BBA - General Subjects (v.1622, #3)

The biological activity and tissue distribution of 2′,3′-dihydrophylloquinone in rats by Toshiro Sato; Rumi Ozaki; Shuichi Kamo; Yusuke Hara; Satoru Konishi; Yosuke Isobe; Sanshiroh Saitoh; Hiroshi Harada (145-150).
2′,3′-Dihydrophylloquinone (dihydro-K1) is a hydrogenated form of vitamin K1 (K1), which is produced during the hydrogenation of K1-rich plant oils. In this study, we found that dihydro-K1 counteracts the sodium warfarin-induced prolonged blood coagulation in rats. This indicates that dihydro-K1 functions as a cofactor in the posttranslational γ-carboxylation of the vitamin K-dependent coagulation factors. It was also found that dihydro-K1 as well as K1 inhibits the decreasing effects of warfarin on the serum total osteocalcin level. In rats, dihydro-K1 is well absorbed and detected in the tissues of the brain, pancreas, kidney, testis, abdominal aorta, liver and femur. K1 is converted to menaquinone-4 (MK-4) in all the above-mentioned tissues, but dihydro-K1 is not. The unique characteristic of dihydro-K1 possessing vitamin K activity and not being converted to MK-4 would be useful in revealing the as yet undetermined physiological function of the conversion of K1 to MK-4.
Keywords: Phylloquinone; 2′,3′-Dihydrophylloquinone; Menaquinone; Vitamin K; Tissue distribution;

Dietary supplementation with docosahexaenoic acid, but not with eicosapentaenoic acid, reduces host resistance to fungal infection in mice by Motoko Oarada; Tsuyoshi Tsuduki; Toshihide Suzuki; Teruo Miyazawa; Takeshi Nikawa; Guan Hong-quan; Nobuyuki Kurita (151-160).
The effect of dietary docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) on host resistance to Paracoccidioides brasiliensis infection was investigated. Mice fed palm oil supplemented with DHA showed reduced antifungal activity in the spleen and liver, as compared with mice fed palm oil or soybean oil without supplementation with DHA. Mice fed DHA-supplemented soybean oil also showed reduced antifungal activity in the liver, but the extent of reduction was less profound. This reduction in antifungal activity was not observed with EPA-supplemented palm or EPA-supplemented soybean oil. These results suggest that two factors, DHA and palm oil in combination, are involved in reducing the host resistance. DHA-enriched palm oil was also responsible for an increase in DHA concentration and a marked decrease in arachidonic acid content in the spleen and liver. However, this group did not show elevated spleen and liver phospholipid hydroperoxide levels compared with the other groups, excluding the possibility that the reduction in antifungal activity observed with DHA-enriched palm oil is due to acceleration of in vivo lipid peroxidation. Greater infection-induced increases in spleen and serum interferon-γ concentrations were observed in mice fed DHA-enriched palm oil compared with the other groups.
Keywords: Docosahexaenoic acid; Eicosapentaenoic acid; Host resistance; IFN-γ; Infection; Palm oil;

The effect of link peptide on proteoglycan synthesis in equine articular cartilage by M.F. Dean; Y.W. Lee; A.M. Dastjerdi; P. Lees (161-168).
The basal rate of in vitro proteoglycan (PG) synthesis in explants of equine articular cartilage was subject to considerable variation in animals of the same age but was greater in younger than older animals. Synthesis of PGs in explant cultures was stimulated by a synthetic link peptide, identical in sequence to the N-terminus of the link protein (LP) of PG aggregates, in a similar manner to that demonstrated previously for human articular cartilage [Biochem. Soc. Trans. 25 (1997) 427; Arthritis Rheum. 41 (1998) 157]. Stimulation occurred in tissue from animals ranging from 1 to 30 years old but older animals required higher concentrations of peptide to produce a measurable response. Synthesis of PGs increased in a concentration-dependent manner and was paralleled by increases in the ability of aggrecan monomers to form aggregates with hyaluronan (HA). In addition to its effect on synthesis of PGs, link peptide also increased synthesis of both aggrecan and LP mRNA. Cartilage explant and chondrocyte cultures secreted small amounts of biologically active interleukin 1 (IL 1) and secretion of this cytokine was reduced considerably by the addition of link peptide. Reduction in the activity of this catabolic cytokine coupled with the increased synthesis of mRNA for aggrecan and link peptide may be the mechanism by which link peptide exerts its positive effect on the rate of PG synthesis in articular cartilage.
Keywords: Link peptide; Proteoglycan; Equine; Articular cartilage;

Simulation model of doxorubicin activity in islets of human breast cancer cells by Jan Lankelma; Rafael Fernández Luque; Henk Dekker; Herbert M. Pinedo (169-178).
During cytotoxic chemotherapy, cancer cells are exposed to a dynamic concentration-versus-time curve. Besides the area under this curve, the shape of this curve may determine the cytotoxic effect. This report describes the concept that cell damage is determined by the molar drug accumulation history inside the tumor cells. Cell numbers of large populations of human MCF-7 cells exposed to three different doxorubicin concentration-versus-time profiles were recorded for 31 days. The drug accumulation history in the cells was calculated using cellular drug transport parameters derived from doxorubicin uptake and efflux measurements on MCF-7 cells attached to culture dishes. Recovery of the proliferation rate of a cell population after drug exposure was described using a mathematical model of cell damage. The model fitted well to the proliferation assays. It allowed for comparison of the effects of changes in doxorubicin concentration-versus-time profiles in vitro. The model was then used to predict the effect of the changes in the doxorubicin concentration profile in vivo, in tumor islets, after a bolus injection of doxorubicin. In the model doxorubicin exposure resulted in less cell damage inside the tumor islets than at the rim.
Keywords: Drug transport; Mathematical model; Target concentration; Pharmacodynamic; Breast cancer;

Monitoring biodistribution of glycoproteins with modified sugar chains by Shinji Takamatsu; Kazuhiro Fukuta; Mineko Asanagi; Reiko Abe; Tomoko Yokomatsu; Yasuhisa Fujibayashi; Tadashi Makino (179-191).
Natural human interferon (hIFN)-γ has mainly biantennary complex-type sugar chains. Previously, we successfully remodeled its sugar chain structure into: (a) highly branched types; or (b) highly sialylated types, by overexpression of: (a) N-acetylglucosaminyltransferase (GnT)-IV and/or GnT-V; or (b) sialyltransferases, in Chinese hamster ovary (CHO) cells. In addition, we prepared asialo hIFN-γs by treatment with sialidase in vitro. In the present study, we assessed the bioactivity of remodeled hIFN-γ in terms of antiviral activity, anticellular activity, and biodistribution. Structural changes to the sugar chains did not have a significant influence on the antiviral and anticellular activities of hIFN-γ, although the attachment of the sugar chain itself affected both activities. However, the biodistribution differed significantly; the number of exposed galactose residues was the major determinant of the specific distribution to the liver and blood clearance rate of hIFN-γ. This phenomenon was considered to be mediated by the hepatic asialoglycoprotein receptor (ASGP-R), and we showed a linear, not exponential, enhancement of the distribution to the liver with an increase in the number of exposed galactose residues. We also confirmed this tendency using fibroblast growth factor (FGF). Our observation is not the same as the “glycoside cluster effect.” We thus provide important information on the character of modified recombinant glycoproteins.
Keywords: Interferon-γ; Fibroblast growth factor; Sugar chain; Remodeling; Biodistribution;

Effect of high pressure and reversed micelles on the fluorescent proteins by Vladislav V. Verkhusha; Alexander E. Pozhitkov; Sergey A. Smirnov; Jan Willem Borst; Arie van Hoek; Natalya L. Klyachko; Andrey V. Levashov; Antonie J.W.G. Visser (192-195).
Two physico-chemical perturbations were applied to ECFP, EGFP, EYFP and DsRed fluorescent proteins: high hydrostatic pressure and encapsulation in reversed micelles. The observed fluorescence changes were described by two-state model and quantified by thermodynamic formalism. ECFP, EYFP and DsRed exhibited similar reaction volumes under pressure. The changes of the chemical potentials of the chromophore in bis(2-ethylhexyl)sulfosuccinate (AOT) micelles caused apparent chromophore protonation changes resulting in a fluorescence decrease of ECFP and EYFP. In contrast to the remarkable stability of DsRed, the highest sensitivity of EYFP fluorescence under pressure and in micelles is attributed to its chromophore structure.
Keywords: Chemical potential; Reaction volume; Chromophore protonation; Thermodynamics;