% over the controls, respectively. Combined application of F. oxysporum and B. firmus was the most effective treatment in controlling R. similis on banana (86.2%), followed by B. firmus alone (63.7%). The compatibility of the biocontrol agents, as well the capacity of F. oxysporum to colonize banana roots in the absence or presence of P. lilacinus was also investigated. P. lilacinus did not adversely affect endophytic colonization by F. oxysporum. Biological control of R. similis in banana can therefore be enhanced via combined applications of antagonists with different modes of action that target different stages in the infection process. Handling editor: Ralf-Udo Ehlers     

The mouse Fc receptor for IgG (Ly-17) : molecular cloning and specificity

A cDNA clone encoding the mouse Ly-17⁺ Fc receptor for IgG, isolated from a myelomonocytic cell line, was sequenced and expression of mRNA and the functional FcR investigated. The receptor is a 301 amino acid transmembrane glycoprotein with two homologous extracellular domains that are also homologous to members of the Ig superfamily. The receptor has four sites of N-linked glycosylation and a long 94 amino acid cytoplasmic tail. Northern analysis, immune complex binding, and serological studies demonstrate that the receptor encoded by the cDNA clone binds mouse IgG_1/2b and rabbit IgG complexes.     

Androgens and FSH affect androgen receptor and aromatase distribution in the porcine ovary

In the present study the authors investigated whether androgens could interact with FSH to induce aromatase and androgen receptor expression in porcine granulosa cells. Dissected whole porcine follicles (small, medium, and large) were incubated for 8 hours in M199 medium supplemented with testosterone (10(-7) M), FSH (100 ng/ml) or both those hormones. After incubation, the follicles were fixed and immunostained to visualise androgen receptor and aromatase. In cultures of granulosa cells isolated from small and large follicles, oestrogen secretion was measured by appropriate RIA. Incubation of follicles with testosterone and FSH increased aromatase immunoreactivity in preantral and early antral (i.e. small) follicles. The immunostaining for androgen receptor was slightly higher in medium follicles, while such hormonal stimulation had no effect on small and large follicles. Moreover, granulosa cells isolated from small follicles cultured with both testosterone and FSH produced more estradiol than control cultures (40 pg vs. 100 pg/10(5) cells). The level was relatively close to that obtained in the culture of control granulosa cells isolated from large preovulatory follicles (105 pg/10(5) cells). These results indicate that testosterone acts synergistically with FSH to increase aromatase expression in the small porcine follicles.     

Pertussis toxin inhibits CAMP-induced desensitization of adenylate cyclase in Dictyostelium discoideum

cAMP binds to surface receptors of Dictyostelium discoideum cells, transducing the signal to adenylate cyclase, guanylate cyclase and to chemotaxis. The activation of adenylate cyclase is maximal after 1 min and then declines to basal levels due to desensitization, which is composed of two components: a rapidly reversible adaptation process, and a slowly reversible down-regulation of cAMP receptors. Adaptation is correlated with receptor phosphorylation.The chemotactic response and the cAMP-induced cGMP response were not significantly altered in D. discoideum cells pretreated with pertussis toxin. The initial increase of cAMP levels was identical in control and toxin treated cells, suggesting that activation of adenylate cyclase was also not affected. However, cAMP synthesis continued in toxin treated cells, due to a strongly diminished desensitization. Pertussis toxin inhibited the adaptation of adenylate cyclase stimulation, but not the down-regulation or phosphorylation of the cAMP receptors. Adenylate cyclase in D. discoideum membranes can be stimulated or inhibited by GTP, depending on the conditions used. Pertussis toxin did not affect the stimulation of adenylate cyclase but nullified the inhibition. In membranes from desensitized control cells, stimulation of adenylate cyclase by GTP was lost, whereas inhibition was retained. Stimulation of adenylate cyclase in membranes from desensitized pertussis toxin treated cells was diminished but not absent. These results indicate that receptor phosphorylation is not sufficient for adaptation of adenylate cyclase, and that a pertussis toxin substrate, possibly Gi, is also involved in this process.Abbreviations used ATPS Adenosine 5-0-(3-Thiotriphosphate) - GTPS Guanosine 5-0-(3-thiotri-phosphate) - (Sp)-cAMPS Adenosine 3,5-monophosphorothioate-Sp-isomer - dcAMP 2-deoxyadenosine 3,5-monophosphate - Hepes N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - DTT Dithiothreitol - buffer A 10 mM KH₂PO₄/Na2HPO₄, pH 6.5 - buffer B 40 mM Hepes/NaOH, 0.5 mM EDTA, 250 mM sucrose, pH 7.7     

Risk Factors for Normal and High-Tension Glaucoma in Poland in Connection with Polymorphisms of the Endothelial Nitric Oxide Synthase Gene

Aim

The purpose of this study was to evaluate the influence of polymorphisms of the eNOS gene on the clinical status of patients with normal and high tension glaucoma.

Methods

266 Polish Caucasian patients with primary open angle glaucoma were studied. Of the 266, 156 had normal tension glaucoma (NTG) and 110 high tension glaucoma (HTG). DNA material was isolated from peripheral venous blood using commercial kits. Real-time PCR reaction was used to amplify the promoter site of the endothelial nitric oxide synthase (eNOS) gene, including the single nucleotide polymorphism (SNP) site T-786C and part of the 7^th exon of eNOS, including G894T SNP. Genotypes were determined with TaqMan SNP Genotyping Assays.

Results

There were no significant differences in frequencies of the allelic variants of both polymorphisms. In G894T SNP, however, the wild GG form was more common in the HTG group. The SNP of the eNOS gene did not significantly influence the progression rate in either of the groups studied. There were no differences in variants of the eNOS gene regarding the necessity for and success of surgery and the progression of the disease. In the NTG group, no statistical correlation was observed between G894T, T786C polymorphism variants, and risk factors such as optic disc haemorrhages, optic disc notches, and peripapillary atrophy. Mean diastolic and systolic pressure during the day and night were lowest in NTG patients with the CC variant of the T786C polymorphism. No statistical correlation was observed between the G894T and T786C polymorphisms and capillaroscopic examination results.

Conclusions

Genotype frequencies are similar for both the eNOS G894T and T-786C polymorphisms in NTG and HTG patients. These polymorphisms do not correlate with risk factors and do not influence the state of the capillary system in NTG patients. Systolic blood pressure is lower in NTG patients with mutated alleles of both polymorphisms.