BBA - General Subjects (v.1526, #2)

MgATP positively regulates the dimerisation reaction, resulting in an increase in the rate of MgATP splitting with increasing MgATP concentration. We investigated the stoichiometry of this dimerisation reaction and found that each subunit in the dimer bound one molecule of MgATP at the dimerisation site. We studied changes with temperature in the MgATPase activity of S1 in the dimeric form for temperatures of 18–25°C. Between 18.0 and 21.2°C, k cat increased steadily with temperature. Between 21.2 and 21.8°C, there was a large decrease in k cat. A strong increase in k cat occurred at temperatures above 21.8°C, corresponding to a new reversible conformation of S1, unable to dimerise. The steep decrease in k cat between 21.2 and 21.8°C is due to a temperature transition in the monomer–dimer equilibrium.
Keywords: Myosin S1; Monomer–dimer; Kinetics; Temperature transition;

The simulated microgravity environment maintains key metabolic functions and promotes aggregation of primary porcine hepatocytes by Konstantinos J Dabos; Leonard J Nelson; Timothy J Bradnock; John A Parkinson; Ian H Sadler; Peter C Hayes; John N Plevris (119-130).
The high aspect ratio vessel allows the culture of primary porcine hepatocytes in an environment of low shear stress and simulated microgravity. Primary porcine hepatocytes have been difficult to maintain in culture long term while preserving their metabolic functions. This study was carried out in order to characterise key metabolic functions of cell aggregates formed by primary porcine hepatocytes cultured in a high aspect ratio vessel for a predetermined period of 21 days. 108 porcine hepatocytes were loaded into the high aspect ratio vessel and continuously rotated during the experiments. 0.7 ml of the culture medium was sampled on days 1, 2, 4, 7, 10, 14 and 21. 1H nuclear magnetic resonance spectroscopy of the culture medium, using the presaturation technique, assessed the following: glucose metabolism, glutamine synthesis and ketogenesis. There was glucose breakdown anaerobically during the first 10 days as manifested by lactate production and pyruvate and threonine consumption. After day 10 there was significantly smaller lactate production (day 1 vs day 10 P<0.01), and significantly smaller pyruvate (day 1 vs day 14 P<0.03) and threonine consumption (day 1 vs day 10 P<0.002), indicative of an aerobic metabolic pattern. Significantly more glutamate was produced after day 10 (day 1 vs day 10 P<0.031), and more glutamine was consumed after day 14. There was a steadily diminishing production of acetate which reached a minimum on day 14 (day 2 vs day 14 P<0.00014). After an initial 10 day period of acclimatisation cell aggregates formed in the high aspect ratio vessel switched from the anaerobic pattern of metabolism to the more efficient aerobic pattern, which was exhibited until the experiments were terminated. The high aspect ratio vessel is suitable for long-term culture of porcine hepatocytes and it is worthwhile carrying out scale-up feasibility studies.
Keywords: High aspect ratio vessel; Bioreactor; Glycolysis; Ketogenesis; Glutamine synthesis; Succinate precursor synthesis;

Isolation and characterization of four bactericidal domains in the bovine β-lactoglobulin by Antonio Pellegrini; Carmen Dettling; Ursula Thomas; Peter Hunziker (131-140).
Proteolytic digestion of bovine β-lactoglobulin by trypsin yielded four peptide fragments with bactericidal activity. The peptides were isolated and their sequences were found as follows: VAGTWY (residues 15–20), AASDISLLDAQSAPLR (residues 25–40), IPAVFK (residues 78–83) and VLVLDTDYK (residues 92–100). The four peptides were synthesized and found to exert bactericidal effects against the Gram-positive bacteria only. In order to understand the structural requirements for antibacterial activity, the amino acid sequence of the peptide VLVLDTDYK was modified. The replacement of the Asp (98) residue by Arg and the addition of a Lys residue at the C-terminus yielded the peptide VLVLDTRYKK which enlarged the bactericidal activity spectrum to the Gram-negative bacteria Escherichia coli and Bordetella bronchiseptica and significantly reduced the antibacterial capacity of the peptide toward Bacillus subtilis. By data base searches with the sequence VLVLDTRYKK a high homology was found with the peptide VLVATLRYKK (residues 55–64) of human blue-sensitive opsin, the protein of the blue pigment responsible for color vision. A peptide with this sequence was synthesized and assayed for bactericidal activity. VLVATLRYKK was strongly active against all the bacterial strains tested. Our results suggest a possible antimicrobial function of β-lactoglobulin after its partial digestion by endopeptidases of the pancreas and show moreover that small targeted modifications in the sequence of β-lactoglobulin could be useful to increase its antimicrobial function.
Keywords: β-Lactoglobulin; Antibacterial activity; Antimicrobial; Antibiotics; Bactericidal peptide;

Chondrocyte deformation within mechanically and enzymatically extracted chondrons compressed in agarose by M.M. Knight; J.M. Ross; A.F. Sherwin; D.A. Lee; D.L. Bader; C.A. Poole (141-146).
Within articular cartilage, the chondron microenvironment will influence chondrocyte behaviour and response to loading. Chondrons were extracted from intact cartilage using either mechanical homogenisation (MC) or enzymatic digestion (EC) and cell and matrix morphology in unstrained and compressed agarose constructs was examined. Isolated chondrocytes (IC) were used for comparison. Immunolocalisation of type VI collagen and keratan sulphate revealed differences in the structure of the pericellular microenvironment such that MC most closely resembled chondrons in situ. The unstrained cell diameters of IC and EC were larger than MC at day 1 and increased significantly over a 7 day culture period. In contrast, cell diameters for MC remained constant. Compression of constructs at day 1 resulted in cell deformation for IC and EC but not MC. The two chondron extraction methods yielded chondrons of differing matrix morphology and associated differences in cell size and cellular response to load. The results indicate that the pericellular microenvironment of MC initially possessed a greater mechanical integrity than that of EC. Although these differences may be reduced with time in culture, characterisation of mechanically isolated chondrons suggests that the stiffness of the chondrons in situ may be greater than previous estimates.
Keywords: Chondron; Chondrocyte; Pericellular matrix; Stiffness; Mechanics;

Matrix metalloproteinase-1, -3, -13 and aggrecanase-1 and -2 are differentially expressed in experimental osteoarthritis by Gilles Bluteau; Thierry Conrozier; Pierre Mathieu; Eric Vignon; Daniel Herbage; Frederic Mallein-Gerin (147-158).
The aim of this study was to characterize the cellular phenotypes of articular cartilage and meniscus in rabbits with experimentally induced osteoarthritis (OA), by histological and molecular biological techniques. OA was induced by severing the anterior cruciate ligament of the knee and rabbits were killed 2, 4 or 9 weeks following surgery. Our histological observations show a progressive destruction of extracellular matrix in both tissues. To determine whether these morphological changes could be related to alterations in the regulation of gene expression for a subset of relevant molecules, levels of mRNA for proteinases and one inhibitor (MMP-1, -3 and -13, aggrecanase-1 and -2 and TIMP-1), matrix molecules and one chaperone (type II and X collagens, aggrecan, osteonectin, βig-h3 and BiP) were assessed by reverse transcription-polymerase chain reaction. Our results indicate that for most markers expression profiles were similar in both tissues. In particular, matrix protein gene expression remained stable or varied little during progression of OA, suggesting a poor repair capacity of the tissues. MMP gene expression increased rapidly whereas aggrecanase gene expression remained stable. These findings suggest that differential regulation of mRNA levels of MMP-1, -3 and -13 on the one hand and aggrecanase-1 and -2 on the other, occurs during OA.
Keywords: Cartilage; Meniscus; Matrix protein; Aggrecanase; Metalloproteinase; Osteoarthritis;

Unusually large numbers of electrons for the oxidation of polyphenolic antioxidants by Hiroki Hotta; Harumi Sakamoto; Satomi Nagano; Toshiyuki Osakai; Yoshio Tsujino (159-167).
Reaction mechanisms of polyphenolic antioxidants were studied using electrochemical methods (flow column electrolysis and cyclic voltammetry). In flow column electrolysis, the numbers (ns) of electrons involved in the oxidation of catechols (chlorogenic acid and caffeic acid) became larger than two (i.e. the number of –OH moieties) at pH>7; the n-values finally reached ca. 4 at pH 10. Other polyphenols including catechin, ellagic acid, and curcumin exhibited higher n-values than the numbers of –OH moieties in the whole pH range studied (4<pH<10). Such unusually large n-values for polyphenols were found to correlate to their irreversible behavior in cyclic voltammetry. A digital simulation analysis of the voltammograms of chlorogenic acid clearly showed that the electrode reaction at higher pHs can be elucidated in terms of a quasi-reversible electron transfer followed by a chemical reaction and also suggested that the chemical reaction is of second order to the concentration of chlorogenic acid, i.e. a dimerization reaction. In a similar manner, polyphenolic antioxidants generally undergo certain chemical reactions on the occasion of their oxidation. As a result, some oxidizable, phenolic –OH moieties are reproduced in the polymeric products. The unusually large n-values of polyphenols and thus their higher radical scavenging activities may be ascribed to such reproduction of –OH moieties by oxidative polymerization.
Keywords: Polyphenol; Antioxidant; Cyclic voltammetry; Flow column electrolysis; Number of electrons; Polymerization;

Characterization of monoclonal antibodies against ascorbate peroxidase isoenzymes: purification and epitope-mapping using immunoaffinity column chromatography by Kazuya Yoshimura; Takahiro Ishikawa; Kei Wada; Toru Takeda; Yoichi Kamata; Toshiji Tada; Keiichiro Nishimura; Yoshihisa Nakano; Shigeru Shigeoka (168-174).
We have developed three monoclonal antibodies against spinach chloroplastic (chl-mAb3 and chl-mAb6) and cytosolic (cyt-mAb1) ascorbate peroxidase (APX) isoenzymes to analyze the cross-reactivity and the structure of the epitopes for each monoclonal antibody. All three antibodies reacted specifically with their respective isoenzymes, but none cross-reacted with the others. Immunoreactive fragments in proteolytic recombinant APX isoenzymes were detected by means of the absorption on the corresponding immunoaffinity column. The cyt-mAb1 reacted with a peptide fragment containing the distal His region obtained by the lysyl endopeptidase digestion. The chl-mAb6 was capable of binding to the fragment, D-I-K-E-K-R, which is consistent with an inherent region of chloroplastic isoenzymes. No fragments reacting to the chl-mAb3 could be found in this study, suggesting that the chl-mAb3 recognizes a conformationally constituted epitope of the chloroplastic APX molecule, which may be destroyed by the enzymatic cleavage. We concluded that the peptides identified as epitopes are characteristic evidence of monoclonal antibodies.
Keywords: Ascorbate peroxidase; Isoenzyme; Recombinant enzyme; Monoclonal antibody; Epitope;

Transferrins (Tfs) are glycoproteins with carbohydrate chains in the C-lobe. Carbohydrate-deficient Tfs (CDTs) with fewer sialic acids increased in several diseases. In this study, the affinity of metals (Al and Fe) to Tfs was compared between native- and asialo-Tf by on-line high-performance liquid chromatography/high-resolution inductively coupled plasma mass spectrometry, to clarify whether the presence of sialic acids influences the metal binding. Fe added as Fe-citrate in the presence of bicarbonate preferred the N-lobe site and the binding affinity was similar between native- and asialo-Tfs. Al-citrate added at Al/Tf=1 also preferred the N-lobe site, while the binding affinity was higher to asialo-Tf than to native-Tf. In Al-oxalate addition, the affinity to the N-lobe site of both Tfs increased further. In the absence of bicarbonate, Al-oxalate showed a preference for the C-lobe site in native-Tf and comparable affinity to both lobes in asialo-Tf. In asialo-Tf, Al2-Tf was the largest peak even at Al/Tf=1. Thus, the lack of sialic acid in glycans and the presence of oxalate enhanced the binding affinity of Al to Tf. Therefore, it was suggested that the binding affinity of Al in patients with CDTs may be enhanced.
Keywords: Transferrin; Iron; Aluminum; Sialidase; Carbohydrate-deficient transferrin; High-resolution inductively coupled plasma mass spectrometry;

Discovery of a non-peptide small molecule that selectively mimics the biological actions of calcitonin by Toyoko Katayama; Mayumi Furuya; Kozo Yamaichi; Kyoko Konishi; Namino Sugiura; Hidenobu Murafuji; Koji Magota; Masayuki Saito; Shoji Tanaka; Shinzo Oikawa (183-190).
Calcitonin (CT), a 32-amino acid peptide hormone secreted mainly from the thyroid gland, plays an important role in maintaining bone homeostasis. To discover non-peptide small molecules with biological actions similar to those of CT, a cell-based screening of an in-house chemical library was performed and a pyridone derivative (SUN B8155) was identified. Like CT, it elevated cyclic AMP (cAMP) levels in T47D and UMR106-06 cells which endogenously express human and rat CT receptor, respectively. SUN B8155 also stimulated cAMP formation in cells expressing recombinant human CT receptor, but not in those expressing human parathyroid hormone/parathyroid hormone-related peptide receptor. Accumulation of cAMP in T47D cells was blocked by a selective antagonist of CT receptor, salmon CT(8–32), whereas SUN B8155 did not displace the specific binding of [125I]CT to the receptor. Our results suggested that the compound selectively interacts with the CT receptor by a mechanism similar to but probably different from that of CT itself. In rats, intraperitoneal administration of SUN B8155 significantly lowered serum calcium levels, like CT. Our results demonstrate, for the first time, that the biological activities of the newly identified small molecule can mimic that of CT, acting via the CT receptor.
Keywords: Calcitonin; Agonist; Non-peptide mimetic; cAMP; SUN B8155; Hypocalcemic activity;

Protective role of superoxide dismutases against ionizing radiation in yeast by Jin Hyup Lee; In Youl Choi; In Sup Kil; Sun Yee Kim; Eun Sun Yang; Jeen-Woo Park (191-198).
The protective role of superoxide dismutases (SODs) against ionizing radiation, which generates reactive oxygen species (ROS) harmful to cellular function, was investigated in the wild-type and in mutant yeast strains lacking cytosolic CuZnSOD (sod1Δ), mitochondrial MnSOD (sod2Δ), or both SODs (sod1Δsod2Δ). Upon exposure to ionizing radiation, there was a distinct difference between these strains in regard to viability and the level of protein carbonyl content, which is the indicative marker of oxidative damage to protein, intracellular H2O2 level, as well as lipid peroxidation. When the oxidation of 2′,7′-dichlorofluorescin was used to examine the hydroperoxide production in yeast cells, the SOD mutants showed a higher degree of increase in fluorescence upon exposure to ionizing radiation as compared to wild-type cells. These results indicated that mutants deleted for SOD genes were more sensitive to ionizing radiation than isogenic wild-type cells. Induction and inactivation of other antioxidant enzymes, such as catalase, glucose 6-phosphate dehydrogenase, and glutathione reductase, were observed after their exposure to ionizing radiation both in wild-type and in mutant cells. However, wild-type cells maintained significantly higher activities of antioxidant enzymes than did mutant cells. These results suggest that both CuZnSOD and MnSOD may play a central role in protecting cells against ionizing radiation through the removal of ROS, as well as in the protection of antioxidant enzymes.
Keywords: Superoxide dismutase; Mutant strain; Ionizing radiation; Antioxidant enzyme; Reactive oxygen species;

Copper complexes of glycyl-histidyl-lysine and two of its synthetic analogues: chemical behaviour and biological activity by Chiara Conato; Riccardo Gavioli; Remo Guerrini; Henryk Koz l ̵ owski; Piotr M l ̵ ynarz; Claudia Pasti; Fernando Pulidori; Maurizio Remelli (199-210).
Copper complex formation equilibria of glycyl-L-histidyl-L-lysine (Gly-His-Lys, GHK) and of two synthetic analogues, where the histidine residue was replaced with a synthetic amino acid (L-spinacine or L-1,2,3,4-tetrahydro-isoquinoline-3-carboxylic acid), have been carefully investigated using different experimental techniques: potentiometry, solution calorimetry, UV-VIS spectrophotometry, circular dichroism and electron paramagnetic resonance spectroscopies. All the ligands formed complexes having different stoichiometries and stabilities; evidence for the formation of binuclear species is also shown. The structures of the main complexes are discussed. It is suggested that the lateral lysine amino group participates in complex formation, but only at alkaline pH values: at physiological pH this group is protonated and available for possible interactions with cellular receptors. The above tripeptides have been tested for their enzymatic stability in human serum: the synthetic compounds showed no significant degradation for at least 3 h. Finally, their activity as growth factor has been studied in vitro. The two synthetic analogues showed an activity comparable to or even higher than that of GHK, thus suggesting their possible use as additives in cell culture media, even in the presence of serum. Relevant information on the GHK action mechanism as cell growth factor has been obtained: the formation of copper complexes, driven by the first (Gly) residue, appears necessary while the second residue (His) does not appear to play a specific role; the presence of the free side chain of the third residue (Lys) appears to be of fundamental importance.
Keywords: Glycyl-histidyl-lysine; Copper complex; Cell growth;

Lsh, a SNF2 family member, is required for normal murine development by Theresa M Geiman; Lino Tessarollo; Miriam R Anver; Jeffrey B Kopp; Jerrold M Ward; Kathrin Muegge (211-220).
Lsh is a member of the SNF2 family of chromatin remodelers, that regulate diverse biological processes such as replication, repair and transcription. Although expression of Lsh is highly tissue specific in adult animals, Lsh mRNA is detectable in multiple tissues during embryogenesis. In order to determine the physiologic role of Lsh during murine development and to assess its unique function in adult mice, we performed targeted deletion of the Lsh gene using homologous recombination in murine embryonic stem cells. Lsh−/− embryos occurred with the expected Mendelian frequency after implantation and during embryogenesis. However, Lsh−/− mice died within a few hours after birth. Furthermore, newborn mice were 22% lower in weight in comparison with their littermates and showed renal lesions. Thus Lsh is a non-redundant member of the SNF2 family and is essential for normal murine development and survival.
Keywords: Lymphoid specific helicase; SNF2; Transcription; Chromatin; Transgenics; Development;

Biliary excretion of polystyrene microspheres depends on the type of receptor-mediated uptake in rat liver by Kentaro Furumoto; Ken-ichi Ogawara; Minoru Yoshida; Yoshinobu Takakura; Mitsuru Hashida; Kazutaka Higaki; Toshikiro Kimura (221-226).
Hepatic uptake and biliary excretion of fluorescein isothiocyanate-labeled polystyrene microspheres with a particle size of 50 nm (MS-50) were studied in rats. Liver perfusion studies revealed that not only apo-E-mediated but also asialoglycoprotein receptor-mediated uptake is involved in the mechanism of the serum protein-dependent uptake of MS-50 in the liver. The uptake of MS-50 mediated by apo-E contributes more to the total uptake of MS-50 by the hepatocytes than that via asialoglycoprotein receptor in the presence of serum in the perfusate. Furthermore, it was found that MS-50 is substantially excreted into the bile by transcytosis. The extent of exocytosis of MS-50 taken up by the hepatocytes was much higher after MS-50 was endocytosed via asialoglycoprotein receptor than after taken up via the process mediated by apo-E. On the basis of these results, a possible regulation of the intracellular sorting of ligands, depending on the receptor-mediated uptake mechanism, was inferred.
Keywords: Polystyrene microsphere; Asialoglycoprotein receptor; Apo-E; Hepato-biliary transport; Intracellular trafficking;