Cottonseed Oil Protects Against Intestinal Inflammation in Dextran Sodium Sulfate-Induced Inflammatory Bowel Disease.

Dietary products may protect against inflammatory bowel disease (IBD) through mechanisms such as forming gut microbiota structures and providing substrates for microbial metabolism. Recently, many studies have been conducted on diets that potentially alleviate or suppress IBD development. To assess the efficacy of dietary oils in treating IBD, we examined the protective effects of olive oil, coconut oil, corn oil, and cottonseed oil in a dextran sodium sulfate (DSS)-induced colitis mouse model. Treatment with cottonseed oil or corn oil ameliorated the severity of DSS-induced colitis, alleviating weight loss and preventing the shortening of the intestine. Moreover, cottonseed oil or corn oil treatment significantly reduced the expression of inflammatory cytokines such as tumor necrosis factor-α, interleukin (IL)-1ß, IL-6, and IL-17, as well as the expression of oxidative stress markers, including 8-hydroxyguanosine and nitrotyrosine in colon sections, compared with vehicle treatment. Cottonseed oil treatment inhibited intestinal fibrosis by reducing the expression of α-smooth muscle actin and type I collagen, compared with vehicle treatment in mice with DSS-induced colitis. Cottonseed oil protects against intestinal inflammation and the development of intestinal fibrosis by reducing inflammatory cytokines and oxidative stress markers, and may therefore be useful as a dietary product with therapeutic benefits for IBD.

A Targeted Multiple Antigenic Peptide Vaccine Augments the Immune Response to Self TGF-ß1 and Suppresses Ongoing Hepatic Fibrosis.

Transforming growth factor (TGF)-ß1 expression is induced upon liver injury, and plays a critical role in hepatic fibrosis. Antibodies against TGF-ß1 represent a novel approach in the treatment of hepatic fibrosis. However, TGF-ß1 is not a suitable antigen due to immunological tolerance. In the current study, we synthesized a multiple antigenic peptide (MAP) vaccine against the dominant B-cell epitope of TGF-ß1. The immunogenicity and potential therapeutic effects of this vaccine were examined using a rat model of hepatic fibrosis. Dominant B-cell epitopes of TGF-ß1 were identified using bioinformatic program. An MAP vaccine corresponding to the 90-98 amino acid domain of TGF-ß1 and containing four dendritic arms was synthesized using a 9-fluorenylmethoxycarbonyl solid phase method. Hepatic fibrosis which was induced in male Sprague-Dawley rats received a high-fat diet and ethanol (1.8 g/kg). Starting from the third week, rats were exposed to 40 % carbon tetrachloride (CCl4; 150 µl/100 g body weight twice weekly, initially 200 µl/100 g) treatment for a duration of 8 weeks. Rats received the MAP vaccine (100 µg) or Freund's adjuvant at weeks 1, 3, 5. A group of rats receiving the fibrosis-inducing regimen alone and a group of healthy rats (receiving an olive oil vehicle alone) were included as controls. At the conclusion of the experiment, serum titre of TGF-ß1 antibody was measured using ELISA and a standard liver functional test panel was examined. The extent of hepatic fibrosis was determined by measuring hydroxyproline content in the liver as well as hematoxylin-eosin (HE) and van Gieson (VG) staining. The expression of TGF-ß1 and alpha-smooth muscle actin (α-SMA) was examined using immunohistochemistry, and presented as positive staining cells. The MAP purity was >90 % upon reverse phase high-performance liquid chromatography, with apparent molecular weight at 4.77 kDa. Serum TGF-ß1 antibody titre was 1:256. The fibrosis-inducing treatment produced significant liver damage, as reflected by increases in liver functional test, HE and VG staining. The MAP vaccine attenuated such damage, as reflected by decreased alanine aminotransferase, aspartate aminotransferase, total bilirubin, and hepatic hydroxyproline (116.78 ± 23.76 vs. 282.71 ± 136.94 IU/L; 319.78 ± 82.48 vs. 495.29 ± 137.13 IU/L; 2.02 ± 0.27 vs. 4.01 ± 0.52 µmol/L; 263.67 ± 41.18 vs. 439.14 ± 43.29 µg/g vs. in model rats, respectively; p < 0.01), as well as fibrosis extent by HE and VG staining. The MAP vaccine reduced TGF-ß1 and α-SMA expression in rats (0.325 ± 0.059 vs. 0.507 ± 0.044 IOD/area; 0.318 ± 0.058 vs. 0.489 ± 0.029 IOD/area vs. model rats, respectively; p < 0.05). The TGF-ß1 MAP vaccine could generate sufficient antibody that suppresses the development of hepatic fibrosis.

Role of AP1 and Gadkin in the traffic of secretory endo-lysosomes.

Whereas lysosome-related organelles (LRO) of specialized cells display both exocytic and endocytic features, lysosomes in nonspecialized cells can also acquire the property to fuse with the plasma membrane upon an acute rise in cytosolic calcium. Here, we characterize this unconventional secretory pathway in fibroblast-like cells, by monitoring the appearance of Lamp1 on the plasma membrane and the release of lysosomal enzymes into the medium. After sequential ablation of endocytic compartments in living cells, we find that donor membranes primarily derive from a late compartment, but that an early compartment is also involved. Strikingly, this endo-secretory process is not affected by treatments that inhibit endosome dynamics (microtubule depolymerization, cholesterol accumulation, overexpression of Rab7 or its effector Rab-interacting lysosomal protein [RILP], overexpression of Rab5 mutants), but depends on Rab27a, a GTPase involved in LRO secretion, and is controlled by F-actin. Moreover, we find that this unconventional endo-secretory pathway requires the adaptor protein complexes AP1, Gadkin (which recruits AP1 by binding to the γ1 subunit), and AP2, but not AP3. We conclude that a specific fraction of the AP2-derived endocytic pathway is dedicated to secretory purposes under the control of AP1 and Gadkin.

Subnuclear compartmentalization and function of actin and nuclear myosin I in plants.

Actins are highly conserved proteins that serve as the basic building blocks of cytoskeletal microfilaments. In animal cells, specific nuclear actin adopts unconventional conformations that are involved in multiple nuclear functions and that associate with nuclear actin binding proteins. However, there is practically no information available about nuclear actin in plants. Indeed, actin has not been detected in the nuclear proteomes of many plants, and orthologs of the main structural nuclear actin-binding proteins have yet to be identified. Here, we have investigated the characteristics, intranuclear compartmentalization, and function of actin in isolated Allium cepa nuclei as well as that of its motor protein nuclear myosin I (NMI). Using conformation-specific antibodies for nuclear actin isoforms, ss-actin, and NMI, the distribution of these proteins was studied in Western blots and by immunocytochemistry. Moreover, the participation of nuclear actin in transcription was analyzed in run on in situ assays and inhibition of RNA polymerases I and II. We show that actin isoforms with distinct solubilities are present in onion nuclei with a consistent subnuclear compartmentalization. Actin and NMI are highly enriched in foci that are similar to transcription foci, although actin is also distributed diffusely in the nucleus and nucleolus as well as accumulating in a subset of the Cajal bodies. Immunogold labeling identified both proteins in the nuclear transcription subdomains and in other subnuclear compartments. In addition, actin and NMI were diffusely distributed in the nuclear matrix.

Ectopic expression of HLA-DO in mouse dendritic cells diminishes MHC class II antigen presentation.

The MHC class II-like molecule HLA-DM (DM) (H-2M in mice) catalyzes the exchange of CLIP for antigenic peptides in the endosomes of APCs. HLA-DO (DO) (H-2O in mice) is another class II-like molecule that is expressed in B cells, but not in other APCs. Studies have shown that DO impairs or modifies the peptide exchange activity of DM. To further evaluate the role of DO in Ag processing and presentation, we generated transgenic mice that expressed the human HLA-DOA and HLA-DOB genes under the control of a dendritic cell (DC)-specific promoter. Our analyses of DCs from these mice showed that as DO levels increased, cell surface levels of A(b)-CLIP also increased while class II-peptide levels decreased. The presentation of some, but not all, exogenous Ags to T cells or T hybridomas was significantly inhibited by DO. Surprisingly, H-2M accumulated in DO-expressing DCs and B cells, suggesting that H-2O/DO prolongs the half-life of H-2M. Overall, our studies showed that DO expression impaired H-2M function, resulting in Ag-specific down-modulation of class II Ag processing and presentation.

Anti-atherogenic effect of citrus flavonoids, naringin and naringenin, associated with hepatic ACAT and aortic VCAM-1 and MCP-1 in high cholesterol-fed rabbits.

The anti-atherogenic effects of the citrus flavonoids, naringin and naringenin, were evaluated in high cholesterol-fed rabbits. At 3 months of age, 30 male New Zealand White (NZW) rabbits were divided into three groups (n = 10 per group). The rabbits were fed a 1% cholesterol diet alone (control group) or a diet supplemented with either 0.1% naringin or 0.05% naringenin for 8 weeks. The plasma lipoprotein levels, total cholesterol, triglyceride, and high-density lipoprotein showed no significant differences in the control and experimental groups. Hepatic acyl-CoA:cholesterol acyltransferase (ACAT) activity was slightly low in naringin (5.0%)- and naringenin (15.0%)-fed rabbits, compared to control group. The aortic fatty streak areas were significantly lower in both the naringin (19.2 +/- 5.6%)- and naringenin (18.1 +/- 6.5%)-supplemented groups than in the control group (60.4 +/- 14.0%). The expression levels of vascular cell adhesion molecule-1 (VCAM-1) and monocyte chemotactic protein-1 (MCP-1), by semiquantitative RT-PCR analysis of the thoracic aorta, were significantly lower in the flavonoids supplemented groups than in the control group. These results suggest that the anti-atherogenic effect of the citrus flavonoids, naringin and naringenin, is involved with a decreased hepatic ACAT activity and with the downregulation of VCAM-1 and MCP-1 gene expression.

A potential role for sterol 27-hydroxylase in atherogenesis.

OBJECTIVE: 27-hydroxycholesterol is the product of the mitochondrial cytochrome P450 sterol 27-hydroxylase, a key enzyme in cholesterol metabolism present in most tissues of the body. 27-hydroxycholesterol increases in abundance with progression of human atherosclerotic lesions, therefore the aim of this study was to determine the pattern of sterol 27-hydroxylase gene expression in normal and diseased arteries and to identify the cell types responsible for its expression. METHODS: Reverse transcription-polymerase chain reaction (RT-PCR) analysis and in situ hybridisation, utilising a sterol 27-hydroxylase cDNA probe, and immunohistochemistry, utilising an antibody to sterol 27-hydroxylase, together with an antibody to smooth muscle cell alpha-actin and an antibody to CD68, a marker for macrophages, were used to study expression of 27-hydroxylase in arterial specimens. In addition, RT-PCR was used to study expression of 27-hydroxylase in cultured macrophages and smooth muscle cells. RESULTS: Semi-quantitative RT-PCR analysis of normal and atherosclerotic human aortas showed that 27-hydroxylase is constitutively expressed in the normal artery wall, and is substantially up-regulated in atherosclerosis. RT-PCR analysis of 27-hydroxylase expression in vitro demonstrated that macrophages constitutively express high levels throughout their differentiation in culture whilst de-differentiated vascular smooth muscle cells express very low levels. In situ hybridisation revealed that in normal artery and fatty streaks, expression of mRNA for 27-hydroxylase was low in the media, but higher in intimal smooth muscle cells. The macrophages of fatty streaks expressed low or undetectable levels of 27-hydroxylase. However in advanced lesions the highest expression of 27-hydroxylase was detectable in macrophages. Immunohistochemistry demonstrated that high levels of 27-hydroxylase protein occurred in macrophages near the shoulder region of plaques, at the edge of the lipid core. CONCLUSIONS: 27-hydroxylase may constitute a protective mechanism for removing cholesterol from macrophages and smooth muscle cells. Genetic heterogeneity resulting in differences in sterol 27-hydroxylase activity between individuals may affect their ability to deal with accumulated cholesterol in the arterial intima, and hence their relative degree of predisposition to atherosclerosis.

Immunocytochemical localization of actin and tubulin in the integument of land crab (Gecarcinus lateralis) and lobster (Homarus americanus).

The crustacean integument consists of the exoskeleton and underlying epithelium and associated tissues. The epithelium, which is composed of a single layer of cells, is responsible for the cyclical breakdown and synthesis of the exoskeleton associated with molting (ecdysis). During premolt (proecdysis) the epithelial cells lengthen and secrete the two outermost layers (epicuticle and exocuticle) of the new exoskeleton while partially degrading the two innermost layers (endocuticle and membranous layer) of the overlying old exoskeleton. This increased cellular activity is associated with increased protein synthesis and a change in cell shape from cuboidal to columnar. The cytoskeleton, composed of microfilaments (actin) and microtubules (tubulin), plays important roles in the intracellular organization and motility of eukaryotic cells. Immunoblot analysis shows that the land crab exoskeleton contains actin, tubulin, and actin-related proteins (Varadaraj et al. 1996. Gene 171:177-184). In the present study, immunocytochemistry of land crab and lobster integument showed that both proteins were localized in various cell types, including epithelia, connective tissue, tendinal cells, and blood vessels. Muscle immunostained for actin and myosin, but not for tubulin. The membranous layer of land crab (the other layers of the exoskeleton were not examined) and membranous layer and endocuticle of lobster also reacted specifically with anti-beta-actin and anti-alpha-tubulin monoclonal antibodies, but not with an anti-myosin heavy chain antibody. During proecdysis immunolabeling of the membranous layer decreased probably due to protein degradation. The staining intensity for actin and tubulin in the proecdysial epithelium was similar to that in the intermolt (anecdysial) epithelium, suggesting that there was a net accumulation of both proteins proportional to the increase in cellular volume. These results support the previous biochemical analyses and, more specifically, localize actin and tubulin in exoskeletal structures, suggesting that they may serve both intracellular and extracellular functions in crustaceans. J. Exp. Zool. 286:329-342, 2000.

The late pollen-specific actins in angiosperms.

The actin gene family of Arabidopsis has eight functional genes that are grouped into two ancient classes, vegetative and reproductive, and into five subclasses based on their phylogeny and mRNA expression patterns. Progress in deciphering the functional significance of this diversity is hindered by the lack of tools that can distinguish the highly conserved subclasses of actin proteins at the biochemical and cellular level. In order to address the functional diversity of actin isovariants, we have used Arabidopsis recombinant actins as immunogens and produced several new anti-actin monoclonal antibodies. One of them, MAb45a, specifically recognizes two closely related reproductive subclasses of actins. On immunoblots, MAb45a reacts strongly with actins expressed in mature pollen but not with actins in other Arabidopsis tissues. Moreover, immunocytochemical studies show that this antibody can distinguish actin filaments in pollen tubes from those in most vegetative tissues. Peptide competition analyses demonstrate that asparagine at position 79 (Asn79) within an otherwise conserved sequence is essential for MAb45a specificity. Actins with the Asn79 epitope are also expressed in the mature pollen from diverse angiosperms and Ephedra but not from lower gymnosperms, suggesting that this epitope arose in an ancestor common to angiosperms and advanced gymnosperms more than 220 million years ago. During late pollen development in angio- sperms there is a switch in expression of actins from vegetative to predominantly reproductive subclasses, perhaps to fulfil the unique functions of pollen in fertilization.

Natural polyreactive secretory immunoglobulin A autoantibodies as a possible barrier to infection in humans.

Secretory immunoglobulin A (S-IgA) was investigated in human secretions for the presence of natural antibodies (Abs) acting as the first "immune barrier" to infection before induction or boosting of specific responses. These molecules could be the secretory counterpart of the natural Abs in serum that were previously shown by our laboratory to be polyreactive to autoantigens. Significant levels of S-IgA Abs to human actin, myosin, tubulin, and spectrin were detected in 10 saliva and 8 colostrum samples from normal subjects. Computer-assisted analysis of immunoblots of extracts from human muscles showed these Abs to react with a large number of autoantigens. Their polyreactivity was confirmed by cross-inhibition and by immunoblotting studies of affinity-purified natural Abs, assayed against a large variety of surface or secreted antigens from Streptococcus pyogenes. The thiocyanate elution method showed that functional affinities of some natural Abs can be of the same order of magnitude as those of tetanus vaccine antitoxins. Moreover, nonimmune binding of these natural Abs to the gut protein Fv (Fv-fragment binding protein) can enhance their effector functions. This demonstrates that human secretions contain polyreactive auto-Abs which can also react with pathogens. These secretory Abs of "skeleton key" specificities are possibly produced by a primordial B-1-cell-associated immune system and can be involved in a plurispecific mucosal protection against pathogens, irrespective of the conventional immune response.

Covisualization in living onion cells of putative integrin, putative spectrin, actin, putative intermediate filaments, and other proteins at the cell membrane and in an endomembrane sheath.

Covisualizations with wide-field computational optical-sectioning microscopy of living epidermal cells of the onion bulb scale have evidenced two major new cellular features. First, a sheath of cytoskeletal elements clads the endomembrane system. Similar elements clad the inner faces of punctate plasmalemmal sites interpreted as plasmalemmal control centers. One component of the endomembrane sheath and plasmalemmal control center cladding is anti-genicity-recognized by two injected antibodies against animal spectrin. Immunoblots of separated epidermal protein also showed bands recognized by these antibodies. Injected phalloidin identified F-actin with the same cellular distribution pattern, as did antibodies against intermediate-filament protein and other cytoskeletal elements known from animal cells. Injection of general protein stains demonstrated the abundance of endomembrane sheath protein. Second, the endomembrane system, like the plasmalemmal puncta, contains antigen recognized by an anti-beta 1 integrin injected into the cytoplasm. Previously, immunoblots of separated epidermal protein were shown to have a major band recognized both by this antibody prepared against a peptide representing the cytosolic region of beta 1 integrin and an antibody against the matrix region of beta 1 integrin. The latter antiboby also identified puncta at the external face of protoplasts. It is proposed that integrin and associated transmembrane proteins secure the endomembrane sheath and transmit signals between it and the lumen or matrix of the endoplasmic reticulum and organellar matrices. This function is comparable to that proposed for such transmembrane linkers in the plasmalemmal control centers, which also appear to bind cytoskeleton and a host of related molecules and transmit signals between them and the wall matrix. It is at the plasmalemmal control centers that the endoplasmic reticulum, a major component of the endomembrane system, attaches to the plasma membrane.

Immunocytochemical localisation of actin and profilin in the generative cell of angiosperm pollen: TEM studies on high-pressure frozen and freeze-substituted Ledebouria socialis Roth (Hyacinthaceae).

Actin was demonstrated for the first time at the EM level in the generative cell of mature angiosperm pollen by using immuno-gold labelling of high-pressure frozen and freeze-substituted Ledebouria socialis Roth anthers. In addition, profilin, an actin-monomer binding protein, is shown to coexist in the generative cell. We attribute the detection of actin and profilin to the applied cryomethods which yield a much better preservation of ultrastructure and antigenicity of delicate cytoskeletal constituents than conventional fixation techniques. Actin labelling was observed within the cytoplasm of the generative cell and became especially clear in close vicinity to microtubular bundles. Filamentous structures congruent with the actin labelling patterns do occur, but are not a frequent feature. Profilin was localised throughout the cytoplasm.

Characterization of a monoclonal antibody prepared against plant actin.

Anti-actin monoclonal antibodies were prepared using phalloidin-stabilized actin that was purified from pea roots by DNase I affinity chromatography. One monoclonal antibody, designated mAb3H11, bound plant actin in preliminary screenings and was further analyzed. Immunoblot analysis showed that this antibody had a high affinity for plant actin in crude and purified preparations but a low affinity for rabbit muscle actin. In immunoblots of plant extracts separated on two-dimensional gels it appeared to bind all actin isoforms recognized by the JLA20 anti-chicken actin antibody. Using immunofluorescent cytochemistry, the antibody was used to observe actin filaments in aldehyde-fixed and methanol-treated tobacco protoplasts. These results indicate that mAb3H11 should be a useful reagent for the study of plant actins.

Autoreactive antibodies in thymus and spleen of neonatal and young adult BALB/c mice: influence of prenatal tolerization.

The repertoire of autoantibody-producing B cells was evaluated in a collection of spleen- and thymus-derived hybridomas from 6- and 28-day-old BALB/c mice, which were untreated or prenatally tolerized with trinitrobenzenesulphonic acid (TNBS). MoAb were tested for their reactivity with TNP-BSA and the autoantigens thyroglobulin (TG), myoglobin (MG), actin (AC), cytochrome C (CY), collagen (CO), transferrin (TF), single-stranded DNA (ssDNA), double-stranded DNA (dsDNA), and bromelain-treated mouse red blood cells (BrMRBC). More than 10% of spleen cell (SC)-derived MoAb from 6- and 28-day-old control mice did bind to AC, ssDNA, dsDNA, MY, and TG, the frequency of MoAb reacting with MY, TG, and BrMRBC increasing with age. Thymus cell (TC)-derived hybridomas contained autoreactive clones too, but only few of them produced multireactive MoAb. MoAb from prenatally TNBS-treated mice were more frequently autoreactive than MoAb from control mice, especially if derived from TC hybridomas. The most remarkable difference in the reactivity pattern as compared with MoAb from untreated mice consisted of a significant increase in the frequency of TG-, My-, ssDNA- and above all dsDNA-reactive MoAb, all TC-derived multireactive MoAb binding to dsDNA. The differences in autoreactivity between MoAb from prenatally untreated and TNBS-treated mice as well as age- and organ-related variations support the interpretation that part of the repertoire of naturally activated B cells is not random but is influenced by and responding to the available panel of self antigens.

Alterations in the cytoskeleton of the goldfish Mauthner cells under various pharmacological treatments.

The ultrastructural changes in the cytoskeleton of the goldfish Mauthner cells (M-cells) at various functional states induced by intracerebral microinjections of biologically active substances were studied. Under the action of kainic acid, a structural analog of the excitatory neurotransmitter of glutamate, the density of the cytoplasmic matrix increased. Cytotoxin II from the cobra toxin, which blocks acetylcholine transmission, had an opposite effect upon the M-cell cytoskeleton. Simultaneously, in some areas of the neural cytoplasm strands of an electron-dense material of various shapes appeared. They had an unique structure which did not resemble any known cytoskeleton element. The molecular composition of the strands is unknown, but similar strands appeared after injections of phalloidin or cytochalasin B, both disturbing the microfilamental component of the cytoskeleton. Decoration with myosin subfragment-1 revealed actin in intact M-cells which was organized as crossing loose filaments and bundles of parallel fibers. The morphology of the fiber bundles resembles the helical part of the strands appearing after the treatment with phalloidin, cytochalasin B, or cytotoxin II. It is suggested that the cytoplasmic matrix of M-cells is a dynamic system which responds to the functional changes by thickening or loosening of its cytoskeletal elements or by formation of new structures.