Melibiosamine, a novel oligosaccharide, suppresses mitogen-induced IL-2 production via inactivation of NFAT and NFκB in Jurkat cells.

d-Glucosamine (GlcNH2) and several of its derivatives are known to possess immunosuppressive activities in various immune cell lines. The novel GlcNH2-containing oligosaccharide Galα1-6GlcNH2 (designated melibiosamine; MelNH2) is expected to be immunosuppressive also. In Jurkat cells (immortalized human T lymphocytes), interleukin 2 (IL-2) production (an index of the T-cell immune response) can be induced by stimulation with a mitogen, such as concanavalin A. Here, we compared the effects of GlcNH2 and MelNH2 on concanavalin A-induced IL-2 production (CIIP) in Jurkat cells and found that GlcNH2 and MelNH2 at millimolar levels both significantly suppressed CIIP without affecting cell viability. When we examined the effects of GlcNH2 and MelNH2 on the activation of the three transcription factors required for CIIP-NFAT (nuclear factor of activated T-cells), NFκB (nuclear factor kappa-light-chain-enhancer of activated B cells), and AP-1 (activator protein 1)-we found that GlcNH2 and MelNH2 both suppressed CIIP by inhibiting the activation of NFAT and NFκB, but, unlike GlcNH2, MelNH2 also promoted the activation of AP-1. These results suggest that MelNH2 may be a potentially useful lead compound for development as an immunosuppressive or anti-inflammatory drug.

Halogen-Substituted Derivatives of Dictyostelium Differentiation-Inducing Factor-1 Suppress Serum-Induced Cell Migration of Human Breast Cancer MDA-MB-231 Cells in Vitro.

Triple-negative breast cancer (TNBC) is highly proliferative and metastatic, and because it lacks three major molecular targets for chemotherapy (estrogen receptor, progesterone receptor, and human epidermal receptor 2), it is extremely refractory. Differentiation-inducing factor 1 (DIF-1) and DIF-3, which are chlorinated alkylphenones, are lead anticancer compounds found in the cellular slime mold Dictyostelium discoideum. Here, we examined the in vitro effects of DIF-1, DIF-3, and 25 DIF derivatives on cell proliferation and serum-induced cell migration in human MDA-MB-231 cells, a model TNBC cell line. We found that Br-DIF-1, a chlorine-to-bromine-substituted derivative of DIF-1, strongly suppressed cell migration (IC50, 3.8 M) with negligible effects on cell proliferation (IC50, >20 M). We then synthesized 18 derivatives of Br-DIF-1 and examined the in vitro effects of these derivatives on cell proliferation and serum-induced cell migration in MDA-MB-231 cells. Among the derivatives, Br-DIF-1(+1), Br-DIF-1(+2), and Br-DIF-3(+2) exhibited strong anti-cell migration activities with IC50 values of 1.5, 1.0, and 3.1 M, respectively, without affecting cell proliferation (IC50, >20 M). These results suggest that these Br-DIF derivatives are good lead compounds for the development of anti-metastatic drugs against TNBC.

Tryptophan nitration of immunoglobulin light chain as a new possible biomarker for atopic dermatitis.

To reduce the incidence and severity of atopic dermatitis, detection and treatment at an early stage are urgently required, but no effective biomarker has been reported. In this study, we attempted to detect a candidate biomarker of early stage atopic dermatitis by focusing on the levels of nitrated residues in the plasma proteins of atopic dermatitis model mice (NC/Nga mice). We found that the immunoglobulin (Ig) light chain was more highly nitrated in the plasma of the animal model than that of control mice. Western blot analysis showed a statistically significant difference between the 6-nitrotryptophan content of the Ig light chain in the NC/Nga mice before onset of atopic dermatitis symptoms and that of the control mice. LC-ESI-MS/MS analysis demonstrated that these light chains contained nitrotryptophan (Trp56) and nitrotyrosine (Tyr66). Immunofluorescence staining revealed a significant increase in manganese superoxide dismutase and inducible nitric oxide synthase production in the skin lesions of the NC/Nga mice. Furthermore, we found protein-bound 6-nitrotryptophan and 3-nitrotyrosine only in the lesioned skin, where their signals partially overlapped with the IgG signal. Our findings suggest that the 6-nitrotryptophan content of Ig light chains could be a new biomarker for detecting early stage atopic dermatitis.

Isolation, Synthesis, and Biological Activity of Chlorinated Alkylresorcinols from Dictyostelium Cellular Slime Molds.

Eight chlorinated alkylresorcinols, monochasiol A-H (1-8), were isolated from the fruiting bodies of Dictyostelium monochasioides. Compounds 1-8 were synthesized to confirm their structures and to obtain sufficient material for performing biological tests. Monochasiol A (1) selectively inhibited the concanavalin A-induced interleukin-2 production in Jurkat cells, a human T lymphocyte cell line. Monochasiols were biogenetically synthesized by the combination of biosynthetic enzymes relating to the principal polyketides, MPBD and DIF-1, produced by Dictyostelium discoideum.

Relationships among plasma granzyme B level, pruritus and dermatitis in patients with atopic dermatitis.

BACKGROUND: Atopic dermatitis (AD) is a multifactorial inflammatory skin disease characterized by skin barrier dysfunction, allergic inflammation and intractable pruritus resistant to conventional antipruritic treatments, including H1-antihistamines. Granzymes (Gzms) are a family of serine proteases expressed by cytotoxic T lymphocytes and natural killer cells that have been shown to modulate inflammation. However, the relationship between Gzms and pathology in AD remains unclear. OBJECTIVE: This study assessed the correlation between plasma GzmB levels and severity of pruritus and dermatitis, in AD patients. METHODS: Plasma was collected from 46 patients with AD, 24 patients with psoriasis, and 30 healthy controls. AD severity was assessed with the scoring atopic dermatitis (SCORAD) index, psoriasis severity with the psoriasis area and severity index (PASI), and degree of pruritus by visual analogue scale (VAS) score. GzmA, GzmB and gastrin releasing peptide (GRP) levels were measured by enzyme-linked immunosorbent assays. RESULTS: Plasma GzmB concentrations were significantly higher in patients with AD and psoriasis than in healthy controls. Correlation analyses showed that plasma GzmB concentrations positively correlated with SCORAD and serum levels of severity markers such as thymus and activation-regulated chemokine, and lactate dehydrogenase in AD patients. Moreover, plasma levels of GRP, an itch-related peptide, were higher in patients with AD, positively correlating with VAS score and plasma GzmB level. In addition, plasma GzmB concentration was significantly lower in the treatment group than the untreated group with AD. Meanwhile, there were no correlations among GzmB levels, VAS score and PASI score in patients with psoriasis. In contrast to the results of plasma GzmB, plasma GzmA levels were unchanged among AD, psoriasis and healthy groups, and showed no correlations with VAS score and SCORAD index in patients with AD. CONCLUSION: Plasma GzmB levels may reflect the degree of pruritus and dermatitis in patients with AD.

Sumoylated α-skeletal muscle actin in the skeletal muscle of adult rats.

Skeletal muscles are composed of two major muscle fiber types: slow-twitch oxidative fibers and fast-twitch glycolytic fibers. The proteins in these muscle fibers are known to differ in their expression, relative abundance, and post-translational modifications. In this study, we report a previously unreported post-translational modification of α-skeletal muscle actin in the skeletal muscles of adult male F344 rats in vivo. Using two-dimensional electrophoresis (2D-PAGE), we first examined the differences in the protein expression profiles between the soleus and plantaris muscles. We found higher intensity protein spots at approximately 60 kDa and pH 9 on 2D-PAGE for the soleus muscle compared with the plantaris muscle. These spots were identified as α-skeletal muscle actin by liquid chromatography-nanoelectrospray ionization-tandem mass spectrometry and western blot analyses. In addition, we found that the 60 kDa α-skeletal muscle actin is modified by small ubiquitin-like modifier (SUMO) 1, using 2D-PAGE and western blot analyses. Furthermore, we found that α-skeletal muscle actin with larger molecular weight was localized in the nuclear and cytosol of the skeletal muscle, but not in the myofibrillar fraction by the combination of subcellular fractionation and western blot analyses. These results suggest that α-skeletal muscle actin is modified by SUMO-1 in the skeletal muscles, localized in nuclear and cytosolic fractions, and the extent of this modification is much higher in the slow muscles than in the fast muscles. This is the first study to show the presence of SUMOylated actin in animal tissues.

Importance of tryptophan nitration of carbonic anhydrase III for the morbidity of atopic dermatitis.

The nitration of proteins results from the vigorous production of reactive nitrogen species in inflammatory disease. We previously reported the proteomic analysis of nitrated tryptophan residues in in vitro model cells for inflammatory diseases using a 6-nitrotryptophan-specific antibody. In this paper, we applied this method to the analysis of a disease model animal and identified the 6-nitrotryptophan-containing proteins in the skin of atopic dermatitis model mice (AD-NC/Nga mice). We found three nitrotryptophan-containing proteins, namely, carbonic anhydrase III (CAIII), α-enolase (α-ENO), and cytoskeletal keratin type II (KTII), and identified the positions of the nitrotryptophan residues in their amino acid sequences: Trp47 and Trp123 in CAIII, Trp365 in α-ENO, and Trp221 in KTII. Among these, the nitration of CAIII was increased not only in the lesional skin of AD-NC/Nga mice but also in the mice that did not present any symptoms. The in vitro nitration of purified CAIII by peroxynitrite reduced its CO2 hydratase activity in a dose-dependent manner. In addition, we found that CAIII was induced during the differentiation of normal human epidermal keratinocytes. Furthermore, we found the presence of CAIII and the formation of 6-nitrotryptophan-containing proteins in both the lesional and the nonlesional sections of the skin of patients with atopic dermatitis through immunohistochemical staining. This study provides the first demonstration of the formation of 6-nitrotryptophan in human tissues and disease.

Long-term study of urinary bisphenol A in elementary school children.

OBJECTIVES: Due to its industrial application and frequent use as a coating material for food containers, bisphenol A (4,4'-isopropylidenediphenol, BPA) is present in abundance in our environment. Data on intake levels of BPA are limited in preadolescent children in Japan. This study was designed to help us better understand the current state of BPA exposure in children in Japan. METHODS: We followed first graders (n = 104) attending school in a Tokyo suburb from 1998 until the sixth grade (2003), during which time we collected a total of three morning urine samples. Urinary BPA was analyzed using high-performance liquid chromatography isotope-dilution tandem mass spectrometry. RESULTS: Ninety-four children were followed for 5 years. Median urinary BPA level was 2.66 ng/mg creatinine (CRE) (range 0.9-38.9) at first grade (1998), 1.52 ng/mg CRE (0.4-11.8) at third grade (2000), and 0.66 ng/mg CRE (0.2-8.5) at sixth grade (2003), showing a significant decrease in urinary BPA levels over a 5-year follow-up study (p < 0.001). No significant difference was seen between boys and girls at each grade. CONCLUSIONS: Urinary levels of BPA were relatively low throughout the study period; however, as the study progressed, we observed a significant decline in levels, the reason behind which is not yet clear.

Antibody isotype responses to paramyosin, a vaccine candidate for schistosomiasis, and their correlations with resistance and fibrosis in patients infected with Schistosoma japonicum in Leyte, The Philippines.

We examined whether antibody isotype responses to paramyosin (PM), a vaccine candidate for schistosomiasis, are associated with age-dependent resistance and pathology in liver fibrosis using human sera collected from 139 individuals infected with Schistosoma japonicum in Leyte, The Philippines. We report that IgA and IgG3 responses to PM showed a positive correlation with age and that the epitopes responsible were localized predominantly within the N-terminal half of PM. In addition, the IgG3 response to PM was associated with serum level of procollagen-III-peptide (P-III-P), an indicator of progression of liver fibrosis. These results imply that IgG3 against PM may not only provoke age-dependent resistance to S. japonicum infection but also enhance liver fibrosis. In contrast, levels of IgE to PM and to multiple PM fragments showed a negative correlation with P-III-P level. Thus, in contrast to IgG3, increases in PM-specific IgE may contribute to suppression of liver pathogenesis in schistosomiasis.

Molecular cloning and characterization of ouabain-insensitive Na(+)-ATPase in the parasitic protist, Trypanosoma cruzi.

Maintaining low intracellular sodium concentrations is vital for almost all organisms. Na(+) efflux is generally governed by P-type ATPases, Na(+)/K(+)-ATPase in animals and Na(+)-ATPase, called ENA, in fungi and plants. Trypanosoma cruzi, which parasitizes mammalian cells, must undergo drastic adaptations to high Na(+) concentrations outside and low Na(+) concentrations inside host cells. However, T. cruzi Na(+) efflux pumps have not been identified. We report here the cloning and characterization of the gene encoding Na(+)-ATPase in T cruzi, which resembled fungal and plant ENAs, termed TcENA. TcENA was a plasma membrane protein expressed throughout the parasite life cycle. The transcription level of TcENA was higher in insect stage epimastigotes and blood stream trypomastigotes than in intracellular amastigotes, probably reflecting the high Na(+) concentration outside the host cells. Biochemical analysis of TcENA expressed heterologously in mammalian cells demonstrated, for the fist time, that the ATPase activity of TcENA is stimulated by both Na(+) and K(+) and is insensitive to ouabain, a specific inhibitor of Na(+)/K(+)-ATPases. Furthermore, epimastigotes overproducing TcENA showed increased tolerance to high Na(+) stress. Our findings suggest that TcENA acts as a sodium pump and provide insights into the regulation of ion homeostasis in the parasitic protist.

Genetic diversity and kinetic properties of Trypanosoma cruzi dihydroorotate dehydrogenase isoforms.

Dihydroorotate dehydrogenase (DHOD) is the fourth enzyme in the de novo pyrimidine biosynthetic pathway and is essential in Trypanosoma cruzi, the parasitic protist causing Chagas' disease. T. cruzi and human DHOD have different biochemical properties, including the electron acceptor capacities and cellular localization, suggesting that T. cruzi DHOD may be a potential chemotherapeutic target against Chagas' disease. Here, we report nucleotide sequence polymorphisms of T. cruzi DHOD genes and the kinetic properties of the recombinant enzymes. T. cruzi Tulahuen strain possesses three DHODgenes: DHOD1 and DHOD2, involved in the pyrimidine biosynthetic (pyr) gene cluster on an 800 and a 1000 kb chromosomal DNA, respectively, and DHOD3, located on an 800 kb DNA. The open reading frames of all three DHOD genes are comprised of 942 bp, and encode proteins of 314 amino acids. The three DHOD genes differ by 26 nucleotides, resulting in replacement of 8 amino acid residues. In contrast, all residues critical for constituting the active site are conserved among the three proteins. Recombinant T. cruzi DHOD1 and DHOD2 expressed in E. coli possess similar enzymatic properties, including optimal pH, optimal temperature, Vmax, and Km for dihydroorotate and fumarate. In contrast, DHOD3 had a higher Vmax and Km for both substrates. Orotate competitively inhibited all three DHOD enzymes to a comparable level. These results suggest that, despite their genetic variations, kinetic properties of the three T. cruziDHODs are conserved. Our findings facilitate further exploitation of T. cruzi DHOD inhibitors, as chemotherapeutic agents against Chagas' disease.

Inhibitory action of marine algae extracts on the Trypanosoma cruzi dihydroorotate dehydrogenase activity and on the protozoan growth in mammalian cells.

Trypanosoma cruzi, the causative agent of Chagas' disease, replicates in mammalian cells and relies on the de novo pyrimidine biosynthetic pathway that supplies essential precursors for nucleic acid synthesis. The protozoan dihydroorotate dehydrogenase (DHOD), the fourth enzyme of the pathway catalyzing production of orotate from dihydroorotate, markedly differs from the human enzyme. This study was thus aimed to search for potent inhibitors against T. cruzi DHOD activity, and a number of methanol extracts prepared from green, brown, and red algae were assayed. The extracts from two brown algae, Fucus evanescens and Pelvetia babingtonii, yielded 59 and 58% decrease in the recombinant DHOD activity, respectively, at the concentration of 50 microg/ml. Inhibition by these extracts was noncompetitive with respect to dihydroorotate, with apparent Ki values of 35.3+/-5.9 and 10.3+/-4.4 microg/ml, respectively. Further, in an in vitro T. cruzi-HeLa cell infection system, ethanol-reconstituted F. evanescens and P. babingtonii extracts at the concentration of 1 microg/ml, respectively, decreased significantly the infection rate of host cells and the average parasite number per infected cell. These results imply that F. evanescens and P. babingtonii contain inhibitor(s) against the T. cruzi DHOD activity and against the protozoan infection and proliferation in mammalian cells. Identification of inhibitor(s) in these two brown algae and further screening of other marine algae may facilitate the discovery of new, anti-trypanosomal lead compounds.