Mechanisms of Ganweikang Tablets against Chronic Hepatitis B: A Comprehensive Study of Network Analysis, Molecular Docking, and Chemical Profiling.

The hepatitis B virus (HBV) is one of the major viral infection problems worldwide in public health. The exclusive proprietary Chinese medicine Ganweikang (GWK) tablet has been marketed for years in the treatment of chronic hepatitis B (CHB). However, the pharmacodynamic material basis and underlying mechanism of GWK are not completely clear. This study is aimed at investigating the pharmacological mechanism of the GWK tablet in the treatment of CHB. The chemical ingredient information was obtained from the Traditional Chinese Medicine Database and Analysis Platform (TCMSP), Traditional Chinese Medicines Integrated Database (TCMID), and Shanghai Institute of Organic Chemistry of CAS. Ingredients and disease-related targets were defined by a combination of differentially expressed genes from CHB transcriptome data and open-source databases. Target-pathway-target (TPT) network analysis, molecular docking, and chemical composition analysis were adopted to further verify the key targets and corresponding active ingredients of GWK. Eight herbs of GWK were correlated to 330 compounds with positive oral bioavailability, and 199 correlated targets were identified. The TPT network was constructed based on the 146 enriched targets by KEGG pathway analysis, significantly associated with 95 pathways. Twenty-five nonvolatile components and 25 volatile components in GWK were identified in UPLC-QTOF/MS and GC-MS chromatograms. The key active ingredients of GWK include ferulic acid, oleanolic acid, ursolic acid, tormentic acid, 11-deoxyglycyrrhetic acid, dibenzoyl methane, anisaldehyde, wogonin, protocatechuic acid, psoralen, caffeate, dimethylcaffeic acid, vanillin, ß-amyrenyl acetate, formonentin, aristololactam IIIa, and 7-methoxy-2-methyl isoflavone, associated with targets CA2, NFKB1, RELA, AKT1, JUN, CA1, CA6, IKBKG, FOS, EP300, CREB1, STAT1, MMP9, CDK2, ABCB1, and ABCG2.

MMTV-cre;Ccn6 knockout mice develop tumors recapitulating human metaplastic breast carcinomas.

Metaplastic breast carcinoma is an aggressive form of invasive breast cancer with histological evidence of epithelial to mesenchymal transition (EMT). However, the defining molecular events are unknown. Here we show that CCN6 (WISP3), a secreted matricellular protein of the CCN (CYR61/CTGF/NOV) family, is significantly downregulated in clinical samples of human spindle cell metaplastic breast carcinoma. We generated a mouse model of mammary epithelial-specific Ccn6 deletion by developing a floxed Ccn6 mouse which was bred with an MMTV-Cre mouse. Ccn6fl/fl;MMTV-Cre mice displayed severe defects in ductal branching and abnormal age-related involution compared to littermate controls. Ccn6fl/fl;MMTV-Cre mice developed invasive high grade mammary carcinomas with bona fide EMT, histologically similar to human metaplastic breast carcinomas. Global gene expression profiling of Ccn6fl/fl mammary carcinomas and comparison of orthologous genes with a human metaplastic carcinoma signature revealed a significant overlap of 87 genes (P=5 × 10-11). Among the shared deregulated genes between mouse and human are important regulators of epithelial morphogenesis including Cdh1, Ck19, Cldn3 and 4, Ddr1, and Wnt10a. These results document a causal role for Ccn6 deletion in the pathogenesis of metaplastic carcinomas with histological and molecular similarities with human disease. We provide a platform to study new targets in the diagnosis and treatment of human metaplastic carcinomas, and a new disease relevant model in which to test new treatment strategies.

Differences between high- and low-motility buffalo sperm identified by comparative proteomics.

This study was undertaken to investigate differences in protein expression between high- and low-motility sperm of swamp buffalo. The research used two-dimensional gel electrophoresis (2DE) coupled to matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry (MALDI-TOF/TOF-MS) to analyse the different proteins. The results showed 18 different expression protein spots between high- and low-motility buffalo sperm; eight of these proteins were up-regulated in low-motility sperm, five were down-regulated, one deleted and four proteins specifically expressed. Finally, four proteins were successfully identified by MS as belonging to three unique proteins; they are outer dense fibre of sperm tails protein 2 (ODF2), ATP synthase subunit alpha (ATP5A1) and succinyl-CoA synthetase subunit beta (SUCLG2). In summary, these results help to develop an understanding of the molecular mechanisms associated with low-motility sperm and provide clues for finding molecular markers associated with sperm motility.

High serum levels of follistatin in patients with ovarian cancer.

OBJECTIVE: This study investigated the potential use of serum follistatin (FST) as a marker for ovarian cancer alongside serum cancer antigen-125 (CA-125). METHODS: Serum samples were collected from patients with ovarian cancer (n = 45), benign ovarian cysts (n = 40) or other cancers (n = 100) and from healthy subjects (n = 60) for the determination of FST and CA-125 levels using enzyme-linked immunosorbent assays. Expression of FST in ovarian tissue was investigated using immunohistochemical staining. RESULTS: Compared with healthy subjects and patients with benign ovarian cysts, serum FST and CA-125 levels were significantly increased in patients with ovarian cancer. Using the 95% confidence interval for the healthy subjects group as the cut-off value, tumour marker sensitivity and specificity in ovarian cancer were 53.3% and 97% for FST and 77.8% and 84% for CA-125, respectively. Tissue expression of FST protein was more pronounced in ovarian cancer than in normal ovary. CONCLUSIONS: The serum FST level was elevated in the peripheral blood of patients with ovarian cancer and has potential as a tumour marker for ovarian cancer diagnosis. It may be particularly useful when combined with CA-125 detection to reduce the number of false-positive results.

Vitamin D receptor gene BsmI polymorphism B allele, but not BB genotype, is associated with systemic lupus erythematosus in a Han Chinese population.

The vitamin D receptor (VDR) gene is a candidate gene for susceptibility to autoimmune disorders. Association studies of VDR polymorphisms and risk of systemic lupus erythematosus (SLE) have often produced conflicting results in different ethnic backgrounds. The aim of this study is to test the association between VDR gene BsmI polymorphism and the genetic susceptibility to SLE in a Han Chinese population. Three hundred and thirty-seven patients with SLE and 239 healthy controls were genotyped for the VDR gene BsmI polymorphism (rs1544410) by polymerase chain reaction and restriction fragment length polymorphism analysis in this study, after which the relationship between BsmI polymorphisms and the mRNA expression of VDR, as well as clinical manifestations in patients with SLE, was evaluated. It was found that the frequency of B allele was significantly increased in SLE relative to the control group (χ(2) = 4.681, p = 0.031), although the distribution of VDR BsmI polymorphism genotype frequencies did not differ significantly between patients and controls (χ(2) = 4.098, p = 0.129). Moreover, VDR B allele was found to be associated with lupus nephritis (p = 0.027) and also with production of anti-nucleosome antibodies (p = 0.037). The mRNA of VDR was markedly down-regulated in patients with VDR B allele compared with that in patients without B allele (p = 0.016). Our results indicate a possible role of genetic factors (the VDR B allele) in influencing disease susceptibility in Han Chinese patients. Also, VDR B allele is associated with the development of nephritis and the down-regulation of VDR mRNA expression in SLE.

Tyrosine phosphorylation of cofilin at Y68 by v-Src leads to its degradation through ubiquitin-proteasome pathway.

Cofilin is a major regulator of actin dynamics involved in the regulation of cell spreading and migration through its actin depolymerizing and severing activities. v-Src is an activated Src tyrosine kinase and a potent oncogene known to phosphorylate a variety of cellular proteins in cell transformation process including altered cell adhesion, spreading and migration. Recently, it has been suggested that cofilin is a potential substrate of v-Src (Rush et al., 2005). Here, we show direct tyrosine phosphorylation of cofilin by v-Src and identify Y68 as the major phosphorylation site. Cofilin phosphorylation at Y68 did not change its activity per se, but induced increased ubiquitination of cofilin and its degradation through the proteosome pathway. Furthermore, the negative effect of cofilin on cellular F-actin contents was inhibited by coexpression of v-Src, whereas that of cofilin mutant Y68F (Y68 mutated to F) was not affected, suggesting that v-Src-mediated cofilin phosphorylation at Y68 is required for the degradation of cofilin in vivo. Lastly, inhibition of cell spreading by v-Src was rescued partially by coexpression of cofilin, and to a greater extent by the Y68F mutant, which is not subjected to v-Src-induced degradation through phosphorylation, suggesting that v-Src-mediated changes in cell spreading is, at least in part, through inhibiting the function of cofilin through phosphorylating it at Y68. Together, these results suggest a novel mechanism by which cofilin is regulated by v-Src through tyrosine phosphorylation at Y68 that triggers the degradation of cofilin through ubiquitination-proteosome pathway and consequently inhibits cofilin activity in reducing cellular F-actin contents and cell spreading.

Lack of association between HLA-G 14-bp polymorphism and systemic lupus erythematosus in a Han Chinese population.

HLA-G is a non-classical HLA-class Ib molecule with multiple immunoregulatory properties. A 14-bp insertion/deletion polymorphism in the HLA-G gene has been suggested to influence the expression of HLA-G and to associate with certain pathological conditions, including autoimmune diseases. We investigated the influence of the 14-bp insertion/deletion polymorphism in the HLA-G gene on disease susceptibility in systemic lupus erythematosus by genotyping this polymorphism in 231 patients with systemic lupus erythematosus and 367 healthy controls and analyzing the levels of soluble HLA-G in a subset of patients with systemic lupus erythematosus and healthy subjects from a Han Chinese population. No statistically significant differences were observed in the frequencies of the 14-bp insertion/deletion HLA-G alleles or genotypes between controls and patients with systemic lupus erythematosus. However, a significant increased expression of soluble HLA-G was noted in patients with systemic lupus erythematosus (mean value = 230.2 U/ml vs 118.3 U/ml in controls, p = 0.0001). Moreover, patients with high levels of soluble HLA-G presented with higher disease activity and had more neurological involvement. Our results do not support the HLA-G 14-bp insertion/deletion polymorphism as a genetic factor influencing systemic lupus erythematosus susceptibility. It is possible that the expression of soluble HLA-G in systemic lupus erythematosus is enhanced as part of a mechanism to try to restore the tolerance process towards auto-antigens and to counteract inflammation. However, the participation of this molecule in the pathological process of the disease also could not be excluded.

Galanin-like peptide promotes feeding behaviour via activation of orexinergic neurones in the rat lateral hypothalamus.

Galanin-like peptide (GALP) is produced in neurones in the hypothalamic arcuate nucleus and is implicated in the neural control of feeding behaviour. Previously, we have reported that GALP immunoreactive fibres were in direct contact with orexin/hypocretin immunoreactive neurones in the rat lateral hypothalamus using double-immunofluorescence. Centrally administered GALP is known to stimulate feeding behaviour. However, the target neurones of this action have not been clarified. The present study aimed to determine features of the GALP-mediated neuronal feeding pathway in rat. Accordingly, at the ultrastructural level, GALP-immunoreactive axon terminals were found to make synapses on orexin/hypocretin immunoreactive cell bodies and dendritic processes in the lateral hypothalamus. c-Fos immunoreactivity was expressed in orexin/hypocretin-immunoreactive neurones but not in melanin concentrating hormone-immunoreactive neurones in the lateral hypothalamus at 90 min after the application of GALP by i.c.v. infusion. Furthermore, to determine whether GALP regulates feeding behaviour via orexin/hypocretin neurones, the feeding behaviour of rats was studied following GALP i.c.v. injection with or without anti-orexin A and B immunoglobulin (IgG) pretreatment. The anti-orexin IgGs markedly inhibited GALP-induced hyperphagia. These results suggest that orexin/hypocretin-containing neurones in the lateral hypothalamus are targeted by GALP, and that GALP-induced hyperphagia is mediated via orexin/hypocretin neurones in the rat hypothalamus.

Electron microscopic examination of the endomorphin 2-like immunoreactive neurons in the rat hypothalamus.

Endomorphins are endogenous opioid peptides with high affinity and selectivity for the mu-opioid receptor. In the present study, we examined the morphology of the endomorphin 2-like immunoreactive (EM2-LI) neurons in the hypothalamus at the light and electron microscopic levels. At the light microscopic level, EM2-LI neurons were found mostly distributed in the regions between the dorsomedial and ventromedial hypothalamic nuclei and the region near the third ventricle. At the electron microscopic level, EM2-LI perikarya could be divided into two groups. Type I perikarya contained relatively undeveloped endoplasmic reticulum and Golgi apparatus while type II perikarya contained well-developed rough-surfaced endoplasmic reticulum and Golgi apparatus. Both type I and type II neurons contained numerous EM2-LI dense-cored vesicles. Type II perikarya and dendrites received synapses and showed immunoreactivity in the endoplasmic reticulum and Golgi apparatus. EM2-LI axon terminals formed synapses with both immunonegative and immunopositive dendrites. In some cases, the axon terminals contained both immunonegative and immunopositive dense-cored vesicles. EM2-LI neurons often had synaptic relationships with neurons containing immunonegative dense-cored vesicles. Myelinated axon shafts containing EM2-LI were also found. This first demonstration of the ultrastructure and synaptic relationships of EM2-LI neurons in the hypothalamus provides morphological evidence that suggests (1) endomorphin 2-containing neurons modulate physiological function through synaptic relationships; (2) endomorphin 2 may coexist with other neurotransmitters in the same neurons; and (3) endomorphin 2-containing neurons could modulate other endomorphin 2-containing neurons as well as those containing other neurotransmitters.

Reciprocal synaptic relationships between orexin- and melanin-concentrating hormone-containing neurons in the rat lateral hypothalamus: a novel circuit implicated in feeding regulation.

OBJECTIVE: Both orexin (ORX)- and melanin-concentrating hormone (MCH) are expressed in different neurons in the lateral hypothalamic area (LH), and are considered to have common effects on stimulating food intake. There are no reports to demonstrate neural interactions between them at the ultrastructural level. We observed these neurons in the LH to evaluate the relationships between them. DESIGN: We used two different types of double immunostaining to reveal the ultrastructure of both the ORX- and MCH-containing neurons. A preembedding double immunostaining technique was used to study the synaptic relationships between the two kinds of neuron. RESULTS: The main new findings are as follows: 1) Both ORX- and MCH-containing neurons received other synaptic input and made synaptic input to other neurons; 2) Reciprocal synaptic relationships were observed between the ORX- and MCH-containing neurons. CONCLUSION: The ORX- and MCH-containing neurons in the lateral hypothalamic area may influence food intake through synapse with each other.

Focal adhesion kinase: protein interactions and cellular functions.

Integrin-mediated cell adhesion to extracellular matrix (ECM) plays important roles in a variety of biological processes. Recent studies suggested that integrins mediate signal transduction across the plasma membrane via activating several intracellular signaling pathways. Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase that has been shown to be a major mediator of integrin signal transduction pathways. Upon activation by integrins, FAK undergoes autophosphorylation as well as associations with several other intracellular signaling molecules. These interactions in the signaling pathways have been shown to regulation a variety of cellular functions such as cell spreading, migration, cell proliferation, apoptosis and cell survival. Recent progress in the understanding of FAK interactions with other proteins in the regulation of these cellular functions will be discussed in this review.

Endomorphin-2 immunoreactivity in the cervical dorsal horn of the rat spinal cord at the electron microscopic level.

Endomorphin-2 is a newly discovered endogenous opioid peptide with high affinity and selectivity for the micro-opioid receptor, and potent analgesic activity, particularly in the spinal cord. Using immunoelectron microscopy, we examined the ultrastructure of the endomorphin-2-like immunoreactive processes and their synaptic relationships in the spinal cord. Endomorphin-2-like immunopositive dense-cored vesicles were observed in many axon terminals, and, in a few cases, were observed together with immunonegative dense-cored vesicles. Immunopositive axons with or without myelination were also observed. The endomorphin-2-like immunoreactive axon terminals formed synapses with both immunopositive and immunonegative processes. Most synapses were asymmetrical, but symmetrical synapses were also found. Examples of axo-dendritic, axo-somatic and axo-axonic contacts were observed. This first demonstration of the ultrastructure and synaptic relationships of endomorphin-2-like immunoreactive axon terminals in the spinal cord dorsal horn provides morphological evidence that this peptide functions as a transmitter regulating pain processes.

Immunocytochemical observation of ghrelin-containing neurons in the rat arcuate nucleus.

Ghrelin is a novel peptide that stimulates the release of growth hormone from the pituitary and is involved in hypothalamic feeding regulation. A pre-embedding immunostaining technique was used to study the ultrastructure and synaptic relationships of ghrelin-containing neurons in the rat arcuate nucleus (ARC). Ghrelin-like immunoreactive (ghrelin-LI) neurons were found in the ARC, and were especially abundant in its ventral part. At the electron microscopic level, ghrelin-LI neurons received afferent synapses from many unknown axon terminals. Ghrelin-LI products in the immunoreactive cell bodies, processes, and axon terminals were detected mainly in dense granular vesicles about 110 nm in diameter. Ghrelin-LI presynaptic axon terminals often made synapses with unknown immunonegative neurons. These results suggest that ghrelin acts to regulate food intake through synaptic connections in hypothalamic neuronal networks.

Transcriptional activation of cyclin D1 promoter by FAK contributes to cell cycle progression.

Integrin-mediated cell adhesion to the extracellular matrix is required for normal cell growth. Cyclin D1 is a key regulator of G1-to-S phase progression of the cell cycle. Our previous studies have demonstrated that integrin signaling through focal adhesion kinase (FAK) plays a role in the regulation of cell cycle progression, which correlates with changes in the expression of cyclin D1 and the cdk inhibitor, p21, induced by FAK. In this report, we first investigated the roles of both cyclin D1 and p21 in the regulation of cell cycle progression by FAK. We found that overexpression of a dominant-negative FAK mutant DeltaC14 suppressed cell cycle progression in p21(-/-) cells as effectively as in the control p21(+/+) cells. Furthermore, we found that overexpression of ectopic cyclin D1 could rescue cell cycle inhibition by DeltaC14. These results suggested that cyclin D1, but not p21, was the primary functional target of FAK signaling pathways in cell cycle regulation. We then investigated the mechanisms underlying the regulation of cyclin D1 expression by FAK signaling. Using Northern blotting and cyclin D1 promoter/luciferase assays, we showed that FAK signaling regulated cyclin D1 expression at the transcriptional level. Using a series of cyclin D1 promoter mutants in luciferase assays as well as electrophoretic mobility shift assays (EMSA), we showed that the EtsB binding site mediated cyclin D1 promoter regulation by FAK. Finally, we showed that FAK regulation of cyclin D1 depends on integrin-mediated cell adhesion and is likely through its activation of the Erk signaling pathway. Together, these studies demonstrate that transcriptional regulation of cyclin D1 by FAK signaling pathways contributes to the regulation of cell cycle progression in cell adhesion.

Immunoelectron microscopic study of beta-endorphinergic synaptic innervation of GABAergic neurons in the dorsal raphe nucleus.

Using a preembedding double immunoreactive technique by immunostaining with antirat beta-endorphin and antisynthetic glutamic acid decarboxylase antisera sequentially, the synaptic relationships between beta-endorphinergic neuronal fibers and GABAergic neurons in the dorsal raphe nucleus of the rat were examined at the ultrastructural level. Although both beta-endorphin-like immunoreactive fibers and glutamic acid decarboxylase-like immunoreactive neurons can be found in the mediodorsal and medioventral parts of the dorsal raphe nucleus, the synapses between them were found only in the mediodorsal part. Most of the beta-endorphin-like immunoreactive neuronal fibers contained many dense-cored vesicles. The synapses made by beta-endorphin-like immunoreactive neuronal axon terminals on glutamic acid decarboxylase-like immunoreactive neurons were both symmetrical and asymmetrical, with the latter predominant, especially in the axo-dendritic synapses. Perikarya with beta-endorphin-like immunoreactivity were found only in the ventrobasal hypothalamus. These findings suggest the possibility that the beta-endorphin-producing neurons in the ventrobasal hypothalamus could influence GABAergic neurons in the dorsal raphe nucleus directly by synaptic relationships.

The Grb7 family proteins: structure, interactions with other signaling molecules and potential cellular functions.

Grb7 family adaptor molecules consist of Grb7, Grb10 and Grb14, each of which has several splicing variants. Like other adaptor molecules, Grb7 family proteins function to mediate the coupling of multiple cell surface receptors to downstream signaling pathways in the regulation of various cellular functions. They share significant sequence homology with each other and a conserved molecular architecture including an amino-terminal proline-rich region, a central segment termed the GM region (for Grb and Mig) which includes a PH domain and shares sequence homology with the Caenorhabditis elegans protein, Mig-10, involved in embryonic migration, and a carboxyl-terminal SH2 domain. Grb7 family proteins are differentially expressed in a variety of tissues. They are phosphorylated on serine/threonine as well as tyrosine residues, although the kinases responsible have not been well characterized. Grb7 family proteins are mainly localized in the cytoplasm, but have been observed at the plasma membrane, focal contacts, or mitochondria under certain conditions. A large number of receptor tyrosine kinases and other signaling molecules can associate with Grb7 family proteins, mostly through the SH2 domains. Various isoforms of Grb10 have been shown to regulate cell proliferation and apoptosis, whereas Grb7 has been found to regulate cell migration and also implicated in tumor progression. Future studies of interests will include identification of potential downstream effectors of Grb7 family proteins as well as understanding of the mechanisms of specificity of the different family members in signal transduction.

The nonreceptor tyrosine kinase ACK2, a specific target for Cdc42 and a negative regulator of cell growth and focal adhesion complexes.

ACK2 (activated Cdc42-associated tyrosine kinase-2) is a nonreceptor tyrosine kinase that is a specific target/effector for the GTP-binding protein Cdc42. Thus far the biological function of this tyrosine kinase has not been determined. Using an inducible eukaryotic expression system in fibroblasts, we demonstrate that ACK2 can strongly influence cell shape and growth as well as focal complex formation. ACK2 was found to associate with the focal adhesion complex components talin and vinculin, but not with the focal adhesion kinase (FAK), in a kinase-independent manner. The tyrosine kinase activity of FAK was also inhibited in cells overexpressing both wild-type and kinase-defective ACK2. This may be due to a competition between ACK2 and FAK for Src, which is an essential cofactor for FAK activation, as we have found that ACK2 specifically binds Src in cells. The ACK2-Src interaction appears to be mediated by the SH3 domain of Src, and the phosphorylation of ACK2 is enhanced in cells overexpressing the hyperactivated Src(Y527F) mutant. Overexpression of both wild-type and kinase-defective ACK2 also results in a severe inhibition of cell growth. In addition, ACK2 dissolves actin stress fibers and disassembles focal complexes but in a kinase-dependent manner. These results, taken together with previous studies demonstrating an association of ACK2 with integrin beta(1) (Yang, W., Lin, Q., Guan, J.-L., Cerione, R. A. (1999) J. Biol. Chem. 274, 8524-8530) and clathrin (Yang, W., Lo, C. G., Dispenza, T., and Cerione, R. A. (2001) J. Biol. Chem. 276, 17468-17473), suggest that the binding and protein tyrosine kinase activities of ACK2 coordinate changes in cell morphology and growth with the disassembly of focal adhesion sites, perhaps to organize new integrin complexes that are required for endocytosis and/or for cellular differentiation.

Differential regulation of cell migration and cell cycle progression by FAK complexes with Src, PI3K, Grb7 and Grb2 in focal contacts.

Focal adhesion kinase (FAK) is a key mediator of integrin signaling, which has been implicated in the regulation of cell migration and cell cycle progression. Using chimeric molecules that fuse the focal adhesion targeting (FAT) sequence directly to several signaling molecules, we investigated the potential role of FAK recruitments of signaling molecules to focal contacts in the regulation of cell migration and cell cycle progression. We found that fusion of FAT to Src, the p85 subunit of phosphatidylinositol 3-kinase, Grb7 and Grb2 resulted in the efficient focal adhesion targeting of these signaling molecules. We showed that expression of Src-FAT, p85-FAT, or Grb7-FAT, but not Grb2-FAT, each stimulated cell migration. Interestingly, tyrosine phosphorylation of paxillin, but not p130cas, was induced by expression of Src-FAT, suggesting a potential role of paxillin in mediating stimulation of cell migration by the chimeric molecule. In contrast, targeting of Grb2, but not Src, p85, or Grb7, to focal contacts increased cell cycle progression. Biochemical analyses correlated Erk activation by Grb2-FAT with its stimulation of cell cycle progression. Together, these results suggest that at least part of the role of FAK interaction with these signaling molecules is to recruit them to focal contacts and that distinct FAK signaling complexes are involved in the regulation of cell migration vs. cell cycle progression.

Reciprocal synaptic relationships between angiotensin II-containing neurons and enkephalinergic neurons in the rat area postrema.

A preembedding double immunostaining technique was used to study synaptic relationships between angiotensin-II-like immunoreactive and enkephalin-like immunoreactive neurons in the rat area postrema. The angiotensin-II-like immunoreactive neurons were detected by silver-gold intensification of the DAB reaction results while the enkephalin-like immunoreactive neurons were detected by simple ABC-DAB reaction. The synaptic relationships were reciprocal between the two neurons. Most of the synapses found between these two neurons were the presynaptic enkephalin-like immunoreactive axon terminals that made synapses on the angiotensin-II-like immunoreactive perikarya and dendrites. Both the axo-somatic and axo-dendritic synapses were symmetrical. However, although angiotensin-II-like immunoreactive axon terminals also made synapses on enkephalin-like perikarya and dendrites, the axo-somatic synapses were symmetrical, while the axo-dendritic synapses were asymmetrical. The present results confirm the presence of angiotensin-II-like immunoreactive neurons in the area postrema and suggest that these angiotensinergic neurons in the area postrema may play a role in the regulation of blood pressure via coordinated synaptic interactions with enkephalinergic neurons.

Regulation of the PH-domain-containing tyrosine kinase Etk by focal adhesion kinase through the FERM domain.

Etk/BMX, a member of the Btk family of tyrosine kinases, is highly expressed in cells with great migratory potential, including endothelial cells and metastatic carcinoma cell lines. Here, we present evidence that Etk is involved in integrin signalling and promotes cell migration. The activation of Etk by extracellular matrix proteins is regulated by FAK through an interaction between the PH domain of Etk and the FERM domain of FAK. The lack of Etk activation by extracellular matrix in FAK-null cells could be restored by co-transfection with wild-type FAK. Disrupting the interaction between Etk and FAK diminished the cell migration promoted by either kinase. Furthermore, inhibiting Etk expression in metastatic carcinoma cell lines with an antisense oligonucleotide blocks integrin-mediated migration of these cells. Taken together, our data indicate the essential role of the interaction of the PH domain of Etk and the FERM domain of FAK in integrin signalling.