BBA - General Subjects (v.1525, #1-2)

Novel functions of complex carbohydrates elucidated by the mutant mice of glycosyltransferase genes by Koichi Furukawa; Kogo Takamiya; Masahiko Okada; Masahiro Inoue; Satoshi Fukumoto; Keiko Furukawa (1-12).
Complex carbohydrates consist of carbohydrate moieties and protein or lipid portions, resulting in the formation of glycoproteins, proteoglycans or glycosphingolipids. The polymorphic carbohydrate structures are believed to contain profound biological implications which are important in cell–cell or cell–extracellular matrix interactions. A number of studies to delineate the roles of carbohydrates have been performed, and demonstrated definite changes in their profiles, cellular phenotypic changes or, sometimes, morphological and functional changes in tissues after modification of their structures. Recent successes in the isolation of glycosyltransferase genes and their modification enzyme genes has enabled clearer demonstrations of the roles of complex carbohydrates. In particular, genetic modification of glycosyltransferase genes in mice can elucidate the biological significances of their products in vivo. Here, we summarize recent advances in the understanding of the roles of complex carbohydrates provided from studies of gene knock-out mice of glycosyltransferase and modification enzyme genes focusing on novel functions which had not been expected.
Keywords: Carbohydrate; Complex carbohydrate; Gene knock-out; Glycosyltransferase; Sulfotransferase; Glycoconjugate;

Occurrence of an unusual phosphorylated N-acetyllactosamine in horse colostrum by Tadashi Nakamura; Satoko Amikawa; Teruyoshi Harada; Tadao Saito; Ikichi Arai; Tadasu Urashima (13-18).
The colostrum of horses (thoroughbreds) was extracted and fractionated to yield Gal(β1-4)GlcNAcα1-phosphate, which has not previously been detected in any mammalian milk or colostrum, as well as Neu5Ac(α2-3)Gal(β1-4)Glc. The structures of these saccharides were established by NMR spectroscopy and MALDI-TOF mass spectrometry.
Keywords: Phosphorylated N-acetyllactosamine; α2-3N-Acetylneuraminyllactose; Horse colostrum; Oligosaccharide structure; NMR datum;

Aspergillus niger produces an extracellular β-galactofuranosidase, which can specifically hydrolyse β-D-galactofuranose (Galf) from glycoconjugates. The production of this enzyme can be induced by the addition of a Galf-containing A. niger mycelial wall extract. However, on other carbon sources accumulation occurred only during the starvation conditions of the late stationary phase. Extracellular glucoamylases from this stage of cultivation possessed significantly lower levels of Galf than those from the earlier exponential growth phase when β-galactofuranosidase is absent, suggesting in situ β-galactofuranosidic hydrolysis. The β-galactofuranosidase responsible was subsequently purified to homogeneity and characterised. It is a glycoprotein of 90 kDa (determined by SDS–PAGE) with activity against β-linked Galf residues, with a K m of 4 mM against p-nitrophenyl-β-D-galactofuranoside and a pH optimum of 3–4. The preparation did not contain other contaminating glycosidase activities; p-nitrophenyl-β-D- and -α-D-galactopyranose, and α-D-methyl-Galf were not hydrolysed. Results are presented to show that this enzyme could be employed as a useful tool for the analysis of glycoconjugates containing biologically important Galf components.
Keywords: Galactofuranose; Glycoconjugate analysis; (Aspergillus niger);

An investigation of the action of porcine pancreatic α-amylase on native and gelatinised starches by Suzanne L Slaughter; Peter R Ellis; Peter J Butterworth (29-36).
The action of pancreatic α-amylase (EC 3.2.1.1) on various starches has been studied in order to achieve better understanding of how starch structural properties influence enzyme kinetic parameters. Such studies are important in seeking explanations for the wide differences reported in postprandial glycaemic and insulinaemic indices associated with different starchy foodstuffs. Using starches from a number of different sources, in both native and gelatinised forms, as substrates for porcine α-amylase, we showed by enzyme kinetic studies that adsorption of amylase to starch is of kinetic importance in the reaction mechanism, so that the relationship between reaction velocity and enzyme concentration [E0] is logarithmic and described by the Freundlich equation. Estimations of catalytic efficiencies were derived from measurements of k cat/K m performed with constant enzyme concentration so that comparisons between different starches were not complicated by the logarithmic relationship between E0 and reaction velocity. Such studies reveal that native starches from normal and waxy rice are slightly better substrates than those from wheat and potato. After gelatinisation at 100°C, k cat/K m values increased by 13-fold (waxy rice) to 239-fold (potato). Phosphate present in potato starch may aid the swelling process during heating of suspensions; this seems to produce a very favourable substrate for the enzyme. Investigation of pre-heat treatment effects on wheat starch shows that the relationship between treatment and k cat/K m is not a simple one. The value of k cat/ K m rises to reach a maximum at a pre-treatment temperature of 75°C and then falls sharply if the treatment is conducted at higher temperatures. It is known that amylose is leached from starch granules during heating and dissolves. On cooling, the dissolved starch is likely to retrograde and become resistant to amylolysis. Thus the catalytic efficiency tends to fall. In addition, we find that the catalytic efficiency on the different starches varies inversely with their solubility and we interpret this finding on the assumption that the greater the solubility, the greater is the likelihood of retrogradation. We conclude that although α-amylase is present in high activity in digestive fluid, the enzymic hydrolysis of starch may be a limiting factor in carbohydrate digestion because of factors related to the physico-chemical properties of starchy foods.
Keywords: α-Amylase; Starch; Catalytic efficiency; Retrogradation;

Gossypol isomers bind specifically to blood plasma proteins and spermatozoa of rainbow trout fed diets containing cottonseed meal by Konrad Dabrowski; Kyeong-Jun Lee; Jacques Rinchard; Andrzej Ciereszko; Joost H Blom; Joseph S Ottobre (37-42).
We investigated the role of gossypol isomers binding to blood plasma, seminal plasma and spermatozoa to elucidate gossypol anti-fertility action in the teleost fish, rainbow trout (Oncorhynchus mykiss). Growth and hematological indicators of males were depressed when fish meal protein in diets was completely replaced with cottonseed meal. The cottonseed meal contained equal proportions of (−) (47.8±1.6%) and (+) gossypol isomers. Concentrations of spermatozoa were decreased with increasing proportions of gossypol in diets (from 0.22% to 0.95%); however, sperm motility and fertilizing ability were not affected. In contrast to mammals, steroid hormone concentrations were not suppressed in fish given diets with gradual increase of gossypol level. Gossypol concentrations were 100-fold higher in blood plasma than in seminal plasma, confirming a barrier in gossypol transfer between the general circulation and the testis. Spermatozoa accumulated predominantly (+) enantiomer (65–75%) with decreasing proportions as dietary gossypol concentrations increased. Spermatozoa bound most of the gossypol contained in the semen; however, this did not result in impairment of the sperm motility apparatus. Teleost fish sperm rely on ATP stores that accumulate during maturation as a source of energy during activation. In addition, the duration of sperm movement is short in these fish. As such, we hypothesize that the major action of gossypol on mammalian sperm, which is uncoupling of oxidative phosphorylation, does not impair the energy supply required for flagellar beating in fish spermatozoa.
Keywords: Cottonseed; Gossypol; Sperm; Anti-fertility; Steroid; Lactate dehydrogenase; (Fish);

Interaction of enkephalin derivatives with reactive oxygen species by Raffaella Coccia; Cesira Foppoli; Carla Blarzino; Carlo De Marco; Maria Anna Rosei (43-49).
The oxidation of opioid peptides by tyrosinase in the presence of an excess of a thiol gives rise to cysteinyldopa derivatives. The major products arising from the reaction between Leu-enkephalin and cysteine are represented by 5-S-cysteinyldopaenkephalin (5-CDenk) and 2-S-cysteinyldopaenkephalin (2-CDenk). The interaction of 5-CDenk and 2-CDenk with reactive oxygen species (ROS) has been studied. These compounds are able to scavenge superoxide anion, hydroxyl and peroxyl radicals as well as to reduce the lipid peroxidation rate induced by ABAP. The scavenging activities in all instances are dose-dependent. In some cases CDenks are more active than compounds recognized as strong radical scavengers, such as Trolox and mannitol. As a result of the action of the Fenton system, the CDenks (as well as the Enks) are oxidized into pigmented derivatives. The possible implications of the interaction of CDenks and Enks with ROS on melanization process in Parkinson’s disease are discussed.
Keywords: Enkephalin; Reactive oxygen species; Cysteinyldopa; Pheomelanin; Scavenger; Cysteinyldopaenkephalin;

Binding specificities of ABO blood group-recognizing lectins toward blood group antigens on neoglycoproteins, glycoproteins and complex-type oligosaccharides were studied by lectin-blotting analysis, enzyme linked immunosorbent assay and lectin-conjugated agarose column chromatography. Human serum albumin conjugated with A- and B-trisaccharides was clearly recognized by Helix pomatia (HPA), Phaseolus lunatus, Dolichos biflorus agglutinins, and Griffonia simplicifolia I agglutinin B4, respectively. Almost the same results were obtained for human group A and B ovarian cyst and A-active hog gastric mucins, but Glycine max agglutinin only reacted to the group A hog mucin. When human plasma von Willebrand factor (vWF), having Asn-linked blood group antigens, was tested, HPA was highly sensitive to blood group A antigen on the vWF. Ulex europaeus agglutinin I (UEA-I) preferentially bound to the vWF from blood group O plasma. Within the GalNAc-recognizing lectins examined, a biantennary complex-type oligosaccharide having the blood group A structure retarded on an HPA–agarose column, and the affinity was diminished after digestion with α-N-acetylgalactosaminidase. This product bound to UEA-I agarose column. These results indicate that HPA and UEA-I are most sensitive for detection of glycoproteins possessing small amounts of blood group A and H antigens and also useful for fractionation of complex-type oligosaccharides with blood group A and H antigens, respectively.
Keywords: ABO blood group; Lectin; Complex-type oligosaccharide; Von Willebrand factor; Helix pomatia agglutinin; Ulex europaeus agglutinin I;

Characterization of blood group ABO(H)-active gangliosides in type AB erythrocytes and structural analysis of type A-active ganglioside variants in type A human erythrocytes by Yasunori Kushi; Motomasa Shimizu; Kiyohiro Watanabe; Takeshi Kasama; Shinobu Watarai; Toshio Ariga; Shizuo Handa (58-69).
Several monosialogangliosides containing the type A-active epitope have been detected in type A erythrocytes on immunological analysis with a monoclonal antibody, and three of them were purified by repeated silica bead column chromatography and by scraping from the TLC plate. Two of these A-active gangliosides were characterized by methylation analysis by GC/MS, negative SIMS, MALDI-TOF/MS, proton nuclear magnetic resonance spectroscopy, and immunological assays, and their structures were concluded to be as follows. A-active ganglioside I:A-active ganglioside II:The reactivity of the purified gangliosides to the anti-A monoclonal antibodies (mAbs) exhibited enhancement after removal of the sialic acid. Therefore, the sialic residue has been shown to inhibit the binding to the terminal A-active epitope through the formation of an immune complex. To confirm the presence of A- (including S-A-I, -II and -III) and B-active gangliosides, the reactivity of anti-A and -B mAbs were investigated using total gangliosides from type A, -B and -AB erythrocytes on TLC plate. The results were that the gangliosides from types A and AB showed positive reaction to anti-A mAbs, whereas in the anti-B mAbs binding the gangliosides from types B and AB were positive. Thus, it revealed that A-active gangliosides were present in type A and -AB, and B-active gangliosides in types B and AB. As there was no difference in respective gangliosides on type AB erythrocytes of 22 individuals, both A- and B-active gangliosides are equally present in type AB erythrocytes. The biological significance of these A- and B-active ganglioside variants remains vague at present. As these molecules exhibit different reactivities to the anti-A mAbs, it is very likely that they can regulate the antigenicity of the A-epitope on the cell surface.
Keywords: ABO(H)-active glycosphingolipid; Blood type antigen; Human erythrocyte membrane; A-active ganglioside; Cold agglutinin; Ley structure;

Cellular sites of H2O2-induced damage and their protection by nitroxides by A.M Samuni; W DeGraff; M.C Krishna; J.B Mitchell (70-76).
While the exact mechanism of H2O2-induced cytotoxicity is unknown, there is considerable evidence implicating DNA as a primary target. A recent study showed that a cell-impermeable nitroxide protected mammalian cells from H2O2-induced cell killing and suggested that the protection was mediated through cell membrane-bound or extracellular factors. To further define the protective properties of nitroxides, Chinese hamster V79 cells were exposed to H2O2 with or without cell-permeable and impermeable nitroxides and selected metal chelators. EPR spectroscopy and paramagnetic line broadening agents were used to distinguish between intra- and extracellular nitroxide distribution. To study the effectiveness of nitroxide protection, in the absence of a cell membrane, H2O2-mediated damage to supercoiled plasmid DNA was evaluated. Both deferrioxamine and Tempol cross the cell membrane, and inhibited H2O2-mediated cell killing, whereas the cell-impermeable DTPA and nitroxide, CAT-1, failed to protect. Similar protective effects of the chelators and nitroxides were observed when l-histidine, which enhances intracellular injury, was added to H2O2. In contrast, when damage to plasmid DNA was induced (in the absence of a cell membrane), both nitroxides were protective. Collectively, these results do not support a role for membrane-bound or extracellular factors in mediating H2O2 cytotoxicity in mammalian cells.
Keywords: Cytotoxicity; DNA; Double strand break; Histidine;

Cinnamophilin as a novel antiperoxidative cytoprotectant and free radical scavenger by George Hsiao; Che-Ming Teng; Joen-Rong Sheu; Yu-Wen Cheng; Kwok-Keung Lam; Yen-Mei Lee; Tian-Shung Wu; Mao-Hsiung Yen (77-88).
The antioxidant properties of cinnamophilin were evaluated by studying its ability to react with relevant reactive oxygen species, and its protective effect on cultured cells and biomacromolecules under oxidative stress. Cinnamophilin concentration-dependently suppressed non-enzymatic iron-induced lipid peroxidation in rat brain homogenates with an IC50 value of 8.0±0.7 μM and iron ion/ADP/ascorbate-initiated rat liver mitochondrial lipid peroxidation with an IC50 value of 17.7±0.2 μM. It also exerted an inhibitory activity on NADPH-dependent microsomal lipid peroxidation with an IC50 value of 3.4±0.1 μM without affecting microsomal electron transport of NADPH-cytochrome P-450 reductase. Both 1,1-diphenyl-2-picrylhydrazyl and 2,2′-azo-bis(2-amidinopropane) dihydrochloride-derived peroxyl radical tests demonstrated that cinnamophilin possessed marked free radical scavenging capacity. Cinnamophilin significantly protected cultured rat aortic smooth muscle cells (A7r5) against alloxan/iron ion/H2O2-induced damage resulting in cytoplasmic membranous disturbance and mitochondrial potential decay. By the way, cinnamophilin inhibited copper-catalyzed oxidation of human low-density lipoprotein, as measured by fluorescence intensity and thiobarbituric acid-reactive substance formation in a concentration-dependent manner. On the other hand, it was reactive toward superoxide anions generated by the xanthine/xanthine oxidase system and the aortic segment from aged spontaneously hypertensive rat. Furthermore, cinnamophilin exerted a divergent effect on the respiratory burst of human neutrophil by different stimulators. Our results show that cinnamophilin acts as a novel antioxidant and cytoprotectant against oxidative damage.
Keywords: Antioxidant; Lipid peroxidation; Peroxyl radical; Cytoprotectant; Superoxide anion; Cinnamophilin;

The technique of pulse radiolysis with spectrophotometric detection has been used to investigate the possibility of electron transfer reactions between oxidizing sulfur–sulfur three-electron-bond complexes (Met2/S∴S+), or reducing α-amino radicals (CH3SCH2CH2CHNH2) derived from reaction of methionine with OH radicals and hydroxycinnamic acid (HCA) derivatives, riboflavin (RF) or flavin adenine dinucleotide (FAD), respectively. The HCA derivatives, such as caffeic acid, ferulic acid, sinapic acid and chlorogenic acid, widely distributed phenolic acids in fruit and vegetables, have been identified as good antioxidants previously can rapidly and efficiently repair oxidizing three-electron-bond complexes via electron transfer. RF and FAD can oxidize reducing α-amino radicals derived from methionine. The electron transfer rate constants ∼109 dm3 mol−1 s−1 were determined by following the build-up kinetics of species produced.
Keywords: Methionine; Hydroxycinnamic acid derivative; Riboflavin; Flavin adenine dinucleotide; Electron transfer; Pulse radiolysis;

Many solutions have been used to investigate the swelling properties of the mammalian corneal stroma but few of the solutions resemble the expected extracellular matrix fluid of the corneal stroma, and little information is available on whether incubation ex vivo causes significant changes in the gross composition of the stroma. From quality-selected recent post-mortem eyes of adult cattle, stroma preparations were cut from the central part of the cornea. The time-dependent changes in wet mass were assessed over 9 h at 37°C, and the preparations then dried. Various solutions of known pH (6.88–8.32) and osmolality (<50–327 mosmol/kg) were used, and were assayed for protein and proteoglycan after the incubation. The rates and extent of stromal swelling were lowest in a glucose-supplemented mixed salts solution containing 35 mM bicarbonate (0.5% CO2) solution, marginally greater in a mixed salts solution containing 35 mM bicarbonate (5% CO2) or similar non-bicarbonate mixed salts solutions (including BSS), and progressively greater in phosphate-buffered saline (PBS), various phosphate buffers (10–67 mM) and saline solutions (0.025–1%), and greatest in water. The initial rates of swelling ranged from 44 to 451 mg/h and the secondary rates from 9 to 106 mg/h. In all solutions, protein and proteoglycans were detected, but these ranged from around 1 to 10% of the samples with the bicarbonate-buffered solutions, to around 30% with the use of some phosphate buffers or saline.
Keywords: Corneal edema; Corneal stroma; Hydration; Extracellular matrix; pH; Proteoglycan;

The purpose of this study was to verify the concept of non-equilibrium facilitated oxygen diffusion. This work succeeds our previous study, where facilitated oxygen diffusion by hemoglobin was measured at conditions of chemical equilibrium, and which yielded diffusion coefficients of hemoglobin and of oxygen. In the present work chemical non-equilibrium was induced using very thin diffusion layers. As a result, facilitation was decreased as predicted by theory. Thus, this work presents the first experimental demonstration of non-equilibrium facilitated oxygen diffusion. In addition, association and dissociation rate parameters of the reaction between oxygen and bovine and human hemoglobin were calculated and the effect of the homotropic and heterotropic interactions on each rate parameter was demonstrated. The results indicate that the homotropic interaction – which leads to increasing oxygen affinity with increasing oxygenation – is predominantly due to an increase in the association rate. The heterotropic interaction – which leads to decreasing oxygen affinity by anionic ligands – appears to be effected in two ways. Cl increases the dissociation rate. In contrast, 2,3-diphosphoglycerate decreases the association rate.
Keywords: Hemoglobin; Oxygen; Facilitated diffusion; Non-equilibrium; Reaction rate;

It is still unknown how insulin-like growth factor-I (IGF-I) regulates cancer cell growth in the condition of the limited availability of key nutrients, such as glutamine. We investigated the effects of IGF-I on cell growth and amino acid transport in a glutamine-deprived human neuroblastoma cell line, SK-N-SH. Cell growth was measured, and 3H-labeled amino acid transport was assayed after treatment with or without IGF-I (50 ng/ml) in 2 mM (control) and 100 μM glutamine concentrations. Cell growth rates were dependent on glutamine concentrations. IGF-I stimulated cell growth in both 2 mM and 100 μM glutamine. IGF-I stimulated glutamine transport in 100 μM glutamine with the mechanism of increasing carrier V max, but had no effect in 2 mM glutamine. IGF-I also stimulated leucine, glutamate and 2-(methylamino)isobutyric acid transport in 100 μM glutamine. There were significant increases in [3H]thymidine and [3H]leucine incorporation in IGF-I-treated cells in both 2 mM and 100 μM glutamine. These data suggest that IGF-I stimulates cell growth by increasing amino acid transport in the condition of low glutamine levels in a human neuroblastoma cell line. This mechanism may allow to maintain cell growth even in nutrient-deprived tumor tissues.
Keywords: Insulin-like growth factor-I; Amino acid transport; Glutamine; Neuroblastoma;

Effect of bile acids on the uptake of irinotecan and its active metabolite, SN-38, by intestinal cells by Kunihiko Kobayashi; Susan Ceryak; Yasushi Matsuzaki; Shoji Kudoh; Bernard Bouscarel (125-129).
The intestinal transport of irinotecan (CPT-11) and its active metabolite, SN-38, has been previously reported (K. Kobayashi et al., Int. J. Cancer, 83 (1999) 491–496). In the present study, the effect of the two major primary bile acids, cholic acid (CA) and taurocholic acid (TCA), on the uptake of CPT-11 and SN-38 by hamster intestinal epithelial cells was investigated. These two bile acids at concentrations up to 200 μM did not directly alter the cellular uptake of CPT-11 and SN-38. However, under physiologically acidic intestinal pH conditions, micelle formation induced by 20 mM TCA significantly reduced the cellular uptake of CPT-11 and SN-38 by 60% and 80%, respectively.
Keywords: Irinotecan; 7-Ethyl-10-hydroxycamptothecin; Intestinal absorption; Bile acid; Taurocholic acid; Micelle;

Skeletal muscle myosin displays two independent and equivalent binding sites for 1,N 6 ethenoadenosine diphosphate, with a dissociation constant of 24.7 μM. MgADP, 10 to 40 μM, behaves as a pure competitive type inhibitor (K SI=8-9 μM) for the binding of 1,N 6 ethenoadenosine diphosphate to skeletal muscle myosin. On the contrary, the inhibition by MgADP, 0.11–1.54 mM, is neither competitive nor non-competitive nor mixed, as is revealed by the analysis with the general kinetic equation (K.J. Laidler, P.S. Bunting, The Chemical Kinetics of Enzyme Action, 2nd ed., Clarendon, Oxford, 1973, p. 94). To explain our finding we propose that MgADP operates a complex type of inhibition, acting both directly as a competitor for myosin active sites, and indirectly by perturbing the regions of the solvent near to the protein.
Keywords: Myosin; Adenosine triphosphate; 1,N 6 Ethenoadenosine diphosphate; Anomalous binding; Solvent effect;

Oxidative changes in brain pyridine nucleotides and neuroprotection using nicotinamide by Lori K. Klaidman; Suman K. Mukherjee; James D. Adams (136-148).
Pyridine nucleotides are critical during oxidative stress due to their roles in reductive reactions and energetics. The aim of the present study was to examine pyridine nucleotide changes in six brain regions of mice after an intracerebroventricular injection of the oxidative stress inducing agent, t-butyl hydroperoxide (t-BuOOH). A secondary aim was to investigate the correlation between NAD+ levels and DNA fragmentation. Here, we demonstrate that t-BuOOH induced a rapid oxidation of NADPH and a slow depletion of NAD+ in most brain regions. A slight increase in NADH also occurred in five brain regions. NAD+ depletion was associated with increased DNA fragmentation. This suggests the initiation of a death cascade involving poly(ADP-ribose) polymerase (PARP), NAD+, ATP depletion and consequent cell death in brain tissue. PARP activity was accelerated in some brain regions after 20 min of oxidative stress. To counteract oxidative stress induced toxicity, NAD+ levels were increased in the brain using an intraperitoneal injection of nicotinamide. A surplus of brain NAD+ prevented DNA fragmentation in some brain regions. Nicotinamide administration also resulted in higher brain NADH, NADP+ and NADPH levels in some regions. Their synthesis was further upregulated during oxidative stress. Nicotinamide as a precursor for NAD+ may provide a useful therapeutic strategy in the treatment of neurodegeneration.
Keywords: Nicotinamide; Oxidative stress; NAD; NADPH; DNA fragmentation; PARP;

Prostate-specific antigen (PSA) is a glycosylated chymotrypsin-like serine protease and is found mainly in prostatic tissue and seminal fluid. We purified two forms of PSA (PSA-A and PSA-B) from human seminal fluid with pI values of approx. 7.2 and approx. 6.9, respectively. To characterize the N-glycans of the two isoforms, the sugar chains were liberated by hydrazinolysis followed by N-acetylation, and derivatized with 2-aminobenzamide. Both PSA-A and PSA-B contained mono- and disialylated sugar chains, although PSA-B had a much higher content of the latter. After removal of sialic acid residues by sialidase digestion, mono- and biantennary N-glycans and three outer chain moieties (Galβ1-4GlcNAcβ1-, GlcNAcβ1-, GalNAcβ1-4GlcNAcβ1-) were found in both samples. However, the ratios of each N-glycan were different. These results indicate that PSA-A and PSA-B differ not only in their sialic acid contents, but also in their outer chain features.
Keywords: Seminal fluid; Carbohydrate structure; Prostate-specific antigen; Prostate cancer; Exoglycosidase sequencing;

Raman vibrational spectroscopy, at 298 K, has been used to study the hydration of betaine hydrochloride and betaine in the concentration range 0.5–2 M. The observed changes in the internal vibrations of the solutes, namely, in the CO, COO and C–H stretchings, and in the components of the O–H stretching band are consonant with anionic water–betaine and betaine hydrochloride dimeric species involving simultaneously hydrogen-bonding between two solute and water molecules. In both cases, betaine hydrochloride and ‘zwitterionic’ betaine behave like structure-makers promoting a larger association in the ‘bulk’ liquid water.
Keywords: Betaine; Betaine hydrochloride; Raman spectroscopy;

Pathophysiological significance of in vivo ESR signal decay in brain damage caused by X-irradiation by Yuri Miura; Kazunori Anzai; Jun-Ichi Ueda; Toshihiko Ozawa (167-172).
X-irradiation of mice decreased the decay rate of the in vivo ESR signal in the head region to 75% of the control when 3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine-1-yloxy (MCPROXYL), a lipophilic and blood–brain barrier-permeable spin probe, was used. We attempted to identify the specific factor responsible for the decrease in the signal decay rate caused by X-irradiation. The signal decay of MCPROXYL in the head region depends on the following three factors: (1) blood concentration of MCPROXYL, (2) reduction to the corresponding hydroxylamine in the brain tissue, and (3) effusion of MCPROXYL from the brain tissue. Irradiation at 15 Gy did not significantly change the rate of decrease of blood concentration of MCPROXYL at 1 h post-irradiation. The reducing activity of the brain homogenate was not changed by the X-irradiation (15 Gy). The contents of MCPROXYL and its hydroxylamine derivative in the brain of 15 Gy-irradiated mice remained higher than in non-irradiated mice. These findings suggest that the effect of X-irradiation observed by in vivo ESR is attributable not to the redox reaction of MCPROXYL in the brain but to the change of the efflux rate of the MCPROXYL from the brain.
Keywords: In vivo electron spin resonance; Nitroxyl radical; Radiation; Brain; 3-Methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine-1-yloxy;

To determine the reductive process of extracellular dehydroascorbic acid (DHA), molecules (homocysteine, homocysteine thiolactone, methionine, cysteine, and homoserine) were tested to identify those with the potential to reduce DHA to ascorbic acid (AA). Homocysteine (Hcy) was the most potent of the molecules tested. The efficacy of Hcy was compared with that of other molecules able to reduce DHA (reduced glutathione (GSH) and cysteine (Cy)). Although all three molecules were able to reduce DHA, GSH and Cy were not to reduce DHA to AA at concentrations lower than 100 μmol/l, and only less than 5% DHA was reduced to AA at concentrations of 200–300 μmol/l. In contrast, Hcy reduced DHA to AA stoichiometrically at concentrations as low as 10 μmol/l. In Jurkat and U937 cells, the increasing concentrations of extracellular Hcy suppressed intracellular dehydroascorbic acid uptake, indicating that extracellular reduction of DHA by Hcy leads to decreasing extracellular DHA available for its intracellular uptake. Simultaneous oxidation and reduction of Hcy and DHA were accelerated extracellularly in the presence of quercetin, an inhibitor of DHA uptake, suggesting that extracellular ascorbic acid concentration increased via blocking DHA uptake by quercetin and reducing extracellular DHA by Hcy. The effect of homocysteine on DHA reduction and uptake was confirmed with human umbilical vein endothelial cells. The oxidation of Hcy also prevented the decrease in DNA synthesis in human umbilical vein endothelial cells, which would occur following exposure to Hcy.
Keywords: Dehydroascorbic acid; Ascorbic acid (vitamin C); Homocysteine; Jurkat and U937 cells; Human umbilical vein endothelial cells; Flavonoids;

Development of proteo-chemometrics: a novel technology for the analysis of drug-receptor interactions by Maris Lapinsh; Peteris Prusis; Alexandrs Gutcaits; Torbjörn Lundstedt; Jarl E.S. Wikberg (180-190).
A novel method for the analysis of drug receptor interactions has been developed and used to explore mechanisms involved in the binding of 4-piperidyl oxazole antagonists to α1a-, α1b- and α1d-adrenoceptors. The method exploits affinity data for a series of organic chemical compounds binding to wild-type and artificially mutated receptors. The receptor sequences and compounds are assigned predictor variables that are correlated to the measured pharmacological activities using partial least-squares projections to latent structures. The predictor variables consist of one descriptor block derived from the chemical properties of the receptors’ primary amino acid sequences and another descriptor block derived from the chemical properties of the organic compounds. The cross-terms generated from the two descriptor blocks are also derived. Using this approach, very sturdy models were generated describing the interactions of the chemical compounds with the receptors. Models are useful to predict binding affinity and receptor subtype selectivity of compounds prior to their synthesis, and may find use in rational drug design. Moreover, models also give quantitative information about the interactions of the amino acids of the receptors with the ligands, thereby giving an insight into the molecular mechanisms involved in ligand binding.
Keywords: Proteo-chemometrics; Modeling; Partial least squares; Partial least-squares projection to latent structure; Chimeric receptor; Site directed mutagenesis; Ligand binding;

Construction of tumor-specific cells expressing a membrane-anchored single-chain Fv of anti-ErbB-2 antibody by Masataka Suzuki; Masashige Shinkai; Hiroyuki Honda; Masamichi Kamihira; Shinji Iijima; Takeshi Kobayashi (191-196).
Cells expressing a membrane-anchored single-chain fragment variable (scFv) domain against a tumor-specific antibody were fabricated. These cells were able to bind to cells of a human colon cancer line (BM314) expressing the erbB-2 proto-oncogene. A plasmid, pMFverbB, was first constructed in which the anti-ErbB-2 scFv gene was cloned in-frame between a signal peptide sequence and the platelet-derived growth factor receptor (PDGFR) transmembrane domain gene to express scFv on the cell surface. An African green monkey cell line, COS-1, was stably transfected with pMFverbB. Immunofluorescence assay experiments and microscopic observation showed that the cells expressing scFv bound to the human tumor cells overexpressing the ErbB-2 protein as well as to cells of a mouse fibroblast line (NIH-3T3) transfected with the erbB-2 gene. The cells expressing scFv could take up magnetite cationic liposomes as a model of particle-type drug and retained the ability to target ErbB-2-expressing cells. The fabricated cells have the potential to serve as drug carriers in drug targeting applications.
Keywords: Drug carrier; Targeting; Magnetite; Single chain fragment antibody;