Neuronal effects of orexins: relevant to sympathetic and cardiovascular functions

Orexin A and B, also called hypocretin 1 and 2, were recently discovered in the hypothalamus. This organ, in which a number of neuropeptides have been demonstrated to stimulate or suppress food intake, is considered important for the regulation of appetite and energy homeostasis. Orexins were initially reported as a regulator of food intake. More recent reports suggest their possible important roles in the multiple functions of neuronal systems, such as narcolepsy, a sleep disorder. Orexins and their receptors are distributed in neural tissue and brain regions involved in the autonomic and neuroendocrine control. Functional studies have shown that these peptides evoke changes in cardiovascular and sympathetic responses. The data from our in vivo and in vitro studies suggest that the peptide acting on neurons in the hypothalamic paraventricular nucleus increases the cardiovascular responses. This review will focus on the neural effects of orexins and how these peptides may participate in the regulation of cardiovascular and sympathetic functions.     

Differential regulation of cell migration,actin stress fiber organization,and cell transformation by functional domains of Crk-associated substrate

The Crk-associated substrate (Cas) is a unique docking protein that possesses a repetitive stretch of tyrosine-containing motifs and an Src homology 3 (SH3) domain. Embryonic fibroblasts lacking Cas demonstrated resistance to Src-induced transformation along with impaired actin bundling and cell motility, indicating critical roles of Cas in actin cytoskeleton organization, cell migration, and oncogenesis. To gain further insight into roles of each domain of Cas in these processes, a compensation assay was performed by expressing a series of Cas mutants in Cas-deficient fibroblasts. The results showed that motifs containing YDxP were indispensable for actin cytoskeleton organization and cell migration, suggesting that CrkII-mediated signaling regulates these biological processes. The C-terminal Src-binding domain played essential roles in cell migration and membrane localization of Cas, although it was dispensable in the organization of actin stress fibers. Furthermore, the Src-binding domain was also a prerequisite for Src transformation possibly, because of its crucial role in the phosphorylation of Cas during transformation. Overall, differential uses of the Cas domains in individual biological processes were demonstrated.     

Influenza B virus BM2 protein is transported through the trans-Golgi network as an integral membrane protein

A bicistronic mRNA transcribed from the influenza B virus RNA segment 7 encodes two viral proteins, matrix protein M1 and uncharacterized small protein BM2. In the present study, we focused on the cytoplasmic transport and cellular membrane association of BM2. Immunofluorescence studies of virus-infected cells indicated that BM2 accumulated at the Golgi apparatus immediately after synthesis and then was transported to the plasma membrane through the trans-Golgi network. Localization of a set of BM2 deletion mutants revealed that the N-terminal half of BM2 (residues 2 to 50) was crucial for its transport; in particular, the deletion of residues 2 to 23, deduced to be a transmembrane domain, resulted in diffused distribution of the protein throughout the entire cell. Sucrose gradient flotation and biochemical analyses of the membrane showed that BM2 was tightly associated with cellular membranes as an integral membrane protein. Oligomerization of BM2 was demonstrated by coprecipitation of differentially epitope-tagged BM2 proteins. Taken together, these results strongly suggest that BM2 is integrated into the plasma membrane at the N-terminal hydrophobic domain as fourth membrane protein, in addition to hemagglutinin, neuraminidase, and NB, of the influenza B virus.     

Cooperative roles of Fyn and cortactin in cell migration of metastatic murine melanoma

Src family kinases are major regulators of various integrin-mediated biological processes, although their functional roles and substrates in cancer metastasis are unknown. We explored the roles of Src family tyrosine kinases in cell migration and the spread of K-1735 murine melanoma cell lines with low or high metastatic potential. Corresponding to elevated cell motility and spreading ability, Fyn was selectively activated among Src family kinases, and the cell motility was blocked by an inhibitor of Src family kinases. Significant tyrosine phosphorylation of cortactin, stable complex formation between activated Fyn and cortactin, and co-localization of cortactin with Fyn at cell membranes were all observed only in cells with high metastatic potential. Both integrin-mediated Fyn activation and hyperphosphorylation of cortactin were observed 2-5 h after stimulation in highly metastatic cells, and they required de novo protein synthesis. We demonstrate that cortactin is a specific substrate and cooperative effector of Fyn in integrin-mediated signaling processes regulating metastatic potential.     

Primary response of naive CD4(+) T cells to amino acid-substituted analogs of an antigenic peptide can show distinct activation patterns: Th1- and Th2-type cytokine secretion, and helper activity for antibody production without apparent cytokine secretion

Naive CD4(+) T cells differentiate into two types of helper T cells showing an interferon-gamma-predominant (Th1) or an interleukin-4-predominant (Th2) cytokine secretion profile after repeated antigenic stimulation. Their differentiation can be influenced by slight differences in the interaction between the T cell receptor (TCR) and its ligand at the time of primary activation. However, the primary response of freshly isolated naive CD4(+) T cells to altered TCR ligands is still unclear. Here, we investigated the primary response of splenic naive CD4(+) T cells derived from transgenic mice expressing TCR specific for residues 323-339 of ovalbumin (OVA323-339) bound to I-A(d) molecules. Naive CD4(+) T cells secreted either Th1- or Th2-type cytokines immediately after stimulation with OVA323-339 or its single amino acid-substituted analogs. Helper activity for antibody secretion by co-cultured resting B cells was also found in the primary response, accompanied by either low-level Th2-type cytokine secretion or no apparent cytokine secretion. Our results clearly indicate that dichotomy of the Th1/Th2 cytokine secretion profile can be elicited upon primary activation of naive CD4(+) T cells. We also demonstrate that the helper activity of naive CD4(+) T cells for antibody production does not correspond to the amounts of the relevant cytokines secreted.     

Peroxynitrite production by TNF-alpha and IL-1beta: implication for suppression of osteoblastic differentiation

To determine the roles of nitric oxide (NO) and its metabolite, peroxynitrite (ONOO(-)), on osteoblastic activation, we investigated the effects of a NO donor [ethanamine, 2, 2'-(hydroxynitrosohydrazono)bis- (dNO)], an O(-2) donor (pyrogallol), and an ONOO(-) scavenger (urate) on alkaline phosphatase (ALPase) activity and osteocalcin gene expression, which are indexes of osteoblastic differentiation. dNO elevated ALPase activity in the osteogenic MC3T3-E1 cell line. The combination of dNO and pyrogallol reduced both ALPase activity and osteocalcin gene expression. Because both indexes were recovered by urate, ONOO(-), unlike NO itself, inhibited the osteoblastic differentiation. Furthermore, treatment with a combination of the proinflammatory cytokines tumor necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta) was found to yield ONOO(-) as well as NO and O(-2). The reductions in ALPase activity and osteocalcin gene expression were also restored by urate. We conclude that ONOO(-) produced by TNF-alpha and IL-1beta, but not NO per se, would overcome the stimulatory effect of NO on osteoblastic activity and inhibit osteoblastic differentiation.     

Optimal combination of soluble factors for tissue engineering of permanent cartilage from cultured human chondrocytes

Since permanent cartilage has poor self-regenerative capacity, its regeneration from autologous human chondrocytes using a tissue engineering technique may greatly benefit the treatment of various skeletal disorders. However, the conventional autologous chondrocyte implantation is insufficient both in quantity and in quality due to two major limitations: dedifferentiation during a long term culture for multiplication and hypertrophic differentiation by stimulation for the redifferentiation. To overcome the limitations, this study attempted to determine the optimal combination in primary human chondrocyte cultures under a serum-free condition, from among 12 putative chondrocyte regulators. From the exhaustive 2(12) = 4,096 combinations, 256 were selected by fractional factorial design, and bone morphogenetic protein-2 and insulin (BI) were statistically determined to be the most effective combination causing redifferentiation of the dedifferentiated cells after repeated passaging. We further found that the addition of triiodothyronine (T3) prevented the BI-induced hypertrophic differentiation of redifferentiated chondrocytes via the suppression of Akt signaling. The implant formed by the human chondrocytes cultured in atelocollagen and poly(l-latic acid) scaffold under the BI + T3 stimulation consisted of sufficient hyaline cartilage with mechanical properties comparable with native cartilage after transplantation in nude mice, indicating that BI + T3 is the optimal combination to regenerate a clinically practical permanent cartilage from autologous chondrocytes.