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PubMed2005

Characterization of the delta muH+-sensitive ubisemiquinone species (SQ(Nf)) and the interaction with cluster N2: new insight into the energy-coupled electron transfer in complex I.

In this report, we describe the electron paramagnetic resonance (EPR) spectroscopic characterizations of the fast-relaxing ubisemiquinone (SQ(Nf)) species associated with NADH-ubiquinone oxidoreductase (complex I) detected in tightly coupled submitochondrial particles (SMP). The signals of SQ(Nf) are observed only in the presence of delta muH+, whereas other slowly relaxing SQ species, SQ(Ns) and SQ(Nx), are not sensitive to delta muH+. In this study, we resolved the EPR spectrum of the delta muH+-sensitive SQ(Nf), which was trapped during the steady-state NADH-Q1 oxidoreductase reaction, as the difference between coupled and uncoupled SMP. Thorough analyses of the temperature profile of the resolved SQ(Nf) signals have revealed previously unrecognized spectra from delta muH+-sensitive SQ(Nf) species. This newly detected SQ(Nf) signals are observable only below 25 K, similar to the cluster N2 signals, and exhibit a doublet signal with a peak-to-peak separation (deltaB) of 56 G. In this work, we identify the partner to the interacting cluster N2. We have analyzed the g = 2.00 and g = 2.05 splittings using a computer simulation program that includes both exchange and dipolar interactions as well as the g-strain effect. Computer simulation of these interaction spectra showed that cluster N2 and fast-relaxing SQ(Nf) species undergo a spin-spin interaction, which contains both exchange (55 MHz) and dipolar interaction (16 MHz) with an estimated center-to-center distance of 12 A. This finding delineates an important functional role for this coupled [(N2)(red)-SQ(Nf)] structure in complex I, which is discussed in connection with electron transfer and energy coupling.

Animals↗
PubMed1996

Molecular cloning of human phosphomevalonate kinase and identification of a consensus peroxisomal targeting sequence.

Two overlapping cDNAs which encode human liver phosphomevalonate kinase (PMKase) were isolated. The human PMKase cDNAs predict a 191-amino acid protein with a molecular weight of 21,862, consistent with previous reports for mammalian PMKase (Mr = 21,000-22,500). Further verification of the clones was obtained by expression of PMKase activity in bacteria using a composite 1024-base pair cDNA clone. Northern blot analysis of several human tissues revealed a doublet of transcripts at approximately 1 kilobase (kb) in heart, liver, skeletal muscle, kidney, and pancreas and lower but detectable transcript levels in brain, placenta, and lung. Analysis of transcripts from human lymphoblasts subcultured in lipid-depleted sera (LDS) and LDS supplemented with lovastatin indicated that PMKase gene expression is subject to regulation by sterol at the level of transcription. Southern blotting indicated that PMKase is a single copy gene covering less than 15 kb in the human genome. The human PMKase amino acid sequence contains a consensus peroxisomal targeting sequence (PTS-1), Ser-Arg-Leu, at the C terminus of the protein. This is the first report of a cholesterol biosynthetic protein which contains a consensus PTS-1, providing further evidence for the concept that early cholesterol and nonsterol isoprenoid biosynthesis may occur in the peroxisome.

Amino Acid Sequence↗
PubMed1997

Phosphorylation of Opaque2 changes diurnally and impacts its DNA binding activity.

In the maize endosperm, the Opaque2 (O2) basic leucine zipper transcriptional activator regulates the expression of a subset of the zein seed storage protein gene family. Immunodetection of wild-type or mutant O2 polypeptides fractionated by SDS-PAGE resolved a closely spaced doublet migrating in the 68- to 72-kD range, whereas by using isoelectric focusing, seven to nine isoforms were detected for each allele. Phosphatase treatment simplified the protein patterns to a single band corresponding to the nonphosphorylated component. In vivo and in vitro labeling confirmed that O2 can be phosphorylated. In protein gel blots probed with DNA, only the nonphosphorylated and hypophosphorylated O2 polypeptides were able to bind an oligonucleotide containing the O2 binding sequence. Upon in situ dephosphorylation of the focused isoforms by phosphatase treatment of the isoelectric focusing filter, the hyperphosphorylated forms acquired DNA binding activity. The ratio among the various isoforms remained constant throughout the developmental stages of endosperm growth but changed from daytime to nighttime, with a significant increase of the hyperphosphorylated forms during the night period. These results indicate that O2 exists in vivo as a pool of differently phosphorylated polypeptides and demonstrate that O2 DNA binding activity is modulated by a phosphorylation/dephosphorylation mechanism that appears to be influenced by environmental conditions.

Circadian Rhythm↗
PubMed1993

Nicein (BM-600) in junctional epidermolysis bullosa: polyclonal antibodies provide new clues for pathogenic role.

We have raised polyclonal antibodies against each of three subunits of the new basement membrane component nicein (formerly BM-600), the antigen recognized by the monoclonal antibody GB3 (Biochem Biophys Acta 942:45-56, 1988). Preparation of such antibodies was achieved from gel electrophoresis purification of nicein isolated by immuno-affinity chromatography. These antibodies were reactive with each transblotted denatured nicein subunit and recognized the native protein both in cultured keratinocytes and in all normal human basement membranes where the GB3 antigen is located. A reciprocal immuno-cross-reactivity was detected with the antibodies directed against the 100-kD and 150-kD (sometimes resolved as a 146-150-kD doublet) subunits of nicein, showing that they share some identical epitopes. In tissues and keratinocyte cultures from patients with the Herlitz form of junctional epidermolysis bullosa (H-JEB), GB3 is unable to recognize nicein, and the question arises whether this is due to an absence of synthesis or a structural abnormality of the protein. We report here that the polyclonal antibody directed against the 150-kD subunit of nicein binds its antigen in H-JEB patients (although usually less intensely than in control skin), whereas the other two antibodies either do not recognize or recognize only weakly their respective antigen subunits. These data suggest that nicein is present but structurally altered in basement membranes from H-JEB tissues. Furthermore, in non-Herlitz junctional and dystrophic types of epidermolysis bullosa, all three polyclonal antibodies recognize their antigens normally. Consequently, such antibodies should serve as potentially useful molecular tools for studying the expression of nicein in H-JEB.

Antibodies↗
PubMed1996

Detection of a stable free radical in the B2 subunit of the manganese ribonucleotide reductase (Mn-RRase) of Corynebacterium ammoniagenes.

Ribonucleotide reductases catalyze the irreversible reductive formation of 2'-deoxyribonucleotides required for DNA replication and cell proliferation, and a radical mechanism was assumed to be involved in this reaction. In order to search for a radical in the aerobic manganese ribonucleotide reductase (Mn-RRase) by electron paramagnetic resonance (EPR) the native metal-containing 100 kDa B2 subunit was deliberately prepared from the wild type strain Corynebacterium ammoniagenes ATCC 6872. Enrichment by 2'5'-ADP Sepharose 4B affinity chromatography, fast protein liquid chromatography (FPLC) with SuperoseTM12 and concentration by vacuum evaporation allowed for the first time the detection of a stable free radical by EPR spectroscopy at 77 K. The EPR spectrum exhibits an easily saturable doublet of 1.8 mT splitting and a line width of 1.3 mT at g = 2.0040. The EPR signal intensity showed a clear correlation with the enzymatic activity upon long-time storage at ambient temperature (294 K) and inactivation by the specific RRase inhibitor hydroxyurea (HU). This leads to the assumption of a protein-linked radical, with functional significance, in the metal-containing 100 kDa B2 subunit of the MnRRase of Corynebacterium ammoniagenes.

Bacterial Proteins↗
PubMed2005

Expression and intracellular localization of Pyk2 in normal and v-src transformed chicken epiphyseal chondrocytes.

The expression and localization of prolin-rich tyrosine kinase 2 (Pyk2) were studied in chick embryo epiphyseal chondrocytes. Two immunoreactive bands were detected in chondrocytes, a major band with an apparent Mr of 123 kDa and a minor band with an apparent Mr of 68 kDa. The major band appears to migrate as a doublet with apparent Mr of 116/123 kDa. Increased levels of the three forms of Pyk2 were observed in v-src transformed chondrocytes as compared to control uninfected chondrocytes. Immunofluorescent staining shows that Pyk2 is clearly visible in the cytosol and in the perinuclear region of control and v-src-chondrocytes and displays a pattern very similar to the distribution of the mitochondrial marker Mito Tracker. More, immunofluorescent staining shows that Pyk2 is nuclear in most chondrocytes. By subcellular fractionation, the p116/123 Pyk2 doublet, was found to be accumulated mainly in the cytoplasm while the p68 Pyk2 form, was found to be accumulated exclusively in the nucleus. The differential nuclear/cytoplasmic distribution of the Pyk2 forms remains unchanged after v-Src-induced transformation. The p68 Pyk2 form could no longer be detected by using a N-terminus domain-specific anti-Pyk2 antibody. Consistently, Pyk2 immunoreactivity was restricted to the cytoplasm of control and v-src transformed chondrocytes. Thus it appears that the p68 Pyk2 form that accumulates in the nucleus has a deletion in the N-terminus region.

Animals↗
PubMed1999

Direct EPR detection of the carbonate radical anion produced from peroxynitrite and carbon dioxide.

The biological effects of peroxynitrite have been recently considered to be largely dependent on its reaction with carbon dioxide, which is present in high concentrations in intra- and extracellular compartments. Peroxynitrite anion (ONOO-) reacts rapidly with carbon dioxide, forming an adduct, nitrosoperoxocarboxylate (ONOOCO2-), whose decomposition has been proposed to produce reactive intermediates such as the carbonate radical (CO-3). Here, by the use of rapid mixing continuous flow electron paramagnetic resonance (EPR), we directly detected the carbonate radical in flow mixtures of peroxynitrite with bicarbonate-carbon dioxide over the pH range of 6-9. The radical was unambiguously identified by its EPR parameters (g = 2.0113; line width = 5.5 G) and by experiments with bicarbonate labeled with 13C. In this case, the singlet EPR signal obtained with 12C bicarbonate splits into the expected doublet because of 13C (a(13C)= 11.7 G). The singlet spectrum of the unlabeled radical was invariant between pH 6 and 9, confirming that in this pH range the detected radical is the carbonate radical anion (CO-3). Importantly, in addition to contributing to the understanding of nitrosoperoxocarboxylate decomposition pathways, this is the first report unambiguously demonstrating the formation of the carbonate radical anion at physiological pHs by direct EPR spectroscopy.

Carbon Dioxide↗
PubMed1988

The identification of biologically important thiols.

A new mass spectral method has been developed and used to identify biologically important thiols. This method is based upon the use of C-14 labeled N-para-bromophenylmaleimide (BPM) to selectively derivatize thiols. The isotopic label facilitates the isolation and purification of trace quantities of the maleimide thiol adducts by high performance liquid chromatography (HPLC) with radioactivity detection. The use of the bromine atom in the thiol derivative greatly enhances the detectability of the parent and fragment ions containing the BPM moiety because of the generation of the characteristic N + 2 doublet ions. Several examples of the use of this method to identify non-volatile thiol peptides such as glutathione (GSH) and homoglutathione (hGSH) by fast atom bombardment mass spectrometry (FAB-MS) are described in this report.

Carbon Radioisotopes↗
PubMed1995

Onchocerca spp: a "family" of secreted acidic proteins expressed by infective larvae in blackflies.

Biosynthetic labeling of developing larvae of Onchocerca in blackflies has been used to characterize a group of stage-specific, secreted proteins produced by vector-stage parasites. These are highly acidic molecules (pI 4.4-5.1) present in at least three members of the genus (O. volvulus, O. lienalis, O. ochengi) that exhibit minor heterogeneity among species in apparent molecular mass (between 18 and 23 kDa). In O. volvulus, there are two polypeptides that run as a doublet of 18 and 20 kDa. In O. lienalis and O. ochengi, single polypeptides of 23 and 20 kDa were detected. The processes of synthesis and secretion appear to be temperature-sensitive and dissociated events. Experiments with O. volvulus in Simulium damnosum sl revealed that synthesis is initiated in second stage larvae and increases in infective-stage parasites: Secretion occurs when larvae leave the vector and enter the phase of development associated with the vertebrate host. Third-stage larvae of O. lienalis were shown to continue to express and accumulate the 23-kDa protein with age. The primary organ of secretion, as indicated by dissection, was the glandular esophagus. These data point to an important biological role for this group of molecules and suggest that they may belong to a family of related products. Because they have the distinctive characteristics of being secreted larval acidic proteins, we propose the acronym SLAP pending further insights into their functional properties.

Animals↗
PubMed1992

Analysis by immunoblotting of Toxoplasma gondii exo-antigens and comparison with somatic antigens.

Two soluble Toxoplasma gondii antigen preparations were compared using the immunoblotting technique. The first preparation, which is commonly employed in ELISA tests, corresponds to a lytic extract of the parasite. The second known as exo-antigen, has yielded good results in cell immunity research and is obtained from Toxoplasma gondii culture supernatants. In both preparations, components exhibiting same molecular weight of 57-52 (doublet), 43, 38, 35, 30 and 20 kDa were revealed. In addition, two major 27- and 78-kDa components were detected in exo-antigens. The 30 kDa protein was intensely recognised by all sera, confirming the advantage of its use as an antigen in serological reactions. Exo-antigens could be employed as a soluble antigenic solution because they are easy to obtain, they display good antigenicity and their antigenic composition has been defined. Moreover their preparation does not require mouse inoculation. An evaluation of the enzyme-linked immunosorbent assays (ELISAs) involving these antigens has yet to be performed.

Animals↗
PubMed1997

Paramagnetic NMR shifts in cyanoferricytochrome c. Investigation of thermal stability and deviations from Curie law behaviour.

The paramagnetic shifts of 13C nuclei positioned alpha to the haem in cyanoferricytochrome c are reported and analysed in terms of molecular orbitals based on D4h symmetry with a rhombic perturbation. The temperature dependence of the Fermi contact and dipolar shifts of the haem and axial histidine ligand show deviations from Curie Law behaviour which are explained by a Boltzmann distribution between partially filled 3e(pi) molecular orbitals and the ground and first excited state Kramers doublets. The comprehensive explanation of the temperature dependence of the paramagnetic shifts leads to the conclusion that there is no detectable temperature dependence of the haem orientation or that of the His ligand orientation. This work also provides evidence for the role of the axial His ligand in determining the orientation of the magnetic z-axis.

Cytochrome c Group↗
PubMed1994

Vasoactive intestinal peptide induces tyrosine phosphorylation in PC12h cells.

Vasoactive intestinal peptide (VIP) is a neuropeptide that induces neuronal differentiation through a cAMP-dependent mechanism. We have previously shown that VIP induces tyrosine phosphorylation and activation of MAP kinases in PC12h cells [J. Biochem. 115, 304-308 (1994)]. In the present study, we showed by Western blotting with anti-phosphotyrosine antibodies that in PC12h cells VIP induced tyrosine phosphorylation of proteins of 140, 120, 110, and 70 kDa in addition to MAP kinases. The immunoprecipitates with anti-phosphotyrosine antibody from VIP-treated cells contained high activity of protein kinase phosphorylating poly(glu-tyr) and enolase; the activity from VIP-stimulated cells was 1.5-2 times higher than that from unstimulated cells. In vitro kinase reaction without extrinsic substrates resulted in tyrosine phosphorylation of doublet proteins which migrated slower than pp125FAK on SDS-PAGE. An increase in kinase activity of the immunecomplex was detected when the cells were stimulated with forskolin. These results suggest that protein tyrosine phosphorylation is involved in differentiation of neuronal cells stimulated by VIP and that it is regulated by a cAMP-dependent mechanism.

Animals↗
PubMed1997

The expression of the secreted protein acidic and rich in cysteine (SPARC) is associated with the neoplastic progression of human melanoma.

SPARC (secreted protein acidic and rich in cysteine) is an extracellular protein associated with tissues exhibiting high rates of cell proliferation and matrix remodeling. The current work shows that the human melanoma cell lines IIB-MEL-LES, IIB-MEL-IAN, and IIB-MEL-J and different human metastatic melanomas expressed high levels of SPARC mRNA and protein. By western blot analysis we detected a single secreted 42-kDa band in human diploid fibroblasts-conditioned medium and a 45- to 40-kDa doublet in the three melanoma cell lines and all the metastatic melanomas tested. Part of the melanoma samples and cell lines showed an additional doublet of 36-34 kDa. SPARC mRNA was expressed by the three established cell lines, 14 metastatic melanoma samples, and tumors raised in nude mice, and no spliced variants were found. The heterogeneous pattern of SPARC secreted by human melanoma cells is the result of post-translational glycosylation and a specific extracellular leupeptin-inhibitable cleavage. Unlike human fibroblasts, melanoma cells did not overexpress SPARC on addition of TGF-beta. Immunohistochemical analysis showed that SPARC was strongly expressed in 100% of primary melanomas (7 of 7) and metastatic melanomas (29 of 29), moderately expressed in most of the positive dysplastic nevi (13 of 14), and only weakly expressed in nevocellular nevi (4 of 25). Normal melanocytes did not express SPARC. The data suggest that the expression of SPARC is associated with the neoplastic progression of human melanoma.

Cell Transformation, Neoplastic↗
PubMed2003

Localization of a symbiosis-related protein, Sym32, in the Anthopleura elegantissima-Symbiodinium muscatinei Association.

Cnidarian-dinoflagellate symbioses are widespread in the marine environment. Growing concern over the health of coral reef ecosystems has revealed a fundamental lack of knowledge of how cnidarian-algal associations are regulated at the cellular and molecular level. We are interested in identifying genes that mediate interactions between the partners, and we are using the temperate sea anemone Anthopleura elegantissima as a model. We previously described a host gene, sym32, encoding a fasciclin domain protein, that is differentially expressed in symbiotic and aposymbiotic A. elegantissima. Here, we describe the subcellular localization of the sym32 protein. In aposymbiotic (symbiont-free) hosts, sym32 was located in vesicles that occur along the apical edges of gastrodermal cells. In symbiotic hosts, such vesicles were absent, but sym32 was present within the symbiosome membranes. Sym32 (or a cross-reactive protein) was also present in the accumulation bodies of the symbionts. Although the anti-sym32 antiserum was not sufficiently specific to detect the target protein in cultured Symbiodinium bermudense cells, Western blots of proteins from two Symbiodinium species revealed a protein doublet of 45 and 48 kDa, suggesting that the symbionts may also produce a fasciclin domain protein. We suggest that host sym32 is relocated from gastrodermal vesicles to the symbiosome membrane when symbionts are taken into host cells by phagocytosis.

Animals↗
PubMedDate not supplied

A mechanical cycling phenomenon in electrofused eryhrocytes.

We have characterized a reciprocating mechanical oscillation that can easily be detected in a large fraction of electrofused erythrocytes. Under our conditions, up to about 30% of all electrofusion products (e.g. doublet, triplet, and higher) show at least one cycle and up to 10% show only two cycles. A much smaller fraction (about 1-2%) show 5-10 or more cycles before stopping. In fused doublets the oscillation appears as a roughly linear and slow expansion of the diameter of the 'hourglass constriction' or fusion zone which proceeds simultaneously with a slow contraction of the pole-to-pole length. At what appears to be a threshold, the fusion zone diameter shrinks simultaneously with an expansion in the pole-to-pole length. This takes place rapidly (within a few video frames). The change in length is about 10% and is easily observable by video light microscopy. The periodicity is variable (5-60 s) and subsequent periods often decrease substantially in length. For the range studied, the characteristics of the reciprocation do not appear to be dependent on the strength of the electric field pulse. To our knowledge this phenomenon was originally discovered, but not characterized, in another laboratory. Using our protocol, cells were observed to undergo fusion from as soon as 6, to as late as 84 s after the fusogenic electric pulse was applied.

Animals↗
PubMed1976

[Membrane glycoproteins and platelet protein glycosylation in chronic myeloid leukemia].

SDS-polyacrylamide gel electrophoresis of solubilized whole platelets and isolated platelet membranes from patients with chronic myeloid leukemia (CML) as compared to the normal platelet-Schiff-stained glycoproteins (GP) (noted GP I, II, III of app. MW 155, 130 and 100.10(3) daltons, respectively), showed the following modifications: the decrease of GP I; the presence of the GP II and GP IIx doublet; the moderate and variable decrease of the GP III; the increase of the platelet glycolipid. No significant differences were detectable on the polypeptide patterns from CML platelets in respect to the normal 20-25 protein constituents. Using a radio-isotopic method it has been measured as an elevated catalytic transfer of the galactosyl and N-acetylgalactosaminyl residues from nucléotide 14C sugar derivatives onto the endogenous acceptors of platelets from CML. The results obtained suggested that CML platelets with recognized abnormal functions, have modifications at the level of the macromolecular surface glycoprotein constituents and/or changes of the glycoconjugates reactive structures involved in the devopment of the periodic acid-Schiff reaction as also in the transfer reaction of the sugar residues.

Acetylgalactosamine↗
PubMed1993

Phenotype analysis of cycling and postcycling thymocytes: evaluation of detection methods for BrdUrd and surface proteins.

We present a comparison of two different methods for simultaneous detection of bromodeoxyuridine and cell surface markers. Both methods use enzymatic generation of single-strand DNA with nuclease. The biological system used is the murine thymus, in which in vivo DNA synthetizing cells were labeled by injection of BrdUrd and analyzed at different time points after the nucleoside pulse. The surface proteins detected were CD4 and CD8 differentiation markers and the T-cell receptor. Extraction of DNA-associated proteins with 0.1N HCl and detergent is necessary for the action of EcoR1 and Exonuclease III, but this treatment destroys phycocyanins and induces cell aggregation, as shown using the doublet-discrimination module. For DNAse I action, cells could be treated with paraformaldehyde and a low concentration of Tween 20, and this treatment was adequate for surface staining preservation (even with phycocyanins) and BrdUrd detection. Both methods were adequate for cell cycle studies, but only 7-amino-actinomycin D could be used as total DNA dye after DNAse action, and good results needed long (48-72 h) incubation in the fixative-detergent mixture. The DNAse I method now allows three-color staining (two surface markers and Brd-Urd), analyzed in a one laser-cytometer for the study of the phenotype of cycling cells, and of their progeny, in vivo and in cell cultures. It also allows the quantitative analysis of cell surface receptor densities in conditions similar to fresh cells.

Animals↗
PubMed2004

Stable-isotope trapping and high-throughput screenings of reactive metabolites using the isotope MS signature.

A highly efficient method has been developed to detect and identify reactive metabolites, using stable-isotope trapping combined with ESI-MS/MS neutral loss scanning. A mixture of glutathione (GSH, gamma-glutamylcysteinylglycine) and the stable-isotope labeled compound (GSX, gamma-glutamylcysteinylglycine-(13)C(2)-(15)N) was used at an equal molar ratio to trap reactive metabolites generated in microsomal incubations. Samples resulting from incubations were cleaned and concentrated by SPE, followed by LC-MS/MS analyses using constant neutral loss scanning for 129 Da (the gamma-glutamyl moiety) to detect formed GSH conjugates. Unambiguous identification of glutathione adducts was greatly facilitated by the presence of a unique MS signature of a prominent isotopic doublet that differs in mass by 3 Da. Further structural characterization of conjugates was achieved with high confidence by subsequently acquiring MS/MS spectra that were featured by neutral losses of 75 and 129 Da for GSH adducts and 78 and 129 Da for isotopic GSX adducts. The reliability of this method was vigorously validated using a number of compounds known to form reactive metabolites. Superior sensitivity was demonstrated by the capability of the current approach to identify reactive metabolites at low abundance. Because of the unique isotopic MS signature, ultrafast analyses of reactive metabolites were accomplished by direct injection of cleaned samples into mass spectrometers for neutral loss scanning. More importantly, this study has demonstrated the feasibility of the current method for completely automated detection of reactive metabolites via computer-assisted pattern recognition.

Carbon Isotopes↗