Pituitary adenylate cyclase-activating peptide (PACAP) stimulates production of interleukin-6 in rat Müller cells

Pituitary adenylate cyclase-activating peptide (PACAP) is known to regulate not only neurons but also astrocytes. Here, we investigated, both in vitro and in vivo, the effects of PACAP38 on rat Müller cells, which are the predominant glial element in the retina. Müller cells isolated from juvenile Wistar rats were treated with PACAP38 or PACAP6-38, a PACAP selective antagonist. Cell proliferation was determined by measuring the incorporation of bromodeoxyuridine with ELISA. Interleukin-6 (IL-6) levels in the culture medium were determined by a bioassay using B9 cells, IL-6 dependent hybridoma. In adult Wistar rats, the expression of IL-6 in the retina after intravitreal injection of PACAP38 (10 pmol) was assessed by immunohistochemistry. PACAP38 stimulated IL-6 production in Müller cells at a concentration as low as 10(-12) M, which did not induce cell proliferation. This elevation of IL-6 production was inhibited by PACAP6-38. Radial IL-6 expression was observed throughout the retina at 2 and 3 days after PACAP38 injection. These data demonstrate that Müller cells are one of the target cells for PACAP. IL-6, which is released from Müller cells with stimulation by PACAP, may play a significant role in the retina.     

MAPKAPK-2-mediated LIM-kinase activation is critical for VEGF-induced actin remodeling and cell migration

Vascular endothelial growth factor-A (VEGF-A) induces actin reorganization and migration of endothelial cells through a p38 mitogen-activated protein kinase (MAPK) pathway. LIM-kinase 1 (LIMK1) induces actin remodeling by phosphorylating and inactivating cofilin, an actin-depolymerizing factor. In this study, we demonstrate that activation of LIMK1 by MAPKAPK-2 (MK2; a downstream kinase of p38 MAPK) represents a novel signaling pathway in VEGF-A-induced cell migration. VEGF-A induced LIMK1 activation and cofilin phosphorylation, and this was inhibited by the p38 MAPK inhibitor SB203580. Although p38 phosphorylated LIMK1 at Ser-310, it failed to activate LIMK1 directly; however, MK2 activated LIMK1 by phosphorylation at Ser-323. Expression of a Ser-323-non-phosphorylatable mutant of LIMK1 suppressed VEGF-A-induced stress fiber formation and cell migration; however, expression of a Ser-323-phosphorylation-mimic mutant enhanced these processes. Knockdown of MK2 by siRNA suppressed VEGF-A-induced LIMK1 activation, stress fiber formation, and cell migration. Expression of kinase-dead LIMK1 suppressed VEGF-A-induced tubule formation. These findings suggest that MK2-mediated LIMK1 phosphorylation/activation plays an essential role in VEGF-A-induced actin reorganization, migration, and tubule formation of endothelial cells.     

Generation and characterization of B7-H4/B7S1/B7x-deficient mice

Members of the B7 family of cosignaling molecules regulate T-cell proliferation and effector functions by engaging cognate receptors on T cells. In vitro and in vivo blockade experiments indicated that B7-H4 (also known as B7S1 or B7x) inhibits proliferation, cytokine production, and cytotoxicity of T cells. B7-H4 binds to an unknown receptor(s) that is expressed on activated T cells. However, whether B7-H4 plays nonredundant immune regulatory roles in vivo has not been tested. We generated B7-H4-deficient mice to investigate the roles of B7-H4 during various immune reactions. Consistent with its inhibitory function in vitro, B7-H4-deficient mice mounted mildly augmented T-helper 1 (Th1) responses and displayed slightly lowered parasite burdens upon Leishmania major infection compared to the wild-type mice. However, the lack of B7-H4 did not affect hypersensitive inflammatory responses in the airway or skin that are induced by either Th1 or Th2 cells. Likewise, B7-H4-deficient mice developed normal cytotoxic T-lymphocyte reactions against viral infection. Thus, B7-H4 plays a negative regulatory role in vivo but the impact of B7-H4 deficiency is minimal. These results suggest that B7-H4 is one of multiple negative cosignaling molecules that collectively provide a fine-tuning mechanism for T-cell-mediated immune responses.     

Nutritional significance of the selective ingestion of Albizia zygia gum exudate by wild chimpanzees in Bossou, Guinea

The selective ingestion of plant gum exudates by chimpanzees has been frequently observed at various study sites. At Bossou, Guinea, chimpanzees also frequently ingest Albizia zygia gum exudate. A functional explanation for this behavior is lacking, so we evaluated its possible contribution of energy in the form of short-chain fatty acids (SCFA) as well as minerals. An in vitro fermentation study of A. zygia gum using the fecal bacteria of a Bossou chimpanzee showed that carboxylic acids were produced with a 6-hr lag phase up to 44 mmol/l by 18 hr of incubation. Acetate was the most abundant acid produced, followed by lactate and propionate. The energy supplied from the fermentation of a piece of gum exudate (20-30 g) was negligible in comparison with the estimated daily energy requirements of chimpanzees in the wild. However, A. zygia gum exudate (20-30 g) can supply sufficient amounts of calcium, manganese, magnesium, and potassium to fulfill the daily requirements for these minerals in chimpanzees.     

Efficacy of adjuvant Immunochemotherapy with polysaccharide K for patients with curatively resected colorectal cancer: a meta-analysis of centrally randomized controlled clinical trials

The benefits of immunochemotherapy employing the biological response modifier polysaccharide K (PSK) for patients with curatively resected colorectal cancer was reassessed by means of a meta-analysis of data with center randomization from 1,094 patients enrolled in three clinical trials. In all three trials, patients were followed up for at least 5 years after surgery and enrollment of the last patient and outcomes for standard chemotherapy were compared with those for chemotherapy plus PSK. The endpoints were overall survival and disease-free survival; and intent-to-treat analysis was performed without patient exclusion. Data were analyzed using the weighted average of the individual log hazard ratios. The overall survival risk ratio for all eligible patients was 0.71 (95% confidence interval (CI) : 0.55–0.90; P=0.006), and the disease-free survival risk ratio was 0.72 (95% CI: 0.58–0.90; P=0.003). The results of this meta-analysis suggest that adjuvant immunochemotherapy with PSK can improve both survival and disease-free survival of patients with curatively resected colorectal cancer.     

The involvement of the 67 kDa laminin receptor-mediated modulation of cytoskeleton in the degranulation inhibition induced by epigallocatechin-3-O-gallate

Recently, we have reported that (-)-epigallocatechin-3-O-gallate (EGCG) acts as an inhibitor of degranulation. However, the inhibitory mechanism for degranulation is still poorly understood. Here we show that suppression of exocytosis-related myosin II regulatory light chain phosphorylation and alteration of actin remodeling are involved in the inhibitory effect of EGCG on the calcium ionophore-induced degranulation from human basophilic KU812 cells. Surface plasmon resonance assay also revealed that EGCG binds to the cell surface, and the disruption of lipid rafts resulted in reduction of EGCG's ability. We have previously identified the raft-associated 67kDa laminin receptor (67LR) as an EGCG receptor on the cell surface. Treatment of the cells with anti-67LR antibody or RNA interference-mediated downregulation of 67LR expression abolished the effects of EGCG. These findings suggest that EGCG-induced inhibition of the degranulation includes the primary binding of EGCG to the cell surface 67LR and subsequent modulation of cytoskeleton.     

The Caenorhabditis elegans muscle specific serpin, SRP-3, neutralizes chymotrypsin-like serine peptidases

Members of the intracellular serpin family may help regulate apoptosis, tumor progression, and metastasis. However, their in vivo functions in the context of a whole organism have not been easily defined. To better understand the biology of these serpins, we initiated a comparative genomics study using Caenorhabditis elegans as a model organism. Previous in silico analysis suggested that the C. elegans genome harbors nine serpin-like sequences bearing significant similarities to the human clade B intracellular serpins. However, only five genes appear to encode full-length serpins with intact reactive site loops. To determine if this was the case, we have cloned and expressed a putative inhibitory-type C. elegans serpin, srp-3. Analysis of SRP-3 inhibitory activity indicated that SRP-3 was a potent inhibitor of the serine peptidases, chymotrypsin and cathepsin G. Spatial and temporal expression studies using GFP and LacZ promoter fusions indicated that SRP-3 was expressed primarily in the anterior body wall muscles, suggesting that it may play a role in muscle cell homeostasis. Combined with previous studies showing that SRP-2 is an inhibitor of the serine peptidase, granzyme B, and lysosomal cysteine peptidases, these data suggested that C. elegans expressed at least two inhibitory-type serpins with nonoverlapping expression and inhibitory profiles. Moreover, the profiles of these clade L serpins in C. elegans share significant similarities with the profiles of clade B intracellular serpin members in higher vertebrates. This degree of conservation suggests that C. elegans should prove to be a valuable resource in the study of metazoan intracellular serpin function.     

Regulation of IL-27p28 gene by lipopolysaccharide in dendritic DC2.4 cells

To elucidate the regulation of IL-27p28 gene, we analyzed the promoter region of the gene in DC2.4 cells with or without lipopolysaccharide (LPS)-treatment. The results indicate that a region (-648 to -364) of p28 promoter was responsible for LPS-induction. EMSA with DNA probes within the region reveals that binding of GATA motif bound proteins was decreased by LPS-treatment. We identified one of the proteins as non-POU domain-containing octamer binding protein (NonO). Taken together, LPS-induced activation of IL-27p28 gene can be accounted for by the displacement of bound NonO protein from the IL-27p28 promoter.     

Characterization of the protease activity that cleaves the extracellular domain of beta-dystroglycan

Dystroglycan (DG) complex, composed of alphaDG and betaDG, provides a link between the extracellular matrix (ECM) and cortical cytoskeleton. Although the proteolytic processing of betaDG was reported in various physiological and pathological conditions, its exact mechanism remains unknown. In this study, we addressed this issue using the cell culture system of rat schwannoma cell line RT4. We found that the culture medium of RT4 cells was enriched with the protease activity that degrades the fusion protein construct of the extracellular domain of betaDG specifically. This activity was suppressed by the inhibitor of matrix metalloproteinase-2 (MMP-2) and MMP-9, but not by the inhibitors of MMP-1, MMP-3, MMP-8, and MMP-13. Zymography and RT-PCR analysis showed that RT4 cells secreted MMP-2 and MMP-9 into the culture medium. Finally, active MMP-2 and MMP-9 enzymes degraded the fusion protein construct of the extracellular domain of betaDG. These results indicate (1) that RT4 cells secrete the protease activity that degrades the extracellular domain of betaDG specifically and (2) that MMP-2 and MMP-9 may be involved in this process.