Novel and simple loading procedure of cisplatin into liposomes and targeting tumor endothelial cells.

Although intravenous administration of high levels of cisplatin (CDDP) are limited due to its severe side effects, efficient delivery of CDDP directly to the tumor should improve the therapeutic response while potentially by-passing significant side effects. High loading of CDDP into liposomes is one technique that could be used as a potential drug delivery system. Since cis-diamminedinitratoplatinum (CDDP3) is highly soluble in water and converts to CDDP in the presence of chloride ions, we encapsulated CDDP3 into liposomes in the absence of chloride ions and supplemented chloride ions to prepare CDDP-encapsulated liposomes (CDDP-Lip) resulting in a significantly improved loading efficiency of CDDP. We further conjugated the CDDP-Lip with Sialyl Lewis(X) (CDDP-SLX-Lip) because we previously demonstrated Sialyl Lewis(X) enhanced efficient accumulation of liposomes into tumors in vivo. CDDP-SLX-Lip treated mice showed a survival rate of 75% at 14 days even if a lethal level of CDDP was injected into mice. Loss of body weight was negligible and no histological abnormality was found in a variety of normal tissues. Accumulation of CDDP-SLX-Lip was about 6 times more than that of CDDP-Lip or CDDP. As the result, there was better antitumor activity of CDDP-SLX-Lip than that of CDDP-Lip with significantly less toxic effects in normal tissues.

Physiological roles of glycerol-transporting aquaporins: the aquaglyceroporins.

A subclass of aquaporin (AQP) water channels, termed aquaglyceroporins, are also able to transport glycerol and perhaps urea and other small solutes. Although extensive data exist on the physiological roles of aquaporin-facilitated water transport, until recently the biological significance of glycerol transport by the mammalian aquaglyceroporins has been unknown. There is now compelling evidence for involvement of aquaglyceroporin- facilitated glycerol transport in skin hydration and fat cell metabolism. Mice deficient in AQP3 have dry skin, reduced skin elasticity and impaired epidermal biosynthesis. Mice lacking AQP7 manifest progressive adipocyte fat accumulation and hypertrophy. These skin and fat phenotypes are attributable to impaired glycerol transport. A potential implication of these findings is the possibility of modulation of aquaglyceroporin expression or function in the therapy of skin diseases and obesity.

Tissue-specific regulation of erythropoietin production in the murine kidney, brain, and uterus.

Erythropoietin (Epo) produced by the kidney regulates erythropoiesis. Recent evidence suggests that Epo in the cerebrum prevents neuron death and Epo in the uterus induces estrogen (E(2))-dependent uterine angiogenesis. To elucidate how Epo expression is regulated in these tissues, ovariectomized mice were given E(2) and/or exposed to hypoxia, and the temporal patterns of Epo mRNA levels were examined. Epo mRNA levels in the kidney and cerebrum were elevated markedly within 4 h after exposure to hypoxia. Although the elevated level of Epo mRNA in the kidney decreased markedly within 8 h despite continuous hypoxia, the high level in the cerebrum was sustained for > or = 24 h, indicating that downregulation operates in the kidney but not in the brain. E(2) transiently induced Epo mRNA in the uterus but not in the kidney and cerebrum. Interestingly, the uterine Epo mRNA was hypoxia inducible only in the presence of E(2). Thus Epo expression appears to be regulated in a tissue-specific manner, endorsing the tissue-specific functions of Epo.

Catalytic activity of anion-exchange resins modified with metal-porphine in oxidative reactions of phenols.

Anion-exchange resins modified with metal-porphine (M-Pr) have been investigated to develop a solid catalyst in the oxidative reaction of phenols by O2 in air. Co-Pr, which is easily prepared and separable from the reaction mixture, has been proved to accelerate the oxidative reaction of phenols such as 3,5-di-tertbutyl-4-hydroxyanisole. The resulting main oxidative products were identified to be quinones by using the GC-MS method.

The oviduct produces erythropoietin in an estrogen- and oxygen-dependent manner.

Previously, we showed that erythropoietin (Epo) is produced in the mouse uterus, where Epo is indispensable for estrogen (E(2))-dependent angiogenesis. Expression of uterine Epo mRNA is stimulated by E(2) and hypoxia. The hypoxic induction requires the presence of E(2). In the present study, we examined other female reproductive organs in the mouse with respect to Epo mRNA expression and its stimuli (E(2) and hypoxia)-induced changes. Although Epo mRNA expression was seen in the ovary and oviduct, the E(2)-induced stimulation of Epo mRNA was found only in the oviduct. The E(2)-induced stimulation in the oviduct was transient and rapidly downregulated. Epo mRNA expression in the oviduct was hypoxia inducible, in both the presence and the absence of E(2). E(2)-dependent production of Epo and its mRNA expression were also found by use of cultured oviducts. The E(2) action is probably mediated through the E(2) receptor, and de novo protein synthesis is not required for E(2) induction of Epo mRNA. In the oviduct, the ampulla and isthmus regions produce Epo.

New antitumor-active azole-bridged dinuclear platinum(II) complexes: synthesis, characterization, crystal structures, and cytotoxic studies.

Three new derivatives of the cytotoxic azole-bridged dinuclear platinum(II) complex [(cis-Pt(NH3)2)2(mu-OH)(mu-pz)][NO3]2 (1) have been prepared and structurally characterized. Their formulas are [(cis-Pt(NH3)2)2(mu-OH)(mu-1,2,3-ta)][NO3]2 (2) (1,2,3-ta = 1,2,3-triazolate), [(Pt(R,R-dach))(mu-OH)(mu-pz)(Pt(S,S- dach))][NO3]2 (3) (dach = 1,2-diaminocyclohexane, pz = pyrazolate), and [(Pt(R,R-dach))(mu-1,2,3- ta)2(Pt(S,S-dach))][NO3]2 (4). The compounds were characterized by 1H, 13C, and 195Pt NMR spectroscopy, and elemental analysis, and their crystal structures were determined. Relevant data for 2: triclinic, space group P1, a = 8.5225(15) A, b = 9.1977(18) A, c = 9.9771(7) A, alpha = 66.988(10) degrees, beta = 75.423(9) degrees, gamma = 67.321(13) degrees, Z = 2. 3: orthorhombic, space group Pca2(1), a = 17.7653(3) A, b = 12.4076(3) A, c = 10.7091(3) A, Z = 4. 4: orthorhombic, space group Pbca, a = 13.8944(1) A, b = 17.8668(1) A, c = 20.7647(2) A, Z = 8. In the crystal structures of 2, and 3, the intramolecular distances between the two Pt atoms are 3.4411(6) and 3.4873(5) A, and the dihedral angles between the platinum coordination planes are 14.1(3) and 9.3(4) degrees, respectively. In 2, an intramolecular hydrogen bond is observed between N9 of the ammine ligand and the noncoordinated nitrogen atom (N3) of the triazole ring (N9...N3: 2.962(10) A). 4 has a boat-form structure, and the two coordination planes cross at 83.64(10) degrees. A cytotoxicity assay of these dinuclear platinum(II) compounds on human tumor cell lines was performed. In most of the cell lines, 1 and 2 showed much higher cytotoxicity than those of cisplatin. On the other hand, 3 was found to be moderately active, and 4 was found only marginally cytotoxic. Implications of these findings are discussed in the context of a structure-activity relationship.

Heparin-selenocystamine conjugate with selenol groups.

Heparin-selenocystamine conjugate, which was intended to mimic the heparin-selenoprotein P complex, was prepared. The conjugate had glutathione peroxidase-like activity and activity was observed toward hydrogen peroxide, tert-butyl hydroperoxide, and cumene hydroperoxide. The ultraviolet spectrum of an aqueous solution of the conjugate was stable and had a similar shape to that observed transiently when selenocystamine was reduced by sodium cyanoborohydride; this suggests that the diselenide bond of selenocystamine introduced into heparin was cleaved during conjugate preparation and the selenol group is preserved. The conjugate reacted to the same degree as cysteine with 5,5'-dithiobis (2-nitrobenzoic acid) (DTNB) releasing thionitro-benzoic acid, which indicated that the selenium in the conjugate is present as selenol. However, the reaction rate of the conjugate was slower than cysteine which may be due to partially restricted access of DTNB to the selenol group in the conjugate. This conjugate had 1,1-diphenyl-2-picryl-hydrazyl(DPPH) radical scavenging activity as well as superoxide anion scavenging activity. These results indicate that the conjugate serves as a useful model compound with a stable selenol group having a range of biological activities, and suggest a possible antioxidant defensive role for the complex of endogenous heparin-like substance and selenoprotein P.

Estrogen-dependent production of erythropoietin in uterus and its implication in uterine angiogenesis.

Although erythropoietin (Epo) has been shown to possess in vitro angiogenic activity, its physiological significance has not been demonstrated. Normally angiogenesis does not occur actively in adults but an exception is the female reproductive organ. In the uterine endometrium, angiogenesis takes place actively for supporting the endometrial growth that occurs during transition from the diestrus to estrous stage. This transition is under control of 17beta-estradiol (E2), an ovarian hormone, and can be mimicked by injection of E2 to ovariectomized (OVX) mouse. Thus, the uterus is a pertinent site to examine the Epo function in angiogenesis. We found that Epo protein and its mRNA were produced in an E2-dependent manner, when the uterus from OVX mouse was cultured in vitro. The de novo protein synthesis was not needed for E2 induction of Epo mRNA. Administration of E2 to OVX mouse induced a rapid and transient increase in Epo mRNA in the uterus. Injection of Epo into the OVX mouse uterine cavity promoted blood vessel formation in the endometrium. Furthermore, injection of the soluble Epo receptor capable of binding with Epo into the uterine cavity of non-OVX mouse in diestrus stage inhibited the endometrial transition to proestrus stage, whereas heat-inactivated soluble Epo receptor allowed the transition to occur. These results, combined with our finding that the endothelial cells in uterine endometrium express Epo receptor, strongly suggest that Epo is an important factor for the E2-dependent cyclical angiogenesis in uterus.

Embryonal carcinoma P19 cells produce erythropoietin constitutively but express lactate dehydrogenase in an oxygen-dependent manner.

Embryonic stem cells and embryonal carcinoma P19 cells produce erythropoietin (Epo) in an oxygen-independent manner, although lactate dehydrogenase A (LDHA) is hypoxia-inducible. To explore this paradox, we studied the operation of cis-acting sequences from these genes in P19 and Hep3B cells. The Epo gene promoter and 3' enhancer from P19 cells conveyed hypoxia-inducible responses in Hep3B cells but not in P19 cells. Together with DNA sequencing and the normal transcription start site of P19 Epo gene, this excluded the possibility that the noninducibility of Epo gene in P19 cells was due to mutation in these sequences or unusual initiation of transcription. In contrast, reporter constructs containing LDHA enhancer and promoter were hypoxia inducible in P19 and Hep3B cells, and mutation of a hypoxia- inducible factor 1 (HIF-1) binding site abolished the hypoxic inducibility in both cells, indicating that HIF-1 activation operates normally in P19 cells. Neither forced expression of hepatocyte nuclear factor 4 in P19 cells nor deletion of its binding site from the Epo enhancer was effective in restoring Epo enhancer function. P19 cells may lack an unidentified regulator(s) required for interaction of the Epo enhancer with Epo and LDHA promoters.

Insulin-like growth factors and insulin stimulate erythropoietin production in primary cultured astrocytes.

Erythropoietin (EPO) is established as a major regulator of erythropoiesis. However, we and others have shown that neurons express erythropoietin receptor (EPO-R), that astrocytes produce EPO and that EPO may act as a neurotrophic factor in the CNS. We also found that EPO production is activated by insulin and insulin-like growth factors (IGFs) in astrocytes in a dose-dependent manner and that IGF-I was the most potent activator. The concentrations required for half-maximal activation were 3 nM IGF-I, 10 nM IGF-II and 100 nM insulin. The oxygen concentration regulates EPO production; hypoxia stimulates EPO production in astrocytes. The stimulatory effect of IGFs and insulin on EPO production in astrocytes was not affected by the oxygen concentration of astrocyte culture. Insulin and IGFs did not increase the total protein synthesis of astrocytes but increased EPO mRNA levels, indicating that EPO production is stimulated at the mRNA level. It appeared that the growth factor-induced accumulation of EPO mRNA in astrocytes was caused by activation of the tyrosine kinase-signal transduction pathway, because tyrosine phosphorylation of receptors for IGF-I and insulin was activated when astrocytes were stimulated by these growth factors.

Determination of trace amounts of albumin in human bronchoalveolar lavage fluids by fluorometry with chromazurol S.

Fluorometry using chromazurole S (CAS) was applied to determine trace amounts of albumin in human bronchoalveolar lavage fluids (BALF). The calibration curve was linear in the range of 5-60 micrograms/ml of albumin. The CAS method was proven to be much more selective for albumin than for IgG. Freezing of BALF samples did not affect albumin analysis by the CAS method after storage at -20 degrees C for 80 days. This finding suggests that albumin in the BALF samples is stable under these conditions. The correlation was highly linear (r = 0.966) between the albumin levels in concentrated BALF samples (n = 47) determined by the CAS method and by radial immunodiffusion. The CAS method is sensitive enough to determine albumin levels in unconcentrated BALF samples, whereas radial immunodiffusion often requires concentration. The former method is more suitable for measuring albumin in BALF samples than the latter, because concentration by ultrafiltration results in poor reproducibility. The concentration of albumin in BALF samples of healthy volunteers (n = 5) and patients with sarcoidosis (n = 32) was determined by the CAS method. There was a statistically significant difference (P < 0.01) in the albumin levels in BALF samples between healthy subjects and patients with sarcoidosis at a clinically active state (n = 15). This finding shows that the determination of albumin levels in BALF samples is useful for investigating lung diseases and that the CAS method is promising in the determination of trace albumin in BALF samples, because it is simple, sensitive and precise.

Determination of human immunoglobulin A and secretory immunoglobulin A in bronchoalveolar lavage fluids by solid phase enzyme immunoassay.

Solid phase enzyme immunoassay methods for the determination of secretory immunoglobulin A (IgA) and the total amount of serum and secretory IgA in bronchoalveolar lavage fluids (BALF) were developed. The solid phase was prepared by immobilizing rabbit anti-human IgA. Horseradish peroxidase-conjugated goat anti-secretory component or horseradish peroxidase-conjugated goat anti-human IgA (Fc) were used as labeled antibodies. The minimum detectable amounts of secretory IgA and total IgA were 2 and 0.5 ng/well, respectively. These assay methods were successfully applied to the determination of secretory and total IgA levels in BALF samples obtained from 44 subjects including healthy non-smokers, smokers and patients with the following lung diseases: idiopathic pulmonary fibrosis, sarcoidosis and hypersensitivity pneumonitis. The secretory and total IgA levels in BALF collected from healthy non-smokers (n = 9) were 10.5 +/- 3.6 and 25.4 +/- 15.5 (S.D.) micrograms/ml, respectively. In healthy smokers, the secretory IgA concentration was significantly decreased and in idiopathic pulmonary fibrosis, the total IgA was increased. These results indicate that the quantitation of secretory and total IgA may be useful for the investigation of lung disease.

Uricase-like catalytic activity of ion-exchange resins modified with metalloporphyrins.

The uricase-like catalytic activity of the ion-exchange resins modified with metalloporphyrins has been investigated through the oxidation of uric acid. The anion-exchange resins modified with Mn(3+)-tetrakis(sulfophenyl)porphine and the cation-exchange resin modified with Mn(3+)-tetrakis(1-methylpyridinium-4-yl)porphine exhibited the highest uricase-like activity among the modified resins tested. The fact that these resins accelerated the oxidation of uric acid even after ten cycles of use indicates that the modified resins act as catalysts in the reaction catalysed by uricase. Some of the modified resins may be effectively used for the determination of uric acid in place of uricase.

Separation and determination of theophylline from paraxanthine in human serum by reversed-phase high-performance liquid chromatography.

A sensitive and highly specific method for the determination of theophylline in serum by high-performance liquid chromatography (HPLC) has been developed. Theophylline was completely separated from paraxanthine, a major metabolite of caffeine which has been known to interfere with most isocratic reversed-phase HPLC methods, with a mixture of acetonitrile/tetrahydrofuran/acetate buffer (10 mM, pH 5.0; 5:1:94, v/v %) as the mobile phase using a C18 bonded reversed-phase column. Neither the other xanthine and uric acid derivatives nor numerous drugs tested were found to interfere. The proposed method was applied to therapeutic monitoring utilizing its excellent precision, reproducibility and high specificity for theophylline.

Thiol exchange reactions involving selenotrisulfides.

The occurrence of in vitro thiol exchange reactions involving selenotrisulfides has been documented by HPLC analyses of reaction solutions. Asymmetric selenotrisulfide (RSSeSR') (R,R' = penicillamine, cysteine, glutathione) was formed by the reactions between (i) a mixture of thiols and selenite, (ii) thiol (R'SH) and symmetric selenotrisulfide (RSSeSR), and (iii) symmetric selenotrisulfides (RSSeSR and R'SSeSR'). Further reaction of an asymmetric selenotrisulfide with thiol (R'SH) produced another symmetric selenotrisulfide (R'SSeSR'). These thiol exchange reactions may offer significant information to elucidate intake and metabolism of selenium in vivo.

Determination of hydrogen peroxide with N,N-diethylaniline and 4-aminoantipyrine by use of an anion-exchange resin modified with manganese-tetrakis(sulphophenyl)porphine, as a substitute for peroxidase.

Amberlite IRA 900 anion-exchange resin modified with manganese-tetrakis(sulphophenyl)-porphine has been used as a catalyst instead of peroxidase for the determination of hydrogen peroxide by the reaction 2H(2)O(2) + N,N-diethylaniline + 4-aminoantipyrine (catalyst)--> quinonoid dye (lambda(max) 550 nm) + 4H(2)O. The apparent molar absorptivity for hydrogen peroxide was 1.1 x 10(4) 1.mole(-1).cm(-1), coefficient of variation 0.7%. This value is approximately 84% of that obtained by the use of peroxidase as catalyst. Similar conditions to those in the enzymatic reaction were suitable for use of the modified resin as catalyst, and the results show it to be a good substitute for peroxidase in this reaction system.

Sequential collection of selenium(IV) and selenium(VI) by the use of an anion-exchange resin loaded with bismuthiol-II sulphonic acid.

A new ligand-loaded anion-exchange resin has been developed which allows determination of Se(VI) and Se(IV) in mixtures of the two. The ligand is a sulphonic acid derivative of bismuthiol-II.