Connective tissue growth factor induces the proliferation, migration, and tube formation of vascular endothelial cells in vitro, and angiogenesis in vivo.

Connective tissue growth factor (CTGF) is a novel cysteine-rich, secreted protein. Recently, we found that inhibition of the endogenous expression of CTGF by its antisense oligonucleotide and antisense RNA suppresses the proliferation and migration of vascular endothelial cells. In the present study, the following observations demonstrated the angiogenic function of CTGF in vitro and in vivo: (i) purified recombinant CTGF (rCTGF) promoted the adhesion, proliferation and migration of vascular endothelial cells in a dose-dependent manner under serum-free conditions, and these effects were inhibited by anti-CTGF antibodies; (ii) rCTGF markedly induced the tube formation of vascular endothelial cells, and this effect was stronger than that of basic fibroblast growth factor or vascular endothelial growth factor; (iii) application of rCTGF to the chicken chorioallantoic membrane resulted in a gross angiogenic response, and this effect was also inhibited by anti-CTGF antibodies. (iv) rCTGF injected with collagen gel into the backs of mice induced strong angiogenesis in vivo. These findings indicate that CTGF is a novel, potent angiogenesis factor which functions in multi-stages in this process.     

Liposomal membranes. XIV. Fusion of liposomal membranes induced by polyisoprenoids as monitored by fluorescence quenching method

Fusion of the single-walled liposomes of egg phosphatidylcholine as induced by the polyisoprenoids such as solanesol, trans-ethyl decaprenoate (EDP), coenzyme Q10, and dolichol has been investigated adopting the fluorescence quenching method. Relative efficiency of the polyisoprenoids employed on the induced fusion of liposomes was a sequence of solanesol less than or equal to EDP much less than CoQ10, dolichol, which was consistent with the result previously obtained by the dye-release method.     

Fertilization Membrane Formation in Sea Urchin Eggs Induced by Drugs Known to Cause Ca2+Release from Isolated Sarcoplasmic Reticulum

Ryanodine, miconazole, clotrimazole, doxorubicin, quercetin, halothane, caffeine and chloroform, which activate Ca²⁺-induced Ca²⁺release from Ca²⁺stores, induced Ca²⁺release from a particulate fraction isolated from sea urchin eggs, Ca²⁺influx into eggs and formation of a fertilization membrane in an appreciable number of eggs. Their minimum effective concentrations for inducing a fertilization membrane increased in the order of these drugs listed above, and this order was also the same as that of their minimum effective concentrations for inducing Ca²⁺release from the isolated particulate fraction. Their effect in inducing a fertilization membrane was blocked by ruthenium red and procaine, which inhibit Ca²⁺release from Ca²⁺stores. Thus these drugs probably induced sufficient Ca²⁺release to make the cytosolic Ca²⁺level high enough in many eggs for formation of a fertilization membrane. In the absence of external Ca²⁺, fewer eggs treated with these drugs formed a fertilization membrane and more eggs did so on further treatment with either A23187 or carbonylcyanide-p-trifluoromethoxy-phenylhydrazone (FCCP). Thus, a high level of Ca²⁺is probably derived from Ca²⁺release through Ca²⁺releasing channels (by A23187), from mitochondria (by FCCP) and its transport from the external medium.     

Identification of osteopontin in isolated rabbit osteoclasts.

Bone remodeling is a complex process coupling bone formation and resorption. Osteoblasts, the bone-forming cells, are known to produce various bone matrix proteins and cytokines; however, little is known about protein factors produced by osteoclasts or bone-resorbing cells. A method utilizing the high affinity of osteoclasts for tissue culture dishes was developed to isolate a large number of pure osteoclasts from rabbit long bones. A cDNA library was then constructed from these isolated osteoclasts, and differential cDNA screening was performed between osteoclasts and spleen cells. Two clones representing osteoclast-specific clones, named OC-1 and OC-2, were isolated. By Northern blot analysis, OC-1 was expressed in osteoclasts and in kidneys, whereas OC-2 was specific for osteoclasts. OC-1 was found to encode osteopontin from its nucleotide sequence, and therefore, osteopontin may have other functions for osteoclastic bone resorption besides osteoclast attachment to bone.     

Polynucleotides. XXXII. Further studies on the synthesis of oligonucleotides containing 8,2''-S-cycloadenosine.

A dinucleoside monophosphate, 8,2'-anhydro-8-mercapto-9-beta-D-arabinofuranosyladenine phosphoryl-(3'-5')-inosine (AspI) was synthesized by the condensation of protected 8-mercapto-adenosine 2',3'-cyclic phosphate and 2',3'-isopropylideneinosine with diphenylphosphorochloridate. 8-Mercaptoadenosine 2',3'-cyclic phosphate was polymerized by using tetraphenyl pyrophosphate as the condensing reagent. As oligonucleotides, thus obtained, contained some uncyclized 8-mercaptoadenosine residues and were cleaved at these sites with 0.3N KOH. As 5'-phosphate was synthesized and polymerized with DCC to give oligonucleotides with chain lengths 2 to 9.     

Effect of High Temperature on the Development of Cyanide-Sensitive Respiration in the Germination Process of Spinach Seeds

Germination of spinach (Spinacia oleracea L. var. grabra cv.Nobel) seeds was inhibited at a high temperature (35?C). Effectsof KCN on the respiration of seeds incubated at 20 and 35?Cwere compared in order to investigate the mechanism of inhibitionof seed germination by high temperature. Respiration of germinatingseeds incubated at 20?C was inhibited about 50% by 5 mM. KCNsolution, whereas it hardly inhibited the weak respiration ofthe seeds at 35?C. Germination of seeds was delayed by exogenousKCN. When the KCN solution was renewed daily, germination wascompletely inhibited. Pericarp removal promoted germinationat 35?C, but atypical germination (cotyledons emerging earlierthan a radicle) took up more than half of the total germination.The inhibitory action of KCN on the respiration of seeds wasnot altered by pericarp removal. A KCN addition, even at 20?C,elicited atypical germination in the pericarp-less seeds. Theseresults show that cyanide-sensitive respiration is needed toinduce typical spinach seed germination (root emergence), butis rendered inoperative by high temperatures thus bringing aboutpoor germination and atypical germination. (Received December 1, 1984; Accepted February 8, 1985)