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BBA - General Subjects (v.1726, #2)
Controlling N-linked glycan site occupancy by Jullian Jones; Sharon S. Krag; Michael J. Betenbaugh (pp. 121-137).
N-linked glycosylation, a common co-translational modification in eukaryotic cells, involves the transfer of a lipid-linked oligosaccharide onto asparagine residues in a tripeptide sequon on a nascent protein in the lumen of the endoplasmic reticulum. The attachment of an oligosaccharide unit to the polypeptide at the site of occupancy can enhance solubility, improve folding, facilitate secretion, modulate antigenicity, and increase in vivo half-life of the glycoprotein. A number of proteins exhibit variable site occupancy. The efficiency of protein N-glycosylation is dependent on the kinetics of the individual steps in the biosynthesis of the dolichol-linked oligosaccharide and the transfer of the oligosaccharide from the lipid donor substrate to the nascent polypeptide. In this review, we will discuss the role of N-linked glycan site occupancy and give an overview of the possible limitations associated with variable site occupancy. The characterization of the dolichol pyrophosphate biosynthetic pathway and the recent identification of potential rate limiting enzymes in yeast and mammalian cells has made it possible to investigate their role in site occupancy. Genetic and biochemical characterization of oligosaccharide transferase (OST) complex in yeast and mammalian cells have demonstrated the importance of specific OST subunits in protein N-glycosylation. In addition, insights into the location and residues in and around the acceptor tripeptide sequon suggest an influence on N-glycan site occupancy. Insights from these characterizations are being used to elucidate methodologies to control N-glycosylation site heterogeneity.
Keywords: Macroheterogeneity; Site occupancy; N; -linked Glycosylation; Dolichol; Oligosaccharide transferase (OST); Glycoprotein; Eukaryotes
Comparative thermal stabilities of recombinant adenoviruses and hexon protein by Peter M. Ihnat; Gary Vellekamp; Linda J. Obenauer-Kutner; Jenny Duan; Michelle A. Han; Leonore C. Witchey-Lakshmanan; Michael J. Grace (pp. 138-151).
Differential scanning calorimetry was used to identify the thermal stability profile of the replication deficient and protein IX deleted recombinant adenovirus type 5 that contains the p53 transgene (rAd/p53) in phosphate buffered saline (vPBS) or 10% glycerol (TRIS/phosphate buffer). The wildtype adenovirus (Ad/WT) and purified hexon protein (major capsid protein) were also evaluated in 10% glycerol (TRIS/phosphate buffer) as controls. The thermal profile of rAd/p53 revealed three endothermic transitions ( T1, T2 and T3) occurring between 25 °C and 90 °C. T1, which occurred at 46.7 °C in vPBS and 49.4 °C in TRIS/PO4 10% glycerol buffer, was irreversible following repeated scanning and attributed to the degradation of the intact vector. The latter two endothermic transitions, T2 and T3, occurring at 69 °C and 78 °C, respectively, corresponded with the two transitions of purified hexon in temperature and amount of heat absorbed. The thermal profile of Ad/WT revealed four endothermic transitions at 51.5 °C ( T1), 70.5 °C ( T2A), 73.6 °C ( T2B), and 77.4 °C ( T3). The higher temperature of degradation as well as additional transition was attributed to the presence of protein IX associated with the hexon. The positions and excess molar heat capacities of the intact rAds were found to be affected by pH, glycerol, vector concentration and the presence or absence of protein IX in the capsid. Irreversibility of T1 implied that the degradation of the intact virus may follow first-order kinetics. The thermal scan rate dependence of T1 further confirmed that degradation of the intact virus may be first-order. The apparent activation energies for the degradation of the intact vectors were determined from the scan rate dependence of T1 and shown to be affected by protein IX in the capsid and solution conditions. Analysis of rAd samples incubated at 45 °C by Field Emission Electron Microscopy (FESEM) confirmed that loss of single particles was first-order. Although aggregates were observed in the samples, degradation appeared to be the dominant reaction leading to disappearance of single virions from the aqueous matrix. Based on thermal and FESEM analysis, an empirical model was proposed that accounted for the disappearance of single rAd particles. At or near T1, degradation of rAd particles followed a unidirectional, pseudo-first order reaction. However, at lower temperatures, disappearance of single virions resulted from competing irreversible degradation and aggregation reactions.
Keywords: Recombinant adenovirus; p53; Hexon; Thermal stability; Differential scanning calorimetry; Transition temperature; Activation energy; Degradation; Aggregation; First-order kinetics
Effects of short-term supplementation with folic acid on different oxidative stress parameters in patients with hypertension by Pablo Stiefel; Sandro Argüelles; Sonia García; Luis Jiménez; Reyes Aparicio; Joaquín Carneado; Alberto Machado; Antonio Ayala (pp. 152-159).
Oxidative stress seems to play an important role in the pathophysiology of essential hypertension. Due to its antioxidant features, we studied the protective action of folic acid in hypertensive patients, their food supplemented for 2 weeks with this vitamin. Several oxidative stress parameters were measured in the serum of these patients. The results show that folic acid supplementation improves levels of oxidative stress markers in individuals with hypertension, overall in those patients whose initial parameter values were highest.
Keywords: Abbreviations; HC; homocysteine; TBARS; thiobarbituric reactive substances; LP; lipid peroxidesFolic acid; Hypertension; Oxidative stress; Homocysteine; Oxidative stress marker; TBARS; Carbonyl group; Lipid peroxide
Characterisation of uterine sarcoma cell lines exhibiting MDR phenotype by vibrational spectroscopy by C. Murali Krishna; G. Kegelaer; I. Adt; S. Rubin; V.B. Kartha; M. Manfait; G.D. Sockalingum (pp. 160-167).
Multidrug resistance (MDR) enables cancer cells to escape cytotoxic insults of anticancer drugs. Rapid identification of cells exhibiting the MDR phenotype is very important since it can lead to an effective and individual patient based treatment plan. We have investigated a combined vibrational spectroscopic approach, using both micro-Raman and FTIR techniques, in order to characterise a sensitive human uterine sarcoma cell line MES-SA and its multidrug-resistant derivative Garf. In this study, these two complementary methods have been evaluated via the use of principal components analysis (PCA), for discrimination of cells exhibiting the MDR phenotype. Our results indicate that, though they inherently have different sensitivities, both Raman and IR methods can provide a good differentiation of cell phenotypes.
Keywords: Multidrug resistance; MES-SA; Garf; Micro-Raman; FTIR; Spectroscopy; PCA
Kinetic studies on the interactions of heparin and complement proteins using surface plasmon resonance by Haining Yu; Eva M. Muñoz; R. Erik Edens; Robert J. Linhardt (pp. 168-176).
Heparin is a naturally occurring polysaccharide known to interact with complement proteins and regulate multiple steps in the complement cascade. Quantitative information, in the form of affinity constants for heparin-complement interactions, is not generally available and there are no reports of a comprehensive analysis using the same interaction method. Such information should improve our understanding of how exogenously administered pharmaceutical heparin and the related endogenous polysaccharide, heparan sulfate, regulate complement activation. The current study provides the first comprehensively analysis of the binding of various complement proteins to heparin using surface plasmon resonance (SPR). Complement proteins C1, C2, C3, C4, C5, C6, C7, C8, C9, C1INH, factor I, factor H, factor B and factor P all bind heparin but exhibit different binding kinetics and dissociation constants ( Kd) ranging from 2 to 320 nM. By taking into account these Kd values and the serum concentrations of these complement proteins, the percentage of each binding to exogenously administered heparin was calculated and found to range from 2% to 41%. This study provides essential information required for the rational design of new therapeutic agents capable of regulating the complement activation.
Keywords: Complement; Polysaccharide; Interaction; SPR; Heparin
Inhibitory effects of U73122 and U73343 on Ca2+ influx and pore formation induced by the activation of P2X7 nucleotide receptors in mouse microglial cell line by Takato Takenouchi; Kazumasa Ogihara; Mitsuru Sato; Hiroshi Kitani (pp. 177-186).
P2X7 receptors are ATP-gated ion channels and play important roles in microglial functions in the brain. Activation of P2X7 receptors by ATP or its agonist BzATP induces Ca2+ influx from extracellular space, followed by the formation of non-selective membrane pores that is permeable to larger molecules, such as fluorescent dye. To determine whether phospholipase C (PLC) is involved in the activation of P2X7 receptors in microglial cells, U73122, a specific inhibitor of PLC, and its inactive analogue U73343 were examined on ATP and BzATP-induced channel and pore formation in an immortalized C57BL/6 mouse microglial cell line (MG6-1). ATP induced both a transient and a sustained increase in the intracellular Ca2+ concentration ([Ca2+]i) in MG6-1 cells, whereas BzATP evoked only a sustained increase. U73122, but not U73343, inhibited the transient [Ca2+]i increase involving Ca2+ release from intracellular stores through PLC activation. In contrast, both U73122 and U73343 inhibited the sustained [Ca2+]i increase either prior or after the activation of P2X7 receptor channels by ATP and BzATP. In addition, these U-compounds inhibited the influx of ethidium bromide induced by ATP and BzATP, suggesting possible PLC-independent blockage of the process of P2X7-associated channel and pore formations by U-compounds in C57BL/6 mouse microglial cells.
Keywords: Cytosolic calcium; U73122; U73343; P2X7 receptor; ATP
Inhibition of several protein phosphatases by a non-covalently interacting microcystin and a novel cyanobacterial peptide, nostocyclin by C. James Hastie; Emma B. Borthwick; Louise F. Morrison; Geoffrey A. Codd; Patricia T.W. Cohen (pp. 187-193).
Microcystins produced by cyanobacterial ‘blooms’ in reservoirs and lakes pose significant public health problems because they are highly toxic due to potent inhibition of protein serine/threonine phosphatases in the PPP family. A dehydrobutyrine (Dhb)-containing microcystin variant [Asp3, ADMAdda5, Dhb7]microcystin-HtyR isolated from Nostoc sp. was found to potently inhibit PP1, PP2A, PPP4 and PPP5 with IC50 values similar to those of microcystin-LR. However, in contrast to microcystin-LR, which forms a covalent bond with a cysteine residue in these protein phosphatases, Asp,ADMAdda,Dhb-microcystin-HtyR did not form any covalent interaction with PP2A. Since the LD50 for Asp,ADMAdda,Dhb-microcystin-HtyR was 100 μg kg−1 compared to 50 μg kg−1 for microcystin-LR, the data indicate that the non-covalent inhibition of protein phosphatases accounts for most of the harmful effects of microcystins in vivo. A 3-amino-6-hydroxy-2-piperidone containing cyclic peptide, nostocyclin, also isolated from Nostoc sp., was non-toxic and exhibited more than 500-fold less inhibitory potency towards PP1, PP2A, PPP4 and PPP5, consistent with the conclusion that potent inhibition of one or more these protein phosphatases underlies the toxicity of microcystins, both lacking and containing Dhb.
Keywords: Microcystin; Cyanobacterial peptide; Protein serine/threonine phosphatase; Tumour promoter; Toxins in water supplies
Cytochrome P450 2E polymorphism in feline liver by Nagako Tanaka; Raku Shinkyo; Toshiyuki Sakaki; Masahiko Kasamastu; Susumu Imaoka; Yoshihiko Funae; Hiroshi Yokota (pp. 194-205).
Only one isoform of cytochrome P450 (CYP) 2E subfamily was known in human and various animals. Three cDNAs corresponding to CYP 2E subfamily members (CYP2E-a, CYP2E-b and CYP2E-c) were obtained from feline liver. These cDNAs each had a 1488-bp nucleotide coding region encoding a predicted amino acid sequence of 495 residues. Eleven amino acid substitutions were observed between CYP2E-a and CYP2E-b, but only one substitution between CYP2E-b and CYP2E-c. The CO difference spectrums about 450 nm wave length and similar values of Vmax and Km of 6-hydoxygenase activity toward chlorzoxazone were observed in all three isoforms expressed in AH22 yeast cells. By PCR-RFLP, mRNA of the CYP2E-a was found to be expressed in liver, mononuclear cells, kidney, lung, stomach, intestine and pancreas, whereas CYP2E-b and CYP2E-c were expressed mainly in the liver and mononuclear cells. Expression of CYP2E-a was observed in the livers of all felines tested, but CYP2E-b and CYP2E-c were not expressed in all cats. The sequences of two different introns between exons I and II and between exons VII and VIII were obtained in genomic DNA from the feline liver.Based on these results, we conclude that cats have two highly similar CYP2E genes.
Keywords: Cat; Cytochrome P450 (CYP); CYP2E1; CYP2E2; Polymorphism
Glycosylation of internal sugar residues of oligosaccharides catalyzed by α-galactosidase from Aspergillus fumigatus by Vladimír Puchart; Peter Biely (pp. 206-216).
Purified α-galactosidase from a thermotolerant fungus Aspergillus fumigatus IMI 385708 was found to catalyze efficiently transgalactosylation reactions using 4-nitrophenyl α-d-galactopyranoside as glycosyl donor. Self-transfer reactions with this substrate afforded in low yields several 4-nitrophenyl galactobiosides. Monosaccharides also served as poor glycosyl acceptors. Disaccharides and particularly higher oligosaccharides of α-1,4-gluco- (maltooligosaccharides), β-1,4-gluco- (cellooligosaccharides) and β-1,4-manno-series were efficiently galactosylated, the latter being the best acceptors that were also doubly galactosylated. With mannooligosaccharides product yields increased with polymerization degree of acceptors reaching 50% at DP of 4–6. Longer oligosaccharide acceptors were galactosylated at internal sugar residues. All galactosyl residues were transferred exclusively to the primary hydroxyl group(s) at C-6 position of oligosaccharide acceptors. This is in accordance with the inability of the enzyme to transfer galactose to β-1,4-linked xylooligosaccharides. This is the first report of glycosyl transfer reaction to internal sugar residues of oligosaccharides catalyzed by a glycosidase. High affinity to oligosaccharide acceptors also opens a way toward enzymatic glycosylation of polysaccharides, thus modulating their physico-chemical and biological properties.
Keywords: Abbreviations; Galα6Man; α-; d; -galactopyranosyl-1,6-; d; -mannopyranose; Gal; 1; Man; 2; β-; d; -mannopyranosyl-1,4-(6-; O; -α-; d; -galactopyranosyl)-; d; -mannopyranose; Gal; 2; Man; 2; α-; d; -galactopyranosyl-1,6-β-; d; -mannopyranosyl-1,4-; d; -mannopyranose; Gal; 1; Man; 3; β-; d; -mannopyranosyl-1,4-β-; d; -mannopyranosyl-1,4-(6-; O; -α-; d; -galactopyranosyl)-; d; -mannopyranose; Man; 2; through Man; 6; β-1,4-mannobiose through β-1,4-mannohexaose; NPGal; 4-nitrophenyl α-; d; -galactopyranosideα-galactosidase; Aspergillus fumigatus; Galactomannan; Transgalactosylation; Internal sugar residues
Time-course of hormonal induction of mitochondrial glycerophosphate dehydrogenase biogenesis in rat liver by T. MráÄ?ek; P. JeÅ¡ina; P. KÅ™iváková; R. Bolehovská; Z. ÄŒervinková; Z. Drahota; J. HouÅ¡tÄ›k (pp. 217-223).
Thyroid hormones are important regulators of mitochondrial metabolism. Due to their complex mechanism of action, the timescale of different responses varies from minutes to days. In this work, we studied selective T3 induction of the inner mitochondrial membrane enzyme–glycerophosphate dehydrogenase (mGPDH) in liver of euthyroid rats. We correlated the kinetics of the T3 level in blood, the mRNA level in liver, the activity and amount of mGPDH in liver mitochondria after a single dose of T3. The T3 level reached maximum after 1 h (80 nmol/l) and subsequently rapidly decreased. mGPDH mRNA increased also relatively fast, reaching a maximum after 12 h and fell to the control level after 72 h. An increase of mGPDH activity could be already found after 6 h and reached a maximum after 24 h in accordance with the increase in mGPDH content (2.4-fold vs. 2.7-fold induction). After 72 h, the mGPDH activity showed a significant 30% decrease. When the rats received three subsequent doses of T3, the increase of mGPDH activity was 2-fold higher than after a single T3 dose. The results demonstrate that mGPDH displays rapid induction as well as decay upon disappearance of a hormonal stimulus, indicating a rather short half-life of this inner mitochondrial membrane enzyme.
Keywords: Abbreviations; T; 3; triiodothyronine; TH; thyroid hormone; TR; thyroid receptor; mGPDH; mitochondrial glycerophosphate dehydrogenase; COX; cytochrome; c; oxidase; CS; citrate synthase; ROS; reactive oxygen speciesLiver; Mitochondrial GPDH; Triiodothyronine; Mitochondrial protein turnover