Interleukin-1 production and antigen presentation by normal human peritoneal macrophages

Human peritoneal macrophages from healthy females have been investigated for their capability to produce interleukin-1 (IL-1), their expression of HLA-DR and -DQ, and for their antigen-presenting capacity in concanavalin A, tetanus toxoid (TT), and autologous T-cell proliferative responses. Fifteen out of thirty macrophage populations produced IL-1 but the activity was 1/5 to 1/10 that of peripheral blood mononuclear cells stimulated under similar conditions. High levels of HLA-DR were expressed on all macrophages while lower and more variable levels of DQ were found. All macrophages induced mitogen-dependent T-cell proliferation while the ability to induce a proliferative response to TT was variable, 12/23 tests were positive. In five samples stimulatory capacity of macrophages in the absence of TT was as strong as in the presence of the stimulus, suggesting that in vivo processed immunogen could be responsible for the proliferative response. The surface density of HLA-D-region-determined antigens was not indicative of the macrophages' ability to induce antigen-specific proliferation. IL-1 production, however, correlated with this function. Antigen presentation was not confined exclusively to peritoneal populations consisting of recently immigrant monocyte-like cells, nor were all young macrophages able to present antigen. This may reflect on the diversion of macrophage function by the local environment.     

G-protein dissociation, GTP-GDP exchange and GTPase activity in control and PMA treated neutrophils stimulated by fMet-Leu-Phe

The addition of the chemotactic factor fMet-Leu-Phe to cell homogenates causes a decrease in the pertussis toxin catalyzed ADP-ribosylation of a 41 kDa protein. The fMet-Leu-Phe induced decrease is not abolished in homogenates prepared from phorbol 12-myristate 13-acetate treated neutrophils. This decreased ribosylation probably reflects a dissociation of the GTP-binding protein oligomer that is not followed by association, possibly because of the release of the alpha-subunit into the suspending medium. Furthermore, fMet-Leu-Phe stimulates the binding of radiolabelled guanylylimidodiphosphate to membrane preparations. Again, the stimulated binding of guanylylimidodiphosphate is not affected by treating the intact neutrophils with phorbol 12-myristate 13-acetate. In addition leukotriene B4, platelet activating factor and fMet-Leu-Phe activate a high-affinity GTPase in membrane preparations. The basal level of this GTPase activity is dramatically inhibited in membrane preparations isolated from cells treated with phorbol 12-myristate 13-acetate. On the other hand, the fMet-Leu-Phe stimulated component is only marginally reduced. The present findings suggest that PMA does not prevent receptor G-protein interaction.     

Effects of tobacco glycoprotein (TGP) on the immune system. III. The effect of aging on the mitogenic response of human peripheral blood lymphocytes to TGP

We have shown that the mitogenic response of human peripheral blood lymphocytes (PBL) to tobacco glycoprotein (TGP), a glycoprotein rich in rutin or rutinlike polyphenol moieties, which is isolated from cured tobacco leaves, does not decrease with age. In contrast, the proliferative response to lipopolysaccharide (LPS) tends to decline with age, and a significant decrease is observed in the mitogenic response to rutin-bovine serum albumin (R-BSA). LPS and R-BSA are similar in some aspects to TGP, the former in that TGP and LPS are both T-independent B-cell mitogens for mice and are both highly negatively charged, and the latter in that covalently bound polyphenol groups are present on both R-BSA and TGP. Although the kinetics of the mitogenic responses to these three mitogens are similar (T. Francus, R. F. Klein, L. Staiano-Coico, G. W. Siskind, and C. G. Becker, Effects of tobacco glycoprotein (TGP) on the immune system. II. TGP is a mitogen for human peripheral blood lymphocytes. Submitted for publication.), multiple regression analyses show no correlation in the mitogenic responses of PBL from young donors to TGP and LPS, to TGP and R-BSA, or to LPS and R-BSA. In contrast, there are significant correlations between the proliferative responses to these mitogens by PBL from old donors. The results suggest that only a small subpopulation of the cells which are stimulated by R-BSA and LPS are not altered with age, and these are most likely the cells that are stimulated by the three mitogens studied.     

Sulfinyl radical formation from the reaction of cysteine and glutathione thiyl radicals with molecular oxygen

Using Electron Spin Resonance spectroscopy at low temperatures, we find that thiyl radicals resulting from irradiation of frozen aqueous solutions of a variety of thiols, including cysteine, glutathione, and penicillamine react with oxygen to form sulfinyl (RSO.) radicals. The identity of the cysteine sulfinyl radical has been confirmed by the use of molecular oxygen isotopically labeled with 17O. Previous workers have suggested the reaction of thiyl radicals and molecular oxygen resulted in the formation of the potentially damaging thiol peroxyl radical, RSOO.; our work shows no evidence for this species. The sulfinyl radicals are suggested to result from a direct reaction between thiyl radicals and molecular oxygen. This reaction results in the cleavage of the dioxygen bond.     

Diel variation of phytoplankton functional groups in a subtropical reservoir in southern Brazil during an autumnal stratification period

A knowledge of diel variation and the vertical distribution of phytoplankton communities may contribute to a better understanding of the driving factors of key species. Applying functional-group classification provides important information on the causes of species selection in the pelagic community. The diel variation of phytoplankton functional groups was analysed during an autumnal stratification period with the aim of understanding their changes in the vertical position related to light, mixing regime and grazing pressure. Phytoplankton and zooplankton communities were sampled every 4 h during a 24-h period in a vertical profile in a subtropical meso-eutrophic reservoir. Strong stratification during a 24-h cycle and a mixed clear epilimnion with partial atelomixis marked the autumn season in the Faxinal reservoir, southern Brazil. The highest phytoplankton densities and biomass were found during the second part of the day, a general pattern reported in the literature, and may be explained by zooplankton dynamics. During the 24-h cycle, phytoplankton functional groups lacking a self-regulating capacity and those able to regulate their vertical position were vertically segregated in the lake. The diel behaviour of both groups was driven by the mixing regime (including atelomixis), light and zooplankton grazing pressure.     

Regulation of NF-κB signaling by oxidized glycerophospholipid and IL-1β induced miRs-21-3p and -27a-5p in human aortic endothelial cells

Exposure of endothelial cells (ECs) to agents such as oxidized glycerophospholipids (oxGPs) and cytokines, known to accumulate in atherosclerotic lesions, perturbs the expression of hundreds of genes in ECs involved in inflammatory and other biological processes. We hypothesized that microRNAs (miRNAs) are involved in regulating the inflammatory response in human aortic endothelial cells (HAECs) in response to oxGPs and interleukin 1β (IL-1β). Using next-generation sequencing and RT-quantitative PCR, we characterized the profile of expressed miRNAs in HAECs pre- and postexposure to oxGPs. Using this data, we identified miR-21-3p and miR-27a-5p to be induced 3- to 4-fold in response to oxGP and IL-1β treatment compared with control treatment. Transient overexpression of miR-21-3p and miR-27a-5p resulted in the downregulation of 1,253 genes with 922 genes overlapping between the two miRNAs. Gene Ontology functional enrichment analysis predicted that the two miRNAs were involved in the regulation of nuclear factor κB (NF-κB) signaling. Overexpression of these two miRNAs leads to changes in p65 nuclear translocation. Using 3′ untranslated region luciferase assay, we identified 20 genes within the NF-κB signaling cascade as putative targets of miRs-21-3p and -27a-5p, implicating these two miRNAs as modulators of NF-κB signaling in ECs.     

Catalysis of covalent Lp(a) assembly: evidence for an extracellular enzyme activity that enhances disulfide bond formation

The assembly of lipoprotein(a) (Lp(a)) particles occurs via a two-step mechanism in which noncovalent interactions between apolipoprotein(a) (apo(a)) and the apolipoproteinB-100 component of low density lipoprotein precede the formation of a single disulfide bond. Although we have previously demonstrated that the rate constant for the covalent step of Lp(a) assembly can be enhanced by altering the conformational status of apo(a), the resultant rates of covalent Lp(a) particle formation measured in vitro are relatively slow. The large excess of Lp(a) (over apo(a)) observed in vivo can be accounted for by a preferential clearance of apo(a) over Lp(a) and/or a sufficiently high rate of covalent Lp(a) assembly. In the present study, we report that cultured human hepatoma cells secrete an oxidase activity that dramatically enhances the rate of covalent Lp(a) assembly. This activity is likely possessed by a protein because it is heat-sensitive and is retained in the concentrate following ultrafiltration through a 5 kDa cutoff filter. However, a small molecule cofactor for the activity is suggested by the observation that the activity is lost upon dialysis. Plots of Lp(a) assembly rate versus input apo(a) concentration gave rectangular hyperbolae; the reaction displayed an unusual dependence on the concentration of apoB-100, with increasing concentrations of apoB-100 resulting in slower rates of Lp(a) assembly at low concentrations of apo(a), an effect that was alleviated by higher apo(a) concentrations. Interestingly, V(max(app))/K(m(app)) ratios were insensitive to apoB-100 concentration, which is diagnostic of a ping-pong reaction mechanism. In this way, the putative Lp(a) oxidase may be functionally analogous to protein disulfide isomerase, which exhibits a similar mechanism during the catalysis of disulfide bond formation during protein folding, although we have ruled out a role for this enzyme in Lp(a) assembly.     

Central insulin‐like growth factor‐1 (IGF‐1) restores whole‐body insulin action in a model of age‐related insulin resistance and IGF‐1 decline

Low insulin‐like growth factor‐1 (IGF‐1) signaling is associated with improved longevity, but is paradoxically linked with several age‐related diseases in humans. Insulin‐like growth factor‐1 has proven to be particularly beneficial to the brain, where it confers protection against features of neuronal and cognitive decline. While aging is characterized by central insulin resistance in the face of hyperinsulinemia, the somatotropic axis markedly declines in older humans. Thus, we hypothesized that increasing IGF‐1 in the brain may prove to be a novel therapeutic alternative to overcome central insulin resistance and restore whole‐body insulin action in aging. Utilizing hyperinsulinemic‐euglycemic clamps, we show that old insulin‐resistant rats with age‐related declines in IGF‐1 level demonstrate markedly improved whole‐body insulin action, when treated with central IGF‐1, as compared to central vehicle or insulin (P < 0.05). Furthermore, central IGF‐1, but not insulin, suppressed hepatic glucose production and increased glucose disposal rates in aging rats (P < 0.05). Taken together, IGF‐1 action in the brain and periphery provides a ‘balance’ between its beneficial and detrimental actions. Therefore, we propose that strategies aimed at ‘tipping the balance’ of IGF‐1 action centrally are the optimal approach to achieve healthy aging and longevity in humans.     

Radiolabel-transfer cross-linking demonstrates that protein 4.1 binds to the N-terminal region of beta spectrin and to actin in binary interactions

Erythrocyte protein 4.1 plays a major role in stabilizing the spectrin-actin junction of the erythrocyte membrane skeleton. The particular sites on spectrin responsible for the binding of actin and protein 4.1 have not been specifically defined, although the general region of the 'tail' end, opposite the self-association site, has been deduced by electron microscopy. Using a photoactivatable, radiolabel-transfer cross-linker, 1-[N-(2-hydroxy-5-azidobenzoyl)-2-aminoethyl]-4-(N-hydroxysuccinimidyl)- succinate, we have determined that the binding site for protein 4.1 on spectrin resides in the N-terminal region of beta spectrin within a sequence homologous to the actin-binding region of alpha actinin. Moreover, this technique provided clear evidence for a direct binding interaction between actin filaments and protein 4.1 that was confirmed by rapid-sedimentation assays. In summary, use of radiolabel-transfer cross-linking has enabled assignment of the protein-4.1-binding site on erythrocyte spectrin and has identified a previously ill-defined binary interaction between protein 4.1 and F-actin.