Suppression of membrane-type 1 matrix metalloproteinase (MMP)-mediated MMP-2 activation and tumor invasion by testican 3 and its splicing variant gene product, N-Tes.

Using expression cloning to screen a human fetal kidney cDNA library for regulator(s) of pro-matrix metalloproteinase (MMP)-2 processing mediated by membrane-type (MT) 1 MMP, we isolated a cDNA whose product interfered with pro-MMP-2 activation. It encodes the NH(2)-terminal 313-amino acid region of a calcium-binding proteoglycan, testican 3, with a 3-amino acid substitution at the COOH terminus and thus was named N-Tes. N-Tes comprises a signal peptide, a unique domain, a follistatin-like domain, and a Ca(2+)-binding domain but lacks a COOH-terminal thyroglobulin domain and two putative glycosaminoglycan attachment sites of testican 3. Pro-MMP-2 activation by MT3-MMP was also inhibited by the coexpression of N-Tes. Immunoprecipitation analysis demonstrated direct interaction of N-Tes with either MT1-MMP or MT3-MMP. Expression of testican 1 or testican 3 but not testican 2 also inhibited pro-MMP-2 activation by either MT1-MMP or MT3-MMP. Deletion and substitution of amino acids residues in N-Tes revealed that the unique NH(2)-terminal domain of N-Tes is responsible for the inhibition of pro-MMP-2 activation by MT-MMPs. Expression of N-Tes and testican 3 was detected in normal brain but down-regulated in glioma tissues. Transfection of either the N-Tes or testican 3 gene into U251 glioma cells or Madin-Darby canine kidney cells transformed by erbB2 suppressed their invasive growth in collagen gel. These results suggest that both N-Tes and testican 3 would interfere with tumor invasion by inhibiting MT-MMPs.

A new halogenated antidiabetic vanadyl complex, bis(5-iodopicolinato)oxovanadium(IV): in vitro and in vivo insulinomimetic evaluations and metallokinetic analysis.

A new vanadyl complex, bis(5-iodopicolinato)oxovanadium(IV), VO(IPA)2, with a VO(N2O2) coordination mode, was prepared by mixing 5-iodopicolinic acid and VOSO4 at pH 5, with the structure characterized by electronic absorption, IR, and EPR spectra. Introduction of the halogen atom on to the ligand enhanced the in vitro insulinomimetic activity (IC50 = 0.45 mM) compared with that of bis(picolinato)oxovanadium(IV) (IC50 = 0.59 mM). The hyperglycemia of streptozotocin-induced insulin-dependent diabetic rats was normalized when VO(IPA)2 was given by daily intraperitoneal injection. The normoglycemic effect continued for more than 14 days after the end of treatment. To understand the insulinomimetic action of VO(IPA)2, the organ distribution of vanadium and the blood disposition of vanadyl species were investigated. In diabetic rats treated with VO(IPA)2, vanadium was distributed in almost all tissues examined, especially in bone, indicating that the action of vanadium is not peripheral. Vanadyl concentrations in the blood of normal rats given VO(IPA)2 remain significantly higher and longer than those given other complexes because of its slower clearance rate. VO(IPA)2 binds with the membrane of erythrocytes, probably owing to its high hydrophobicity in addition to its binding with serum albumin. The longer residence of vanadyl species shows the higher normoglyceric effects of VO(IPA)2 among three complexes with the VO(N2O2) coordination mode. On the basis of these results, VO(IPA)2 is indicated to be a preferred agent to treat insulin-dependent diabetes mellitus in experimental animals.

Long circulating emulsion carrier systems for highly lipophilic drugs.

With the aim of developing of emulsion carrier systems for lipophilic drugs with the potential for prolonged circulation in the blood or hepatic targeting, the in vivo disposition of four model compounds, i.e., [3H]prostaglandin E1, [3H]retinoic acid, [14C]cholesterol, and [14C]cholesteryl oleate with calculated log PC(oct) values of 2.15, 6.61, 9.46, and 18.3, respectively, injected with various emulsion formulations, were studied in mice. Small sized emulsions of about 100 nm in diameters, with compositions of egg phosphatidylcholine (PC): soybean oil = 1:1 (small PC emulsion) and PC: egg sphingomyelin (SM): soybean oil = 0.7:0.3:1 (small SM emulsion), and a conventional emulsion with a diameter of about 250 nm and a composition of PC: soybean oil = 1:1 (large PC emulsion) were compared. Highly lipophilic [14C]cholesteryl oleate, a marker of emulsion particles, indicated diverse in vivo behaviors; i.e., the small SM emulsion produced prolonged circulation in the blood, and the small PC emulsion followed this, while the large PC emulsion was rapidly uptake by the liver. Thus, a reduction in size and coating with SM on the surface of oil droplets resulted in avoidance of the reticuloendothelial system (RES). Disposition profiles of other test compounds differed, depending on their lipophilicities: [14C]cholesterol showed disposition patterns in all formulations similar to those of [14C]cholesteryl oleate, but moderately lipophilic [3H]prostaglandin E1 and [3H]retinoic acid showed common disposition profiles, regardless of emulsion types, suggesting their rapid release from the emulsion carriers. These results suggest that small SM emulsion and large PC emulsion can act respectively as long circulating and liver targeting carriers for highly lipophilic drugs with log PC(oct) larger than 9.

Role of free radical scavengers on phenylhydrazine induced hemolysis in rat.

The mechanism of the phenylhydrazine induced oxidative hemolysis was studied on the point of role of the free radical scavengers in rats. Phenylhydrazine resulted in the degradation of hemoglobin and the lipid peroxidation of the erythrocyte membrane. Otherwise, the elevation of coenzyme Q9, endogenous CoQ in rats, levels in plasma was observed against the phenylhydrazine induced oxidative stress. Supplementation of coenzyme Q10, exogenous CoQ in rats, inhibited the phenylhydrazine induced hemolysis according to the suppression of both the degradation of hemoglobin and the lipid peroxidation of the erythrocyte membrane. These results suggest that free radical scavengers such as coenzyme Q9 and coenzyme Q10 have important roles on the phenylhydrazine induced hemolysis in rats.