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PubMed2005

Laminin gamma3 chain binds to nidogen and is located in murine basement membranes.

Recently a novel laminin gamma3 chain was identified in mouse and human and shown to have the same modular structure as the laminin gamma1 chain. We expressed two fragments of the gamma3 chain in mammalian cells recombinantly. The first, domain VI/V, consisting of laminin N-terminal (domain VI) and four laminin-type epidermal growth factor-like (domain V) and laminin N-terminal modules, was shown to be essential for self-assembly of laminins. The other was domain III3-5, which consists of three laminin-type epidermal growth factor-like modules and is predicted to bind to nidogens. The gamma3 VI/V fragment was a poor inhibitor for laminin-1 polymerization as was the beta2 VI/V fragment. The gamma3 III3-5 fragment bound to nidogen-1 and nidogen-2 with lower affinity than the gamma1 III3-5 fragment. These data suggested that laminins containing the gamma3 chain may assemble networks independent of other laminins. Polyclonal antibodies raised against gamma3 VI/V and gamma3 III3-5 showed no cross-reaction with homologous fragments from the gamma1 and gamma2 chains of laminin and allowed the establishment of gamma chain-specific radioimmunoassays and light and electron microscopic immunostaining of tissues. This demonstrated a 20-100-fold lower content of the gamma3 chain compared with the gamma1 chain in various tissue extracts of adult mice. The expression of gamma3 chain was highly tissue-specific. In contrast to earlier assumptions, the antibodies against the gamma3 chain showed light microscopic staining exclusively in basement membrane zones of adult and embryonic tissues, such as the brain, kidney, skin, muscle, and testis. Ultrastructural immunogold staining localized the gamma3 chain to basement membranes of these tissues.

Animals↗

PubMed1995

Kinetics of nucleotide-induced changes in the tryptophan fluorescence of the molecular chaperone Hsc70 and its subfragments suggest the ATP-induced conformational change follows initial ATP binding.

The kinetics of nucleotide-induced changes of tryptophan fluorescence have been measured for recombinant bovine 70 kDa heat shock cognate protein (Hsc70), a 60 kDa subfragment (amino acid residues 1-554) which has ATPase and peptide binding activities, and a 44 kDa subfragment (residues 1-386) which has only ATPase activity. The fluorescence changes resulting from ATP binding to Hsc70 and the 60 kDa fragment are biphasic, and can be interpreted as arising from a two-step process in which ATP initially binds in a bimolecular reaction, followed by a conformational change of the protein-MgATP complex. Fluorescence changes resulting from ADP binding indicate a single-step, bimolecular process. Under single-cycle conditions of the ATPase reaction, a fluorescence change is observed whose rate constant correlates with product release in Hsc70, and with product release/ATP hydrolysis (which are kinetically indistinguishable under single-cycle conditions) in the 60 kDa fragment. These data support a scheme for Hsc70 in which a conformational transition is induced after initial ATP binding but prior to hydrolysis, and the reverse transition is induced by product release. The 60 kDa fragment shows behavior that is quantitatively similar to that of Hsc70. The 44 kDa ATPase fragment does not show biphasic kinetics for ATP binding, and does not show fluorescence changes that suggest conformational changes of the type seen in Hsc70 and the 60 kDa fragment.

Adenosine Triphosphate↗

PubMed2006

Nuclear translocation of endonuclease G and apoptosis-inducing factor during acetaminophen-induced liver cell injury.

Mitochondrial dysfunction and internucleosomal DNA fragmentation are well-recognized features of acetaminophen (AAP)-induced hepatocyte cell death. However, the endonucleases responsible for this effect have not been identified. Apoptosis-inducing factor (AIF) and endonuclease G are nucleases located in the intermembrane space of mitochondria. AIF is thought to trigger chromatin condensation and induce cleavage of DNA into high molecular weight fragments (50-300 kb), and endonuclease G can produce oligonucleosomal DNA fragments. Therefore, the objective of this investigation was to test the hypothesis that endonuclease G and AIF could be involved in AAP-induced nuclear DNA fragmentation. Using immunofluorescence microscopy, it was shown that in primary cultured mouse hepatocytes, endonuclease G and AIF translocated to the nucleus between 3 and 6 h after exposure to 5 mM AAP. In contrast, other mitochondrial intermembrane proteins such as cytochrome c or the second mitochondria-derived activator of caspases (Smac) did not accumulate in the nucleus. The translocation of AIF and endonuclease G correlated with mitochondrial dysfunction as indicated by the progressive loss of the mitochondrial membrane potential (measured with the JC-1 assay) and the appearance of nuclear DNA fragments in the cytosol (determined by an anti-histone ELISA). Pretreatment with 20mM N-acetylcysteine prevented mitochondrial dysfunction, the nuclear translocation of endonuclease G and AIF, and the nuclear DNA fragmentation. The data support the conclusion that endonuclease G and AIF translocate to the nucleus in response to AAP-induced mitochondrial dysfunction and may be responsible, at least in part, for the initial DNA fragmentation during AAP hepatotoxicity.

Acetaminophen↗

PubMed2002

Structural influence of hanatoxin binding on the carboxyl terminus of S3 segment in voltage-gated K(+)-channel Kv2.1.

The voltage-sensing domains of voltage-gated potassium channels Kv2.1 (drk1) contain four transmembrane segments in each subunit, termed S1 to S4. While S4 is known as the voltage sensor, the carboxyl terminus of S3 (S3C) bears a gradually broader interest concerning the site for gating modifier toxins like hanatoxin and thus the secondary structure arrangement as well as its surrounding environment. To further examine the putative three-dimensional (3-D) structure of S3C and to illustrate the residues required for hanatoxin binding (which may, in turn, show the influence on the S4 in terms of changes in channel gating), molecular simulations and dockings were performed. These were based on the solution structure of hanatoxin and the structural information from lysine-scanning results for S3C fragment. Our data suggest that several basic and acidic residues of hanatoxin are electrostatically and stereochemically mapped onto their partner residues on S3C helix, whereas some aromatic or hydrophobic residues located on the same helical fragment interact with the hydrophobic patch of the toxin upon binding. Therefore, a slight distortion of the S3C helix, in a direction toward the N-terminus of S4, may exist. Such conformational change of S3C upon toxin binding is presented as a possible explanation for the observed shift in hanatoxin binding-induced gating.

Animals↗

PubMed1998

Assessment and requirements of nuclear reaction databases for GCR transport in the atmosphere and structures.

The transport properties of galactic cosmic rays (GCR) in the atmosphere, material structures, and human body (self-shielding) are of interest in risk assessment for supersonic and subsonic aircraft and for space travel in low-Earth orbit and on interplanetary missions. Nuclear reactions, such as knockout and fragmentation, present large modifications of particle type and energies of the galactic cosmic rays in penetrating materials. We make an assessment of the current nuclear reaction models and improvements in these model for developing required transport code data bases. A new fragmentation data base (QMSFRG) based on microscopic models is compared to the NUCFRG2 model and implications for shield assessment made using the HZETRN radiation transport code. For deep penetration problems, the build-up of light particles, such as nucleons, light clusters and mesons from nuclear reactions in conjunction with the absorption of the heavy ions, leads to the dominance of the charge Z = 0, 1, and 2 hadrons in the exposures at large penetration depths. Light particles are produced through nuclear or cluster knockout and in evaporation events with characteristically distinct spectra which play unique roles in the build-up of secondary radiation's in shielding. We describe models of light particle production in nucleon and heavy ion induced reactions and make an assessment of the importance of light particle multiplicity and spectral parameters in these exposures.

Aerospace Medicine↗

PubMed1975

The primary structure of allergen M from cod.

The complete primary structure of allergen M of cod (Gadus callarias L.) is presented. The amino acid sequence of fragment TM1, the NH2-terminal peptide of allergen M, was elucidated by the dansyl-Edman method. It consists of 75 amino acids and 1 glucose residue (mol. wt. 8,492). By summation of the sequence data of fragment TM1 and the previously reported fragment TM2, the intact allergen M has 113 residues (mol. wt. 12,328). Fragment TM1 of cod shows less homology (30.6 percent) with the corresponding fragments of other reported fish species than does fragment TM2 (42.1 percent); the intact allergen M shows 34.5 percent homology. The single half cystine of allergen M was shown to be blocked. Gas chromatographic analysis of the reduced and nonreduced allergen M suggested that the glucose is bound to Cys 18 through an S-glucosidic bond.

Allergens↗

PubMed2004

SUR2A C-terminal fragments reduce KATP currents and ischaemic tolerance of rat cardiac myocytes.

C-terminal fragments of the sulphonylurea receptor SUR2A can alter the functional expression of cloned ATP-sensitive K(+) channels (K(ATP)). To investigate the protective role of K(ATP) channels during metabolic stress we transfected SUR2A fragments into adult rat cardiac myocytes. A fragment comprising residues 1294-1358, the A-fragment, reduced sarcolemmal K(ATP) currents by over 85% after 2 days (pinacidil-activated current densities were: vector alone 7.04 +/- 1.22; and A-fragment 0.94 +/- 0.07 pA pF(-1), n= 6,6, P < 0.001). An inactive fragment (1358-1545, current density 6.30 +/- 0.85 pA pF(-1), n= 6) was used as a control. During metabolic inhibition (CN and iodoacetate) of isolated myocytes stimulated at 1 Hz, the A-fragment delayed action potential shortening and contractile failure, but accelerated rigor contraction and increased Ca(2+) loading. On reperfusion, A-fragment-transfected cells also showed increased intracellular Ca(2+) and the proportion of cells recovering contractile function was reduced from 40.0 to 9.5% (P < 0.01). The protective effect of pretreatment with 2,4-dinitrophenol, measured from increased functional recovery and reduced Ca(2+) loading, was abolished by the A-fragment. Our data are consistent with a role for K(ATP) channels in causing action potential failure and reduced Ca(2+) loading during metabolic stress, and with a major role in protection by preconditioning. The effects of the A-fragment may arise entirely from reduced expression of the sarcolemmal K(ATP) channel, but we also discuss the possibility of mitochondrial effects.

2,4-Dinitrophenol↗

PubMed1975

Cleavage of DNA in nuclei and chromatin with staphylococcal nuclease.

Treatment of either rat liver chromatin or intact nuclei with the enzyme staphylococcal nuclease results in the conversion of about half of the DNA to acid-soluble oligonucleotides. As previously described, mild digestion of nuclei results in the liberation of a series of nucleoprotein particles containing DNA fragments which are all integral multiples of a unit length DNA 185 base pairs in length. Analysis of the kinetics of appearance of these fragments suggests that at least 85% of the nuclear DNA is involved in the formation of the repeating subunit profile. More extensive digestion of nuclei however results in the generation of a series of eight unique DNA fragments containing 160 to 50 base pairs. The series of smaller molecular weight DNA is virtually identical with the profile obtained upon limit digestion of isolated chromatin. By velocity centrifugation we have obtained highly purified preparations of the monomeric nucleoprotein particle. Digestion of this monomeric subunit results in the solubilization of 46% of the DNA and analysis of the resistant DNA again reveals the set of eight lower molecular weight fragments. These data suggest that the initial site of nuclease cleavage in chromatin resides within the DNA bridging the repeating monomeric subunits. Further attack results in cleavage at a set of sites within the monomer liberating a pattern of smaller DNA fragments which probably represents the points of intimate contact between the histones and DNA.

Animals↗

PubMed1994

Detection of DNA point mutations with DNA mismatch repair enzymes.

We have developed a simple and reliable procedure to screen gene mutations using DNA mismatch repair (MR) specific mut Y enzyme of Escherichia coli and thymidine DNA glycosylase from HeLa cells. The mut Y enzyme cleaves A of G/A mismatches in DNA duplex and thymidine glycosylase cleaves T at G/T mismatches. Previously, we showed the determination of G:C-->T:A mutations in the N-ras gene in two human tumor samples with mut Y G/A MR enzyme. As low as 1-2% mutant DNAs in a sample of mutant and wild-type DNA can be detected with a synthetic DNA to create G/A mispairing for the assay. In this paper, we simplify the assay, include G/T MR thymidine glycosylase from HeLa cells and evaluate the application for screening DNA point mutations of p53 and ras genes. In this method, DNA fragments amplified from normal and mutated genes by polymerase chain reaction (PCR) were mixed and annealed to create DNA mismatches for cleavage by mismatch repair enzymes. The cleaved products and the substrates were separated by gel electrophoresis and detected by autoradiography. In theory, the enzymes that cut G/A or G/T mispairs will detect the mutations of G:C-->A:T, A:T-->G:C, G:C-->T:A and T:A-->G:C. Several human tumor samples were examined for p53 or K-ras mutations with G/A and G/T mismatch repair enzymes. The reliability of mutation detection was evaluated by comparing the results with reported mutations or confirmed by DNA sequencing of the same PCR-amplified DNA fragments. Our data showed that, following mismatch repair enzyme cleavage, all mutated DNA samples yielded cleaved products with sizes as expected. In addition, our assay is able to characterize the nature of mutation by 5' end-labeling of 32P on mutant or wild-type DNA fragments. The low background, reliability and the determination of the sites of mutations as well as the types of DNA base changes indicate the advantages of the method over other techniques in testing DNA mutants.

Base Sequence↗

PubMed1976

Why sleep?

Alert clinicians realize that much more data are fragmentedly available than has coherently been assimilated for practical application to the understanding of sleep--its vital function, its vicissitudes and its values. Now may be the time to add to sleep research, a dimension of field trips away from the labs. Cross cultural data might also be correlated, bringing social anthropologists and sleep physiologists together for didactic exchange.

Enuresis↗

PubMed2006

ESI-QqTOF-MS/MS and APCI-IT-MS/MS analysis of steroid saponins from the rhizomes of Dioscorea panthaica.

Using high-resolution quadrupole time-of-flight mass spectrometry along with an electrospray ionization source (ESI-QqTOF-MS), accurate molecular weights of 13 steroid saponins extracted from the rhizomes of Dioscorea panthaica were acquired and the corresponding molecular formulae obtained. In order to elucidate the fragmentation pathways of steroid saponins in D. panthaica, 10 authentic samples were investigated using ESI-QqTOF-MS/MS. In addition, atmospheric pressure chemical ionization mass spectrometry combined with ion trap tandem mass spectrometry (APCI-IT-MS/MS) was used to analyze the structures of 13 steroid saponins in D. panthaica. Through the analysis of their tandem mass data, diagnostic fragment ions of the spirostanol and furostanol steroid saponins in D. panthaica were detected as m/z 271.2056 and 253.1951. In addition, four pairs of isomers were detected and the possible structures of four unknown steroid saponins in D. panthaica speculated. ESI-TOF and APCI-MS(n) have proved to be effective tools for research on fragmentation mechanism of steroid saponins and the rapid determination of native steroid saponins in extract mixture, thereby avoiding tedious derivation and separation steps.

Carbohydrate Sequence↗

PubMed1996

Endothelial cells synthesize and process apolipoprotein B.

We reported previously that a 116-kDa lipoprotein lipase (LPL)-binding protein from endothelial cells has sequence homology to the amino-terminal region of apolipoprotein (apo) B. We now tested whether endothelial cells synthesize apoB mRNA and protein. Primers were designed to the human apoB cDNA sequence and reverse transcription polymerase chain reaction was performed using total RNA isolated from bovine and human endothelial cells. With primers to the 5' region of the apoB mRNA (amino-terminal region of apoB protein) expected size PCR products were generated from both bovine and human endothelial cells as well as from mouse liver RNA, which was used as a control. Primers designed to the 3' region of apoB mRNA generated PCR products from human endothelial cells and HepG2 cells but not from bovine or mouse cells. These data suggest that endothelial cells contain full-length apoB mRNA and that the 5' or the amino-terminal region of apoB is highly conserved from mouse to human. This was confirmed by direct sequencing of the mouse and bovine PCR products. To test whether apoB protein was produced, bovine endothelial cell proteins were metabolically labeled with [35S]methionine/cysteine or [3H]leucine and immunoprecipitated with anti-human apoB antibodies. Using extracts from cells labeled for 1 h, monoclonal antibody 47, directed to the low density lipoprotein receptor binding region of apoB, precipitated a protein of approximate molecular mass 550,000, the size of full-length apoB. Immunoprecipitation of the 550-kDa protein was abolished in the presence of added unlabeled low density lipoprotein. From cells labeled for 16 h, a 116-kDa protein was immunoprecipitated by polyclonal anti-apoB antibodies. This protein was partly released from cells by heparin treatment. Pulse-chase analysis showed that the 116-kDa fragment appeared at the same time as the full-length apoB began disappearing. The immunoprecipitated 116-kDa fragment also bound labeled LPL on ligand blot, further suggesting that it is an amino-terminal fragment of apoB. Incubation of endothelial cells with oleic acid (0.25 and 0.5 mM) did not significantly alter the production of either the full-length apoB or the 116-kDa fragment. These data show that endothelial cells synthesize apoB. The full-length apoB appears to be cleaved to form a 116-kDa fragment that can function as a LPL-binding protein.

Amino Acid Sequence↗

PubMed1987

Proteolysis of smooth muscle myosin light chain kinase. Formation of inactive and calmodulin-independent fragments.

Proteolysis by trypsin of gizzard myosin light chain kinase in the absence of Ca2+-calmodulin causes a biphasic effect on kinase activity. During the initial phase of proteolysis, Ca2+-calmodulin-dependent kinase activity is reduced over a thousand-fold. Further proteolysis, in the second phase, causes an increase in activity that is independent of Ca2+-calmodulin. Loss of activity is associated with the formation of a 64,000-dalton fragment. Calmodulin-independent activity is associated with the formation of a 61,000-dalton fragment. Procedures for the isolation of each fragment are outlined. Tryptic hydrolysis of the isolated 64,000-dalton peptide generates the 61,000-dalton peptide and increases calmodulin-independent activity. Km values for ATP and light chains for the native kinase and two fragments are the same, i.e. approximately 100 and 5 microM, respectively. Neither fragment binds to F-actin. Amino acid analyses of both fragments are given. Synthetic peptides corresponding to the calmodulin-binding regions of the smooth and skeletal muscle kinases are potent inhibitors of the 61,000-dalton fragment. These data demonstrate the existence of an inhibitory region that is suggested to be located between the active site and the calmodulin-binding site. Whether it is distinct from or at the N-terminal end of the calmodulin-binding site cannot be determined from these data.

Amino Acid Sequence↗

PubMed2000

Epidemiological typing of Bacillus cereus by amplified fragment length polymorphism.

A simple amplified fragment length polymorphism method was developed for the epidemiological typing of Bacillus cereus. The method was applied to 21 cultures from seven food poisoning and eight non-food poisoning incidents. Results were compared with those obtained by conventional serotyping using flagellar antigens and assessed in relation to epidemiological data. Amplified fragment length polymorphism was found to be highly reproducible and 16 different profiles (each unique to the 15 incidents) were recognized. The method was also able to discriminate three subtypes within serotype H1, which is responsible for the majority of the emetic type of B. cereus food poisoning in England and Wales.

Animals↗

PubMed1998

Identification and characterization of protease-resistant SecA fragments: secA has two membrane-integral forms.

We have identified and characterized the protease-resistant SecA fragments (X. Chen, H. Xu, and P. C. Tai, J. Biol. Chem. 271:29698-29706, 1996) through immunodetection with region-specific antibodies, chemical extraction, and sequencing analysis. The 66-, 36-, and 27-kDa proteolytic fragments in the membranes all start at Met1, whereas the 48-kDa fragment starts at Glu361. The overlapping of the sequences of the 66- and 48-kDa fragments indicates that they are derived from different SecA molecules. These two fragments were generated differently in response to ATP hydrolysis and protein translocation. Furthermore, the presence of membrane is required for the generation of the 48-kDa fragment but not for that of the 66-kDa fragment. These data suggest that there are two different integral forms of SecA in the membrane: SecA(S) and SecA(M). The combination of these two forms of SecA has several membrane-interacting domains. Both forms of SecA are integrated in the membrane, since both the 48- and 66-kDa fragments could be derived from urea- or Na2CO3-washed membranes. Moreover, all fragments are resistant to extraction with a high concentration of salt or with heparin, but the membrane-specific 48-kDa SecA domain is more sensitive to Na2CO3 or urea extraction. This suggests that this domain may interact with other membrane proteins in an aqueous microenvironment and therefore may form a part of the protein-conducting channel.

Adenosine Triphosphatases↗

PubMed1980

A novel human pituitary peptide containing the gamma-MSH sequence.

It is well established that ACTH and beta-lipotropin (LPH) originate from a common precursor molecule. Recently the complete complementary DNA sequence of the bovine precursor was reported, and within the cryptic sequence of this molecule is a third melanocyte stimulating hormone (MSH) region tentatively named gamma-MSH. The signal peptide of this molecule consists of 26 amino acids in both the rat and mouse. Pulse-chase experiments using both rat and mouse pituitary cells, showed the gradual maturation of this common precursor to proceed via the initial cleavage of the carboxy terminal beta-LPH, followed by release of ACTH, leaving an NH2-terminal extension of about 105 amino acids, which does not seem to undergo appreciably further maturation. It is within the sequence of this NH2-terminal extension that gamma-MSH is located. It is not yet clear what the biological role of this molecule is and whether gamma-MSH itself is released. Recently, it was shown that a synthetic 12 amino acid bovine gamma-MSH fragment possessed very little melanophore-stimulating activity as compared to alphs-MSH. We report here the successful purification of the human NH2 terminal cryptic peptide, its amino acid composition and present some of its tryptic fragments. The data show that human and bovine gamma-MSH have indentical amino acid composition.

Amino Acid Sequence↗

PubMed1985

Scatter analysis of discrete-sized chromatin fragments favours a cylindrical organization.

Fragments of chromatin containing 23 +/- 2.5 nucleosomes have been fractionated after light nuclease treatment of chicken erythrocyte nuclei. Low-angle scattering measures the total z-average radius of gyration of the already well-defined particles and the shape of scatter curves can be compared with three-dimensional analysis as opposed to cross-section analysis of long chromatin fragments. The data show that the particles are not spherical, have no detectable hole in the center of the structure and are best represented by a solid rod-like shape such as that generated by a coil of nucleosomes with the centre perhaps filled with linker DNA and histone H1/H5. 23 nucleosome fragments, where the DNA is partially fragmented, have near-identical scatter curves to the above-defined intact particles, indicating the primary importance of histone proteins in maintaining the integrity of the chromatin higher-order structure. Neutron scattering shows the radii of gyration to be contrast-independent, which fits in with the model calculations for solenoids. Particles with fragmented DNA and the intact particles, therefore, behave as sections of a solenoidal higher-order structure and possibly are observed as "superbeads' only during the folding and unfolding pathways of nucleosome multimers.

Animals↗

PubMed1998

H2O2 induces apoptosis of pig thyrocytes in culture.

Apoptosis might be involved in the reduction of the thyroid cell population in physiopathological situations such as goitre involution and autoimmune deleterious processes. Up to now, little attention has been paid to the apoptotic phenomenon in the normal thyroid gland the specialized metabolism of which is expected to generate reactive oxygen species. Indeed, thyroid cells have the capacity to synthesize H2O2. In this study, we have analyzed the capacity of H2O2 to trigger apoptosis of pig thyrocytes in culture to try to determine whether thyrocytes exhibit a particular resistance to apoptosis induced by an oxidative stress. We show that exposure of thyrocytes cultured as monolayers to exogenous H2O2 induced cell death with characteristics of apoptosis. The effect of H2O2 was concentration-dependent; apoptotic cells were already observed after exposure to 50 micro M H2O2. At high concentrations (millimolar range), H2O2 exerted toxic effects leading to rapid cell disruption. Within the first hour after the onset of exposure to 50-300 micro M H2O2, early signs of apoptosis, i.e. DNA fragmentation, appeared in a low (0.1-1%) but definite fraction of thyrocytes. The proportion of adherent cells exhibiting DNA fragmentation remained fairly constant after 6, 15 and 24 h. During the 24-h period, an increasing number of cells detached from the culture dish and up to 30-40% of cells in suspension displayed apoptotic features. The fraction of cells that lost contact with the culture dish amounted to up to 25% 24 h after addition of 300 micro M H2O2. In conclusion, as reported for other cell types, low H2O2 concentrations are capable of triggering apoptosis in thyrocytes cultured as monolayers. Thyrocytes that undergo apoptosis secondarily lose contact with neighbour cells and the substratum; cell detachment from the monolayer probably happens within 1-2 h after initiation of DNA fragmentation. Our data show that the apoptotic commitment can take place many hours after initiation of the oxidative stress.

Animals↗