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PubMed1975

Membrane-localized pyruvate kinase of red blood cells in hemolytic anemia associated with pyruvate kinase deficiency.

Pyruvate kinase activity of red blood cell membranes, which is normally masked, has been determined after mechanical disruption of the membranes in normal individuals and in three homozygous patients with pyruvate kinase deficiency. Although patients 1 and 2, who were siblings, had relatively high enzyme activities in their hemolysates, they had the severest form of the disorder. The activities of their membrane fragments were decreased to seven per cent of fragments of normal membranes. Patient 3 had a mild form of hemolytic anemia despite a low enzyme activity of his hemolysates. The membrane fragments of this patient contained 28 per cent of the pyruvate kinase activity of normal fragments. The data suggest a relationship between the amount of membrane-localized pyruvate kinase and the severity of the clinical disorder. The reduced production of ATP by the enzyme portion localized within the membrane may cause an impairment of membrane functions in pyruvate kinase deficiency.

Aged↗

PubMed1989

Direct effects of serotonin and ketanserin on the functional morphology of embryonic chick skin in vitro.

Different organotypical culture methods are used to test direct effects of serotonin and ketanserin, a S2, alpha 1, and H1 receptor antagonist in vascular tissue, on fibroblasts and epidermal cells of embryonic chick skin in vitro. From light microscopic and electron microscopic analyses, we learn that serotonin enhances keratinization and differentiation, whereas ketanserin reduces differentiation in comparison to the control cultures. Incorporation data of fragments cultured with [3H]thymidine show that ketanserin, within a dose range from 0.05 to 5 micrograms/ml, stimulates proliferation. Serotonin at a concentration of 10 micrograms/ml slightly slows down proliferation, whereas lower doses of 0.1 and 1 microgram/ml result in tritium activities that do not differ from control cultures.

Animals↗

PubMed1997

DNA typing in forensic medicine and in criminal investigations: a current survey.

Since 1985 DNA typing of biological material has become one of the most powerful tools for personal identification in forensic medicine and in criminal investigations [1-6]. Classical DNA "fingerprinting" is increasingly being replaced by polymerase chain reaction (PCR) based technology which detects very short polymorphic stretches of DNA [7-15]. DNA loci which forensic scientists study do not code for proteins, and they are spread over the whole genome [16, 17]. These loci are neutral, and few provide any information about individuals except for their identity. Minute amounts of biological material are sufficient for DNA typing. Many European countries are beginning to establish databases to store DNA profiles of crime scenes and known offenders. A brief overview is given of past and present DNA typing and the establishment of forensic DNA databases in Europe.

Civil Rights↗

PubMed2002

Species relationships in Fagopyrum revealed by PCR-based DNA fingerprinting.

Random amplified polymorphic DNA (RAPD) markers were used to distinguish between 28 different accessions belonging to 14 species and two sub-species of Fagopyrum. Of the 75 random 10-mer primers tested, only 19 generated robust, easily interpretable amplification products. A total of 364 bands were observed with an average of 19.15 bands per primer, of which 99.45% were polymorphic. Primer OPN-08 produced the maximum number of fragments and UBC-183 produced the minimum number of fragments. The data were utilized to elucidate genetic relationships among 14 species and two sub-species of Fagopyrum. Cluster analysis using the unweighted paired group method of arithmetic means (UPGMA) showed four main clusters, two each of the cymosum and urophyllum groups. The results showed that Fagopyrum tataricum is closer to its wild ancestor F. tataricum ssp. potanini Batalin, closely followed by Fagopyrum giganteum. Cultivated common buckwheat ( Fagopyrum esculentum) showed affinity with its putative wild ancestor F. esculentum ssp. ancestrale and the other closely related diploid species Fagopyrum homotropicum. In the urophyllum group, Fagopyrum macrocarpum and Fagopyrum pleioramosum formed one cluster, whereas Fagopyrum capillatum, Fagopyrum gracilipes and Fagopyrum gilessii clustered separately. Except for a few cases, our results correspond with previously reported studies on Fagopyrum using the isozyme, RFLP and RAPD methods. Species-diagnostic amplification products specific to some species in the cymosum and urophyllum groups were identified. Our results show that RAPDs can be successfully used to analyze species relationships in Fagopyrum and also for constructing linkage maps.

Journal Article↗

PubMed2006

Molecular mapping of genes for resistance to the bean pod weevil (Apion godmani Wagner) in common bean.

The bean pod weevil (Apion godmani Wagner) is a serious insect pest of common beans (Phaseolus vulgaris L.) grown in Mexico and Central America that is best controlled by host-plant resistance available in Durango or Jalisco genotypes such as J-117. Given unreliable infestation by the insect, the use of marker-assisted selection is desirable. In the present study, we developed a set of nine molecular markers for Apion resistance and mapped them to loci on chromosomes 2, 3, 4 and 6 (linkage groups b01, b08, b07 and b11, respectively) based on genetic analysis of an F (5:10) susceptible x resistant recombinant inbred line population (Jamapa x J-117) and two reference mapping populations (DOR364 x G19833 and BAT93 x JaloEEP558) for which chromosome and linkage group designations are known. All the markers were derived from randomly amplified polymorphic DNA (RAPD) bands that were identified through bulked segregant analysis and cloned for conversion to sequence tagged site (STS) markers. One of the markers was dominant while four detected polymorphism upon digestion with restriction enzymes. The other markers were mapped as RAPD fragments. Phenotypic data for the population was based on the evaluation of percentage seed damage in replicated trials conducted over four seasons in Mexico. In single point regression analysis, individual markers explained from 3.5 to 22.5% of the variance for the resistance trait with the most significant markers overall being F10-500S, U1-1400R, R20-1200S, W9-1300S and Z4-800S, all markers that mapped to chromosome 2 (b01). Two additional significant markers, B1-1400R and W6-800R, were mapped to chromosome 6 (b11) and explained from 4.3 to 10.2% of variance depending on the season. The latter of these markers was a dominant STS marker that may find immediate utility in marker-assisted selection. The association of these two loci with the Agr and Agm genes is discussed as well as the possibility of additional resistance genes on chromosome 4 (b07) and chromosome 3 (b08). These are among the first specific markers developed for tagging insect resistance in common bean and are expected to be useful for evaluating the mechanism of resistance to A. godmani.

Animals↗

PubMed2004

Biodegradation of alachlor by soil streptomycetes.

Streptomycetes resistant to the herbicide alachlor [2-chloro-2',6'-diethyl- N-(methoxymethyl) acetanilide] were used in degradation assays to characterize the products of alachlor biodegradation. Of six strains tested, Streptomyces sp. LS166, LS177, and LS182 were able to grow at an alachlor concentration of 144 mg l(-1) and degraded approximately 60-75% of the alachlor in 14 days, as evaluated by high performance liquid chromatography. The alachlor biodegradation products were identified by gas chromatography-mass spectrometry based on mass spectral data and fragmentation patterns. All compounds detected in these assays were similar for all streptomycetes strains tested, and involved dechlorination with subsequent N-dealkylation and cyclization of the remaining N-substituent with one of the ethyl groups to produce indole and quinoline derivatives. The enzymatic pathway used by Streptomyces sp. LS182 did not generate DEA (2',6'-diethylaniline), a carcinogenic derivative of alachlor reported in other studies. Given the high degradation rates observed here, the Streptomyces strains tested may be useful in the degradation/detoxification processes of alachlor.

Acetamides↗

PubMed1998

Role of yeast Rth1 nuclease and its homologs in mutation avoidance, DNA repair, and DNA replication.

The RTH1(RAD27) gene of Saccharomyces cerevisiae encodes a structure-specific endonuclease that cleaves 5'-ended single-stranded DNA at its junction with duplex DNA. Genetic and biochemical studies have indicated a role of Rth1 nuclease in the removal of RNA primers formed during DNA replication. The rth1Delta mutation confers temperature-sensitive lethality, and increases sensitivity to alkylating agents. The instability of repetitive DNA is greatly enhanced in the rth1Delta mutant. The conditional lethality of the rth1Delta mutation indicates that another nuclease can function in DNA replication in the absence of RTH1. RAD2, a homolog of RTH1, is required for nucleotide-excision repair. Here, we examine three other homologs of RTH1/RAD2 - YEN1, EXO1, and DIN7. Deletion of any of these genes in the rth1Delta strain has no effect on cell viability, suggesting the involvement of another, and as yet unidentified, nuclease in the maturation of Okazaki fragments. Our data also indicate that only RTH1 functions in the repair of alkylation damage. Deletions of YEN1, EXO1, DIN7, or RAD2, either singly or when combined with one another and with the rth1Delta mutation, have no effect on the rate of instability of dinucleotide repeats or on the rate of formation of large duplications in the CAN1 gene. These data provide evidence of a high degree of specificity for the role of RTH1 in DNA replication and in base-excision repair, and for the requirement of RAD2 in nucleotide-excision repair. The possibility that both Rth1 and Exo1 function in DNA mismatch repair is discussed.

Alkylation↗

PubMed1998

Phospholipid rearrangement of apoptotic membrane does not depend on nuclear activity.

The behaviour of plasma membrane was studied in UV-treated cells to investigate its involvement in apoptosis. It was studied in HL60 cells, in which DNA oligonucleosomic cleavage occurs, and in Molt-4 cells, which are characterised by a different fragmentation pattern. During the early stages of apoptosis, a membrane lipid rearrangement occurs, which involves phosphatidylserine translocation from the inner to the outer leaflet. This molecular alteration was investigated by annexin V-FITC binding, analysed by flow cytometry and confocal microscopy. It was correlated with transmission electron microscopy, subdiploid peak appearance and DNA fragmentation. Our data indicate that the plasma membrane represents an early apoptotic target, even if its alterations are not detectable by ultrastructural analysis, which indicates its good preservation until late apoptotic stages. In addition, the study of apoptotic cells with absent or inactivated endonuclease demonstrates the independence of this membrane mechanism from nuclear activity.

Apoptosis↗

PubMed2004

Inhibition of the arachidonic acid metabolism blocks endothelial cell migration and induces apoptosis.

Previous studies have demonstrated that inhibitors of the arachidonic acid metabolism block migration and sensitise human glioma cells to treatment inducing apoptosis. This paradigm may provide a new concept for anti-invasive treatment strategies targeting invasive glioma cells. However, the effect of such treatment on other cellular elements in glial tumours such as endothelial cells is unknown. In this study we have analysed the expression of metabolites of the arachidonic acid pathway in endothelial cells in vitro and in vivo and we have assessed the influence of inhibitors of this pathway on motility, capillary like tube formation, and apoptosis in human endothelial cells. Human endothelial cells (HUVEC) in culture showed expression for thromboxane synthase and both isoforms of cyclo-oxygenase, COX-1 and COX-2. Immunostaining demonstrated low levels of COX-1 expression in capillaries and larger vessels of normal brain and moderately elevated levels of this enzyme in small vessels of brain tumours of various grades. Both thromboxane synthase and COX-2 expression was limited to endothelial cells found in anaplastic gliomas and glioblastomas. Thromboxane synthase inhibitors strongly decreased endothelial cell migration in HUVEC in vitro and capillary like tube formation was strongly inhibited by the compound at a similar dose range. The non-selective cyclo-oxygenase inhibitor ASA and the selective COX-2 inhibitor sulindac only had a minor effect on endothelial cell migration, however, the COX-2 inhibitor sulindac showed a synergistic effect with the thromboxane synthase inhibitor. Thromboxane synthase inhibitors induced apoptosis in endothelial cells as demonstrated by intracellular histone-complexed DNA fragmentation. These data suggest that inhibitors of thromboxane synthase influence migration and apoptosis in both human glioma cells and human endothelial cells. An anti-invasive treatment strategy using this class of compounds may therefore not only sensitise glioma cells to conventional treatments inducing apoptosis but may also be supported by an anti-angiogenic effect.

Apoptosis↗

PubMed2000

Discrimination between Atlantic and Pacific Subspecies of Northern Bluefin Tuna (Thunnus thynnus) by Magnetic-Capture Hybridization Using Bacterial Magnetic Particles.

The previously developed magnetic-capture hybridization technique employing bacterial magnetic particles was applied to discriminate between Atlantic and Pacific subspecies of the northern bluefin tuna (Thunnus thynnus) using specific DNA sequences. Nucleotide sequences of a 925-bp fragment (ATCO) flanking the mitochondrial ATPase and cytochrome oxidase subunit III genes in these two subspecies were compared. Two regions having single-nucleotide and three-nucleotide differences between the subspecies were adopted to design DNA probes (NR1, 21-mer; NR2, 29-mer), and two internal primer sets were designed to amplify DNA fragments containing these regions. The DNA probes were immobilized on bacterial magnetic particles via streptavidin-biotin conjugation and subjected to magnetic-capture hybridization with the digoxigenin-labeled fragments amplified using the internal primers. The luminescence intensities of DNA on bacterial magnetic particles obtained by hybridization between the probes and the complementary fragments were higher than those obtained by hybridization with noncomplementary fragments. These data suggest that this system employing DNA on bacterial magnetic particles may be useful for discrimination of these two subspecies by recognizing a single-nucleotide difference.

Journal Article↗

PubMed2003

Immediate control of a methicillin-resistant Staphylococcus aureus outbreak in a neonatal intensive care unit.

An outbreak of methicillin-resistant Staphylococcus aureus (MRSA) colonization occurred from November 2001 in the neonatal intensive care unit (NICU) of our hospital. Since the establishment of our NICU in 1991, some MRSA has been detected in NICU patients. For MRSA infection preventive measures, utilization of the following items was implemented: mupirocin ointment, diluted povidone iodine, methylrosaniline chloride, and disposable rubber gloves. Patients in whom MRSA was detected received intranasal administration of the mupirocin ointment three times daily and were bathed in, or their entire body was wiped with diluted povidone iodine once daily for the first 3 days in each week. In addition, they received an intraoral application of methylrosaniline chloride daily. All therapy was done until MRSA strains were undetectable for 3 continuous weeks. Genotypes of 13 MRSA strains isolated from eight inpatients and one mother were analyzed by pulsed-field gel electrophoresis (PFGE). All PFGE patterns were identical, except for one, which had one distinct migrating fragment. These data suggested that this MRSA outbreak was caused by the same strain, which was derived from the mother of a low-birth-weight infant born on October 30, 2001. Gradually, the number of inpatients carrying MRSA decreased, until finally MRSA was no longer observed, in April 2002. Fortunately, we controlled the MRSA outbreak immediately, and none of the inpatients developed severe MRSA infection. We think that in our NICU, which is isolated from other hospital wards, it is important to prevent the entrance of MRSA-carrying mothers.

Cross Infection↗

PubMed1988

Characterization of type I collagen from the skin of blue grenadier (Macruronus novaezelandiae).

The skin collagen of a fish, blue grenadier (Macruronus novaezelandiae), has been purified and characterized. The fish skin was readily soluble in dilute acetic acid, with no pepsin treatment needed. The collagen was purified by salt precipitation. Skin samples from fish of various ages showed that even in the oldest sample, more than 8 years of age, the collagen was still readily acid soluble. The purified collagen had a melting temperature of 22 degrees C; the shrinkage temperature for the skin was 48 degrees C. Its tissue distribution, examined by immunohistology, and its chemical properties indicated a close homology to mammalian type I collagen. However, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed that three distinct alpha-chains were present. These were purified by ion-exchange chromatography on CM-cellulose and by gel permeation chromatography on Superose 6. The three purified alpha-chain fractions were examined by amino acid analysis and by SDS-PAGE of their cyanogen bromide fragments. These data indicated that the additional chain was genetically distinct, and most closely related to the alpha 1-chain, from which it was poorly resolved on SDS-PAGE.

Age Factors↗

PubMed1978

Anion transport in relation to proteolytic dissection of band 3 protein.

Sulfate efflux was measured in inside-out vesicles obtained from human red cells. Inhibition was observed in vesicles derived from cells pretreated with DIDS (4,4'-diisothiocyano-2,2'-stilbene disulfonate) or after addition of dipyridamole to the vesicles, both agents being specific and potent inhibitors of anion transport in cells. Trypsinization of the cytoplasmic side of the membrane in order to release a 40 000 dalton fragment from band 3 (the purported anion transport protein) had no effect on sulfate efflux. Further degradation of band 3 to a 17 000 dalton segment, by trypsinization of inside-out vesicles derived from cells that had been pretreated with chymotrypsin, also showed little reduction in transport activity. Furthermore, such vesicles derived from DIDS pretreated cells were inhibited by over 90%. In DIDS-treated cells, the agent is highly localized in band 3. In trypsinized inside-out vesicles, it is largely found in a 55000 fragment and in trypsinized vesicles derived from cells pretreated with chymotrypsin it is largely located in the 17 000 fragment. The data suggest that both the anion transport and inhibitor binding sites are located in a 17 000 transmembrane segment of band 3.

Biological Transport↗

PubMed1988

Analysis of the major integral membrane proteins of peroxisomes from mouse liver.

Two major proteins with subunit molecular masses of 68 and 70 kDa were isolated from the integral membrane protein fraction of peroxisomes purified from mouse liver. The two proteins were shown to be distinct proteins by two criteria: first, immunoblot analysis demonstrated that antisera against the 68 kDa protein did not cross-react with the 70 kDa protein, and vice versa; and second, the partial peptide maps resulting from proteinase digestion of the proteins were different. Immunoblot analyses to test the specificities of the antisera demonstrated that only the expected molecular mass species in purified peroxisomes, and in membranes prepared from these organelles, were recognized; there was no identification of proteins from purified mitochondrial or microsomal fractions. The concentrations of both of these proteins were increased in livers of mice treated with clofibrate, a hypolipidemic drug and peroxisome proliferator, with the effect being greater for the 70 kDa component. The localization of the 68 kDa protein was shown to be completely integral to the peroxisome membrane. Although some 70 kDa protein was integral to the membrane, a significant proportion was released from the membrane by some procedures believed to detach peripheral proteins. The 70 kDa protein was also particularly susceptible to degradation during isolation - in particular, addition of EDTA to media used for isolation of peroxisomes resulted in membranes in which this protein was degraded to smaller immunoreactive fragments. These data have been discussed in relation to the significant clarification which they have provided of the status and characteristics of the major protein components of peroxisomal membranes.

Animals↗

PubMed1989

Activation of matrix metalloproteinase 3 (stromelysin) and matrix metalloproteinase 2 ('gelatinase') by human neutrophil elastase and cathepsin G.

The ability of human neutrophil elastase and cathepsin G to activate matrix metalloproteinase 3 (MMP-3 = stromelysin) and MMP-2 ('gelatinase') purified from human rheumatoid synovial fibroblasts in culture was examined. The zymogen of MMP-3 (proMMP-3) was activated to full activity with elastase and cathepsin G by limited proteolysis of the molecule into two active forms of Mr approximately 45,000 and Mr approximately 25,000. In contrast, proMMP-2 was not activated at all by these neutrophil serine proteinases, although it was degraded into small fragments. These data suggest that neutrophil elastase and cathepsin G may play an important role in the activation of proMMP-3 in vivo in various inflammatory conditions, but proMMP-2 may be activated in different ways.

Cathepsin G↗

PubMed1984

Preliminary crystallographic data for a monoclonal Fab fragment specific for HPr of the phosphoenolpyruvate: sugar phosphotransferase system of Escherichia coli.

Crystals for Fab fragments from a monoclonal antibody to HPr of the phosphoenopyruvate:sugar phosphotransferase system of Escherichia coli have been obtained from 14% polyethylene glycol 6000, 5 mM-Tris X HCl, 50 mM-sodium phosphate and 0.2 M-sodium chloride at pH 8.0. The space group is P2(1) with a = 110.85 A, b = 66.18 A, c = 67.21 A, beta = 113.0 degrees and Z = 4. The crystals exhibit the forms [100], [011] and [011] and the solvent content is 47%.

Antibodies, Monoclonal↗

PubMed1976

Radiation-induced chromosomal aberrations in human lymphocytes. I. Dependence on the dose of gamma-rays and an anomaly at low doses.

Cultures of human lymphocytes obtained from blood of healthy adult donors were irradiated with different doses of 60Co gamma-rays and the irradiated cells were analysed in metaphase 50 h after irradiation. The effect (total yield of aberrations of chromosome type, or total yield of exchange type aberrations) produced by the lowest dose (5 rad) appears to be statistically significant in a sample of 1500 cells. In the usual dose range (25-400 rad), both parabolic and linear-quadratic equations give a satisfactory fit of experimental data (dicetrics, fragments, or all aberrations of chromosome type). Low doses of gamma-rays, however, produced more aberrations than expected, if one extrapolates dose-effect curves from higher doses. Both relations should be considered, therefore, merely as empirical equations. Dicentrics show at low doses (10-30 rad) a plateau which appears to be statistically significant. Some indications are obtained that the total number of chromosome-type aberrations is a more reliable criterion of cytogenetic damage than the usually accepted yield of dicentrics and rings.

Cells, Cultured↗

PubMed1985

Effects of pre-treatment with sodium butyrate on the frequencies of X-ray-induced chromosomal aberrations in human peripheral blood lymphocytes.

The effects of sodium butyrate-mediated alterations in chromatin structure on the yields of X-ray-induced chromosomal aberrations were studied in human peripheral blood lymphocytes. Unstimulated (G0) lymphocytes were pre-treated with sodium butyrate (5 mM) for 24 h, X-irradiated and then stimulated to pass through the cell cycle. Cells in their first post-radiation metaphase were scored for chromosomal aberrations. In parallel biochemical experiments nucleoid sedimentation technique was used to examine the induction and repair of DNA-strand breaks. The results show that sodium butyrate pre-treatment leads to a significant increase in the frequencies of dicentrics and rings, but not of fragments. The data from biochemical studies suggest that the numbers and rates of repair of X-ray-induced DNA-strand breaks are the same in butyrate-treated and untreated cells. We therefore suggest that the observed effect is probably a consequence of butyrate-induced conformational changes in the chromatin of G0 lymphocytes.

Butyrates↗