Ubiquitous expression of a resident K-Ras(G12V) oncogene in adult mice revealed that most tissues are resistant to K-Ras oncogenic signals. Indeed, K-Ras(G12V) expression only induced overt tumors in lungs. To identify these transformation-permissive cells, we induced K-Ras(G12V) expression in a very limited number of adult lung cells (0.2%) and monitored their fate by X-Gal staining, a surrogate marker coexpressed with the K-Ras(G12V) oncoprotein. Four weeks later, 30% of these cells had proliferated to form small clusters. However, only SPC(+) alveolar type II (ATII) cells were able to form hyperplastic lesions, some of which progressed to adenomas and adenocarcinomas. In contrast, induction of K-Ras(G12V) expression in lung cells by intratracheal infection with adenoviral-Cre particles generated hyperplasias in all regions except the proximal airways. Bronchiolar and bronchioalveolar duct junction hyperplasias were primarily made of CC10(+) Clara cells. Some of them progressed to form benign adenomas. However, only alveolar hyperplasias, exclusively made up of SPC(+) ATII cells, progressed to yield malignant adenocarcinomas. Adenoviral infection induced inflammatory infiltrates primarily made of T and B cells. This inflammatory response was essential for the development of K-Ras(G12V)-driven bronchiolar hyperplasias and adenomas, but not for the generation of SPC(+) ATII lesions. Finally, activation of K-Ras(G12V) during embryonic development under the control of a Sca1 promoter yielded CC10(+), but not SPC(+), hyperplasias, and adenomas. These results, taken together, illustrate that different types of lung cells can generate benign lesions in response to K-Ras oncogenic signals. However, in adult mice, only SPC(+) ATII cells were able to yield malignant adenocarcinomas.

T cells play an important role in cancer immunosurveillance and tumor destruction. However, tumor cells alter immune responses by modulating immune cells through antigen stimulation and immunoregulatory cytokines. A better understanding of the interplay between tumor cells and T cells might provide new strategies to enhance antitumor immunity. Through an antigen-screening approach using colorectal tumor-reactive T cells, we identified an HLA-DR11-restricted T-cell epitope encoded by KIAA0040 as well as MHC-unrestricted human galectin-3 (Gal-3) expressed by tumor cells. Although the biological function of KIAA0040 remains to be determined, we found that Gal-3 functioned as an immune regulator for direct T-cell activation and function. T-cell activation induced by Gal-3 resulted in T-cell apoptosis. We showed that a high level of expression of Gal-3 promoted tumor growth in vitro and in vivo. Using a mouse tumor model, we showed that delivery of high doses of Gal-3 inhibited tumor-reactive T cells and promoted tumor growth in mice receiving tumor-reactive CD8(+) T cells. These findings suggest that Gal-3 may function as an immune regulator to inhibit T-cell immune responses and promote tumor growth, thus providing a new mechanism for tumor immune tolerance.

In Hodgkin lymphoma (HL), the malignant Hodgkin Reed-Sternberg cells interact with the host microenvironment to create an immunosuppressive network that protects the lymphoma from immune attack. These mechanisms are not fully understood. We examined the role of the immunomodulatory protein galectin-1 (Gal-1) on Epstein-Barr virus-specific CD8(+) T cell responses in HL. Initial studies indicated Gal-1 expression in all in vitro established Hodgkin Reed-Sternberg cell lines. In situ analysis revealed Gal-1 expression in 26 of 42 classic HL, whereas Gal-1 was uniformly negative in nodular lymphocyte predominant HL. Gal-1(hi) expression was associated with male gender, older patients, reduced CD8(+) T cell infiltration at the tumor site, and most importantly, an impaired latent membrane protein 1 and 2-specific CD8(+) T-cell responses. In vitro exposure to recombinant Gal-1 inhibited proliferation and interferon-gamma expression by Epstein-Barr virus-specific T cells. These observations provide an important link between the Gal-1-mediated immunomodulatory networks and loss of antigen-specific T-cell function in classic HL.

A variety of pulmonary disorders, including cystic fibrosis, are potentially amenable to treatment in which a therapeutic gene is directly transferred to the bronchial epithelium. This is difficult to accomplish because the majority of airway epithelial cells replicate slowly and/or are terminally differentiated. Adenovirus vectors may circumvent this problem, since they do not require target cell proliferation to express exogenous genes. To evaluate the diversity of airway epithelial cell targets for in vivo adenovirus-directed gene transfer, a replication deficient recombinant adenovirus containing the Escherichia coli lacZ (beta-galactosidase [beta-gal]) gene (Ad.RSV beta gal) was used to infect lungs of cotton rats. In contrast to uninfected animals, intratracheal Ad.RSV beta gal administration resulted in beta-gal activity in lung lysate and cytochemical staining in all cell types forming the airway epithelium. The expression of the exogenous gene was dose-dependent, and the distribution of the beta-gal positive airway epithelial cells in Ad.RSV beta gal-infected animals was similar to the normal cell differential of the control animals. Thus, a replication deficient recombinant adenovirus can transfer an exogenous gene to all major categories of airway epithelial cells in vivo, suggesting that adenovirus vectors may be an efficient strategy for in vivo gene transfer in airway disorders such as cystic fibrosis.

Although plants contain substantial amounts of arabinogalactan proteins (AGPs), the enzymes responsible for AGP glycosylation are largely unknown. Bioinformatics indicated that AGP galactosyltransferases (GALTs) are members of the carbohydrate-active enzyme glycosyltransferase (GT) 31 family (CAZy GT31) involved in N- and O-glycosylation. Six Arabidopsis GT31 members were expressed in Pichia pastoris and tested for enzyme activity. The At4g21060 gene (named AtGALT2) was found to encode activity for adding galactose (Gal) to hydroxyproline (Hyp) in AGP protein backbones. AtGALT2 specifically catalyzed incorporation of [(14)C]Gal from UDP-[(14)C]Gal to Hyp of model substrate acceptors having AGP peptide sequences, consisting of non-contiguous Hyp residues, such as (Ala-Hyp) repetitive units exemplified by chemically synthesized (AO)7 and anhydrous hydrogen fluoride-deglycosylated d(AO)51. Microsomal preparations from Pichia cells expressing AtGALT2 incorporated [(14)C]Gal to (AO)7, and the resulting product co-eluted with (AO)7 by reverse-phase HPLC. Acid hydrolysis of the [(14)C]Gal-(AO)7 product released (14)C-radiolabel as Gal only. Base hydrolysis of the [(14)C]Gal-(AO)7 product released a (14)C-radiolabeled fragment that co-eluted with a Hyp-Gal standard after high performance anion-exchange chromatography fractionation. AtGALT2 is specific for AGPs because substrates lacking AGP peptide sequences did not act as acceptors. Moreover, AtGALT2 uses only UDP-Gal as the substrate donor and requires Mg(2+) or Mn(2+) for high activity. Additional support that AtGALT2 encodes an AGP GALT was provided by two allelic AtGALT2 knock-out mutants, which demonstrated lower GALT activities and reductions in β-Yariv-precipitated AGPs compared with wild type plants. Confocal microscopic analysis of fluorescently tagged AtGALT2 in tobacco epidermal cells indicated that AtGALT2 is probably localized in the endomembrane system consistent with its function.

Escherichia coli express fimbriae-associated adhesins through which they attach to mucosal cells and activate a cytokine response. The receptors for E. coli P fimbriae are the globoseries of glycosphingolipids; Gal alpha 1-->4Gal beta-containing oligosaccharides bound to ceramide in the outer leaflet of the lipid bilayer. The receptors for type 1 fimbriae are mannosylated glycoproteins rather than glycolipids. This study tested the hypothesis that P-fimbriated E. coli elicit a cytokine response through the release of ceramide in the receptor-bearing cell. We used the A498 human kidney cell line, which expressed functional receptors for P and type 1 fimbriae and secreted higher levels of interleukin (IL)-6 when exposed to the fimbriated strains than to isogenic nonfimbriated controls. P-fimbriated E. coli caused the release of ceramide and increased the phosphorylation of ceramide to ceramide 1-phosphate. The IL-6 response to P-fimbriated E. coli was reduced by inhibitors of serine/threonine kinases but not by other protein kinase inhibitors. In contrast, ceramide levels were not influenced by type 1-fimbriated E. coli, and the IL-6 response was insensitive to the serine/threonine kinase inhibitors. These results demonstrate that the ceramide-signaling pathway is activated by P-fimbriated E. coli, and that the receptor specificity of the P fimbriae influences this process. We propose that this activation pathway contributes to the cytokine induction by P-fimbriated E. coli in epithelial cells.

Suzuki, K., Mori, I., Nakayama, Y., Miyakoda, M., Kodama, S. and Watanabe, M. Radiation-Induced Senescence-like Growth Arrest Requires TP53 Function but not Telomere Shortening. Normal human diploid cells irradiated with X rays showed permanent cell cycle arrest and exhibited senescence-like phenotypes including the expression of senescence-associated beta-galactosidase (SA-beta-gal). X irradiation caused persistent phosphorylation of TP53 at Ser 15 and accumulation of the TP53 protein, followed by the induction of CDKN1A (also known as p21(Waf1/Cip1)) and CDKN2A (also known as p16), preceded the expression of SA-beta-gal. NCI-H1299 human lung carcinoma cells, in which no TP53 protein was expressed, were irradiated with X rays with or without the exogenous expression of TP53 gene. Although induction of TP53 protein alone could induce SA-beta-gal expression, the frequency of SA-beta-gal-positive cells was significantly increased when TP53-induced H1299 cells were exposed to X rays. The mean terminal restriction fragment length in normal human cells was approximately 12 kb and did not change in SA-beta-gal-positive cells. These results indicate that ionizing radiation induces senescence-like growth arrest that is dependent on TP53 function but independent of telomere shortening. Our findings suggest that cells harboring irreparable DNA damage are programmed to undergo premature senescence to maintain the integrity of the genome.

Approximately 25 years have passed since the first publication suggesting the Flinders sensitive line (FSL) rat as an animal model of depression. At least 6 years of research on these rats was completed before that seminal paper, and there has been a steady stream of publications (130+) over the years. The present review will focus on several issues not previously covered in earlier reviews, summarize the several lines of ongoing investigations, and propose a novel mechanism that accounts for a number of previously unexplained observations. A key observation in the FSL rat relates to the antidepressant (AD)-like effects of known and putative antidepressants. The FSL rat typically exhibits an AD-like effect in behavioral tests for AD-like activity following chronic (14 days) treatment, although some studies have found AD-like effects after fewer days of treatment. In other observations, exaggerated swim test immobility in the FSL rat has been found to have a maternal influence, as shown by cross-fostering studies and observations of maternal behavior; the implications of this finding are still to be determined. Ongoing or recently completed studies have been performed in the laboratories of Marko Diksic of Canada, Aleksander Mathé of Sweden, Gregers Wegener of Denmark, Brian Harvey of South Africa, Paul Pilowsky and Rod Irvine of Australia, and Gal Yadid of Israel. Jennifer Loftis of Portland, Oregon, and Lynette Daws of San Antonio, Texas, have been working with the FSL rats in the United States. A puzzling feature of the FSL rat is its sensitivity to multiple chemicals, and its greater sensitivity to a variety of drugs with different mechanisms of action. It has been recently shown that each of these drugs feeds through G protein-coupled receptors to potassium-gated channels. Thus, an abnormality in the potassium channel could underlie the depressed-like behavior of the FSL rats.

Galectin-1 (Gal-1), a β-galactoside-binding protein, can alter fate and effector function of Th cells; however, little is known about how Gal-1 induces Th cell differentiation. In this article, we show that both uncommitted and polarized Th cells bound by Gal-1 expressed an immunoregulatory signature defined by IL-10. IL-10 synthesis was stimulated by direct Gal-1 engagement to cell surface glycoproteins, principally CD45, on activated Th cells and enhanced by IL-21 expression through the c-Maf/aryl hydrocarbon receptor pathway, independent of APCs. Gal-1-induced IL-10(+) T cells efficiently suppressed T cell proliferation and T cell-mediated inflammation and promoted the establishment of cancer immune-privileged sites. Collectively, these findings show how Gal-1 functions as a major glycome determinant regulating Th cell development, inflammation, and tumor immunity.

The bioavailability of commercial carbamazepine talbets with and without meals was compared to a propylene glycol solution respect to extent of absorption in 6 normal humans after a dose of 6 MG/KG. The presence of dose-dependent kinetics within a clinically sigificant range was also investigated. Serum and urine samples were assayed by gal-liquid chromatography (GLC). Carbamazepine is rapidly absorbed from the propylene glycol solution. Eight per cent of the dose was absorbed from the commercial tablet, resulting in therapeutic serum concentrations(30 to 6 mcg/ni). The data were consitent with disolution rate-limited absorption. Mean half-lives ranged from 31 to 35 hr. No dose-dependent kinetics were observed following administration of does of 3. 6. or 9 mg/kg. The fraction of dose abosrbed, the fraction excredted unchanged in urine, the time of maxium serum concentration, and absorption and elimination half-lives appear to be independent of dose. The time course of side effects could not be correlated with serum carbamazepine levels, suggesting that metabolities contributed to side effects.

Fragmentation behavior of fucosylated N-glycans in both protonated and sodiated form was studied by low-energy collision-induced dissociation with an ion trap mass spectrometer as well as by laser-induced dissociation with matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry (MALDI-TOF/TOF-MS). Diantennary, core-(alpha1-6)-fucosylated N-glycans with Lewis X (Gal(beta1-4)[Fuc(alpha1-3)]GlcNAcbeta1-) and/or fucosylated LacdiNAc antennae (GalNAc(beta1-4)[Fuc(alpha1-3)]GlcNAcbeta1-) were obtained from the human parasite Schistosoma mansoni and used as model substances, after labeling with 2-aminobenzamide, or as native reducing glycans. While fragment spectra of sodiated as well as protonated species obtained in both mass spectrometers resulted in B- and Y-type ions, fragmentation of proton adducts additionally gave rise to various fragment ions which had acquired fucose residues from other parts of the molecule. In particular, fucose was transferred efficiently to the Lewis X antennae suggesting the occurrence of difucosylated antennae, which could erroneously be interpreted as Lewis Y epitopes. By studying two additional model substances, this fucose gain was shown to occur by transfer of fucose between the antennae, but not by transfer of a core-(alpha1-6)-fucose. Despite the drastically different lifetimes of the ions, protonated species analyzed on the ion trap (millisecond range) and by MALDI-TOF/TOF-MS (microsecond range) showed similar rearrangement patterns, suggesting that the fucose mobility goes hand in hand with decomposition. Notably, permethylation of the model N-glycans seemed to completely preclude fucose migration. This study indicates that caution should be applied with the interpretation of tandem mass spectrometric (MS/MS) data of protonated glycoconjugates, including glycopeptides, because of the potential occurrence of fucose rearrangements.

Amoebiasis (a human intestinal infection affecting 50 million people every year) is caused by the protozoan parasite Entamoeba histolytica. To study the molecular mechanisms underlying human colon invasion by E. histolytica, we have set up an ex vivo human colon model to study the early steps in amoebiasis. Using scanning electron microscopy and histological analyses, we have established that E. histolytica caused the removal of the protective mucus coat during the first two hours of incubation, detached the enterocytes, and then penetrated into the lamina propria by following the crypts of Lieberkühn. Significant cell lysis (determined by the release of lactodehydrogenase) and inflammation (marked by the secretion of pro-inflammatory molecules such as interleukin 1 beta, interferon gamma, interleukin 6, interleukin 8 and tumour necrosis factor) were detected after four hours of incubation. Entamoeba dispar (a closely related non-pathogenic amoeba that also colonizes the human colon) was unable to invade colonic mucosa, lyse cells or induce an inflammatory response. We also examined the behaviour of trophozoites in which genes coding for known virulent factors (such as amoebapores, the Gal/GalNAc lectin and the cysteine protease 5 (CP-A5), which have major roles in cell death, adhesion (to target cells or mucus) and mucus degradation, respectively) were silenced, together with the corresponding tissue responses. Our data revealed that the signalling via the heavy chain Hgl2 or via the light chain Lgl1 of the Gal/GalNAc lectin is not essential to penetrate the human colonic mucosa. In addition, our study demonstrates that E. histolytica silenced for CP-A5 does not penetrate the colonic lamina propria and does not induce the host's pro-inflammatory cytokine secretion.

Inhibition of bacterial adherence to bladder cells has been assumed to account for the beneficial action ascribed to cranberry juice and cranberry juice cocktail in the prevention of urinary tract infections (A. E. Sobota, J. Urol. 131:1013-1016, 1984). We have examined the effect of the cocktail and juice on the adherence of Escherichia coli expressing surface lectins of defined sugar specificity to yeasts, tissue culture cells, erythrocytes, and mouse peritoneal macrophages. Cranberry juice cocktail inhibited the adherence of urinary isolates expressing type 1 fimbriae (mannose specific) and P fimbriae [specific for alpha-D-Gal(1----4)-beta-D-Gal] but had no effect on a diarrheal isolate expressing a CFA/I adhesin. The cocktail also inhibited yeast agglutination by purified type 1 fimbriae. The inhibitory activity for type 1 fimbriated E. coli was dialyzable and could be ascribed to the fructose present in the cocktail; this sugar was about 1/10 as active as methyl alpha-D-mannoside in inhibiting the adherence of type 1 fimbriated bacteria. The inhibitory activity for the P fimbriated bacteria was nondialyzable and was detected only after preincubation of the bacteria with the cocktail. Cranberry juice, orange juice, and pineapple juice also inhibited adherence of type 1 fimbriated E. coli, most likely because of their fructose content. However, the two latter juices did not inhibit the P fimbriated bacteria. We conclude that cranberry juice contains at least two inhibitors of lectin-mediated adherence of uropathogens to eucaryotic cells. Further studies are required to establish whether these inhibitors play a role in vivo.

Efforts were directed to identify the specific mutations in the alpha-galactosidase A (alpha-Gal A) gene which cause Fabry disease in families of Japanese origin. By polymerase-chain-reaction-amplification of DNA from reverse-transcribed mRNA and genomic DNA, different point mutations were found in two unrelated Fabry hemizygotes. A hemizygote with classic disease manifestations and no detectable alpha-Gal A activity had a G-to-A transition in exon 1 (codon 44) which substituted a termination codon (TAG) for a tryptophan codon (TGG) and created an NheI restriction site. This point mutation would predict a truncated alpha-Gal A polypeptide, consistent with the observed absence of enzymatic activity and a classic Fabry phenotype. In an unrelated Japanese hemizygote who had an atypical clinical course characterized by late-onset cardiac involvement and significant residual alpha-Gal activity, a G-to-A transition in exon 6 (codon 301) resulted in the replacement of a glutamine for an arginine residue. This amino acid substitution apparently altered the properties of the enzyme such that sufficient enzymatic activity was retained to markedly alter the disease course. Identification of these mutations permitted accurate molecular heterozygote diagnosis in these families.

In the present study, we examined the supplementation of paeonol extracted from Moutan cortex of Paeonia suffruticosa Andrews (MC) or the root of Paeonia lactiflora Pall (PL) on reducing oxidative stress, cognitive impairment and neurotoxicity in d-galactose (D-gal)-induced aging mice. The ICR mice were subcutaneously injected with D-gal (50 mg/(kg day)) for 60 days and administered with paeonol (50, 100 mg/(kg day)) simultaneously. The results showed that paeonol significantly improved the learning and memory ability in Morris water maze test and step-down passive avoidance test in D-gal-treated mice. Further investigation showed that the effect of paeonol on improvement of cognitive deficit was related to its ability to inhibit the biochemical changes in brains of D-gal-treated mice. Paeonol increased acetylcholine (Ach) and glutathione (GSH) levels, restored superoxide dismutase (SOD) and Na(+), K(+)-adenosine triphosphatase (Na(+), K(+)-ATPase) activities, but decreased cholinesterase AChe activity and malondialdehyde (MDA) level in D-gal-treated mice. Furthermore, paeonol ameliorated neuronal damage in both hippocampus and temporal cortex in D-gal-treated mice. These results suggest that paeonol possesses anti-aging efficacy and may have potential in treatment of neurodegenerative diseases.

Previously, we showed that the addition of human erythrocyte glycosphingolipids (GSLs) to nonhuman CD4(+) or GSL-depleted human CD4(+) cells rendered those cells susceptible to HIV-1 envelope glycoprotein-mediated cell fusion. Individual components in the GSL mixture were isolated by fractionation on a silica-gel column and incorporated into the membranes of CD4(+) cells. GSL-supplemented target cells were then examined for their ability to fuse with TF228 cells expressing HIV-1LAI envelope glycoprotein. We found that one GSL fraction, fraction 3, exhibited the highest recovery of fusion after incorporation into CD4(+) nonhuman and GSL-depleted HeLa-CD4 cells and that fraction 3 contained a single GSL fraction. Fraction 3 was characterized by MS, NMR spectroscopy, enzymatic analysis, and immunostaining with an antiglobotriaosylceramide (Gb3) antibody and was found to be Gal(alpha1-->4)Gal(beta1-->4)Glc-Cer (Gb3). The addition of fraction 3 or Gb3 to GSL-depleted HeLa-CD4 cells recovered fusion, but the addition of galactosylceramide, glucosylceramide, the monosialoganglioside, GM3, lactosylceramide, globoside, the disialoganglioside, GD3, or alpha-galactosidase A-digested fraction 3 had no effect. Our findings show that the neutral GSL, Gb3, is required for CD4/CXCR4-dependent HIV-1 fusion.

Tim-3 was initially identified on activated Th1, Th17, and Tc1 cells and induces T cell death or exhaustion after binding to its ligand, Gal-9. The observed relationship between dysregulated Tim-3 expression on T cells and the progression of many clinical diseases has identified this molecule as an important target for intervention in adaptive immunity. Recent data have shown that it also plays critical roles in regulating the activities of macrophages, monocytes, dendritic cells, mast cells, natural killer cells, and endothelial cells. Although the underlying mechanisms remain unclear, dysregulation of Tim-3 expression on these innate immune cells leads to an excessive or inhibited inflammatory response and subsequent autoimmune damage or viral or tumor evasion. In this review, we focus on the expression and function of Tim-3 on innate immune cells and discuss (1) how Tim-3 is expressed and regulated on different innate immune cells; (2) how it affects the activity of different innate immune cells; and (3) how dysregulated Tim-3 expression on innate immune cells affects adaptive immunity and disease progression. Tim-3 is involved in the optimal activation of innate immune cells through its varied expression. A better understanding of the physiopathological role of the Tim-3 pathway in innate immunity will shed new light on the pathogenesis of clinical diseases, such as autoimmune diseases, chronic viral infections, and cancer, and suggest new approaches to intervention.

Galectin-9 (gal-9), widely expressed in many tissues, regulates Th1 cells and induces their apoptosis through its receptor, T-cell Ig mucin 3, which is mainly expressed on terminally differentiated Th1 cells. Type 1 diabetes is a Th1-dominant autoimmune disease that specifically destroys insulin-producing beta cells. To suppress the Th1 immune response in the development of autoimmune diabetes, we overexpressed gal-9 in NOD mice by injection of a plasmid encoding gal-9. Mice treated with gal-9 plasmid were significantly protected from diabetes and showed less severe insulitis compared with controls. Flow cytometric analyses in NOD-T1/2 double transgenic mice showed that Th1-cell population in spleen, pancreatic lymph node and pancreas was markedly decreased in gal-9 plasmid-treated mice, indicating a negative regulatory role of gal-9 in the development of pathogenic Th1 cells. Splenocytes from gal-9 plasmid-treated mice were less responsive to mitogenic stimulation than splenocytes from the control group. However, adoptive transfer of splenocytes from gal-9-treated or control mice caused diabetes in NOD/SCID recipients with similar kinetics, suggesting that gal-9 treatment does not induce active tolerance in NOD mice. We conclude that gal-9 may downregulate Th1 immune response in NOD mice and could be used as a therapeutic target in autoimmune diabetes.

Endothelial cell surface expression of VCAM-1 is one of the initial steps in the pathogenesis of atherosclerosis. The inflammatory response transcription factor nuclear factor (NF)-kappaB plays an important role in the regulation of VCAM-1 expression by various stimuli including tumor necrosis factor (TNF)-alpha. Other transcription factors may modulate this response through interaction with NF-kappaB factors. Since c-Fos/c-Jun (activating protein-1 (AP-1)) are expressed in vascular endothelium during proinflammatory conditions, we investigated the role of AP-1 proteins in the expression of VCAM-1 by TNF-alpha in SV40 immortalized human microvascular endothelial cells (HMEC). TNF-alpha induced expression of both early protooncogenes, c-fos and c-jun. The ability of TNF-alpha to activate the kappaB-motif (kappaL-kappaR)-dependent VCAM-1 promoter-chloramphenicol acetyltransferase (CAT) reporter gene lacking a consensus AP-1 element was markedly inhibited by co-transfection of the expression vector encoding c-fos ribozyme, which decreases the level of c-fos by degrading c-fos mRNA, or c-fos or c-jun oligonucleotides. Conversely, co-transfection of c-Fos and c-Jun encoding expression vectors potentiated the p65/NF-kappaB-mediated transactivation of the VCAM-1 promoter-CAT reporter gene. Furthermore the c-Fos encoding expression vector potentiated by 2-fold the transactivation activity of a chimeric transcriptional factor Gal/p65 (containing the transactivation domain of p65 and the DNA binding domain of the yeast transcriptional factor Gal-4). Consistent with the promoter studies, curcumin and NDGA, inhibitors of AP-1 activation, markedly inhibited the ability of TNF-alpha to activate the expression of VCAM-1 mRNA levels at concentrations that did not inhibit the activation of NF-kappaB. In gel mobility supershift assays, the antibodies to c-Fos or c-Jun inhibited the binding of TNF-alpha-activated nuclear NF-kappaB to the kappaL-kappaR, suggesting that both c-Fos and c-Jun interacted with NF-kappaB. These results suggest that AP-1 proteins may mediate the effect of TNF-alpha in the regulation of VCAM-1 expression through interaction with NF-kappaB factors in endothelial cells.

Studies of the properties of immune complexes (IC) in the circulation, urine, and mesangium of IgA nephropathy (IgAN) patients have provided data relevant to the pathogenesis of this disease. IC contain predominantly polymeric IgA1 molecules which are deficient in galactose (Gal) residues on O-linked glycan chains in the hinge region (HR) of their heavy (H) chains. As a result of this aberrancy, a novel antigenic determinant(s) involving N-acetylgalactosamine (GalNAc) and perhaps sialic acid (SA) of O-linked glycans is generated and recognized by naturally occurring GalNAc-specific antibodies. Thus, IC in IgAN consist of Gal-deficient IgA1 molecules as an antigen, and GalNAc-specific IgG and/or IgA1 as an antibody. IgG antibodies to Gal-deficient IgA1 are probably induced by cross-reactive microbial antigens; they are present at variable levels not only in humans with or without IgAN but also in many phylogenetically diverse vertebrate species. Incubation of human mesangial cells with IC from sera of IgAN patients indicated that stimulation of cellular proliferative activity was restricted to the large (>800 kDa) complexes. These findings suggest that experimental approaches that prevent the formation of large Gal-deficient IgA1-IgG IC may be applied ultimately in an immunologically mediated therapy.

The protective functions that have been ascribed to anthocyanins in leaves can be performed as effectively by a number of other compounds. The possibility that anthocyanins accumulate most abundantly in leaves deficient in other phytoprotective pigments has been tested. Pigment concentrations and their histological distribution were surveyed for a sample of 1000 leaves from a forest population of Quintinia serrata, which displays natural polymorphism in leaf colour. Eight leaf phenotypes were recognized according to their patterns of red coloration. Anthocyanins were observed in almost all combinations of every leaf tissue, but were most commonly located in the vacuoles of photosynthetic cells. Red leaves contained two anthocyanins (Cy-3-glc and Cy-3-gal), epicuticular flavones, epidermal flavonols, hydroxycinnamic acids, chlorophylls, and carotenoids. Green leaves lacked anthocyanins, but had otherwise similar pigment profiles. Foliar anthocyanin levels varied significantly between branches and among trees, but were not correlated to concentrations of other pigments. Anthocyanins were most abundant in older leaves on trees under canopies with south-facing gaps. These data indicate that anthocyanins are associated with photosynthesis, but do not serve an auxiliary phytoprotective role. They may serve to protect shade-adapted chloroplasts from brief exposure to high intensity sunflecks.

Galectin-3 (Gal-3), a member of a family of highly conserved carbohydrate-binding proteins, has recently emerged as a novel cellular modulator at inflammatory foci. Here we investigated the effects of Gal-3 on central effector functions of human neutrophils, including phagocytosis, exocytosis of secretory granules, and survival. We examined the effects of Gal-3 alone or in combination with soluble fibrinogen (sFbg), an extracellular mediator that plays a key role during the early phase of the inflammatory response through binding to integrin receptors. In addition we evaluated the intracellular signals triggered by these mediators in human neutrophils. Human neutrophils incubated with recombinant Gal-3 alone increased their phagocytic activity and CD66 surface expression. In contrast to the known antiapoptotic effect of Gal-3 on many cellular types, Gal-3 enhanced PMN apoptotic rate. Preincubation with Gal-3 primed neutrophils to the effects of sFbg, resulting in a synergistic action on degranulation. On the other hand, Gal-3 and sFbg had opposite effects on PMN survival, and the simultaneous action of both agonists partially counteracted the proapoptotic effects of Gal-3. In addition, although sFbg induced its effects through the activation of the ERKs, Gal-3 led to p38 phosphorylation. Disruption of this signaling pathway abrogated Gal-3-mediated modulation of neutrophil degranulation, phagocytosis, and apoptosis. Together, our results support the notion that Gal-3 and sFbg are two physiological mediators present at inflammatory sites that activate different components of the MAPK pathway and could be acting in concert to modulate the functionality and life span of neutrophils.

We have developed an enzyme-nanoparticle sensor array where the sensitivity is amplified through enzymatic catalysis. In this approach cationic gold nanoparticles are electrostatically bound to an enzyme (beta-galactosidase, beta-Gal), inhibiting enzyme activity. Analyte proteins release the beta-Gal, restoring activity and providing an amplified readout of the binding event. Using this strategy we have been able to identify proteins in buffer at a concentration of 1 nM, substantially lower than current strategies for array-based protein sensing. Moreover, we have obtained identical sensitivity in studies where the proteins are spiked into the complex protein matrix provided by desalted human urine ( approximately 1.5 muM total protein; spiked protein concentrations were 0.067% of the overall protein concentration), demonstrating the potential of the method for diagnostic applications.

Yeast ubiquinone or coenzyme Q(6) (Q(6)) is a redox active lipid that plays a crucial role in the mitochondrial electron transport chain. At least nine proteins (Coq1p-9p) participate in Q(6) biosynthesis from 4-hydroxybenzoate (4-HB). We now show that the mitochondrial ferredoxin Yah1p and the ferredoxin reductase Arh1p are required for Q(6) biosynthesis, probably for the first hydroxylation of the pathway. Conditional Gal-YAH1 and Gal-ARH1 mutants accumulate 3-hexaprenyl-4-hydroxyphenol and 3-hexaprenyl-4-aminophenol. Para-aminobenzoic acid (pABA) is shown to be the precursor of 3-hexaprenyl-4-aminophenol and to compete with 4-HB for the prenylation reaction catalyzed by Coq2p. Yeast cells convert U-((13)C)-pABA into (13)C ring-labeled Q(6), a result that identifies pABA as a new precursor of Q(6) and implies an additional NH(2)-to-OH conversion in Q(6) biosynthesis. Our study identifies pABA, Yah1p, and Arh1p as three actors in Q(6) biosynthesis.