The role of lipolysis in mediating the proinflammatory effects of very low density lipoproteins in mouse peritoneal macrophages

Hypertriglyceridemia is an important risk factor for atherosclerosis, especially in obesity. Macrophages are one of the primary cell types involved in atherogenesis and are thought to contribute to lesion formation through both lipid accumulation and proinflammatory gene expression. In this study, we sought to determine the direct impact of triglyceride (TG)-rich VLDL-induced lipid accumulation on macrophage proinflammatory processes. Incubation of mouse peritoneal macrophages with 100 microg/ml VLDL for 6 h led to 2.8- and 3.7-fold increases in intracellular TGs and FFAs, respectively (P < 0.05). The inflammatory proteins tumor necrosis factor-alpha, interleukin-1beta, monocyte chemoattractant protein-1, intercellular adhesion molecule-1, matrix metalloproteinase 3 (MMP3), and macrophage inflammatory protein-1alpha (MIP-1alpha) were all upregulated by at least 2-fold (P < 0.05) in a dose-dependent manner in VLDL-treated macrophages. The increase in inflammatory gene expression coincided with the phosphorylation of the mitogen-activated protein kinase (MAPK) pathway members extracellular signal-regulated kinase (ERK) 1/2, stress-activated protein kinase/c-Jun NH2-terminal kinase, and p38 MAPK and was ameliorated by U0126, an inhibitor of ERK1/2. Inhibition of extracellular TG hydrolysis with tetrahydrolipstatin (Orlistat) resulted in the absence of intracellular TG and FFA accumulation and was accompanied by the amelioration of ERK1/2 phosphorylation and MIP-1alpha gene expression. These data indicate that VLDL hydrolysis, and the subsequent accumulation of intracellular FFAs and TGs, plays a substantive role in mediating the proinflammatory effects of VLDL. These data have important implications for the direct proatherogenic effects of VLDL on macrophage-driven atherosclerosis.     

The role of membrane phosphoinositides in mediating hormonal effects

The present notions on the pathways of phosphoinositide metabolism in a cell and on the functional significance of the given process are reported. The enzymic systems providing catabolism of phosphoinositides are analyzed. The data indicating the dose interrelation between the stimulation of the phosphatidyl inosite and polyphosphoinositides metabolism by hormones and the Ca2+ transport into a cell are generalized. Universality, biochemical mechanisms and functional significance of the "phosphatidyl inosite" response are discussed. The phosphoinositide metabolism is considered from the standpoint of its significance for other cell processes: synthesis of eicosanoids, provision of Ca2+-dependent processes in the synapse, cell proliferation, activation and the "anchoring" of enzymes on membranes.     

Effect of low-frequency ultrasound on the chemotactic and phagocytic activity of peritoneal macrophages in rats

The influence of low-frequency ultrasound on the chemotactic, ingestive and digestive activity of peritoneal macrophages in rats was studied. The intraoperative treatment of the peritoneum with ultrasound enhanced chemotactic activity 3.3-fold in comparison with that in the control animals. The digestive function of peritoneal macrophages considerably increased, the stimulation of their ingestive capacity also occurred. The activation of the phagocytic function of macrophages was observed within 7 days after a single sonar treatment. The authors believe that the stimulation of the macrophage system is probably one of the mechanisms of the sanative action of ultrasound which is used at present in purulent surgery.     

The role of adipose tissue in mediating the beneficial effects of dietary fish oil

Fish oil improves several features of metabolic syndrome (MetS), such as dyslipidemia, insulin resistance and hepatic steatosis. Fish oil may mediate some of its beneficial effects by modulating the storage and/or secretory functions of adipose tissue (AT). The storage of triglycerides in AT is regulated by the availability of free fatty acids and the degree of lipolysis in AT. Fish oil has been shown to reduce lipolysis in several studies, indicating improved triglyceride storage. Importantly, AT secretes a variety of adipokines and fish oil feeding is associated with remarkable changes in the plasma levels of two key adipokines, adiponectin and leptin. Much attention has been focused on the contribution of adiponectin in fish oil-mediated improvements in MetS. However, emerging evidence also indicates a role of leptin in modulating the components of the MetS upon fish oil feeding. In addition to improving the storage and secretory functions of AT, fish oil, and the n-3 fatty acids found in fish oil, has been shown to reduce inflammation in AT. These effects may be in part a result of activation of peroxisome proliferator-activated receptor γ or inhibition of Toll-like receptor 4. Thus, there is compelling evidence that fish oil mediates its beneficial effects on MetS by improving AT storage and secretory functions and by reducing inflammation.     

Differential effects of lectins mediating erythrocyte attachment and ingestion by macrophages

Lectin-mediated interaction of erythrocytes and macrophages was brought about in two steps. Step I involved macrophage treatment with lectin, and step II is the incubation of lectin-treated macrophages with mouse erythrocytes. The extent and nature of lectin-mediated macrophage erythrocyte interaction was studied using concanavalin A (ConA), wheat germ (WGA), soybean (SBA) and waxbean (WBA) agglutinins. The parameters affecting the interaction were studied in detail with the first two lectins.Under comparable conditions of lectin interaction with macrophages (step I), WGA mediates rosette formation involving interaction with several times the number of erythrocytes than those interacting with ConA-treated macrophages. The interaction mediated by WGA reaches, at 37 °C, a saturation value after 30 min of step II, whereas that mediated by ConA is still linear and exhibits half the amount of attached erythrocytes at 60 min. ConA-mediated attachment of erythrocytes is highly temperature-dependent being at 37 °C twice that observed at 24 °C. The temperature dependence of attachment is not affected by changes of either ConA concentration (5–40 μg/ml) or the temperature in step I. An optimum is observed, however, when the temperature of incubation in step I ranges between 14–18 °C. WGA-mediated attachment of erythrocytes is markedly less temperature-sensitive, exhibiting 70% of optimal attachment already at 8 °C. Only when the attachment phase follows incubation with a low concentration of WGA (2 μg/ml) high temperature sensitivity is exhibited. At 37 °C, however, the number of attached erythrocytes is the same for macrophages treated with WGA at concentrations of 2, 5, 10 and 40 μg/ml.ConA-mediated erythrocyte-macrophage interaction does not lead to erythrophagocytosis. When mediated by WGA, the attachment step is followed by a temperature-dependent ingestion step, i.e. 10% and 50% of the erythrocytes that attach to macrophages during the 60 min incubation at 24 °C and 37 °C, respectively, are ingested. There is a lag period of 10–20 min between attachment and ingestion implicating involvement of additional cellular processes preceding engulfment. Electron microscope images of areas of interaction of attached erythrocytes with macrophages indicate a significantly tighter binding (a thinner gap at membrane-membrane apposition areas) in the case of WGA-mediated rosette formation as compared with that established in ConA-mediated rosettes. Attachment via WGA is followed by a rapid change in the relative position of the attached erythrocytes on the macrophage, from a primary attachment at the distal peripheral regions of the cell, to a perinuclear position. In contrast, erythrocytes attached via ConA remain at the primary attachment point (at 37 °C) for extended periods. This differential behaviour does not stem from effects of ConA on macrophages, since when yeast cells were attached to ConA treated macrophages, the yeast cells showed the same movement as that exhibited by erythrocyte when attached via WGA.The different interaction patterns of erythrocytes with macrophages coated with ConA and WGA can be fitted into the following working hypothesis: the number of WGA-binding sites on the plasma membrane of macrophages is at least three times that of ConA-binding sites. Stable cell-cell interactions involve multibridge formation at the contact area of the two cells and this involves a delicate balance between number of lectin-receptor conjugates and their aggregation state within the membrane phase. A certain amount of clustering is a prerequisite for attachment, while a high degree of clustering reduces the chance of fruitful interactions. The engulfment step depends on the ability of membrane areas adjacent to primary contact area to establish additional stable bridges in the entire circumference of the attached cell. ConA-receptor conjugates appear to be less abundant and more aggregated within the membrane plane, preventing the completion of fruitful circumferential interaction of the adjacent membrane. WGA-receptor conjugates, being more abundant and apparently less aggregated are available at membrane areas needed for cell enclosure and provide the additional bridging without which engulfment does not take place. Change in relative position of attached erythrocytes seems to be a step in the manifold events occurring from attachment to ingestion.     

Immunocorrecting properties of low-frequency ultrasound

The possibility to correct the secondary immunodeficient state by means of two- and three-fold low frequency ultrasound effect on the spleen has been demonstrated on mice of CBA strain. It makes it possible to use low-frequency ultrasound on a large scale in the clinical practice.     

The role of plant resistance and tolerance to herbivory in mediating the effects of introduced herbivores

While the generally negative consequences of introduced species are well known, little is appreciated on the role of the evolutionary history of plants with herbivores in mediating the indirect impacts of herbivory. We examined how variation in plant resistance and tolerance traits can mediate the effects of herbivory and can have differential indirect impacts on other species and processes. We used two examples of a native and an introduced herbivore, Castor canadensis (American beaver) and Cervus elaphus (Rocky Mountain elk) with Populus spp. to test a conceptual model regarding possible outcomes of species interactions with native and exotic mammalian herbivores. Using these two herbivore test cases, we make two predictions to create testable hypotheses across systems and taxa: First, adaptive traits of tolerance or resistance to herbivory will be fewer when exotic species feed on plant species with which they have no evolutionary history. Second, historical constraints of species interactions will allow for negative feedbacks to stabilize the effects of herbivory by a native species. Overall, these two case studies illustrate that plant resistance and tolerance traits can mediate the indirect consequences of herbivory on associated interacting species. Specifically, when there is no evolutionary history between the plants and herbivores, which is often the case with species introductions, the effects of herbivory are more likely to reduce genetic variation and habitat mosaics, thus indirectly affecting associated species.     

The role of glucose 6-phosphate in mediating the effects of glucokinase overexpression on hepatic glucose metabolism

Pharmacological activation or overexpression of glucokinase in hepatocytes stimulates glucose phosphorylation, glycolysis and glycogen synthesis. We used an inhibitor of glucose 6-phosphate (Glc6P) hydrolysis, namely the chlorogenic derivative, 1-[2-(4-chloro-phenyl)-cyclopropylmethoxy]-3, 4-dihydroxy-5-(3-imidazo[4,5-b]pyridin-1-yl-3-phenyl-acryloyloxy)-cyclohexanecarboxylic acid (also known as S4048), to determine the contribution of Glc6P concentration, as distinct from glucokinase protein or activity, to the control of glycolysis and glycogen synthesis by glucokinase overexpression. The validity of S4048 for testing the role of Glc6P was supported by its lack of effect on glucokinase binding and its nuclear/cytoplasmic distribution. The stimulation of glycolysis by glucokinase overexpression correlated strongly with glucose phosphorylation, whereas glycogen synthesis correlated strongly with Glc6P concentration. Metabolic control analysis was used to determine the sensitivity of glycogenic flux to glucokinase or Glc6P at varying glucose concentrations (5-20 mm). The concentration control coefficient of glucokinase on Glc6P (1.4-1.7) was relatively independent of glucose concentration, whereas the flux control coefficients of Glc6P (2.4-1.0) and glucokinase (3.7-1.8) on glycogen synthesis decreased with glucose concentration. The high sensitivity of glycogenic flux to Glc6P at low glucose concentration is consistent with covalent modification by Glc6P of both phosphorylase and glycogen synthase. The high control strength of glucokinase on glycogenic flux is explained by its concentration control coefficient on Glc6P and the high control strength of Glc6P on glycogen synthesis. It is suggested that the regulatory strength of pharmacological glucokinase activators on glycogen metabolism can be predicted from their effect on the Glc6P content.     

The role of A-cells in affecting the immunostimulating effect of F(ab')2-fragments

The immunoregulatory effect of F(ab')2 fragments on normal rabbit IgG and that preincubated with A-cells from spleen have been compared. Both products were tested for their ability to enhance primary immune response of rabbit spleen cells to SRBC. It was demonstrated that low molecular mass product appeared after F(ab')2 fragments incubation with A-cells at 37 degrees C and possessed immunostimulating activity similar to that of initial F(ab')2 fragments. In addition, it was shown that F(ab')2 reduction to monovalent Fab' fragment with the following alkylation of SH-group abolished the ability of Fab' fragment to enhance the immune response. It may signify that half cystein Fab' fragment residue is essential for processing of the fragment in A-cells and (or) for immune response enhancement.     

The role of the mitochondria in mediating cytotoxicity of anti-cancer therapies

Optimal cytotoxic anticancer therapy, at the cellular level, requires effective and selective induction of cell death to achieve a net reduction of biomass of malignant tissues. Standard cytotoxic chemotherapeutics have been developed based on the observations that mitotically active cancer cells are more susceptible than quiescent normal cells to chromosomal, microtubular or metabolic poisons. More recent development of molecularly targeted drugs for cancer focuses on exploiting biological differentials between normal and transformed cells for selective eradication of cancers. The common thread of “standard” and “novel” cytotoxic drugs is their ability to activate the apoptosis-inducing machinery mediated by mitochondria, also known as the intrinsic death signaling cascade. The aim of this article is to provide an overview of the role of the mitochondria, an energy-generating organelle essential for life, in mediating death when properly activated by cytotoxic stresses.     

The role of loop 7 in mediating calcineurin regulation

Calcineurin (CN) is a heterodimer protein consisting of a 61 kDa catalytic subunit A and a 19 kDa regulatory subunit B. It plays a critical role in T-cell activation and is involved in many cellular processes. Regulation of CN is rather complex, including a number of factors such as divalent metal ions (primarily Ca(2+) and Mn(2+)), calmodulin (CaM) and autoinhibition (AI) segment. Previously, we reported that a loop 7 deletion mutant (V314) in subunit A exhibited high phosphatase activity, although the mechanism for the surprising activity enhancement and whether the activity change applies to other loop 7 residues were not known. In order to probe the role of loop 7, we have carried out extensive mutagenesis experiments, followed by systematic activity assays under a number of regulatory conditions. All mutants, including single deletion mutants Y315, N316 and double deletion mutant V314Y315, showed increased phosphatase activity. Significantly, activities of the mutants containing the V314 deletion, namely V314 and V314Y315, were no longer regulated by regulatory subunit B. These results, along with the structure analysis, suggest that loop 7 as a whole plays an important role in mediating CN's regulation through bridging the regulatory subunit and catalytic core and interaction with the AI segment of CN.     

The mediating role of classroom quality on peer effects and language gain in pre-kindergarten ECSE classrooms

The current study was designed to determine whether classroom quality (i.e., the quality of teacher–child interactions) served as a mediator of peer effects with respect to preschool children’s gain in language skills over an academic year. Participants were 670 preschool-age children (64.6% boys; M?=?4 years, 4 months, SD?=?7 months) enrolled in 83 inclusive early childhood special education (ECSE) classrooms in multiple school districts in a single Midwestern state. Measures included an index of classroom quality, average level of classmates’ language skills, and fall and spring language assessments for each child. Results showed that both classroom quality and peer effects contributed to children’s language gain over an academic year, showing that both instructional quality and peers are influential to language development. Results did not support a mediating role for classroom quality: that is, classroom quality did not mediate the effects of peers on children’s language gain.     

Critical role of effector macrophages in mediating CD4-dependent alloimmune injury of transplanted liver parenchymal cells

Despite the recognition that humoral rejection is an important cause of allograft injury, the mechanism of Ab-mediated injury to allograft parenchyma is not well understood. We used a well-characterized murine hepatocellular allograft model to determine the mechanism of Ab-mediated destruction of transplanted liver parenchymal cells. In this model, allogeneic hepatocytes are transplanted into CD8-deficient hosts to focus on CD4-dependent, alloantibody-mediated rejection. Host serum alloantibody levels correlated with in vivo allospecific cytotoxic activity in CD8 knockout hepatocyte rejector mice. Host macrophage depletion, but not CD4(+) T cell, NK cell, neutrophil, or complement depletion, inhibited in vivo allocytotoxicity. Recipient macrophage deficiency delayed CD4-dependent hepatocyte rejection and inhibited in vivo allocytotoxicity without influencing alloantibody production. Furthermore, hepatocyte coincubation with alloantibody and macrophages resulted in Ab-dependent hepatocellular cytotoxicity in vitro. These studies are consistent with a paradigm of acute humoral rejection in which CD4(+) T cell-dependent alloantibody production results in the targeting of transplanted allogeneic parenchymal cells for macrophage-mediated cytotoxic immune damage. Consequently, strategies to eliminate recipient macrophages during CD4-dependent rejection pathway may prolong allograft survival.     

Regulation of B-lymphocyte production in the bone marrow: role of macrophages and the spleen in mediating responses to exogenous agents

B-Lymphocyte production in mouse bone marrow can be stimulated by administering a variety of foreign materials in vivo. The nature and location of cells mediating this effect have now been studied, using assays of lymphocyte renewal and pre-B-cell proliferation. Pretreatment of mice with silica, to depress macrophage function, abolished the stimulation of small lymphocyte renewal produced by administering either sheep red blood cells (SRBC) or mineral oil and reduced the response to bovine serum albumin. The response was still abolished when silica was given 6 or 24 hr, but not 48 hr, after SRBC. Splenectomy prevented the stimulation of marrow lymphocyte renewal when performed either 4 weeks before or up to 72 hr after SRBC injection. The stimulation of pre-B-cell proliferation was similarly prevented by pretreatment with either silica or splenectomy. The results indicate that the wave of increased B-lymphocyte production after SRBC injection depends for the first 2-3 days upon silica-sensitive, spleen-dependent mechanisms, suggesting an early mediation by splenic macrophages. Primary B-lymphocyte production in vivo may thus normally be stimulated by exposure to external environmental agents acting indirectly on bone marrow B-cell progenitors via cellular reactions in peripheral lymphoid tissues.     

The role of neurosteroids and nongenomic effects of progestins in the ventral tegmental area in mediating sexual receptivity of rodents

Progesterone (P(4)) in the ventromedial hypothalamus (VMH) and ventral tegmental (VTA) is important for facilitation of lordosis; however, P(4)'s actions in these brain areas are different. Using lordosis in rodents as in vivo experimental models, we have examined the effects progestins exert in the midbrain and hypothalamus. Localization and blocker studies indicate that P(4)'s actions in the VMH require intracellular progestin receptors (PRs) but in the VTA they do not. Progestins that have rapid, membrane effects, and/or are devoid of affinity for PRs, facilitate lordosis when applied to the VTA. Manipulation of GABA and/or GABA(A)/benzodiazepine receptor complexes (GBRs) in the VTA alter lordosis, which suggests that progestins may interact with GBRs to facilitate receptivity by enhancing the function of GABAergic neurons. Interfering with P(4)'s metabolism to 5 alpha-pregnan-3 alpha-ol-20-one (3 alpha,5 alpha-THP), the most effective endogenous positive modulator of GBRs, or the biosynthesis of the neurosteroid 3 alpha,5 alpha-THP in the VTA attenuates female sexual behavior in rodents. Stimulation of mitochondrial benzodiazepine receptors (MBRs), which enhance neurosteroid production, by infusions of a MBR agonist to the VTA enhances lordosis. 3 alpha,5 alpha-THP is increased in the midbrain of mated > proestrous > diestrous rodents. These data suggest that 3 alpha,5 alpha-THP has a proximate modulatory role on lordosis. In summary, the actions of P(4) in the VTA are different from those in the VMH that involve PRs. In the VTA, P(4) may facilitate lordosis following metabolism to and/or biosynthesis of 3 alpha,5 alpha-THP, which may have subsequent actions at GBRs and/or MBRs to acutely modulate female sexual behavior in rodents.     

Differential role of melanocortins in mediating leptin's central effects on feeding and reproduction

Leptin serves as a humoral link coupling the status of energy reserves to the functional activity of the reproductive system. Leptin is thought to act through melanocortinergic pathways in the brain to regulate ingestive behaviors; however, whether melanocortins mediate leptin's actions on the neuroendocrine-reproductive axis is unknown. We tested this hypothesis first by determining whether the effects of leptin on feeding behavior and reproduction in the ob/ob mouse could be blocked by the melanocortin receptor (MC-R) antagonist SHU9119 and second, by examining the effects of the MC-R agonist MTII on feeding and the endocrine-reproductive system. Administered by intracerebroventricular injections, leptin inhibited food intake, raised plasma gonadotropin levels, and increased seminal vesicle weights compared with controls; SHU9119 (intracerebroventricularly) attenuated leptin's effects on food intake and body weight but did not alter leptin's stimulatory effect on the reproductive axis. MTII (intracerebroventricularly and intraperitoneally) decreased food intake and increased body temperature compared with controls but had no effect on the reproductive-endocrine axis. These results suggest that although leptin acts centrally through melanocortinergic pathways to inhibit ingestive behaviors and stimulate metabolism, leptin's activational effect on the reproductive axis is likely to be mediated by other, unknown neuroendocrine circuits.     

A role for fibroblasts in mediating the effects of tobacco-induced epithelial cell growth and invasion

Cigarette smoke and smokeless tobacco extracts contain multiple carcinogenic compounds, but little is known about the mechanisms by which tumors develop and progress upon chronic exposure to carcinogens such as those present in tobacco products. Here, we examine the effects of smokeless tobacco extracts on human oral fibroblasts. We show that smokeless tobacco extracts elevated the levels of intracellular reactive oxygen, oxidative DNA damage, and DNA double-strand breaks in a dose-dependent manner. Extended exposure to extracts induced fibroblasts to undergo a senescence-like growth arrest, with striking accompanying changes in the secretory phenotype. Using cocultures of smokeless tobacco extracts-exposed fibroblasts and immortalized but nontumorigenic keratinocytes, we further show that factors secreted by extracts-modified fibroblasts increase the proliferation and invasiveness of partially transformed epithelial cells, but not their normal counterparts. In addition, smokeless tobacco extracts-exposed fibroblasts caused partially transformed keratinocytes to lose the expression of E-cadherin and ZO-1, as well as involucrin, changes that are indicative of compromised epithelial function and commonly associated with malignant progression. Together, our results suggest that fibroblasts may contribute to tumorigenesis indirectly by increasing epithelial cell aggressiveness. Thus, tobacco may not only initiate mutagenic changes in epithelial cells but also promote the growth and invasion of mutant cells by creating a procarcinogenic stromal environment.     

Antitumor and immunostimulating effects of Anoectochilus formosanus Hayata.

The water extract of Anoectochilus formosanus Hayata showed a potent tumor inhibitory activity in BALB/c mice after subcutaneous transplantation of CT-26 murine colon cancer cells. The tumor-inhibition ratios of mice pre-administered with A. formosanus for 2 days before tumor transplantation, and treated further for 12 consecutive days, were 55.4% and 58.9% at the oral dose of 50 and 10 mg/mouse per day, respectively. Even for the tumor-bearing mice, after oral administration of the water extract of A. formosanus for 12 consecutive days, the tumor inhibition ratios were still 23.8% and 40.5% at doses of 50 and 10 mg/mouse, respectively. Because the low-concentration water extract of A. formosanus does not show direct cytotoxicity in CT-26 tumor cells, we observed further that oral administration of the water extract of A. formosanus may activate murine immune responses, such as stimulating the proliferation of lymphoid tissues and activating the phagocytosis of peritoneal macrophages against Staphylococcus aureus. This study suggests that the antitumor activity of A. formosanus may be associated with its potent immunostimulating effect. It is worth further analyzing the immunomodulating component purified from A. formosanus, and evaluating its potential value for the treatment of human cancers.     

The role of ultrasound in analytical derivatizations

Ultrasound is a type of energy that until recently was rarely used for analytical purposes. In recent years, work in chemical and industrial fields alerted analytical chemists to the great potential of ultrasonic energy to accelerate or improve different steps of the analytical process. One of these steps is derivatization: depolymerization, redox, hydrolysis, esterification, alkylation and complex formation are examples of derivatization reactions, all of which are significantly improved with the aid of ultrasound. This review discusses the valuable characteristics of ultrasound and its influence on a number of derivatization reactions is discussed in this review.