Blockade of the interaction between BMP9 and endoglin on erythroid progenitors promotes erythropoiesis in mice.

Bone morphogenetic protein-9 (BMP9), a member of the transforming growth factor ß (TGFß) superfamily, plays important roles in the development and maintenance of various cell lineages via complexes of type I and type II TGFß receptors. Endoglin is a coreceptor for several TGFß family members, including BMP9, which is highly expressed in a particular stage of differentiation in erythroid cells as well as in endothelial cells. Although the importance of the interaction between BMP9 and endoglin for endothelial development has been reported, the contribution of BMP9 to endoglin-expressing erythroid cells remains to be clarified. To address this point, we prepared an anti-BMP9 antibody that blocks the BMP9-endoglin interaction. Of note, challenge with the antibody promotes erythropoiesis in wild-type mice but not in a mouse model of renal anemia in which erythropoietin (EPO) production in the kidneys is genetically ablated. While endoglin-positive erythroid progenitors are mainly maintained as progenitors when bone marrow-derived lineage-negative and cKit-positive cells are cultured in the presence of EPO and stem cell factor, the erythroid-biased accumulation of progenitors is impeded by the presence of BMP9. Our findings uncover an unrecognized role for BMP9 in attenuating erythroid differentiation via its interaction with endoglin on erythroid progenitors.

Cancer-associated fibroblasts might sustain the stemness of scirrhous gastric cancer cells via transforming growth factor-ß signaling.

Cancer-associated fibroblasts (CAFs) have recently been implicated in tumor growth and metastasis in gastric cancer. Cancer stem cells (CSCs) have been proposed to have an important role in cancer progression. The aim of this study was to clarify the effect of CAFs on CSCs characteristics in gastric carcinoma. Scirrhous gastric cancer cell lines, OCUM-12 and OCUM-2MD3, and non-scirrhous gastric cancer cell lines, MKN-45 and MKN-74, were used. OCUM-12/side population (SP) cells and OCUM-2MD3/SP cells were sorted by flow cytometry as CSC-rich cells from the parent cells. CaF-37 was established from the tumoral gastric specimens as CAFs. Flow cytometric analysis of SP fraction, spheroid colony assay, and RT-PCR analysis of CSC markers were performed to identify CSCs properties. Effect of CAFs on the tumorigenicity by OCUM-12/SP cells was examined using nude mice. CAF CM significantly increased the percentages of the SP fraction of OCUM-12/SP and OCUM-2MD3/SP cells, but not that of MKN-45/SP and MKN-74/SP cells. Taken together, CM from CaF-37 significantly increased the number of spheroid colonies and the expression level of CSC markers of OCUM-12/SP and OCUM-2MD3/SP cells. These stimulating-activities by CM were significantly decreased by TGFß inhibitors, but not FGFR and cMet inhibitor. Tumorigenicity by subcutaneous coinoculation of OCUM-12/SP cells with CAFs was significantly high in comparison with that by OCUM-12/SP cells alone. Phospho-Smad2 expression level was significantly increased by co-inoculation with CAFs. These findings suggested that CAFs might regulate the stemness of CSCs in scirrhous gastric cancer by TGFß signaling.

An inhibitor of transforming growth factor beta type I receptor ameliorates muscle atrophy in a mouse model of caveolin 3-deficient muscular dystrophy.

Skeletal muscle expressing Pro104Leu mutant caveolin 3 (CAV3(P104L)) in mouse becomes atrophied and serves as a model of autosomal dominant limb-girdle muscular dystrophy 1C. We previously found that caveolin 3-deficient muscles showed activated intramuscular transforming growth factor beta (TGF-ß) signals. However, the cellular mechanism by which loss of caveolin 3 leads to muscle atrophy is unknown. Recently, several small-molecule inhibitors of TGF-ß type I receptor (TßRI) kinase have been developed as molecular-targeting drugs for cancer therapy by suppressing intracellular TGF-ß1, -ß2, and -ß3 signaling. Here, we show that a TßRI kinase inhibitor, Ki26894, restores impaired myoblast differentiation in vitro caused by activin, myostatin, and TGF-ß1, as well as CAV3(P104L). Oral administration of Ki26894 increased muscle mass and strength in vivo in wild-type mice, and improved muscle atrophy and weakness in the CAV3(P104L) mice. The inhibitor restored the number of satellite cells, the resident stem cells of adult skeletal muscle, with suppression of the increased phosphorylation of Smad2, an effector, and the upregulation of p21 (also known as Cdkn1a), a target gene of the TGF-ß family members in muscle. These data indicate that both TGF-ß-dependent reduction in satellite cells and impairment of myoblast differentiation contribute to the cellular mechanism underlying caveolin 3-deficient muscle atrophy. TßRI kinase inhibitors could antagonize the activation of intramuscular anti-myogenic TGF-ß signals, thereby providing a novel therapeutic rationale for the alternative use of this type of anticancer drug in reversing muscle atrophy in various clinical settings.

Combination effect of a TGF-beta receptor kinase inhibitor with 5-FU analog S1 on lymph node metastasis of scirrhous gastric cancer in mice.

Transforming growth factor-beta (TGF-beta) signals are closely associated with the distant metastases of gastric cancer. The aim of this study was to clarify the effect of a TGF-beta receptor I (TbetaR-I) phosphorylation inhibitor, Ki26894, in combination with anticancer drugs, on the lymph node (LN) metastasis of scirrhous gastric cancer. A novel TbetaR-I kinase inhibitor, Ki26894, inhibits the phosphorylation of Smad2 at the ATP binding site of TbetaR-I. S1 is a 5-fluorouracil analog. The human scirrhous gastric cancer cell line OCUM-2MLN and the human gastric fibroblasts NF-33 were used. OCUM-2MLM cells in the upper well and NF-33 cells in the lower well were co-incubated with or without Ki26894. The proliferation of OCUM-2MLN cells was significantly stimulated by co-culture with NF-33 cells. Ki26894 significantly suppressed the growth interactions between OCUM-2MLN cells and NF-33 cells. Gastric cancer models established by orthotopic inoculation of OCUM-2MLN cells showed diffusely infiltrating gastric adenocarcinoma accompanied by LN metastases. We divided these mice into four groups, (control vehicle, Ki26894, S1, Ki26894 plus S1), and examined the effect of Ki26894 and/or S1 on phosphorylation of Smad2, tumor size, LN metastases, and lymphatic involvements. Ki26894 inhibited the Smad2 phosphorylation of cancer cells and decreased the extent of lymphatic involvement, compared with the control or S1 only group. The Ki26894 plus S1 administration group significantly suppressed tumor growth and decreased LN metastasis more effectively than either alone. These findings suggested that the TbetaR-I kinase inhibitor with S1 is useful for the treatment of scirrhous gastric carcinoma with LN metastasis. (Cancer Sci 2010).

TGF-beta receptor kinase inhibitor enhances growth and integrity of embryonic stem cell-derived endothelial cells.

Recent findings have shown that embryonic vascular progenitor cells are capable of differentiating into mural and endothelial cells. However, the molecular mechanisms that regulate their differentiation, proliferation, and endothelial sheet formation remain to be elucidated. Here, we show that members of the transforming growth factor (TGF)-beta superfamily play important roles during differentiation of vascular progenitor cells derived from mouse embryonic stem cells (ESCs) and from 8.5-days postcoitum embryos. TGF-beta and activin inhibited proliferation and sheet formation of endothelial cells. Interestingly, SB-431542, a synthetic molecule that inhibits the kinases of receptors for TGF-beta and activin, facilitated proliferation and sheet formation of ESC-derived endothelial cells. Moreover, SB-431542 up-regulated the expression of claudin-5, an endothelial specific component of tight junctions. These results suggest that endogenous TGF-beta/activin signals play important roles in regulating vascular growth and permeability.

Synthesis and evaluation of [2-(4-quinolyloxy)phenyl]methanone derivatives: novel selective inhibitors of transforming growth factor-beta kinase.

We synthesized and evaluated various [2-(4-quinolyloxy)phenyl]methanone derivatives. These compounds had novel chemical structures that were distinct from those of previously reported inhibitors. Biological data suggested that these compounds inhibited transforming growth factor-beta signaling by interacting with the ATP-binding pocket of the transforming growth factor-beta type I receptor kinase domain. Here, we report on the synthesis and structure-activity relationships of the compounds in this series.

SB-431542 and Gleevec inhibit transforming growth factor-beta-induced proliferation of human osteosarcoma cells.

Transforming growth factor-beta (TGF-beta) has growth-stimulating effects on mesenchymal cells and several tumor cell lines. The signaling pathway for this effect is, however, not well understood. We examined how TGF-beta stimulates proliferation of MG63 human osteosarcoma cells. Two distinct type I receptors for TGF-beta, ALK-1 and ALK-5, were expressed and functional in MG63 cells. Of these two receptors, ALK-5 appears to be responsible for the growth stimulation because expression of constitutively active ALK-5, but not ALK-1, stimulated proliferation of MG63 cells. SB-431542 (0.3 microM), a novel inhibitor of ALK4/5/7 kinase, suppressed TGF-beta-induced growth stimulation. DNA microarray analysis as well as quantitative real-time PCR analysis of RNAs from TGF-beta-treated cells demonstrated that several growth factors, including platelet-derived growth factor AA, were induced in response to TGF-beta in MG63 cells. Gleevec (1 microM) as well as AG1296 (5 microM) inhibited TGF-beta-induced growth stimulation of MG63 cells, suggesting that platelet-derived growth factor AA was mainly responsible for the growth-stimulatory effect of TGF-beta. We also examined the mechanisms of perturbation of growth-suppressing signaling in MG63 cells. We found that expression of c-Myc, which is down-regulated by TGF-beta in many other cells, was up-regulated in MG63 cells, suggesting that up-regulation of c-Myc expression may be the mechanism canceling growth-suppressing signaling of TGF-beta in MG63 cells.

Smad4-independent regulation of p21/WAF1 by transforming growth factor-beta.

The transforming growth factor-beta (TGF-beta)-Smad signaling pathway inhibits the growth of human epithelial cells and plays a role in tumor suppression. The Smad4 gene is mutated or deleted in 50% of pancreatic cancers. In this study, the Smad4-null pancreatic cancer cell line BxPC-3 was transfected with either the Smad4 expression vector or the empty vector and incubated in the presence or absence of TGF-beta. The cells were analysed using a cDNA microarray, which included 2280 named genes to screen for target genes regulated by TGF-beta in either a Smad4-dependent or -independent manner. The microarray and subsequent quantitative RT-PCR analysis demonstrated that the Smad4-independent and -dependent signaling pathways driven by TGF-beta upregulated only one of the 2280 genes, respectively, suggesting that Smad4-independent signaling downstream of TGF-beta might be as widespread as Smad4-dependent signaling. In this study, we demonstrated that the cyclin-dependent kinase inhibitor p21/WAF1, which has been considered the major effector of the Smad-dependent growth inhibitory signal of TGF-beta, is upregulated in a Smad4-independent manner. The upregulation occurs through Smad2/3-dependent transcriptional activation of the p21/WAF1 promoter region. These results suggest a novel mechanism of gene regulation, that is, a novel signal mediator other than Smad4.

Forelimb vasodilatation induced by hypothalamic stimulation is greatly mediated with nitric oxide in anesthetized cats.

The aim of this study was to examine whether or not stimulation of the hypothalamic defense area is capable of inducing sympathetic vasodilatation of the forelimb vascular bed in anesthetized cats. When the hypothalamic defense area was electrically stimulated, brachial blood flow velocity (brachial BFV) and vascular conductance were increased as well as femoral BFV and vascular conductance. Brachial BFV and vascular conductance increased by 110-139% during hypothalamic stimulation. These increases were blunted to approximately one-fifth of the control responses following i.v. injection of a synthesis inhibitor of nitric oxide, N(omega)-nitro-L-arginine methyl ester (L-NAME). The attenuating effect of L-NAME on forelimb vasodilatation evoked by hypothalamic stimulation was greater than that on hindlimb vasodilatation. The combined administration of L-NAME and atropine sulfate eliminated nearly all of the increases in brachial BFV and vascular conductance during hypothalamic stimulation. From the present results, we conclude that stimulation of the hypothalamic defense area is able to induce neurogenic vasodilatation of the cat forelimb vascular bed, which is greatly mediated with a nitric oxide mechanism. The contribution of nitric oxide to neurogenic vasodilatation seems to be greater in the forelimbs than hindlimbs.

Hypoxia stimulates the EMT of gastric cancer cells through autocrine TGFß signaling.

Epithelial mesenchymal transition (EMT) is considered to be correlated with malignancy of cancer cells and responsible for cancer invasion and metastasis. We previously reported that distant metastasis was associated with hypoxia in gastric cancer. We therefore investigated the effect of hypoxic condition on EMT of gastric cancer cells. Gastric cancer cells were cultured in normoxia (21% O2) or hypoxia (1% O2) for 24 h. EMT was evaluated as the percentage of spindle-shaped cells in total cells. Effect of transforming growth factor ß1 (TGFß1) or tyrosine kinase inhibitors on the EMT was evaluated. The expression level of TGFß1 and TGFßR was evaluated by real time RT-PCR. The TGFß1 production from cancer cells was measured by ELISA. Hypoxia stimulated EMT of OCUM-2MD3 and OCUM-12 cells, but not that of OCUM-2M cells. The expression level of TGFß1 mRNA under hypoxia was significantly higher than that under normoxia in all of three cell lines. The expression level of TGFßR mRNA was significantly increased by hypoxia in OCUM-2MD3 cells, but not in OCUM-2M cells. TGFßR inhibitor, SB431542 or Ki26894, significantly suppressed EMT of OCUM-2MD3 and OCUM-12. TGFß1 production from OCUM-2MD3 and OCUM-12 cells was significantly increased under hypoxia in comparison with that under normoxia. These findings might suggest that hypoxia stimulates the EMT of gastric cancer cells via autocrine TGFß/TGFßR signaling.

Relationship between the cAMP levels in leukocytes and the cytokine balance in patients surviving gram negative bacterial pneumonia.

Lipopolysaccharide-stimulated leukocytes secrete proinflammatory cytokines including tumor necrosis factor-α and interleukin-12. Over-activation of host defense systems may result in severe tissue damage and requires regulation. Granulocyte colony-stimulating factor and interleukin-10 are candidate cytokines for inducing tolerance to lipopolysaccharide re-stimulation. We compared cytokines secreted by lipopolysaccharide-stimulated blood cells from patients who had survived gram negative bacterial pneumonia (Pseudomonas aeruginosa, Escherichia coli or Proteus mirabilis, n = 26) and age-matched healthy volunteers (n = 18). Interleukin-12p70 and tumor necrosis factor-α expression was significantly lower in patients (p = 0.0039 and p<0.001) compared to healthy controls, while granulocyte colony-stimulating factor production was markedly higher in patients (p<0.001). Levels of interleukin-10 were comparable. Granulocyte colony-stimulating factor expression was inversely correlated with interleukin-12p70 (R = -0.71, p<0.001) and tumor necrosis factor-α (R = -0.64, p<0.001) expression; interleukin-10 showed no significant correlation. In unstimulated leukocytes from patients, cAMP levels were significantly raised (p = 0.020) and were correlated inversely with interleukin-12p70 levels (R = -0.81, p<0.001) and directly with granulocyte colony-stimulating factor (R = 0.72, p = 0.0020), matrix metalloproteinase-9 (R = 0.67, p = 0.0067) and interleukin-10 (R = 0.54, p = 0.039) levels. Our results demonstrate that granulocyte colony-stimulating factor production by lipopolysaccharide-stimulated leukocytes is a useful indicator of tolerance induction in surviving pneumonia patients and that measuring cAMP in freshly isolated leukocytes may also be clinically significant.

Variable microsatellite loci isolated from the Asian honeybee, Apis cerana (Hymenoptera; Apidae).

We developed 12 polymorphic microsatellite loci for the Asian honeybee, Apis cerana using the magnetic particle method. Eight of these 12 were highly polymorphic, having four to seven alleles with an expected heterozygosity of 0.38 to 0.78. The primers also produce polymorphic products in related honeybee species such as Apis nigrocincta. These loci can be used to study parameters associated with genetic structure, such as paternity frequency and worker reproduction.

Ki26894, a novel transforming growth factor-beta type I receptor kinase inhibitor, inhibits in vitro invasion and in vivo bone metastasis of a human breast cancer cell line.

Transforming growth factor (TGF)-beta signaling has been shown to promote tumor growth and metastasis in advanced cancer. Use of inhibitors of TGF-beta signaling may thus be a novel strategy for treatment of patients with such cancers. In this study, we investigated the effects of a novel TGF-beta type I receptor (TbetaR-I) kinase inhibitor, Ki26894, on bone metastasis of a highly bone-metastatic variant of human breast cancer MDA-MB-231 cells, termed MDA-MB-231-5a-D (MDA-231-D). Ki26894 blocked TGF-beta signaling in MDA-231-D cells, as detected by suppression of phosphorylation of Smad2 and inhibition of TGF-beta-responsive reporter activity. Moreover, Ki26894 decreased the motility and the invasion of MDA-231-D cells induced by TGF-beta in vitro. Ki26894 also suppressed transcription of plasminogen activator inhibitor-1 (PAI-1), parathyroid hormone-related protein (PTHrP), and interleukin-11 (IL-11) mRNA of MDA-231-D cells, which were stimulated by TGF-beta. X-ray radiography revealed that systemic Ki26894 treatment initiated 1 day before the inoculation of MDA-231-D cells into the left ventricle of BALB/cnu/nu female mice resulted in decreased bone metastasis of breast cancer cells. Moreover, Ki26894 prolonged the survival of mice inoculated with MDA-231-D cells compared to vehicle-treated mice. These findings suggest that TbetaR-I kinase inhibitors such as Ki26894 may be useful for blocking the progression of advanced cancers.

Partial renal ischemia elicits heterogeneous control of renal sympathetic nerve activity to ischemic and nonischemic regions of the kidney.

We tested the hypothesis that renal sympathetic nerve activity (RSNA) to the ischemic and nonischemic regions responded differently during partial ischemia of the kidney in pentobarbital-anesthetized cats. The renal artery divides into two branches at the front of the renal hilus: one branch perfuses predominantly the dorsal half of the kidney, and the other perfuses its ventral half. We identified the innervated area of a renal nerve bundle by supramaximal electrical stimulation and subsequently determined the changes in RSNA in response to occlusion of either renal arterial branch for 3 min. RSNA to the nonischemic region of the kidney gradually decreased by 23 +/- 4% during partial renal ischemia, whereas RSNA to the ischemic region of the same kidney showed no significant change. Crushing either all renal nerve bundles or only the renal nerve bundles terminated to the ischemic region abolished the decrease in RSNA to the nonischemic region. Furthermore, intra-arterial administration of a prostaglandin synthesis inhibitor (meclofenamate, 4 mg/kg) abolished the decrease in RSNA to the nonischemic region of the kidney. Following spinal transection at the level of T7, the inhibitory response in RSNA to the nonischemic region disappeared, whereas the RSNA to the ischemic region was markedly augmented by 47 +/- 17%. Thus it is likely that renal chemoreceptors activated during renal partial ischemia elicit heterogeneous control of renal sympathetic outflows to the ischemic and nonischemic regions of the same kidney, which may be determined by a net output between the supraspinal inhibitory and spinal excitatory reflexes.

Amelioration of diabetic peripheral neuropathy by implantation of hematopoietic mononuclear cells in streptozotocin-induced diabetic rats.

This study was performed in order to evaluate the angiogenic effect of implantation of either peripheral blood mononuclear cells (PBMNCs) or bone marrow mononuclear cells (BMMNCs) on diabetic peripheral neuropathy. Streptozotocin (50 mg/kg) was injected intravenously into 6-week-old male Lewis rats. Four weeks after the induction of diabetes, 6 x 10(7) of PBMNCs or 1 x 10(8) of BMMNCs were implanted into the left hindlimb muscle. Motor nerve conduction velocity (MNCV) was monitored before and after implantation. At the end of the experiment, bilateral nerve blood flow (NBF) was measured by laser Doppler and the number of vessels in the sciatic nerves quantified by Factor VIII staining of the sections. Diabetes resulted in an approximately 20% reduction (P < 0.01) in sciatic MNCV. Four weeks after implantation, MNCV was improved by 54% with PBMNCs and by 67% with BMMNCs (both P < 0.01). Moreover, the effects of implantation were almost abolished by administration of VEGF-neutralizing antibody. Sciatic NBF was reduced by approximately 50% by diabetes (P < 0.05). This reduction in perfusion was improved by 74% by implantation of PBMNCs and by 62% by implantation of BMMNCs (P < 0.05 and P < 0.01, respectively). These effects were observed only in the implanted limb. Immunohistochemical staining of sciatic nerve sections for Factor VIII showed no significant increase in the number of vessels in the sciatic nerve following implantation of either PBMNCs or BMMNCs. These data suggest that implantation of hematopoietic mononuclear cell fractions is associated with an improvement in MNCV as a result of arteriogenic effects in the sciatic nerve, and that VEGF may contribute to this effect. This improvement occurred in the absence of angiogenesis. Implantation of these cell fractions may therefore be a potential new therapeutic method for treating diabetic peripheral neuropathy.

Notch1 oncoprotein antagonizes TGF-beta/Smad-mediated cell growth suppression via sequestration of coactivator p300.

The Notch proteins constitute a family of transmembrane receptors that play a pivotal role in cellular differentiation, proliferation and apoptosis. Although it has been recognized that excess Notch signaling is potentially tumorigenic, little is known about precise mechanisms through which dysregulated Notch signaling induces neoplastic transformation. Here we demonstrate that Notch signaling has a transcriptional cross-talk with transforming growth factor-beta (TGF-beta) signaling, which is well characterized by its antiproliferative effects. TGF-beta-mediated transcriptional responses are suppressed by constitutively active Notch1, and this inhibitory effect is canceled by introduction of transcriptional coactivator p300. We further show that this blockade of TGF-beta signaling is executed by the sequestration of p300 from Smad3. Moreover, in a human cervical carcinoma cell line, CaSki, in which Notch1 is spontaneously activated, suppression of Notch1 expression with small interfering RNA significantly restores the responsiveness to TGF-beta. Taken together, we propose that Notch oncoproteins promote cell growth and cancer development partly by suppressing the growth inhibitory effects of TGF-beta through sequestrating p300 from Smad3.

Functional diversity among Notch1, Notch2, and Notch3 receptors.

To clarify functional diversities among the Notch receptors, we generated truncated forms of Notch1, Notch2, and Notch3 comprising the intracellular domain (aN1, aN2, and aN3) and investigated their transcriptional activities for HES1 and HES5 promoters driving the luciferase reporter gene (HES1-Luc and HES5-Luc). The reporter assays demonstrated that the transcriptional activities of aNs were markedly different from each other and dependent on the promoters examined. Furthermore, relative activities between some aN and another for each promoter were altered by the expression level of RBP-J kappa. We also found that the activities of aN1 and aN3 were reduced by coexpression of aN2. These observations suggest that each Notch receptor has a diverse role in the downstream gene expression and that the levels of HES1 and HES5 gene expression are complexly determined by various factors, such as the type and combination of the Notch receptors which confer the downstream signals and the expression level of RBP-J kappa.

Integrity of intracellular domain of Notch ligand is indispensable for cleavage required for release of the Notch2 intracellular domain.

The biological activity of the soluble form of the Notch ligand (sNL) and requirement of the intracellular domain (ICD) of the Notch ligand have been debated. Here we show that soluble Delta1 (sD1) activates Notch2 (N2), but much more weakly than full-length Delta1 (fD1). Furthermore, tracing the N2 molecule after sD1 stimulation revealed that sD1 has a defect in the cleavage releasing ICD of N2 (intracellular cleavage), although it triggers cleavage in the extracellular domain of N2. This represents the molecular basis of the lower activity of sD1 and suggests the presence of an unknown mechanism regulating activation of the intracellular cleavage. The fact that Delta1 lacking its ICD (D1Delta(ICD)) exhibits the phenotype similar to that exhibited by sD1 indicates that the ICD of D1 (D1(ICD)) is involved in such an as yet unknown mechanism. Furthermore, the findings that D1Delta(ICD) acts in a dominant-negative fashion against fD1 and that the signal-transducing activity of sD1 is enhanced by antibody-mediated cross-linking suggest that the multi merization of Delta1 mediated by D1(ICD) may be required for activation of the N2 intracellular cleavage.

Sympathetic cholinergic vasodilation of skeletal muscle small arteries.

Recently we have studied the direct vasomotor response of the hindlimb extramuscular large arteries (internal diameter, 500-1400 microm) and intramuscular small arteries (internal diameter, 50-500 microm) of in vivo thick skeletal muscle during activation of sympathetic cholinergic nerve in anesthetized cats. The hypothalamic defense area was electrically stimulated so as to induce a profound increase in femoral blood flow mediated by sympathetic cholinergic fibers. To visualize the vascular arrangement from the extramuscular large feeding arteries to small arteries in the triceps surae muscle, we developed a new X-ray TV system. The internal diameter, flow velocity, and volume flow of arterial blood vessels were directly measured before and during stimulation of the hypothalamic defense area. The major new finding is that the hypothalamic stimulation causes an intense increase in the internal diameter of small arteries in skeletal muscle, which is abolished either by cholinergic blockade or by the section of the sciatic nerve, but not by combined alpha- and beta-adrenergic blockade. In contrast, the internal diameter of the extramuscular larger arteries does not change during the hypothalamic stimulation, but their flow velocity and volume flow increase. These findings indicate that sympathetic cholinergic vasodilation occurs at intramuscular small arteries with internal diameter of 50-500 microm, which in turn increases flow velocity and volume flow of upstream blood vessels.

An orphan PRD class homeobox gene expressed in mouse brain and limb development.

We report the cDNA sequence and expression of a mouse homeobox gene, Dmbx1, from the PRD class and comparison to its human orthologue. The gene defines a new homeobox gene family, Dmbx, phylogenetically distinct from the Ptx, Alx, Prx Otx, Gsc, Otp and Pax gene families. The Dmbx1 gene is expressed in the developing mouse diencephalon, midbrain and hindbrain, and has dynamic expression during forelimb and hindlimb development. Unusually for homeobox genes, there is no orthologue in the Drosophila or Caenorhabditis genomes; we argue this reflects secondary loss.