BBA - General Subjects (v.1523, #1)

A procedure is presented that is based on the detection of Cu(II)–asparagine complexes by quantitative ESR, and allows in a very simple and rapid manner to evaluate the changes of asparagine concentration during the entire time range of the growth of Bacillus subtilis in a typical growth medium. The analysis is carried out in terms of the decrease of the intensity of the ESR-active mono- and di-asparagine–copper(II) complexes. It is resulted that at the end of the exponential growth the asparagine concentration was reduced to values as low as 2% of the initial value. The procedure here reported may be the basis of similar methods to be used for other amino acids and prokaryote systems.
Keywords: ESR; Asparagine depletion; Cu(II)–amino acid complexes; Bacillus subtilis;

New long-wavelength perylenequinones: synthesis and phototoxicity of hypocrellin B derivatives by Lei Li; Yanwen Chen; Jianquan Shen; Manhua Zhang; Tao Shen (6-12).
Five new derivatives of hypocrellin B were obtained from the reactions of hypocrellin B with ammonia and ethanolamine. Their photophysical and photochemical properties were investigated. The phototoxicity of one compound on AH cells irradiated with red light (λ=600–700 nm) was also studied. Their significantly enhanced red absorptivities at wavelengths longer than 600 nm and singlet oxygen-generating function qualify them as promising photodynamic therapy agents.
Keywords: Hypocrellin B; Ammonia; Ethanolamine; Singlet oxygen; Photodynamic therapy;

Relative position of the hexahistidine tag effects binding properties of a tumor-associated single-chain Fv construct by Apollina Goel; David Colcher; Ja-Seok Koo; Barbara J.M. Booth; Gabriela Pavlinkova; Surinder K. Batra (13-20).
Hexahistidine tag (His-tag) is the most widely used tag for affinity purification of recombinant proteins for their structural and functional analysis. In the present study, single chain Fv (scFv) constructs were engineered form the monoclonal antibody (MAb) CC49 which is among the most extensively studied MAb for cancer therapy. For achieving efficient purification of scFvs by immobilized metal–ion affinity chromatography (IMAC), a His-tag was placed either at the C-terminal (scFv-His6) or N-terminal (His6-scFv) of the coding sequence. Solid-phase radioimmunoassay for scFv-His6 showed only 20–25% binding whereas both His6-scFv and scFv (no His-tag) showed 60–65% binding. Surface plasmon resonance studies by BIAcore revealed the binding affinity constant (K A) for His6-scFv and scFv as 1.19×106 M−1 and 3.27×106 M−1, respectively. No K A value could be calculated for scFv-His6 due to poor binding kinetics (k on and k off). Comparative homology modeling for scFv and scFv-His6 showed that the C-terminal position of the His-tag partially covered the antigen-binding site of the protein. The study demonstrates that the C-terminal position of His-tag on the CC49 scFv adversely affects the binding properties of the construct. The results emphasize the importance of functional characterization of recombinant proteins expressed with purification tags.
Keywords: Single chain Fv; Yeast; Histidine tag; Immobilized metal affinity chromatography; BIAcore;

Fungal cell wall, mainly composed of chitin, an N-acetylglucosamine polymer, is known to participate in heavy metal detoxification. In the present study, an effort was made to elucidate the sites involved in complexation of cadmium by the chitin material of cell wall of Neurospora crassa. Based on the results of physical techniques, such as solid-state 13C-NMR, X-ray diffraction, IR and molecular modeling, a structure was proposed for the chitin–cadmium complex. The ring and C-3 hydroxyl oxygens of N-acetylglucosamine were implicated in the complexation of cadmium by the chitin of the fungal cell wall. The studies further revealed that the conformation of chitin did not alter after cadmium complexation.
Keywords: Chitin; Cadmium; Neurospora crassa; Metal chelation; Solid-state 13C-NMR;

The di-cysteine substituted hypocrellin B (DCHB) derivative has been found to be a potential phototherapeutic agent and exhibit photosensitized damage to DNA. Electronic paramagnetic resonance (EPR) and spectrophotometry demonstrate that one-electron transfer from calf thymus DNA to triplet DCHB induces the generation of the reduced form of DCHB (DCHB •− radical), followed by the second electron transfer from DNA to DCHB •− or the disproportionation of DCHB •− to form the hydroquinone of DCHB (DCHBH2) in anaerobic conditions. This electron transfer process induces the direct damage to DNA in oxygen-free media and contributes partly to the damage of DNA in aerobic media. Superoxide radical and hydroxyl radical are formed with enhanced efficiencies while singlet oxygen is generated with a reduced efficiency from irradiation of DCHB and DNA solution under aerobic conditions as compared with the case in the absence of DNA. All of three reactive oxygen species play an evident role in the photosensitized damage to DNA in aerobic system in addition to the direct electron-transfer damage.
Keywords: Hypocrellin derivative; Electronic paramagnetic resonance; Electron transfer; Reactive oxygen species; Damage to DNA;

Induction of hepatic antioxidants in freshwater catfish (Channa punctatus Bloch) is a biomarker of paper mill effluent exposure by Iqbal Ahmad; Tariq Hamid; Masroor Fatima; Hitendra S Chand; Swatantra K Jain; Mohammad Athar; Sheikh Raisuddin (37-48).
Enzymatic and non-enzymatic antioxidants serve as an important biological defense against environmental oxidative stress. Information on antioxidant defense in fish is meager despite that fish are constantly exposed to a myriad of environmental stress including the oxidants. This study, therefore, assesses the activities of antioxidant enzymes viz., glutathione peroxidase, catalase and glutathione S-transferase and the non-enzymatic antioxidants viz., glutathione and metallothionein in various tissues of freshwater fish Channa punctatus (Bloch), in response to short-term and long-term exposures to paper mill effluent. The fish were exposed to the effluent at a concentration of 1.0% (v/v) for 15, 30, 60 and 90 days. The exposure caused a time-dependent increase in glutathione level (P<0.001), activities of glutathione peroxidase (P<0.05 to P<0.001), glutathione S-transferase (P<0.001) and a marginal initial decrease in catalase activity in the liver (P<0.01 to P<0.001). Metallothionein was induced in liver after 60 days of exposure. Two isoforms of metallothionein were detected. Catalase activity also increased 60 days afterwards. Antioxidant pattern was different in gill and kidney showing that liver was more resistant to oxidative damage as compared to gills and kidney. Our results demonstrate a pollutant-induced adaptive response in fish. In addition, levels of enzymatic and non-enzymatic tissue antioxidants may serve as surrogate markers of exposure to oxidant pollutants in fish.
Keywords: Freshwater fish; Aquatic pollution; Oxidative stress; Antioxidants; Metallothionein; Adaptive response; Biomarker of pollution;

Role of intestinal surfactant-like particles as a potential reservoir of uropathogenic Escherichia coli by A. Mahmood; M.J. Engle; S.J. Hultgren; G.S. Goetz,; K. Dodson; D.H. Alpers (49-55).
The binding of uropathogenic Escherichia coli is mediated at the tips of pili by the PapG adhesin, which recognizes the Galα(1-4)Gal disaccharide on the uroepithelial surface. These receptors have been identified unequivocally in the human and murine urinary tracts but not in intestinal epithelium, yet uropathogenic E. coli strains are commonly found in normal colonic microflora. The gastrointestinal tract from duodenum to rectum elaborates a phospholipid-rich membrane particle with surfactant-like properties. In these studies, we report that purified murine particles contain a receptor recognized by the class I PapG adhesin because: (1) PapD–PapG complexes and class I pili bound to surfactant-like particles in a solid-phase assay, whereas binding was not detected in microvillous membranes derived from the same tissues, (2) purified PapD–PapG complex bound to a glycolipid receptor detectable in lipid extracts from the particles, and (3) soluble Galα(1-4)Gal inhibited the adhesin by 72% from binding to surfactant-like particles. The Galα(1-4)Gal receptor present in the intestinal surfactant-like particle which overlies the intestinal mucosa could provide one means to establish an intestinal habitat for uropathogenic E. coli.
Keywords: Surfactant-like particle; Microvillous membrane; Bacterial adhesin; Ecology; Escherichia coli;

Keywords: Adrenaline; Adrenaline-quinone; Adrenochrome; Absorption spectroscopy; Fluorescence spectroscopy;

Glucose induces slow oscillations of the cytoplasmic Ca2+ concentration in pancreatic β-cells. In order to elucidate the mechanisms responsible for the slow [Ca2+]i oscillations the effects of various nutrient insulin secretagogues on glucose-induced [Ca2+]i oscillations in intact mouse pancreatic islets and single β-cells were studied. These were the glycolytic intermediates, glyceraldehyde and pyruvate, and the mitochondrial substrate, α-ketoisocaproic acid (KIC). Glucose, at a 10 or 15 mM concentration, induced the typical slow oscillations of [Ca2+]i (0.4 min−1). At higher glucose concentrations the frequency of these oscillations decreased further (0.2 min−1). Glyceraldehyde, an insulin secretagogue like glucose, did not cause slow oscillations of [Ca2+]i in the absence of glucose. However, it exhibited a synergistic action with glucose. Glyceraldehyde, at 3 or 10 mM concentration, induced slow [Ca2+]i oscillations at a substimulatory concentration of 5 mM glucose (0.3–0.4 min−1) and reduced the frequency of the glucose-induced [Ca2+]i oscillations at stimulatory concentrations of 10 or 15 mM glucose (0.2 min−1). KIC (5 or 10 mM) as well as pyruvate (10 mM), the end product of glycolysis, and its ester methyl pyruvate (10 mM), did not cause slow oscillations of [Ca2+]i in the absence of glucose. In contrast to glyceraldehyde, however, all three compounds were capable of preventing the slow [Ca2+]i oscillations induced by glucose. Mannoheptulose (2 mM), an inhibitor of glucokinase and glucose-induced insulin secretion, reversibly blocked any kind of [Ca2+]i oscillation and returned the [Ca2+]i to a basal level through its ability to inhibit glycolytic flux. It can be concluded therefore that only substrates which generate a glucokinase-mediated metabolic flux through glycolysis and produce glycolytic ATP can induce slow [Ca2+]i oscillations in pancreatic β-cells.
Keywords: Cytoplasmic Ca2+; Slow [Ca2+]i oscillation; Glucose; α-Ketoisocaproic acid; Glyceraldehyde; Mouse pancreatic islet;

Permeabilising electric pulses can be advantageously used for DNA electrotransfer in vivo for gene therapy, as well as for drug delivery. In both cases, it is essential to know the electric field distribution in the tissues: the targeted tissue must be submitted to electric field intensities above the reversible permeabilisation threshold (to actually permeabilise it) and below the irreversible permeabilisation threshold (to avoid toxic effects of the electric pulses). A three-dimensional finite element model was built. Needle electrodes of different diameters were modelled by applying appropriate boundary conditions in corresponding grid points of the model. The observations resulting from the numerical calculations, like the electric field distribution dependence on the diameter of the electrodes, were confirmed in appropriate experiments in rabbit liver tissue. The agreement between numerical predictions and experimental observations validated our model. Then it was possible to make the first precise determination of the magnitude of the electric field intensity for reversible (362±21 V/cm, mean±S.D.) and for irreversible (637±43 V/cm) permeabilisation thresholds of rabbit liver tissue in vivo. Therefore the maximum of induced transmembrane potential difference in a single cell of the rabbit liver tissue can be estimated to be 394±75 and 694±136 mV, respectively, for reversible and irreversible electroporation threshold. These results carry important practical implications.
Keywords: Electroporation; Electropermeabilization; Electrode; Electrochemotherapy; Gene therapy; DNA electrotransfer; Finite element modelling;

Nuclear coactivator protein p100 is present in endoplasmic reticulum and lipid droplets of milk secreting cells by Thomas W. Keenan; Stefanie Winter; Hans-Richard Rackwitz; Hans W. Heid (84-90).
We have identified the p100 protein, previously known as a novel cellular coactivator, as a constituent of endoplasmic reticulum and cytosolic lipid droplets from milk secreting cells. Cytosolic lipid droplets of terminally differentiated mammary epithelial cells are secreted as milk lipid globules. However, milk lipid globules did not have detectable amounts of p100 protein. The p100 protein was found also in cytosol from lactating mammary gland, in storage lipid droplets from mouse adipocytes, and in endoplasmic reticulum from liver. Immunofluorescence microscopy of mammary epithelial cells confirmed the presence of p100 in non-nuclear regions of these cells. Partial sequence analysis of tryptic peptides from p100 from cow mammary gland showed extensive homology with the reported sequence of p100 determined from a human cDNA. Antibodies against a peptide synthesized to duplicate a sequence in human p100 recognized a protein of the size of p100 in cow, mouse and rat cell fractions.
Keywords: p100 protein; Cytosolic lipid droplet; Milk lipid droplet; Endoplasmic reticulum; Adipocyte;

The non-homeostatic regulation of blood sugar concentration in the insect Manduca sexta L. was affected by nutritional status. Larvae maintained on diets lacking sucrose displayed low concentrations of trehalose, the blood sugar of insects, which varied from 5 to 15 mM with increasing dietary casein level between 12.5 and 75 g/l. These insects were glucogenic, as demonstrated by the selective 13C enrichment of trehalose synthesized from [3-13C]alanine, and de novo synthesis was the sole source of blood sugar. The distribution of 13C in glutamine established that following transamination of the 13C substituted substrate, [3-13C]pyruvate carboxylation rather than decarboxylation was the principal pathway of Pyr metabolism. The mean blood trehalose level was higher in insects maintained on diets with sucrose. At the lowest dietary casein level blood trehalose was approximately 50 mM, and declined to 20 mM at the highest casein level. Gluconeogenesis was detected in insects maintained on sucrose-free diets at the higher protein levels examined, but [3-13C]pyruvate decarboxylation and TCA cycle metabolism was the principal fate of [3-13C]alanine following transamination, and dietary carbohydrate was the principal source of glucose for trehalose synthesis. Feeding studies established a relationship between nutritional status, blood sugar level and dietary self-selection. Insects preconditioned by feeding on diets without sucrose had low blood sugar levels regardless of dietary casein level, and when subsequently given a choice between a sucrose diet or a casein diet, selected the former. Larvae preconditioned on a diet containing sucrose and the lowest level of casein had high blood sugar levels and subsequently selected the casein diet. Larvae maintained on the sucrose diet with the highest casein level had low blood sugar and self-selected the sucrose diet. When preconditioned on diets with sucrose and intermediate levels of casein, insects selected more equally between the sucrose and the casein diets. It is concluded that blood sugar level may be intimately involved in dietary self-selection by M. sexta larvae, and that in the absence of dietary carbohydrate, gluconeogenesis provides sufficient blood sugar to ensure that larvae choose a diet or diets that produce an optimal intake of dietary protein and carbohydrate.
Keywords: Gluconeogenesis; Glucose; 13C; Invertebrate; Nuclear magnetic resonance spectroscopy; Nutrition; Trehalose; Manduca sexta;

Strength of conjugate binding to plasmid DNA affects degradation rate and expression level in vivo by Patricia M Mullen; Charles P Lollo; Quynh-Chi Phan; Arjang Amini; Mariusz G Banaszczyk; Joanne M Fabrycki; Dongpei Wu; Alison T Carlo; Patrick Pezzoli; Christopher C Coffin; Dennis J Carlo (103-110).
In vitro assays have demonstrated the capability of poly-L-lysine to protect plasmid DNA from serum nucleases and cellular lysates. Our purpose was to evaluate the stability and potency of poly-L-lysine–DNA polyplexes after intravenous injection into mice. Polyplexes consisted of 32P-radiolabeled plasmid DNA complexed with poly-L-lysine at specified charge ratios. Variations in conjugate hydrophobicity and levels of modification with polyethylene glycol were investigated. Our results show that, in contrast to in vitro studies, the systemically administered polyplexes exhibited marked DNA degradation in the vascular compartment within 5 min. Substitution of poly-L-lysine ϵ-amino sites with polyethylene glycol or hydrocarbon chains resulted in faster degradation even when complexed at higher charge (±) ratios. Use of excess cationic charge in the polyplexes (±2.5) diminished degradation rates only slightly. An analysis was made of the strength of the poly-L-lysine:DNA interaction by competition with poly-aspartic acid. Polyplexes with the strongest binding between conjugate and DNA in the competition assay were also the most stable in blood. However, tighter binding was not enough to fully protect the polyplex in vivo and polyplex DNA was substantially degraded within 10 min. Increased polyplex stability did not correlate with improved in vivo transfection efficiency.
Keywords: Degradation; DNA; Expression; In vivo; Polyplex;

Polycarboxylates inhibit the glucan-binding lectin of Streptococcus sobrinus by Choon-Teck Goh; Suwimol Taweechaisupapong; K.G. Taylor; R.J. Doyle (111-116).
Polycarboxylates, such as carboxymethylcellulose and hyaluronan, were found to be reversible inhibitors of the glucan-binding lectin of Streptococcus sobrinus. When the carboxylate groups were coupled to ethylenediamine, or reduced with carbodiimide-borohydride, inhibitory powers were lost. Similarly, N-deacetylated hyaluronan had poor inhibitory powers, probably due to the introduction of positive charges into the polymer. Other polymers, such as chondroitin sulfates, dextran sulfate, fetuin, heparin were not inhibitors. It appears that inhibition is based on repeating carboxylates, free of influence from ammonium groups. Such polymers have the property of complexing with metals. Earlier studies had concluded that the streptococcal lectin depended on manganese for activity. It is likely the carboxymethylcellulose and hyaluronan perturb essential metal coordination centers in the lectin. Polycarboxylates may have value in oral health care by acting on glucan-dependent microbial adhesion and biofilm formation.
Keywords: Lectin; Glucan; Hyaluronate; Carboxymethylcellulose; Streptococcus;

In this study we investigated the potential antioxidant properties of blueberry polyphenolics in vitro and vivo, using red blood cell (RBC) resistance to reactive oxygen species (ROS) as the model. In vitro incubation with anthocyanins or hydroxycinnamic acids (HCA) (0.5 and 0.05 mg/ml) was found to enhance significantly RBC resistance to H2O2 (100 μM) induced ROS production. This protection was also observed in vivo following oral supplementation to rats at 100 mg/ml. However, only anthocyanins were found to afford protection at a significant level, this at 6 and 24 h post supplementation. This protection was not consistent with the measured plasma levels of anthocyanins. Indeed, plasma polyphenolic concentrations were highest after 1 h, declining considerably after 6 h and not detected after 24 h. The difference in absorption between anthocyanins and HCA is likely to have contributed to the observed difference in their abilities to afford protection to RBC. This protection represents a positive role following dietary consumption of polyphenolics from blueberries, against ROS formation within RBC in vivo.
Keywords: Polyphenolic; Anthocyanin; Plasma; Absorption; Oxidative stress; Red blood cell;

The glutaryl 7-aminocephalosporanic acid (GL-7-ACA) acylase from Pseudomonas sp. strain GK16 is an (αβ)2 heterotetramer of two non-identical subunits that are cleaved autoproteolytically from an enzymatically inactive precursor polypeptide. The newly formed N-terminal serine of the β subunit plays an essential role as a nucleophile in enzyme activity. Chemical modification studies on the recombinant enzyme purified from Escherichia coli revealed the involvement of a single arginine and tryptophan residue, per αβ heterodimer of the enzyme, in the catalytic activity of the enzyme. Glutaric acid, 7-aminocephalosporanic acid (7-ACA) (competitive inhibitors) and GL-7-ACA (substrate) could not protect the enzyme against phenylglyoxal-mediated inactivation, whereas except for glutaric acid protection was observed in case of N-bromosuccinimide-mediated inactivation of the enzyme. Kinetic parameters of partially inactivated enzyme samples suggested that while arginine is involved in catalysis, tryptophan is involved in substrate binding.
Keywords: Glutaryl 7-aminocephalosporanic acid acylase; Active site arginine residue; Active site tryptophan residue;

Impaired ability of glycated insulin to regulate plasma glucose and stimulate glucose transport and metabolism in mouse abdominal muscle by Alison C Boyd; Yasser H.A Abdel-Wahab; Aine M McKillop; Helene McNulty; Christopher R Barnett; Finbarr P.M O’Harte; Peter R Flatt (128-134).
Previous studies have shown that glycated insulin is secreted from pancreatic β-cells under conditions of hyperglycaemia. This study has investigated the effects of monoglycated insulin on plasma glucose homeostasis and in vitro cellular glucose transport and metabolism by isolated abdominal muscle of mice. Monoglycated insulin was prepared under hyperglycaemic reducing conditions, purified by RP-HPLC and identified by electrospray ionisation mass spectrometry (5971.1 Da). When administered to mice at an intraperitoneal dose of 7 nmoles/kg body weight, insulin (non-glycated) decreased plasma glucose concentrations and substantially reduced the glycaemic excursion induced by conjoint intraperitoneal injection of 2 g glucose/kg body weight. In comparison, the same dose of monoglycated insulin decreased plasma glucose concentrations to a lesser extent (P<0.05), corresponding to an approx. 20% reduction of glucose lowering potency. Using isolated abdominal muscle, insulin (10−9–10−7 M) stimulated dose-dependent increases in cellular 2-deoxy-d-[1-3H]glucose uptake, d-[U-14C]glucose oxidation and glycogen production. Monoglycated insulin was approx. 20% less effective than native insulin in stimulating glucose uptake and both indices of metabolism, generally requiring 10-fold greater concentrations to achieve significant stimulatory effects. These data indicate that the impaired biological activity of glycated insulin may contribute to glucose intolerance of diabetes.
Keywords: Glycation; Insulin; Biological activity; Glucose transport and metabolism; Isolated muscle; Plasma glucose;