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Recent results suggest that bone marrow (BM)-derived hematopoietic cells are major components of tumor stroma and play crucial roles in tumor growth and angiogenesis. An E-type prostaglandin is known to regulate angiogenesis. We examined the role of BM-derived cells expressing an E-type prostaglandin receptor subtype (EP3) in tumor-induced angiogenesis and tumor growth. The replacement of wild-type (WT) BM with BM cells (BMCs) from green fluorescent protein (GFP) transgenic mice revealed that the stroma developed via the recruitment of BMCs. Selective knockdown of EP3 by recruitment of genetically modified BMCs lacking EP3 receptors was performed by transplantation of BMCs from EP3 knockout (EP3(-/-)) mice. Tumor growth and tumor-associated angiogenesis were suppressed in WT mice transplanted with BMCs from EP3(-/-) mice, but not in mice transplanted with BMCs from either EP1(-/-), EP2(-/-), or EP4(-/-) mice. Immunohistochemical analysis revealed that vascular endothelial growth factor (VEGF) expression was suppressed in the stroma of mice transplanted with BMCs from EP3(-/-) mice. EP3 signaling played a significant role in the recruitment of VEGFR-1- and VEGFR-2-positive cells from the BM to the stroma. These results indicate that the EP3 signaling expressed in bone marrow-derived cells has a crucial role in tumor-associated angiogenesis and tumor growth with upregulation of the expression of the host stromal VEGF together with the recruitment of VEGFR-1/VEGFR-2-positive. The present study suggests that the blockade of EP3 signaling and the recruitment of EP3-expressing stromal cells may become a novel strategy to treat solid tumors.

Gamma-secretase belongs to an atypical class of aspartic proteases that hydrolyzes peptide bonds within the transmembrane domain of substrates, including amyloid-beta precursor protein and Notch. gamma-Secretase is comprised of presenilin, nicastrin, APH-1, and PEN-2 which form a large multimeric membrane protein complex, the three-dimensional structure of which is unknown. To gain insight into the structure of this complex enzyme, we purified functional gamma-secretase complex reconstituted in Sf9 cells and analyzed it using negative stain electron microscopy and 3D reconstruction techniques. Analysis of 2341 negatively stained particle images resulted in the three-dimensional representation of gamma-secretase at a resolution of 48 angstroms. The structure occupies a volume of 560 x 320 x 240 angstroms and resembles a flat heart comprised of two oppositely faced, dimpled domains. A low density space containing multiple pores resides between the domains. Some of the dimples in the putative transmembrane region may house the catalytic site. The large dimensions are consistent with the observation that gamma-secretase activity resides within a high molecular weight complex.

Stimulated neutrophils produced vascular permeability enhancing (VPE) activity in the presence of high molecular weight kininogen (HMWK), which was inhibited mainly by a neutrophil elastase (NE) inhibitor or a bradykinin (BK) B(2)-receptor antagonist. NE (>3 nM) generated VPE activity from kininogens at normal plasma concentrations with the smaller protein being several fold more responsive than the larger protein, through releasing a new VPE peptide (E-kinin), SLMKRPPGFSPFRSSRI. Synthetic E-kinin, SLMKRPPGFSPFRSS and SLMKRPPGFSPFR had VPE and blood pressure lowering activities, which were comparable to the activities of BK and completely inhibited by B(2)-receptor antagonists. Interestingly, E-kinin and SLMKRPPGFSPFRSS did not induce smooth muscle contraction. These results suggest that E-kinin formed in vivo may be processed at the carboxy-terminus to give a peptide that can bind to the B(2)-receptor. The molecular mechanism for neutrophil-associated VPE may be explained by excision of E-kinin from kininogens by NE, followed by further processing of the peptide., ((c) 2002 Elsevier Science (USA).)

It was reported that angiotensin II stimulates angiogenesis in vivo, and angiotensin-converting enzyme (ACE) inhibitors inhibit angiogenesis. We found that an AT1-receptor (AT1-R) antagonist, TCV-116, inhibited tumor growth, tumor-associated angiogenesis, and metastasis in a murine model. Tumor growth of Sarcoma 180 (S-180) cells and of fibrosarcoma (NFSA) cells was strongly inhibited by administration of TCV-116 in the diet at a dose of approximately 100 mg/kg/day. This reduction was accompanied with a marked reduction in tumor-associated angiogenesis. The same treatment also reduced the lung metastasis of intravenously injected Lewis lung carcinoma cells. These effects of TCV-116 were equivalent to those of the ACE inhibitor, lisinopril. In S-180 and NFSA tumor tissues, ACE and AT1a receptor (AT1a-R) mRNAs were expressed when assessed with RT-PCR. AT1b receptor and AT2 receptor, however, were not detected. Immunoreactive AT1-R was detected mainly on the neovascularized vascular endothelial cells in which expression was reduced by TCV-116 and lisinopril. These results suggested that TCV-116 inhibits the angiogenesis, growth, and metastasis of tumors highly dependent on AT1a-R blockade. Blockade of AT1a-R signaling may therefore become an effective novel strategy for tumor chemoprevention., ((c) 2002 Elsevier Science (USA).)

The NGF-family of neurotrophic factors are structurally similar peptides with related functional properties. So far, this family of neurotrophic factors has only been identified in the vertebrate nervous system. We have determined that cultured Drosophila embryonic cells produce and secrete into medium, an activity which stimulates neurite outgrowth of embryonic chick sensory ganglia. This Drosophila activity can be blocked by antibodies to mouse NGF, indicating an immunological relationship between the Drosophila factor, mouse NGF and possibly other vertebrate neurotrophic factors. Addition of mouse NGF to Drosophila embryonic cells in culture results in increased cell number and enrichment of the neuronal phenotype, indicating that Drosophila cells have the ability to respond to the vertebrate factor. In addition, poly(A)+RNA extracted from Drosophila contains a single 1.4 Kb band which cross-hybridizes with a mouse NGF cRNA probe. These results indicate that vertebrate neurotrophic factor-like functions may operate in a genetically defined invertebrate species.

T-kininogen levels in plasma and liver microsomes were measured by radioimmunoassay in female and male rats of various ages. High levels of T-kininogen were found in plasma and liver of 1-day to 1-week old male and female rats and their mothers. The levels in newborns gradually decreased along with their development. In mature male rats the levels were as low as 1/5-1/2 of those in mature female rats. Treatment with estradiol increased the plasma and the liver levels of T-kininogen significantly in both sexes, but testosterone decreased the level in female rats and had no effect in male rats. These results suggest that sex hormones may regulate the physiological level of T-kininogen in rats.

The amino acid sequence around kinin moiety in rat High-Molecular-Weight (HMW) kininogen was determined by isolating a peptide containing bradykinin after cyanogen bromide treatment of the purified kininogen as follows; NH2-Thr-Ser-Val-Ile-Arg-Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg-Ala-Pro-Arg- Val-Lys-Lys-. The data indicated that rat HMW kininogen contains the arginyl-bradykinin moiety, instead of lysyl-bradykinin. Kinins liberated from rat HMW kininogen by rat urinary and submaxillary kallikreins were identified to be bradykinin, not arginyl-bradykinin.