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BBA - General Subjects (v.1760, #5)
Diltiazem, a L-type calcium channel antagonist, suppresses ouabain-enhanced dopamine efflux by 1-methyl-4-phenylpyridinium ion (MPP+) in rat striatum by Toshio Obata (pp. 721-723).
The present study was examined whether diltiazem, a L-type Ca2+ channel antagonist, could suppresses 1 methyl-4-phenylpyridinium ion (MPP+)-induced dopamine (DA) in extracellular fluid of rat striatum. Ouabain (100 μM; 100 μM or 100 pmol/μl per min) significantly enhanced the level of DA by MPP+. However, in the presence of diltiazem (100 μM) significantly suppressed the level of DA release by ouabain and MPP+. These results suggest that diltiazem suppresses Ca2+-dependent release of DA by ouabain-induced Ca2+ overload.
Keywords: Abbreviations; MPP; +; 1-methyl-4 phenylpyridinium ion; ·OH; hydroxyl radical; DA; dopamine; 2,3-DHBA; 2,3-dihydroxybenzoic acidDiltiazem; Ouabain; Ca; 2+; overload; 1-Methyl-4-phenylpyridinium ion (MPP; +; ); Dopamine; Parkinson's disease
Catalase-peroxidase of Mycobacterium bovis BCG converts isoniazid to isonicotinamide, but not to isonicotinic acid: Differentiation parameter between enzymes of Mycobacterium bovis BCG and Mycobacterium tuberculosis by Sung-Koo Kang; Jong-Ho Lee; Young-Choon Lee; Cheorl-Ho Kim (pp. 724-729).
Isonicotinic acid hydrazide (Isoniazid, INH) is one of the major drugs worldwide used in the chemotherapy of tuberculosis. Many investigators have emphasized that INH activation is associated with mycobacterial catalase-peroxidase (katG). However, INH activation mechanism is not completely understood. In this study, katG of M. bovis BCG was separated and purified into two katGs, katG I (named as relatively higher molecular weight than katG II) and katG II, indicating that there is some difference in protein structure between two katGs. The molecular weight of the enzymes of katG I and katG II was estimated to be approximately 150,000 Da by gel filtration, and its subunit was 75,000 Da as determined by SDS-PAGE, indicating that purified enzyme was composed of two identical subunits. The specific activity of the purified enzyme katG I was 991.1 (units/mg). The enzymes were then investigated in INH activation by using gas chromatography mass spectrometry (GC-MS). The analysis of GC-MS showed that the katG I from M. bovis BCG directly converted INH ( Mr, 137) to isonicotinamide ( Mr, 122), not to isonicotinic acid ( Mr, 123), in the presence or absence of H2O2. Therefore, this is the first report that katG I, one of two katGs with almost same molecular weight existed in M. bovis BCG, converts INH to isonicotinamide and this study may give us important new light on the activation mechanism of INH by KatG between M. bovis BCG and M. tuberculosis.
Keywords: Catalase-Peroxidase; Mycobacterium bovis; BCG; Isoniazid; Isonicotinamide; Isonicotinic acid
Measurement of protein turnover rates by heavy water labeling of nonessential amino acids by Robert Busch; Yoo-Kyeong Kim; Richard A. Neese; Valerie Schade-Serin; Michelle Collins; Mohamad Awada; James L. Gardner; Carine Beysen; Michael E. Marino; Lisa M. Misell; Marc K. Hellerstein (pp. 730-744).
In vivo measurements of protein synthesis using isotope-labeled amino acids (AAs) are hampered by the heterogeneity of AA pools and, for slow turnover proteins, the difficulty and expense of long-term labeling. Continuous oral heavy water (2H2O) labeling can safely maintain stable body water2H enrichments for weeks or months.2H is metabolically incorporated into C–H bonds of nonessential AAs (NEAAs) and hence into proteins. No posttranslational label exchange occurs, so2H incorporation into protein NEAAs, in principle, reports on protein synthesis. Here, we show by mass isotopomer distribution analysis (MIDA) of2H2O-labeled rodent tissue proteins that metabolic2H flux into C–H bonds of Ala, Gly, or Gln used for protein synthesis is nearly complete. By2H2O labeling of rodents, turnover of bone and muscle mixed proteins was quantified and stimulation of liver collagen synthesis by CCl4 was detected. Kinetics of several human serum proteins were also measured, reproducing published t1/2 estimates. Plateau enrichments in Ala varied among different proteins. Moderate amounts of protein, isolated chromatographically or electrophoretically, sufficed for kinetic analyses. In conclusion,2H2O labeling permits sensitive, quantitative, operationally simple measurements of protein turnover in vivo by the rise-to-plateau approach, especially for proteins with slow constitutive turnover.
Keywords: Abbreviations; 2; H; 2; O; heavy water; 2; H; deuterium; MIDA; mass isotopomer distribution analysis; tRNA; transfer ribonucleic acid; GC/MS; gas chromatography/mass spectrometry; NEAAs; nonessential amino acids; PMSF; phenylmethylsulfonyl fluoride; EDTA; ethylenediamine tetraacetic acid; LDL; low-density-lipoprotein; PTFE; polytetrafluoroethylene; PFBBr; pentafluorobenzyl bromide; PCI; positive chemical ionization; NCI; negative chemical ionization; SEC; size exclusion chromatography; ELISA; enzyme-linked immunosorbent assay; m/z; mass to charge ratio; AAs; amino acids; SDS-PAGE; sodium dodecyl sulfate-polyacrylamide gel electrophoresis; PBS; Dulbecco's phosphate buffered saline without divalent cations; ApoB; apolipoprotein B; Ig; immunoglobulin; BSA; bovine serum albumin; GCRC; General Clinical Research Center; PFB; pentafluorobenzyl N,N-di(pentafluorobenzyl); VLDL; very low density lipoproteinProtein synthesis/turnover; Stable isotope labeling; Deuterated water; Mass isotopomer distribution analysis; Gas chromatography/mass spectrometry
The calcium- and zinc-responsive regions of calreticulin reside strictly in the N-/C-domain by Yan Tan; Mingnan Chen; Zhenjie Li; Katsuhide Mabuchi; Marlene Bouvier (pp. 745-753).
Calreticulin (CRT) is a chaperone of the endoplasmic reticulum. We dissected CRT into its two structural domains, N-/C-domain and P-domain, to identify its metal ion-responsive region. For this, we constructed bacterial expression systems for the N-/C-domain (1–180 fused by a linker to 290–400) and P-domain (189–280). Circular dichroism (CD) studies showed that calcium ions increased tertiary packing and thermal stability of apo N-/C-domain, whereas zinc ions had a strong destabilizing effect. Interestingly, neither calcium nor zinc ions altered the structural properties of apo P-domain. These results indicate that the calcium- and zinc-responsive regions reside strictly in the N-/C-domain. Analysis of thermal denaturation curves of CRT, N-/C-domain, and P-domain suggested a structural role for the P-domain in CRT. Rotary shadowing electron microscopy (EM) analysis of CRT and calnexin provided convincing evidence for their structural relatedness. This analysis also revealed that apo P-domain adopts various curved shapes suggesting conformational flexibility. EM images of apo N-/C-domain revealed objects having wide gaps suggesting weak interactions between the N- and C-domains. This is consistent with the larger size of apo N-/C-domain on the gel filtration column. Our studies provide a framework for correlating the structural organization of CRT with its metal ion-responsive region.
Keywords: Abbreviations; CD; circular dichroism; CRT; calreticulin; CNX; calnexin; ER; endoplasmic reticulum; GST; glutathione; S; -transferase; LB; Luria Broth; MW; molecular weight; PAGE; polyacrylamide gel electrophoresis; SDS; sodium dodecyl sulfate; T; m; thermal denaturation mid-point temperature; UV; ultravioletCalreticulin; Metal ion; Rotary shadowing electron microscopy; Circular dichroism; Calnexin; Protein stability
Identification of new differentiation inducing factors from Dictyostelium discoideum by Tamao Saito; Graham W. Taylor; Ji-Chun Yang; David Neuhaus; Dmitry Stetsenko; Atsushi Kato; Robert R. Kay (pp. 754-761).
Developing Dictyostelium discoideum amoebae form a stalked fruiting body in which individual cells differentiate into either stalk cells or spores. The major known inducer of stalk cell differentiation is the chlorinated polyketide DIF-1 (1-(3,5-dichloro-2,6-dihydroxy-4-methoxyphenyl)hexan-1-one); however a mutant blocked in the terminal step of DIF-1 biosynthesis still produces one of the prestalk cell subtypes – the pstA cells – as well as some mature stalk cells. We therefore searched for additional stalk cell-inducing factors in the medium supporting development of this mutant. These factors were purified by solvent extraction and HPLC and identified by mass spectroscopy and NMR. The mutant lacked detectable DIF-2 and DIF-3 (the pentanone and deschloro homologues of DIF-1) but four major stalk cell-inducing activities were detected, of which three were identified. Two compounds were predicted intermediates in DIF-1 biosynthesis: the desmethyl, and desmethyl-monochloro analogues of DIF-1 (dM-DIF-1 and Cl-THPH, respectively), supporting the previously proposed pathway of DIF-1 biosynthesis. The third compound was a novel factor and was identified as 4-methyl-5-pentylbenzene-1,3-diol (MPBD) with the structure confirmed by chemical synthesis. To investigate the potential roles of these compounds as signal molecules, their effects on morphological stalk and spore differentiation were examined in cell culture. All three induced morphological stalk cell differentiation. We found that synthetic MPBD also stimulated spore cell differentiation. Now that these factors are known to be produced and released during development, their biological roles can be pursued further.
Keywords: Abbreviations; DIF; Differentiation Inducing Factor, 1-(3,5-dichloro-2,6-dihydroxy-4-methoxyphenyl)hexan-1-one, dM-DIF-1,1-(3,5-dichloro-2,4,6-trihydroxyphenyl)hexan-1-one; Cl-THPH; 1-(3-chloro-2,4,6-trihydroxyphenyl)hexan-1-one; MPBD; 4-methyl-5-pentylbenzene-1,3-diol Dictyostelium; Stalk cell inducer; Differentiation inducing factor (DIF); Natural products
Competing regulation of matrix biosynthesis by mechanical and IGF-1 signalling in elderly human articular cartilage in vitro by Mandy S. Plumb; Katrina Treon; Richard M. Aspden (pp. 762-767).
This study investigates the separate and combined effects of IGF-1 and mechanical loads on chondrocytes in elderly human femoral head articular cartilage. Full depth biopsies of articular cartilage were subjected to either no load, static or cyclic (2s on/2s off) loading in unconfined compression at a stress of 1 MPa for 48 h with or without IGF-1 (300 ng ml−1). Chondrocyte biosynthetic activity was measured using35S-sulphate and3H-leucine during the last 24 h of loading. IGF-1 alone increased the rates of isotope incorporation, by 80% for35S-SO4 and 40% for3H-leucine, whereas loading alone reduced matrix biosynthesis. Applying load (cyclic or static) in the presence of IGF-1 returned the incorporation rates to their unstimulated levels. This study suggests elderly human articular cartilage is responsive to stimulation by IGF-1 but mechanical factors seem to act sufficiently strongly in the opposite direction to cancel this response.
Keywords: Articular cartilage; Elderly human; IGF-1; Matrix biosynthesis; Mechanical load
Branching mode in complex-type triantennary N-glycans as regulatory element of their ligand properties by Sabine André; Shuji Kojima; Gislinde Gundel; Roland Russwurm; Xaver Schratt; Carlo Unverzagt; Hans-Joachim Gabius (pp. 768-782).
We address the question whether the two natural types of branching in complex-type triantennary N-glycans differ in ligand properties. Toward this end, we prepared the set of pergalactosylated undecasaccharides and derivatives with α2,3/6-sialylation by chemoenzymatic synthesis. Conjugation resulted in neoglycoproteins which were tested in assays with lectins/antibodies, cultured cells and animals. Solid-phase assays with galactoside-specific proteins (a plant toxin, galectins and an antibody fraction) disclosed that the branching mode did not significantly affect affinity. However, compared to previous studies under identical conditions increase in antennae number and presence of substitutions in biantennary N-glycans altered KD-values with differences between receptors. Neoglycoprotein binding to cells of eight human tumor lines was sensitive to N-glycan branching. Staining intensity revealed pronounced branch-mode-dependent differences in four cases. Biodistribution profiles in mice uncovered dramatic changes in clearance rates with prolonged serum presence associated with type II branching of sialylated N-glycans and markedly increased uptake of neoglycoproteins with type I-branched N-glycans into liver, spleen, heart and lungs. This part of the study is relevant for rational glycoengineering of pharmaproteins. In general, our study supports the concept to view details of N-glycan structure, here branching, as a means to modulate ligand properties.
Keywords: Agglutinin; Drug targeting; Lectin; Sialylation; Tumor imaging
Ribosome-inactivating proteins in edible plants and purification and characterization of a new ribosome-inactivating protein from Cucurbita moschata by Luigi Barbieri; Letizia Polito; Andrea Bolognesi; Marialibera Ciani; Emanuele Pelosi; Valentina Farini; Ajay K. Jha; Neelam Sharma; Jorge M. Vivanco; Angela Chambery; Augusto Parente; Fiorenzo Stirpe (pp. 783-792).
The basic protein fraction of tissue extracts from 40 edible plants inhibited cell-free protein synthesis and released adenine from herring sperm DNA, thus having adenine glycosylase activity. This suggested the presence of ribosome-inactivating proteins (RIPs) in the plant extracts. This indication was further strengthened by the presence of the two activities after a partial chromatographic purification of three extracts, including that from Lycopersicon esculentum (tomato), which had very low activity. From the extract of Cucurbita moschata (pumpkin), the most active one, a glycoprotein of 30,665 Da was purified which had the properties of a RIP, in that (i) it inhibited protein synthesis by a rabbit reticulocyte lysate with IC50 (concentration giving 50% inhibition) 0.035 nM (1.08 ng ml−1) and by HeLa, HT29 and JM cells with IC50 in the 100 nM range, (ii) deadenylated hsDNA and other polynucleotidic substrates, and (iii) depurinated yeast rRNA at a concentration of 0.1 ng ml−1, all values being comparable to those of other RIPs. The C. moschata RIP gave a weak cross-reaction only with an antiserum against dianthin 32, but not with antisera against other RIPs, and had superoxide dismutase, antifungal and antibacterial activities.
Keywords: Cucurbita moschata; DNA glycosylase; Plant defence; Pumpkin; Ribosome-inactivating protein; Tomato
Oxidative status of valinomycin-resistant normal, β-thalassemia and sickle red blood cells by Johnny Amer; Zipora Etzion; Robert M. Bookchin; Eitan Fibach (pp. 793-799).
Exposure of red blood cells (RBC) to the K+-ionophore valinomycin (val), causes loss of KCl and water, resulting in cell dehydration, manifested by increased cell density. While almost all normal val-treated RBC dehydrate, in sickle cell anemia (SCA) a portion of the RBC fail to dehydrate and maintain a light density, indicating the existence of val-resistant ( val-res) RBC. In thalassemia and sickle cell disease (SCD), although the primary lesion is in the globin genes, damage to the RBC is partly mediated by oxidative stress. We previously showed that such RBC are under oxidative stress, having more reactive oxygen species (ROS) and less reduced glutathione than normal RBC. We now report a relationship between the phenomenon of val-res and the RBC oxidative status: Treatment with oxidants that increase ROS, also increased the frequency of val-res cells. Val-res cells had higher oxidative status than other RBC in the sample. Similar to SCA, thalassemic blood has more val-res cells than does normal blood. Val-res cells in thalassemic and sickle blood showed a higher oxidative status than normal val-res cells. Thus, oxidative stress might be involved in generation of val-res cells. Further studies are required to elucidate the origin and significance of these cells.
Keywords: RBC; Valinomycin; Free radical; Oxidant; Glutathione; Flow cytometry; Thalassemia; Sickle cell disease
Campest-5-en-3-one, an oxidized derivative of campesterol, activates PPARα, promotes energy consumption and reduces visceral fat deposition in rats by Ikuo Ikeda; Rie Konno; Takeshi Shimizu; Takashi Ide; Nobuyuki Takahashi; Teruo Kawada; Koji Nagao; Nao Inoue; Teruyoshi Yanagita; Tadateru Hamada; Yae Morinaga; Hiroko Tomoyori; Katsumi Imaizumi; Kunio Suzuki (pp. 800-807).
Dietary campest-5-en-3-one (campestenone), an oxidized derivative of campesterol, significantly reduced visceral fat weight and the concentration of triacylglycerol in serum and liver of rats. Dietary campestenone dramatically increased the activities and the mRNA expressions of mitochondrial and peroxisomal enzymes involved in β-oxidation in the liver. Campestenone activated human peroxisome proliferator-activated receptor (PPAR) α as determined using the novel GAL4 ligand-binding domain chimera assay system with coactivator coexpression. In contrast, dietary campestenone reduced the activities and the mRNA expressions of enzymes involved in fatty acid synthesis, except for the malic enzyme. Dietary campestenone decreased the sterol regulatory element binding protein-1 (SREBP-1) mRNA level. Energy expenditure was significantly higher in the feeding of campestenone in rats. Dietary campestenone reduced hepatic cholesterol concentration and increased fecal excretion of neutral steroids originated from cholesterol. Lymphatic absorption of cholesterol was reduced by the coadministration of campestenone in rats cannulated in the thoracic duct. These observations suggest a possibility that campestenone has an ability to prevent coronary heart disease by improving obesity and abnormality of lipid metabolism.
Keywords: Campest-5-en-3-one; Campesterol; PPARα; SREBP-1c; Visceral fat
Purification and characterization of a galactose-specific lectin with mitogenic activity from pinto beans by Jack H. Wong; Clarence C.T. Wong; T.B. Ng (pp. 808-813).
A galactose-specific dimeric lectin from pinto beans was purified using a procedure that involved affinity chromatography on Affi-gel blue gel, anion exchange chromatography on Q-Sepharose, fast protein liquid chromatography (FPLC)-ion exchange chromatography on Mono S, and FPLC-gel filtration on Superdex 200. The molecular mass of this homodimeric lectin was 62 kDa and that of each of its subunits was 31 kDa. The hemagglutinating activity of pinto bean lectin was stable within the pH range of 3–12 and the temperature range of 0–70 °C. By using the [3H-methyl]-thymidine incorporation assay, it was shown that the lectin had the ability to evoke a mitogenic response from murine splenocytes but it did not inhibit proliferation of L1210 leukemia cells. The pinto bean lectin inhibited HIV-1 reverse transcriptase with an IC50 of 3 μM.
Keywords: Lectin; Pinto bean; Isolation
A new dinuclear biphenylene bridged copper(II) complex: DNA cleavage under hydrolytic conditions by V. Uma; M. Kanthimathi; J. Subramanian; Balachandran Unni Nair (pp. 814-819).
The dinucleating ligand, tpbpd (tetrapyridyl biphenylenediamine) forms a dicopper complex with practically no electronic coupling between the two copper (II) centres. The EPR spectrum of the complex is consistent with coordination of each copper ion to two nitrogens of the binuclear ligand. Cyclic voltammogram of the complex also reveals that the two copper (II) centres have identical ligating environment. This dimeric copper (II) complex is found to be a very efficient catalyst for the cleavage of plasmid DNA in the absence of any added cofactor. The amount of conversion of supercoiled form (Form I) of plasmid to the open circular form (Form II) depends on the concentration of the complex as well as the duration of incubation of the complex with DNA. The maximum rate of conversion of the supercoiled form to the nicked circular form at pH 7.5 in the presence of 150 μM of the complex is found to be 1.8×10−3 s−1.
Keywords: Copper(II); Dinuclear complex; Biphenylene bridge; DNA hydrolytic cleavage
A carboxylesterase from the thermoacidophilic archaeon Sulfolobus solfataricus P1; purification, characterization, and expression by Young-Jun Park; Soo Young Choi; Hee-Bong Lee (pp. 820-828).
The carboxylesterase, a 34 kDa monomeric enzyme, was purified from the thermoacidophilic archaeon Sulfolobus solfataricus P1. The optimum temperature and pH were 85 °C and 8.0, respectively. The enzyme showed remarkable thermostability: 41% of its activity remained after 5 days of incubation at 80 °C. In addition, the purified enzyme exhibited stability against denaturing agents, including various detergents, urea, and organic solvents. The enzyme has broad substrate specificity towards various PNP esters and short acyl chain triacylglycerols such as tributyrin (C4:0). Among the PNP esters tested, the best substrate was PNP-caprylate (C8) with Km and kcat values of 71 μM and 14,700 s−1, respectively. The carboxylesterase gene consisted of 915 bp corresponding to 305 amino acid residues. We demonstrated that active recombinant S. solfataricus carboxylesterase could be expressed in Escherichia coli. The enzyme was identified as a serine esterase belonging to mammalian hormone-sensitive lipases (HSL) family and contained a catalytic triad composed of serine, histidine, and aspartic acid in the active site.
Keywords: Carboxylesterase; Thermostability; Chemostability; Archaea; Sulfolobus solfataricus
Location and characterization of the O-GlcNAcase active site by Clifford Toleman; Andrew J. Paterson; Jeffrey E. Kudlow (pp. 829-839).
NCOAT is a bifunctional nucleo-cytoplasmic protein with both O-GlcNAcase and histone acetyltransferase domains. The O-GlcNAcase domain catalyzes the removal of O-linked GlcNAc modifications from proteins and we have found that it resides in the N-terminal third of NCOAT. The recognition of the substrate GlcNAc suggests that the O-GlcNAcase is related in structure and catalytic mechanism to chitinases, hexosaminidases and hyaluronidases. These families of glycosidases all possess a catalytic doublet of carboxylate-containing residues, with one providing an acid-base function, and the second acting to orient and use the N-acetyl group of GlcNAc during catalysis. Indeed, we show that the O-GlcNAcase also possesses the catalytic doublet motif shared among these enzymes and that these two essential residues are aspartic acids at positions 175 and 177, respectively, in mouse NCOAT. In addition, a conserved cysteine at 166 and a conserved aspartic acid at 174 were also found to be necessary for fully efficient enzymatic activity. Given this information, we propose that the O-GlcNAcase active site resembles those of the above glycosidases which carry out the hydrolysis of GlcNAc linkages in a substrate-assisted acid–base manner.
Keywords: Post-translational modification; Mutagenesis