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PubMed1987

Abnormal structure of von Willebrand factor in myeloproliferative syndrome is associated to either thrombotic or bleeding diathesis.

The multimeric and subunit patterns of plasma von Willebrand factor (vWF) were analyzed in eight patients with myeloproliferative syndrome (MS) in order to investigate the possible existence of heterogeneity in the "in vivo" proteolytic cleavage of the protein, previously observed in this entity. Six patients lacked large vWF multimers, five of them having normal bleeding times (BT) and clinically documented episodes of thrombotic origin, whereas one patient had long BT and bleeding symptoms. Seven patients showed a relative increase in the 176 kDa subunit fragment while the 189 kDa polypeptide was increased in only one. In addition, another patient (and prior to any therapy) showed the presence of a new fragment of approximately 95 kDa which disappeared after Busulfan therapy. The collection of blood from these patients with proteinase inhibitors did not correct the abnormalities. The infusion of DDAVP to two patients with abnormal vWF was accompanied by: the appearance of larger vWF multimers which disappeared rapidly from plasma; an increase in the relative proportion of the satellite bands of each multimer and a further increase of the 176 kDa fragment. These data point to some heterogeneity in the vWF abnormality present in MS which may be related in part to a variable degree of proteolysis of vWF occurring "in vivo" rather than "in vitro", and which may be associated to either a thrombotic or a bleeding diathesis. They also suggest that despite the presence of abnormal, already proteolyzed vWF, DDAVP-enhanced proteolysis occurs in MS to a similar extent to what is described in normal individuals.

Deamino Arginine Vasopressin↗

PubMed1987

Radioimmunodetection of human glioma xenografts by monoclonal antibody to epidermal growth factor receptor.

Murine IgG2a monoclonal antibody (MAb) 425 specifically detects epidermal growth factor receptor, which is expressed on human gliomas and tumors of other tissue origin but rarely on normal brain tissues, and not at all on bone marrow and peripheral blood cells. 131I-labeled F(ab')2 fragments of this MAb injected into nude mice grafted with U-87 MG glioma cells preferentially localized in tumor tissue compared to normal mouse tissues, as determined by differential tissue counting of radioactivity. The mean tumor-to-tissue ratios of radioactivity ranged between 8.2 (blood) and 55.8 (muscle) at 2 days after the injection of 15 muCi of 131I-425 F(ab')2/mouse. Radiolabeled fragments of an anti-hepatitis virus IgG2a MAb did not localize in tumors. The localization index derived from the ratios of specific antibody to indifferent antibody in tumor tissue relative to blood was 9.94 at 2 days following the MAb injection. The labeled MAb did not localize in a xenograft of colorectal cancer tumor, which does not express the epidermal growth factor receptor. Tumors could be located by whole-body gamma-scintigraphy without background subtraction following the injection of 100 muCi of radiolabeled MAb 425 F(ab')2 fragments. The data suggest that MAb 425 is a likely candidate for clinical diagnostic and radioimmunotherapy trials.

Animals↗

PubMed1978

Long-term preservation of canine bone marrow: in vitro studies.

In vitro studies were performed on canine bone marrow frozen with DMSO and stored in liquid nitrogen for 2 to 6 months. The results are compared with previously reported parallel in vivo experiments that demonstrated no loss of stem cells. When studies were performed immediately after thawing, there was no substantial drop in the count of nucleated cells and, except for megakaryocytes, there was no alteration of the bone marrow morphology. After two washes, and removal of DMSO, the nucleated cell count dropped to 50% of its previous value. Optic and electron microscopy showed severe damage in mature myeloid elements. In some instances, the cells had a condensed nucleus similar to the red-purple inclusion body of LE cells (as observed in systemic lupus erythematosus), and electron microscopy showed heavy chromatin clumping. On the other hand, both optic and electron microscopy showed a good preservation of lymphocytes, plasmocytes, and erythroid precursors. Two-hour DNA synthesis slightly dropped after storage, and this drop appeared more consistent when related to a constant volume of bone marrow (50 microliters) rather than to a constant number of nucleated cells (10(6)). In five instances frozen and thawed bone marrow was grown in short-term cultures, and analysis of 98 metaphases showed no major aberrations of the chromosomes and only 2% of minor aberrations, such as breakages and fragments. These data, compared with the results of previous in vivo experiments that showed no loss of stem cells after 5 months storage, suggest that stem cells are less sensitive to freezing and thawing injury than myeloid elements and/or that it might be safer for the thawed bone marrow not to be manipulated before infusion.

Animals↗

PubMedDate not supplied

[Physicochemical properties of temperate phage DNA and its possible effect on the variability of Mycobacterium lacticolum].

The temperate phage 104 S was isolated from the S variant of Mycobacterium lacticolum, strain 104, and some of its characteristics were studied. The content of GC pairs in the phage DNA was 77 mole% as was calculated from the melting profile or 65 mole% as was calculated from the value of buoyant density in CsCl. The DNA was shown to be composed of 18,000 nucleotide pairs. DNA restriction fragments of M. lacticolum R, S and M variants were subjected for the first time to molecular hybridization with [32P]DNA of the temperate phage. The genome of the three M. lacticolum variants and the genome of a non-dissociating S variant clone were shown to contain sequences homologous to the DNA sequence of phage 104 S. Differences are found among the variants in the hybridizing DNA fragments. These data indicate that the phage DNA may actively be involved in the variability of the culture. Its participation can be realized by the different mode of prophage incorporation into the genome of the variants.

Base Composition↗

PubMed1983

The relationship of the properties of antihemophilic factor (factor VIII) that support ristocetin-induced platelet agglutination (factor VIIIR:RC) and platelet retention by glass beads as demonstrated by a monoclonal antibody.

A monoclonal antibody to human antihemophilic factor (AHF, factor VIII) was derived from BALB/c mouse spleen cells fused with P3x63Ag8 mouse plasmacytoma cells. This antibody, harvested from culture medium or ascites fluid, reacted with purified AHF and with plasmas with normal subjects or classic hemophiliacs, as measured by enzyme-linked immunosorbent assay (ELISA), but not with plasmas from patients with severe von Willebrand's disease. The antibody possessed only IgG, heavy chains and kappa light chains. It blocked ristocetin-induced platelet agglutination and, to a lesser degree, platelet retention by glass bead columns, but it did not inhibit the procoagulant activity of AHF significantly. An amount of rabbit antiserum against AHF that provided equivalent inhibition of ristocetin-induced platelet agglutination inhibited glass bead retention much more effectively than the mouse monoclonal antibody. This difference was exaggerated in studies of the corresponding Fab fragments. These data suggest that the site or sites on the AHF complex molecule that are associated with ristocetin-induced platelet agglutination differ quantitatively or qualitatively from those associated with enhancement of platelet retention by glass beads. ELISA titers of immunoreactive AHF, using the monoclonal antibody, were closely correlated to those using rabbit antiserum against AHF in normal, hemophilic, and most von Willebrand's disease plasma.

Antibodies, Monoclonal↗

PubMed1982

Asymmetric distribution of sites with high affinity for antithrombin III in rat skin heparin proteoglycans.

The distribution of sites with high affinity for antithrombin III in [35S]heparin proteoglycans from rat skin was studied by affinity chromatography of the intact proteoglycans (Mr congruent to 1 x 10(6)) and degradation products. Unfractionated proteoglycan and proteoglycan fractions with low affinity and high affinity separated on antithrombin III-agarose were treated with alkali, releasing heparin chains (Mr congruent to 1 x 10(5)). Each chain preparation was fractionated on antithrombin III-agarose into fractions with low affinity and high affinity. Unfractionated proteoglycan and proteoglycan fractions with low affinity and high affinity were incubated with rat serum at pH 6.0, which gave products of similar size to commercial heparins (Mr congruent to 1 x 10(4)) termed heparin fragments. Each fragment preparation was fractionated on antithrombin III-agarose, yielding fractions with no affinity, low affinity, and high affinity, 40% of the proteoglycan preparation had low affinity, containing 4% high affinity chains and 7% high affinity fragments. The high affinity proteoglycan fraction yielded 40% high affinity chains and 22% high affinity fragments. The data show that the distribution of binding sites with high affinity for antithrombin III in heparin proteoglycans is highly asymmetric. Therefore, the concept that polymer modification reactions occurring during heparin biosynthesis, which must be involved in the formation of high affinity binding sites, occur in a random way must be reappraised.

Animals↗

PubMed1982

[Early changes in template DNA structure following glucocorticoid injection].

The structure of chromatin is changed at early stages of glucocorticoid hormone interaction with rat hepatocytes. These changes consist in: a) increase of actidine orange binding in rate liver nuclei after injection of the hormone; b) decrease of the number of sites in chromatin which are sensitive to nuclease S1; c) inhibition of the nuclei capacity for DNA synthesis in the presence of E. coli DNA polymerase and d) increase of molecular weight of DNA fragments. The data obtained suggest that at early stages of hormonal induction part of template DNA ruptures is reconstituted, which can result in an increase of DNA molecular weights and changes in chromatin superstructure and the template properties of the protein.

Animals↗

PubMedDate not supplied

[Chlorophyll phosphorescence in chloroplasts and their fragments].

Spectral parameters, decay time and quantum yield of low temperature (--196 degrees) delayed luminescence of chloroplasts and chloroplasts fragments have been investigated. It has been shown that the fragments enriched in photosystem 2, photosystem 1 of stroma or grana, obtained by differential centrifugation after digitonin treatment produce a delayed emission with components similar to that of initial chloroplasts, normal leaves and alga; i. e. phosphorescence (maximum 960--980 nm) and delayed fluorescence (maxima at 690 and 740 nm) of chlorophyll and phosphorescence of pigments of different nature (780--820 nm). Excitation spectra of chlorophyll phosphorescence have the maximum at 669 +/- 1 nm and shoulder at 650 nm. The quantum yield of chlorophyll phosphorescence in chloroplast is found to be lower than that of chlorophyll in ethanol by the factor of 200. It increases with destruction of initial structures reaching the maximal value in the most light fragments. The data suggest that the luminescence studied accompanies desactivation of the triplet states of the chlorophyll molecules involved in the photosynthetic pigment complex. On the basis of previous studies it has been proposed that the increase of phosphorescence intensity in the light particles is due to disturbances in the pigment complex leading to a decrease of triplet-triplet energy transfer from chlorophyll to carotenoids.

Carotenoids↗

PubMed1994

[Studies on the molecular evolution of apolipoprotein multigene family].

The apolipoprotein genes represent a large family of genes encoding various binding proteins for plasma lipid transport. Because of their long divergence history, it is not known whether and how these genes have evolved through gene duplication from a common ancestor. To test this possibility and reconstruct a reliable phylogenetic tree, a simple method to evaluate the branch length and its divergence time in unrooted parsimony tree under the condition of non-even evolutionary rate was developed. The tree built from the 26 apolipoprotein sequences by above method clearly shows: (1) The common ancestor of ApoA-I ApoA-II, ApoA-IV, ApoE may appear 460 million years ago in an ordovician vertebrate which may be related with the major apolipoprotein LAL1 and LAL2 in Lamprey from the evidence of sequence alignment; (2) The central role of different selection pressure upon the ancestor gene of apolipoprotein made them evolved into different subgroups; (3) The high evolution rate in rodent ApoE molecules may be related with the existence of a large amount of hidden substitutions and the disruption of synonym codon usage clock in their genome; (4) The evolutionary rate of various branches in parsimony tree is significantly different in which the average UEP of ApoA-I, ApoA-IV is 2.0 MY, ApoA-II 1.7MY, ApoE 2.4MY; (5) The receptor domain in ApoE seems to be more conservative than other fragments. These data suggest a long, complex evolutionary history for apolipoprotein genes in which the gene duplication events of different origins took place.

Amino Acid Sequence↗

PubMed1993

Morphine induces apoptosis in murine thymocytes in vivo but not in vitro: involvement of both opiate and glucocorticoid receptors.

We tested the hypothesis that the previously observed loss of thymic lymphocytes in mice after treatment with time-release morphine pellets was occurring through the process of apoptosis. Apoptosis is a form of cell death, distinct from necrosis, which involves a specific endonuclease that fragments the cell's own DNA. Forty-eight hours after implantation of a time-release morphine pellet in B6C3F1 mice, thymus weight and cellularity was reduced to 30% of that observed in placebo-treated mice. Thymocytes from morphine pellet-treated mice were found to have a significantly greater percentage of their DNA fragmented than did thymocytes from either placebo pellet-implanted or naive control mice. The peak level of DNA fragmentation was found to occur approximately 12 hr postpellet implant. When separated on agarose gels, the sizes of the DNA fragments observed corresponded to the multiples of 180 base pairs which are characteristic of apoptosis. In vivo, the use of either the opiate receptor antagonist naloxone, or the glucocorticoid receptor antagonist RU-38486, was able to block completely the morphine mediated increase in thymocyte apoptosis. In vitro experiments in which thymocytes were cultured with morphine concentrations as high as 10(-4) M showed no evidence of an increased rate of DNA fragmentation. These data indicate that both opiate and glucocorticoid receptors are involved in morphine-induced apoptosis and that the opiate receptor responsible is not located on the thymic lymphocytes.

Animals↗

PubMed1996

Activation of the CD95 (APO-1/Fas) system in T cells from human immunodeficiency virus type-1-infected children.

Increased apoptosis of CD4+ T cells is considered to be involved in CD4+ T-cell depletion in human immunodeficiency virus type-1 (HIV-1)-infected individuals progressing toward acquired immunodeficiency syndrome (AIDS). We have recently shown that CD95 (APO-1/Fas) expression is strongly increased in T cells of HIV-1-infected children. In this report we provide further evidence for a deregulated CD95 system in AIDS. CD95 expression in HIV-1+ children is not restricted to previously activated CD45RO+ T cells but is also increased on freshly isolated naive CD45RA+ T cells. In addition, specific CD95-mediated apoptosis is enhanced in both CD4+ and CD8+ T cells. Furthermore, levels of CD95 ligand mRNA are profoundly increased. Specific T-cell receptor/CD3-triggered apoptosis in HIV-1+ children is more enhanced in CD8+ than in CD4+ T cells. Accelerated activation induced cell death of T cells could partially be inhibited by blocking anti-CD95 antibody fragments. These data suggest an involvement of the CD95 receptor/ligand system in T-cell depletion and apoptosis in AIDS and may open new avenues of rational intervention strategies.

Adult↗

PubMed1996

Phylogeography and population structure of the Atlantic and Mediterranean green turtle Chelonia mydas: a mitochondrial DNA control region sequence assessment.

Mitochondrial (mt) DNA sequences were analysed to resolve the phylogeography and population genetic structure of Atlantic and Mediterranean populations of green turtles (Chelonia mydas). Analysis of sequence variation over 487 base pairs of the control (D-loop) region identified 18 haplotypes among 147 individuals from nine nesting populations. Pairwise comparisons of haplotype frequencies distinguished most nesting colonies, indicating significant genetic differentiation among rookeries and a strong propensity for natal homing behaviour by nesting females. Comparison of control region sequence data to earlier restriction fragment length polymorphism (RFLP) data for the same individuals demonstrates approximately a sixfold higher substitution rate in the 5' end of the control region. The sequence data provide higher resolution both in terms of the number of mtDNA genotype variants and the phylogeographic relationships detected within the Atlantic region, and reveal a gene genealogy that distinguishes two groups of haplotypes corresponding to (i) the western Caribbean and Mediterranean, and (ii) eastern Caribbean, South Atlantic and West Africa. The data suggest that phylogeographic patterns in the Atlantic Ocean may be interpreted in terms of female nest site fidelity and episodic dispersal events. The distribution of mtDNA haplotypes within the region is thus explained by the geological and climatic alternations (glacial and interglacial) over the last million years.

Animals↗

PubMed1977

[Effect of gamma-irradiation on human chromosomes. VIII. Cytogenetic effect of low doses during in vitro irradiation].

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

Cells, Cultured↗

PubMed1997

Fas-independent apoptosis of activated T cells induced by antibodies to the HLA class I alpha1 domain.

In addition to their major function in antigen presentation and natural killer cell activity regulation, HLA class I molecules may modulate T-cell activation and proliferation. Monoclonal antibodies (MoAbs) that recognize distinct epitopes of HLA class I molecules were reported to interfere with T-cell proliferation. We show here that two MoAbs (mouse MoAb90 and rat YTH862) that bind to an epitope of the alpha1 domain of HLA class I heavy chain induce apoptotic cell death of activated, but not resting, peripheral T lymphocytes. Other reference anti-HLA class I antibodies specific for distinct epitopes of the alpha1 (B9.12.1), alpha2 (W6/32), or alpha3 (TP25.99) domains of the heavy chain decreased T-cell proliferation but had little or no apoptotic effect. Apoptosis shown by DNA fragmentation, phosphatidylserine externalization, and decrease of mitochondrial transmembrane potential was observed whatever the type of T-cell activator. Apoptosis did not result from Fas/Fas-L interaction and distinct though partly overlapping populations of activated T cells were susceptible to Fas- and HLA class I-mediated apoptosis, respectively. Induction of apoptosis did not require HLA class I cross-linking inasmuch as it could be observed with monovalent Fab' fragments. The data indicate that MoAb90 and YTH862 directed against the alpha1 domain of HLA class I trigger apoptosis of activated T lymphocytes by a pathway which does not involve Fas-ligand.

Animals↗

PubMed1976

Organization of membrane proteins in the intact myelin sheath. Pyridoxal phosphate and salicylaldehyde as probes of myelin structure.

Pyridoxal phosphate and salicylaldehyde were used as protein-labeling probes to study the organization of membrane proteins in the intact myelin sheath of the cat dorsal column. Both reagents react with protein amino groups to form Schiff's bases which can be reduced with NaB3H4. The relatively membrane-impermeant pyridoxal phosphate labels all proteins of the intact myelin except basic protein. This major protein of myelin is labeled only after loss of membrane integrity. The relatively membrane-permeant probe, salicylaldehyde, was then used to establish that the basic protein is truly located on the cytoplasmic side of the myelin bilayer, and not merely sequestered within the multiple lamellar structure of the sheath. All proteins in the intact myelin are readily labeled by this reagent, with the label distribution pattern identical to that of disrupted myelin fragments. These data suggest a model for myelin structure in which the basic protein is the only major protein component located exclusively on the cytoplasmic side of the membrane (the major period zone of the sheath), with the other major proteins disposed wholly, or in part, in the extracellular half of the membrane bilayer (the intraperiod zone). All proteins, although asymmetrically disposed with respect to membrane sidedness, appear to be randomly distributed throughout the lamellae which comprise the sheath.

Aldehydes↗

PubMed1997

Cellular determinants of resistance to indolocarbazole analogue 6-N-formylamino-12,13-dihydro-1,11-dihydroxy-13(beta-D-glucopyranosyl)- 5H-indolo[2,3-alpha]pyrrolo[3,4-c]carbazole-5,7(6H)-dione (NB-506), a novel potent topoisomerase I inhibitor, in multidrug-resistant human tumor cells.

Membrane protein-associated alterations in cellular drug accumulation have been recently implicated in resistance to topoisomerase I (TOP-I)-interactive drugs. The present study investigated the cellular determinants of resistance to the indolocarbazole compound NB-506 [6-N-formylamino-12,13-dihydro-1,11-dihydroxy-13(beta-D-glucopyranosyl)- 5H-indolo[2,3-alpha]pyrrolo[3,4-c]carbazole-5,7(6H)-dione], a structurally novel TOP-I-interactive drug, in parental and multidrug-resistant tumor cells expressing either the P-170 glycoprotein (Pgp170) or multidrug resistance protein (MRP). MRP-expressing 250-fold doxorubicin-resistant human fibrosarcoma HT1080/DR4 tumor cells were drug sensitive to NB-506 and camptothecin (CPT) (resistance factor: 0.7 and 0.8, respectively) with no alterations of TOP-I parameters including DNA relaxation, expression of TOP-I protein and mRNA. In contrast, doxorubicin-resistant human ovarian A2780/Dx5 tumor cells [pgp170 phenotype] were 6.2-fold resistant to NB-506, whereas resistance to CPT was 2.6-fold. HPLC analysis of cellular NB-506 accumulation showed no significant differences between A2780 and A2780/Dx5 cells (peak intracellular concentrations after 120-min exposure to 10 microM NB-506: 400+/-85.0 and 352+/-95.1 nmol NB-506/mg protein, respectively). However, resistant A2780/Dx5 cells expressed a lower amount of TOP-I mRNA and 29% protein levels of TOP-I compared to parental A2780 cells, resulting in decreased TOP-I catalytic activity (3.17+/-0.02 vs. 1.16+/-0.15 rel.U/microg nuclear protein) and reduced induction of NB-506-mediated cleavable complex formation in A2780/Dx5 cells. Furthermore, the lower induction of NB-506-induced protein-linked DNA breaks (PLDB) in A2780/Dx5 cells correlated with significantly decreased DNA 12.2-440 kb size fragmentation in these cells. The present study demonstrates that expression of MRP and Pgp170 does not confer resistance to NB-506. Resistance to indolocarbazole substance NB-506 in A2780/Dx5 cells was only related to downregulation of TOP-I associated with lower induction of cleavable complex formation and DNA fragmentation. The data reported herein may indicate that the new indolocarbazole compound NB-506 has potent antitumor efficacy in membrane-associated multidrug resistance.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1993

Simplification of high-energy collision spectra of peptides by amino-terminal derivatization.

Four-sector tandem mass spectrometry proves extremely useful for providing sequence information for peptides. The complexity of ion fragmentations, however, makes data interpretation difficult and time consuming. Attachment of a fixed positive charge to the peptide amino terminus forces production of only N-terminal fragment ions to yield simplified, predictable fragmentation. Reaction of a peptide at pH 6 with iodoacetic anhydride selectively modifies the N-terminus by exploiting the pK(a) differences between the alpha-amino group and any lysine side-chain epsilon-amino groups. The iodoacetyl peptide can react with many reagents to form a fixed positive charge. We find reaction with dimethyloctylamine forms a quaternary ammonium derivative with good surface activity properties and concomitant increased sensitivity. The high-energy CAD fragment ion spectra of the N-terminally derivatized peptides show predominantly a(n) and d(n) ions. The abundant d(n) ions permit ready distinction of leucine and isoleucine. Fewer fragment ions make data interpretation simpler and lead to more intense peaks since the ion intensity is spread among fewer peaks. The method is particularly useful for peptides which do not otherwise yield sufficient fragmentation to provide either the complete sequence or the locations of modified amino acids.

Amino Acid Sequence↗

PubMed2003

An assessment of three dinucleotide parameters to predict DNA curvature by quantitative comparison with experimental data.

Curved DNA fragments are often found near functionally important sites such as promoters and origins of replication, and hence sequence-dependent DNA curvature prediction is of great utility in genomics and bioinformatics. In light of this, an assessment of three different dinucleotide step parameters (based on gel retardation as well as crystal structure data) is carried out. These parameters (BMHT, LB and CS) are evaluated quantitatively for their ability to predict correctly the experimental results of a large set of nucleic acid sequences containing A-tracts as well as GC-rich motifs. This set contained around 40 synthetic as well as natural sequences whose solution properties have been well characterized experimentally. All three models could account reasonably well for curvature in the various DNA sequences. The CS model, where dinucleotide parameters are calculated from crystal structure data, consistently shows slightly better correlation with experimental data. Our simple analysis also indicates that presently available trinucleotide parameters fail to predict curvature in some of the well-characterized sequences. The study shows that the dinucleotide parameters with some further refinement can be used to predict sequence-dependent curvature correctly in genomic sequences.

Base Sequence↗