A novel protein-free synthetic medium was developed for the culture of normal human oral keratinocytes. This medium, designated PFM-7, supports the serial cultivation of primary or secondary normal oral keratinocytes in protein-free, chemically defined conditions. Normal oral keratinocytes in PFM-7 exhibited nearly equal growth in mass culture without noticeable changes in morphology, response to added growth factors, or gene expression of growth factors and their receptors, compared to cells in Keratinocyte-SFM containing epidermal growth factor and bovine pituitary extract. Furthermore, PFM-7 supported the serial subcultivation of human squamous cell carcinoma cells and enabled both normal and malignant oral squamous cells derived from the same patient to grow under the same protein-free defined conditions. These results indicate that PFM-7 can be used for precise investigations of growth mechanisms, cell products, and gene expression associated with carcinogenesis of human epidermal cells.

Animals↗

PubMedDate not supplied

Effects of isopropyl unoprostone on rabbit ciliary artery.

PURPOSE: Isopropyl unoprostone (unoprostone), a prostaglandin F(2 alpha) (PG F(2 alpha))-related compound, is widely used for treatment of glaucoma in Japan and is reported to have effects on ocular circulation. To investigate the action of this drug, we have studied the effect of unoprostone on the isolated rabbit ciliary artery. METHODS: Under microscopic observation, ciliary arteries were prepared from rabbit eyes and mounted in a myograph system. The effects of unoprostone on the isolated rabbit ciliary artery were investigated in vitro using isometric tension recordings. RESULTS: Exogenously applied PG F(2 alpha) but not unoprostone evoked contraction in the rabbit ciliary artery. After precontraction with excess-[K](o) solution, unoprostone evoked dose-dependent relaxation. The relaxation was not blocked by 10 microM/L N(G)-nitro-L-arginine methylester (L-NAME), 1 microM/L 8-37 calcitonin gene-related peptide (8-37 CGRP) or 10 microM/L indomethacin. Moreover, unoprostone could induce relaxation even in preparations without endothelium. The relaxation induced by diltiazem was greater in muscle precontracted in excess-[K](o) solution than that precontracted by 10 microM/L histamine. On the other hand, unoprostone induced a similar amplitude of relaxation in muscles precontracted by either drug. CONCLUSIONS: These results indicate that unoprostone acts directly to relax rabbit ciliary artery. The relaxation was not dependent on the endothelium and was not caused by intrinsic prostoglandins CGRP, or nitric oxide. Moreover, the relaxation was different from that caused by a Ca(2)+ antagonist. The mechanism for this relaxation is not yet determined.

Animals↗

PubMedDate not supplied

Further studies on the site-specific protein modification by microbial transglutaminase.

A guinea pig liver transglutaminase (G-TGase)-mediated procedure for the site-specific modification of chimeric proteins was recently reported. Here, an alternative method with advantages over the recent approach is described. This protocol utilizes a microbial transglutaminase (M-TGase) instead of the G-TGase as the catalyst. M-TGase, which has rather broad structural requirements as compared to the G-TGase, tends to catalyze an acyl transfer reaction between the gamma-carboxamide group of a intact protein-bound glutamine residue and various primary amines. To demonstrate the applicability of the M-TGase-catalyzed protein modification in a drug delivery system, we have utilized recombinant human interleukin 2 (rhIL-2) as the target protein and two synthetic alkylamine derivatives of poly(ethyleneglycol) (PEG12; MW 12 kDa) and galactose-terminated triantennary glycosides ((Gal)(3))) as the modifiers. For the M-TGase-catalyzed reaction with PEG12 and (Gal)(3), 1 mol of alkylamine was incorporated per mole of rhIL-2, respectively. Peptide mapping of (Gal)(3)-modified rhIL-2 ((Gal)(3)-rhIL-2) by liquid chromatography-electrospray ionization mass spectrometry (LC-ESI/MS) suggested that the Gln74 residue in rhIL-2 was site specifically modified with (Gal)(3). The PEG12-rhIL-2 and (Gal)(3)-rhIL-2 conjugates retained full bioactivity relative to the unmodified rhIL-2. In pharmacokinetic studies, PEG12-rhIL-2 was eliminated more slowly from the circulation than rhIL-2, whereas (Gal)(3)-rhIL-2 accumulated in the liver via hepatic asialoglycoprotein receptor binding. The results of this study expand the applicability of the TGase-catalyzed methodology for the preparation of protein conjugates for clinical use.

Animals↗

PubMedDate not supplied

Transglutaminase-mediated dual and site-specific incorporation of poly(ethylene glycol) derivatives into a chimeric interleukin-2.

To expand the applications of poly(ethylene glycol) (PEG)-protein conjugates for clinical use, we have developed a novel method for dual and site-specific incorporations of PEG derivatives into proteins using a substrate peptide (AQQIVM, named TG2) and transglutaminase (TGase). In our previous studies, TG2 was shown to be a special peptide with two adjacent Gln substrates for guinea pig liver transglutaminase (G-TGase). We have now constructed a chimeric protein (named rTG2-IL-2) of human interleukin-2 (IL-2), in which TG2 was fused to the N-terminus of IL-2. For the G-TGase-catalyzed reaction, rTG2-IL-2 was dually and site-specifically modified with alkylamine derivatives of PEG (PEG10, average M(r) 10 kDa) at both the Gln2 and Gln3 residues in the appended tag. To demonstrate the effectiveness of the G-TGase-catalyzed PEG-incorporation, we have compared the characteristics and the biological properties of PEG10-rTG2-IL-2 species with two PEG10 molecules attached to rTG2-IL-2 [(PEG10)(2)-rTG2-IL-2] with that of (PEG10)(2)-rhIL-2(R), in which PEG10 was randomly incorporated into rhIL-2 by a general procedure using a N-hydroxysuccinimidyl ester of PEG (PEG10-COOSu) (M(r) 10 kDa). (PEG10)(2)-rTG2-IL-2 was found to be superior in its in vitro bioactivities and equivalent in its pharmacokinetic profiles to (PEG10)(2)-rhIL-2(R). Unlike most previous methods, this approach can place dual PEG chains at designed sites on chimeric proteins without decreasing their bioactivities. Thus, TGase-catalyzed PEG-incorporation would improve the therapeutic utility of PEG-protein conjugates.

Animals↗

PubMedDate not supplied

Hypoaggregability of washed platelets from stroke-prone spontaneously hypertensive rats (SHRSP).

The aggregation properties of washed SHRSP platelets were investigated in comparison with normotensive WKY platelets at prehypertensive (4 weeks), early hypertensive (11 weeks) and late hypertensive (17 weeks) ages in the absence of plasma factors. The number of platelets in SHRSP was markedly lower with the development of hypertension than that in WKY. The thrombin- and collagen-induced aggregation was markedly reduced in the platelets from 11 and 17 week old SHRSP compared with that of age-matched WKY, whereas the degree of platelet aggregation in 4 week old SHRSP showed a tendency to be even greater than that in WKY. The changes in blood pressure and platelet aggregability were correlated inversely. ADP did not induce aggregation in the same system used for thrombin and collagen stimulation but in another system it aggregated washed rat platelets. Aggregation responses to ADP and ionophore A23187 were also significantly lower in 14 week old SHRSP platelets than age-matched WKY platelets. Together with other evidence, these results suggest that defective Ca2+ function, rather than the presence of exhausted platelets, is responsible for hypoaggregability in SHRSP platelets.

Adenosine Diphosphate↗

PubMedDate not supplied

Initial daily interferon administration can gain more eradication of HCV-RNA in patients with chronic hepatitis C, especially with serum intermediate viral load.

BACKGROUND/AIMS: We studied the effect of initial daily administration of interferon for the treatment of chronic hepatitis C, especially in patients with intermediate viral load. METHODOLOGY: Consecutive patients who met the inclusion criteria were randomly enrolled into two groups in this study. All patients analyzed could be treated with interferon-alpha for 6 months. Patients in group A were administered 6 million units of interferon-alpha subcutaneously daily initially for 2 weeks and then thrice weekly. Patients in group B were treated with the same dose of interferon-alpha thrice weekly from the first administration. We decided the criteria of complete remission as the absence of serum HCV-RNA at both points of the end of interferon treatment and 6 months later. RESULTS: Due to the relationship between the efficacy and serum viral load, we decided the criteria of the intermediate load as the quantitative value of serum HCV-RNA to be not lower than 10(5.0) and not higher than 10(6.5) copies/ml. Seventy-six and 78 patients, whose genotype and quantitative value of serum HCV-RNA could be measured before treatment, were analyzed in group A and B, respectively. The rate of complete remission in group A (40.8%) was higher than that in group B (25.6%), significantly (p = 0.046). In the intermediate viral load group, the rate of complete remission in group A (52.3%) was significantly higher than that in group B (29.3%) (p = 0.045). In the patients with genotype 1 b virus, the rate of complete remission had a tendency to be higher in group A (33.3%) than in group B (17.4%) (not significant). In the patients with genotype 2, the rate of complete remission was higher in group A (77.8%) than in group B (41.2%) (significant, p = 0.041). CONCLUSIONS: These results suggest that the initial daily interferon administration is necessary to gain a higher rate of serum HCV-RNA eradication in patients with intermediate viral load in chronic hepatitis C.

Adult↗

PubMedDate not supplied

The importance of initial daily administration of interferon alpha for the eradication of hepatitis C virus in patients with chronic hepatitis C: a multicenter randomized trial.

BACKGROUND/AIMS: We studied the effect of initial daily administration of interferon for the treatment of chronic hepatitis C, to clarify a more effective treatment protocol for the eradication of the hepatitis C virus. METHODOLOGY: Consecutive patients who met the inclusion criteria were randomly enrolled in two groups in this study. One hundred and five patients were randomized and assigned to two groups. Patients, who enrolled in group A, were treated with 6 million units of natural interferon-alpha given subcutaneously daily for an initial two weeks and then thrice a week for 22 weeks. Patients, who were enrolled in group B, were treated with the same dose of interferon-alpha given for 26 weeks thrice a week from the first administration. RESULTS: In groups A and B, 58 and 47 patients were analyzed, respectively. At the end of treatment, 37 patients in group A (63.8%) had negative serum HCV-RNA test, compared with 26 in group B (55.3%), but at 6 months after discontinuation of interferon administration, 27 patients in group A (46.6%), compared with 8 in group B (21.3%). The rate of complete remission in group A (46.6%) was higher than that in group B (21.3%) (P<0.01). In patients with genotype 1b virus, the rate of complete remission was higher in group A (31.3%) than in group B (12.5%) (not significantly), and the relapse rate was lower in group A (9.4%) than in group B (37.5%), significantly (p<0.05). CONCLUSIONS: This study suggests that initial daily interferon administration is necessary to gain a higher rate of serum HCV-RNA eradication in patients with chronic hepatitis C.

Alanine Transaminase↗