B-raf, a new member of the raf family, is activated by DNA rearrangement.

Complementary DNA clones of a putative transforming gene were isolated from NIH 3T3 cells transformed with human Ewing sarcoma DNA. The gene was termed B-raf because it is related to but distinct from c-raf and A-raf. It appears that substitution in the amino-terminal portion of the normal B-raf protein confers transforming activity to the gene.     

Contribution of the neural cell recognition molecule NB‐3 to synapse formation between parallel fibers and Purkinje cells in mouse

The neural cell recognition molecule NB‐3, also referred to as contactin‐6, is expressed prominently in the developing nervous system after birth and its deficiency has been shown to cause impairment in motor coordination. Here, we investigated the contribution of NB‐3 to cerebellar development, focusing on lobule 3 where NB‐3 was expressed in granule cells but not in Purkinje cells. In the developing molecular layer, the neural cell recognition molecules TAG‐1, L1, and NB‐3 formed distinct expression zones from the external granule cell layer to the internal granule cell layer (IGL), respectively. The NB‐3‐immunoreactive zone did not overlap with TAG‐1‐immunoreactive zone. By contrast, the L1‐immunoreactive zone overlapped with both the TAG‐1‐ and NB‐3‐immunoreactive zones. NB‐3‐positive puncta overlapped with vesicular glutamate transporter 1, a presynaptic marker and were apposed close to metabotropic glutamate receptor 1A, a postsynaptic marker, indicating that NB‐3 is localized presynaptically at glutamatergic synapses between parallel fibers and Purkinje cells. In NB‐3 knockout mice, L1 immunoreactive signals were increased in the IGL at postnatal day (P) 5, suggesting the increase in the number of immature granule cells of the IGL. In addition, the density of parallel fiber synaptic terminals was reduced in NB‐3 knockout mice relative to wild‐type mice at P5 to P10. In parallel with these findings, caspase‐dependent cell death was significantly increased in the NB‐ 3‐deficient cerebellum at P15. Collectively, our results indicate that NB‐3 deficiency affects synapse formation during postnatal cerebellar development. © 2009 Wiley Periodicals, Inc. Develop Neurobiol, 2009     

Core3 O-Glycan Synthase Suppresses Tumor Formation and Metastasis of Prostate Carcinoma PC3 and LNCaP Cells through Down-regulation of ��2��1 Integrin Complex

Although there are numerous reports of carbohydrates enriched in cancer cells, very few studies have addressed the functions of carbohydrates present in normal cells that decrease in cancer cells. It has been reported that core3 O-glycans are synthesized in normal gastrointestinal cells but are down-regulated in cancer cells. To determine the roles of core3 O-glycans, we transfected PC3 and LNCaP prostate cancer cells with β3-N-acetylglucosaminyltransferase-6 (core3 synthase) required to synthesize core3 O-glycans. Both engineered cell lines exhibited reduced migration and invasion through extracellular matrix components compared with mock-transfected cells. Moreover we found that α2β1 integrin acquired core3 O-glycans in cells expressing core3 synthase with decreased maturation of β1 integrin, leading to decreased levels of the α2β1 integrin complex, decreased activation of focal adhesion kinase, and reduced lamellipodia formation. Upon inoculation into the prostate of nude mice, PC3 cells expressing core3 O-glycans produced much smaller tumors without metastasis to the surrounding lymph nodes in contrast to robust tumor formation and metastasis seen in mock-transfected PC3 cells. Similarly LNCaP cells expressing core3 O-glycans barely produced subcutaneous tumors in contrast to robust tumor formation by mock-transfected LNCaP cells. These findings indicate that addition of core3 O-glycans to β1 and α2 integrin subunits in prostate cancer cells suppresses tumor formation and tumor metastasis.Cancer cells often express surface carbohydrates different from normal cells (1). One such change is expression of sialyl Lewis X and Lewis B blood group antigens in cancer cells (2, 3). These structural elements are seen as capping oligosaccharides attached to the underlying glycan backbone where they likely function as ligands for cell adhesion molecules.The structure of underlying glycans also changes during malignant transformation and differentiation. In particular, there are several reports that an increase in the β1,6-N-acetylglucosaminyl branch in N-glycans synthesized by β1,6-N-acetylglucosaminyltransferase-V is associated with oncogenic transformation (4–7). Similar structural changes are seen in mucin-type O-glycans, which have N-acetylgalactosamine at the reducing end linked to polypeptide threonine or serine residues. Addition of different carbohydrate residues to N-acetylgalactosamine confers a variety of backbone structures on mucin-type O-glycans; the most abundant of those are classified as core1, core2, core3, and core4 O-glycans (8) (Fig. 1). Among these O-glycans, the synthesis of the core2 branch has been extensively studied particularly because conversion of core1 to core2 O-glycans was observed in T cell activation (9). Expression of core2 branch apparently represents an oncodifferentiation antigen because core2 branched O-glycans are synthesized in early stages of T cell differentiation, down-regulated in mature T cells, and reappear in T cell leukemia and immune deficiencies such as AIDS and Wiskott-Aldrich syndrome (for a review, see Ref. 10). In addition, overexpression of core2 O-glycans is seen in many cancers, including lung and breast carcinoma cells (11, 12).Open in a separate windowFIGURE 1.Biosynthetic pathways of mucin-type O-glycans. N-Acetylgalactosamine is transferred to a serine or threonine residue in a polypeptide. Resultant GalNAcα1→Ser/Thr is converted by core3 synthase (β3GnT-6) to GlcNAcβ1→3GalNAcα1→Ser/Thr (core3). Core3 is then converted to core4 by C2GnT-2 (C2GnT-M). GalNAcα1→Ser/Thr is also converted to core1, Galβ1→3GalNAcα1→Ser/Thr, by core1 synthase. Core1 is then converted to core2 by C2GnT-1, C2GnT-2, and C2GnT-3.By contrast, core3 and core4 O-glycans are synthesized in normal cells but apparently down-regulated in gastric and colorectal carcinoma (13, 14). Core3 O-glycans are synthesized by core3 synthase (β3GnT-6),² which adds β1,3-linked N-acetylglucosamine to N-acetylgalactosamine at the reducing terminus (15) (Fig. 1). Iwai et al. (16) showed that forced expression of core3 synthase in human fibrosarcoma HT1080 FP-10 cells resulted in significant reduction in the formation of lung tumor foci in mice after intravenous injection of tumor cells through a tail vein. However, the same study did not address whether the expression of core3 influences tumor metastasis because the cancer cells were intravenously injected and no primary tumor was formed to spread into the lung as metastasis in contrast to the other studies (17, 18). Core4 O-glycan is synthesized by addition of β1,6-linked N-acetylglucosamine to a core3 acceptor by core2 β1,6-N-acetylglucosamine M type (C2GnT-M) or C2GnT-2 (19, 20) (Fig. 1). Huang et al. (21) reported that C2GnT-M is down-regulated in colonic carcinoma cells and that forced expression of C2GnT-M in HCT116 colonic carcinoma cells significantly decreased cell invasion and subcutaneous tumor formation. How up-regulation of core3 and core4 O-glycans influences the pathophysiology of cells expressing core3 and core4 O-glycans has not been addressed.Cell-extracellular matrix interaction plays an essential role during acquisition of migration and invasive behavior of cancer cells. For example, α2β1 integrin is the major receptor for collagen (22) and most abundantly expressed in prostate cancer cells (23). Glycosylation on integrin is one of the important modulators of integrin functions, and many glycan structures, mainly N-glycans, have been studied. An increase of bisecting GlcNAc structure on α5β1 integrin inhibits the cell spreading and migration (24), and induced β1,6-GlcNAc sugar chains on N-glycans of β1 integrin result in stimulation of cell migration (25). However, it has not been addressed whether changes in O-glycans affect integrin maturation and functions.To determine the role of core3 O-glycans in tumor formation and metastasis, we analyzed PC3 and LNCaP human prostate cancer cells. We found that these cell lines express only small amounts of detectable core3 synthase; thus we transfected the cell lines with core3 synthase. Core3 synthase-transfected PC3 and LNCaP cells expressed increased amounts of core3 O-glycans in α2β1 integrin, showed the reduced maturation of β1 integrin and low levels of α2β1 integrin formation, migrated less efficiently through collagen and other extracellular matrix components, and were less invasive than mock-transfected cells. Moreover those cells exhibited decreased activation of focal adhesion kinase (FAK) compared with mock-transfected cells. Significantly PC3 cells expressing core3 O-glycans produced almost no primary tumors in the prostate and formed much fewer metastases in the draining lymph nodes than mock-transfected cells. Similarly LNCaP cells expressing core3 O-glycans produced much smaller subcutaneous tumors than mock-transfected LNCaP cells. These findings indicate that addition of core3 O-glycans to the α2β1 integrin leads to decreased cell migration and invasion, resulting in decreased prostate tumor formation and metastasis.     

Assembly of ordered DNA-curli fibril complexes during Salmonella biofilm formation correlates with strengths of the type I interferon and autoimmune responses

Deposition of human amyloids is associated with complex human diseases such as Alzheimer’s and Parkinson’s. Amyloid proteins are also produced by bacteria. The bacterial amyloid curli, found in the extracellular matrix of both commensal and pathogenic enteric bacterial biofilms, forms complexes with extracellular DNA, and recognition of these complexes by the host immune system may initiate an autoimmune response. Here, we isolated early intermediate, intermediate, and mature curli fibrils that form throughout the biofilm development and investigated the structural and pathogenic properties of each. Early intermediate aggregates were smaller than intermediate and mature curli fibrils, and circular dichroism, tryptophan, and thioflavin T analyses confirmed the establishment of a beta-sheet secondary structure as the curli conformations matured. Intermediate and mature curli fibrils were more immune stimulatory than early intermediate fibrils in vitro. The intermediate curli was cytotoxic to macrophages independent of Toll-like receptor 2. Mature curli fibrils had the highest DNA content and induced the highest levels of Isg15 expression and TNFα production in macrophages. In mice, mature curli fibrils induced the highest levels of anti-double-stranded DNA autoantibodies. The levels of autoantibodies were higher in autoimmune-prone NZBWxF/1 mice than wild-type C57BL/6 mice. Chronic exposure to all curli forms led to significant histopathological changes and synovial proliferation in the joints of autoimmune-prone mice; mature curli was the most detrimental. In conclusion, curli fibrils, generated during biofilm formation, cause pathogenic autoimmune responses that are stronger when curli complexes contain higher levels of DNA and in mice predisposed to autoimmunity.     

The UVB-Stimulated Expression of Transglutaminase 1 Is Mediated Predominantly via the NFκB Signaling Pathway: New Evidence of Its Significant Attenuation through the Specific Interruption of the p38/MSK1/NFκBp65 Ser276 Axis

The influence of ultraviolet B (UVB) radiation on transglutaminase 1 (TGase 1), a major factor that regulates skin keratinization, has not been sufficiently characterized especially at the gene or protein level. Thus, we determined whether UVB affects the expression of TGase 1 in human keratinocytes and clarified the intracellular stress signaling mechanism(s) involved. Exposure of human keratinocytes to UVB significantly up-regulated the expression of TGase 1 at the gene and protein levels. Treatment with inhibitors of p38, MEK, JNK or NFκB significantly abolished the UVB-stimulated protein expression of TGase 1. Treatment with astaxanthin immediately after UVB irradiation did not attenuate the increased phosphorylation of Ser536/Ser468NFκBp65, c-Jun, ATK-2 and CK2, and did not abrogate the increased or diminished protein levels of c-Jun/c-Fos or I-κBα, respectively. However, the same treatment with astaxanthin significantly abolished the UVB-stimulated expression of TGase 1 protein, which was accompanied by the attenuated phosphorylation of Thr565/Ser376/Ser360MSK1, Ser276NFκBp65 and Ser133CREB. The MSK1 inhibitor H89 significantly down-regulated the increased protein expression of TGase 1 in UVB-exposed human keratinocytes, which was accompanied by an abrogating effect on the increased phosphorylation of Ser276NFκBp65 and Ser133CREB but not Thr565/Ser376/Ser360MSK1. Transfection of human keratinocytes with MSK1 siRNA suppressed the UVB-stimulated protein expression of TGase 1. These findings suggest that the UVB-stimulated expression of TGase 1 is mediated predominantly via the NFκB pathway and can be attenuated through a specific interruption of the p38/MSK1/NFκBp65Ser276 axis.     

Mass Spectra of Indoleacetic Acid and Its Chloroderivatives

In the presence of silver carbonate, primary and secondary alcohols such as γ-hydroxybutyrophenone, 17α-21-dihydroxy-4-pregnene-3,11,20-trione and 11α-hydroxy-17α-methyltestosterone react with alkyl halides in N,N-dimethylformamide under mild reaction conditions to give the corresponding unsymmetrical dialkylcarbonates in good yields.     

Angiotensin II Type 1 Receptor Antagonist Attenuates Lacrimal Gland,Lung, and Liver Fibrosis in a Murine Model of Chronic Graft-Versus-Host Disease

Chronic graft-versus-host disease (cGVHD), a serious complication following allogeneic HSCT (hematopoietic stem cell transplantation), is characterized by systemic fibrosis. The tissue renin-angiotensin system (RAS) is involved in the fibrotic pathogenesis, and an angiotensin II type 1 receptor (AT1R) antagonist can attenuate fibrosis. Tissue RAS is present in the lacrimal gland, lung, and liver, and is known to be involved in the fibrotic pathogenesis of the lung and liver. This study aimed to determine whether RAS is involved in fibrotic pathogenesis in the lacrimal gland and to assess the effect of an AT1R antagonist on preventing lacrimal gland, lung, and liver fibrosis in cGVHD model mice. We used the B10.D2→BALB/c (H-2^d) MHC-compatible, multiple minor histocompatibility antigen-mismatched model, which reflects clinical and pathological symptoms of human cGVHD. First, we examined the localization and expression of RAS components in the lacrimal glands using immunohistochemistry and quantitative real-time polymerase chain reaction (PCR). Next, we administered an AT1R antagonist (valsartan; 10 mg/kg) or angiotensin II type 2 receptor (AT2R) antagonist (PD123319; 10 mg/kg) intraperitoneally into cGVHD model mice and assessed the fibrotic change in the lacrimal gland, lung, and liver. We demonstrated that fibroblasts expressed angiotensin II, AT1R, and AT2R, and that the mRNA expression of angiotensinogen was greater in the lacrimal glands of cGVHD model mice than in controls generated by syngeneic-HSCT. The inhibition experiment revealed that fibrosis of the lacrimal gland, lung, and liver was suppressed in mice treated with the AT1R antagonist, but not the AT2R antagonist. We conclude that RAS is involved in fibrotic pathogenesis in the lacrimal gland and that AT1R antagonist has a therapeutic effect on lacrimal gland, lung, and liver fibrosis in cGVHD model mice. Our findings point to AT1R antagonist as a possible target for therapeutic intervention in cGVHD.     

Synthesis of Brassinolide Analogs with a Modified Ring B and Their Plant Growth-promoting Activity

7-Aza, 7-thia and 6-deoxo analogs of (22S,23S)- and (22R,23R)-homobrassinolide were synthesized. They were found to possess weak bioactivity in a lamina-inclination test.