Exome analysis and functional classification of identified variants in racing Quarter Horses.

The main objectives of this study were to identify and functionally classify SNPs and indels by exome sequencing of animals of the racing line of Quarter Horses. Based on the individual genomic estimated breeding values (GEBVs) for maximum speed index (SImax) obtained for 349 animals, two groups of 20 extreme animals were formed. Of these individuals, 20 animals with high GEBVs for SImax and 19 with low GEBVs for SImax had their exons and 5' and 3' UTRs sequenced. Considering SNPs and indels, 105 182 variants were identified in the expressed regions of the Quarter Horse genome. Of these, 72 166 variants were already known and 33 016 are new variants and were deposited in a database. The analysis of the set of gene variants significantly related (Padjusted  < 0.05) to extreme animals in conjunction with the predicted impact of the changes and the physiological role of protein product pointed to two candidate genes potentially related to racing performance: SLC3A1 on ECA15 and CCN6 on ECA10.

Prevention of arterial thrombosis by a monoclonal antibody against the 100 to 109 amino acid sequence stretch of the beta-subunit of the human platelet fibrinogen receptor: a comparative study with low dose aspirin.

OBJECTIVES: The aim of this study was to compare, in dogs, the antithrombotic activity of aspirin and the murine monoclonal antibody P37, which inhibits platelet aggregation and fibrinogen binding to activated platelets. BACKGROUND: The antithrombotic activity of P37 has been somewhat predictable, given its in vitro platelet antiaggregating activity and localization at or very near the fibrinogen binding site in the platelet fibrinogen receptor, the glycoprotein IIb/IIIa or integrin alpha IIb-beta 3. METHODS: The monoclonal antibody P37 of the immunogamma-globulin-1 isotype was prepared according to previously described immunization and fusion protocols and screening assays. To compare its antiaggregating capacity with that of aspirin, experimental thrombosis was induced in all dogs by means of direct current applied to the carotid artery. Autologous platelets had previously been labeled with indium-111 oxine. The dogs were assigned to three groups: group I (n = 18) was the control group; group II (n = 12) was treated orally with 5 mg of aspirin/kg body weight per day for 7 days before induction of thrombosis, and group III (n = 10) was treated intravenously with a single dose of P37 (0.8 mg/kg). RESULTS: The indium-111 oxine activity deposited in the thrombi was 12.94 +/- 12.83% (mean +/- SD) in group I, 3.55 +/- 2.99% in group II and 0.03 +/- 0.03% in group III. The differences between groups were always statistically significant (p < 0.05). CONCLUSIONS: We conclude that a single dose (0.8 mg/kg) of P37 in a canine model of arterial thrombosis is approximately 100 times more efficient than the administration of aspirin (5 mg/kg per day) in preventing platelet deposition during thrombus formation.

Normal frequencies of the C677T genotypes on the methylenetetrahydrofolate reductase (MTHFR) gene among lymphoproliferative disorders but not in multiple myeloma.

The folate availability seems to be critical for the DNA integrity since it is required for the transfer of methyl groups in the biosynthesis of thymidilate. Although the excessive incorporation of uracils to the DNA can be efficiently removed, this mechanism of reparation produces many double-strand breaks from two opposing nicks. Several chromosomal abnormalities (mainly translocations and deletions perhaps not well understood) are involved in the origin of lymphoproliferative disorders. The TT homozygosity at nucleotide 677 in the gene of methylene tetrahydrofolatereductase (MTHFR), a key enzyme in folate metabolism, was recently linked to a significant protection against colon carcinoma and acute lymphoblastic leukaemia in adults. We analysed the genotype frequencies of C677T-MTHFR in a group of 143 patients with lymphoproliferative disorders (REAL classification) and 200 controls. Overally, the frequencies of the polymorphic allele were similar (35.3% and 32.0% respectively)(P=0.6). We did not find differences between patients and controls except for myeloma/plasmacytoma group (n=26) which showed a CC genotype less than expected (19% vs 46%) (p=0.01) with a frequency ratio of 0.28 (0.10-0.77). Even among the IgG myeloma cases only one patient showed a common genotype (CC) (1/15, 7%) (P=0.003). If these preliminary data are validated with prospective studies, the 677C allele of MTHFR gene could be confirmed as an effective multiple myeloma protective factor (specially for the IgG cases).

Radiosensitization by quaternary salts of 5-nitroimidazole derivatives.

The radiosensitizing effects of five newly synthesized quaternary salts of 5-nitroimidazole derivatives on the survival of TC-SV40 mammalian cells have been measured. A toxicity study was carried out in order to determine the concentrations to be used in the radiosensitizing experiments. The oxygen enhancement ratio (OER) for TC-SV40 cells was 2.74. None of the five 5-nitroimidazole derivatives showed radiosensitizing activity in aerobic conditions, while in hypoxia their dose-modifying factors (DMF) at the concentration of 0.2 mmol dm-3 range from 1.52 to 1.03 in this order: unsubstituted pyridinium greater than carbamoyl pyridinium greater than trimethyl pyridinium greater than t-butyl pyridinium greater than imidazolium. This latter product at the concentration of 2 mmol dm-3 has a DMF of 1.64. As comparison, metronidazole was also tested on this cell line and its DMF at 0.2 mmol dm-3 was 1.35. The response-concentration dependences for the unsubstituted pyridinium 5-nitroimidazole derivative and for metronidazole (comparing charged and uncharged structures) showed the flattening response-concentration curve of quaternary compounds. The electron affinity was evaluated through the CNDO/S theoretical method, and an exponential relationship between these values and the DMFs of the pyridinium derivatives was demonstrated.

Organization of the glycoprotein (GP) IIb/IIIa heterodimer on resting human platelets studied by flow cytometric energy transfer.

Glycoprotein IIb/IIIa is a heterodimer of glycoproteins IIb and IIIa which serves as the inducible receptor for fibrinogen and other adhesive proteins at the surface of platelets. Although a model of the quaternary structure of the GPIIb/IIIa molecule has been constructed in solution by Calvete et al. [Biochem. J. 282 (1992) 523], a corresponding model at the surface of intact platelets is still missing. In the present work conformation and lateral distribution of the GPIIb/IIIa heterodimer were studied at a nanometer resolution on the surface of resting human platelets under physiological conditions. The experiments were based on dual wavelength flow cytometric detection of fluorescence resonance energy transfer and application of a panel of monoclonal antibodies raised against well described binding sites. Monodisperse distribution of the GPIIb/IIIa heterodimer has been observed and a detailed three-dimensional proximity map of antibody binding sites was constructed on the platelet membrane, under physiological conditions, for the first time. Our data support the view that the GPIIb subunit is in a bent conformation. A detailed analysis of the K(d)-values and the number of binding sites for a set of monoclonal antibodies was also carried out giving supplementary data for the topology of the binding sites. Our results provide a refinement of the membrane-topology of the GPIIb/IIIa heterodimer.

[Cesarean section in a patient with a congenital deficit of antithrombin III: apropos of a case]. / Cesárea en una paciente con déficit congénito de antitrombina III: a propósito de un case.

Antithrombin III (AT III) is a physiological inhibitor of coagulation. AT III deficit, whether congenital or acquired, results in a state of hypercoagulability characterized by recurring instances of venous thrombosis in young people. Although AT III levels normally change little during pregnancy, a deficit can be associated to the appearance of recurring thromboembolism and the need to perform cesarean section increases the risk. We report a cesarean section under general anesthesia in a patient with congenital AT III, reviewing the etiology and pathophysiology of this entity as well as its treatment.

Glyoxylic compounds as radiosensitizers of hypoxic cells.

The radiosensitizing effect of five glyoxal derivatives on the survival of TC-SV40 cells has been measured, under aerobic and hypoxic conditions. A toxicity study was previously performed in order to use nontoxic concentrations. The OER for the TC-SV40 cells was 2.74. None of the glyoxylic compounds showed radiosensitizing activity under aerobic conditions while in hypoxia their radiosensitizing factors decreased in the order phenylglyoxylic acid (1.68 at 8 x 10(-3) mole dm-3) greater than phenylglyoxal (1.55 at 5 x 10(-6) mole dm-3) greater than 2-2' furil (1.48 at 5 x 10(-5) mole dm-3) greater than glyoxylic acid (1.39 at 1 x 10(-3) mole dm-3) greater than glyoxal (1.30 at 5 x 10(-5) mole dm-3). The dose-modifying factors were also determined at two equimolar concentrations 5 x 10(-5) and 5 x 10(-6) mole dm-3. A concentration effect was noticed for all the compounds although their relative radiosensitizing activity kept, independently of the concentration, the same order noted above. Glyoxals with aromatic or heterocyclic rings exert a greater radiosensitization than the others. The acidic compounds have less radiosensitizing activity than their aldehydic counterparts. Interaction of these glyoxals with NPSH cellular groups was tested and the low degree of inhibition shows that this mechanism would contribute very little, if any, to the radiosensitization effect.

Proteolytic dissection of the isolated platelet fibrinogen receptor, integrin GPIIb/IIIa. Localization of GPIIb and GPIIIa sequences putatively involved in the subunit interface and in intrasubunit and intrachain contacts.

Human platelet glycoproteins IIb (GPIIb) and IIIa (GPIIIa) form the subunits of the Ca(2+)-dependent heterodimer GPIIb/IIIa, which belongs to the integrin family of phylogenetically related receptors mediating a wide variety of cell-cell and cell-substratum interactions. GPIIb/IIIa plays a central role in haemostasis as a receptor for fibrinogen and other adhesive proteins at the surface of activated platelets. The covalent structure of the subunits is largely known; however, the tertiary and quaternary structures of the heterodimer remain to be determined. To this end, our approach consisted of limited proteolysis of the isolated heterodimer with proteinases of different specificities, followed by protein-chemical and immunochemical analyses of the peptide fragments within each isolated proteolytic product. From the information obtained, we have drawn a rudimentary map which outlines the demarcation of compact domains and the subunit peptide stretches carrying the sequences putatively involved in intrachain, intrasubunit and intersubunit non-covalent connectivity in the heterodimer. Three compact domains have been well defined: one in the heavy (H) chain of GPIIb [GPIIbH-(600-700)], and two in GPIIIa, the N-terminal [GPIIIa-(1-52)] and the core [GPIIIa-(423-622)] domains. Between the latter two domains there is a proteolysis-susceptible region, which is partly involved in ligand binding [GPIIIa-(100-220)] and partly implicated as being in teh subunit interface of the heterodimer. Contrary to GPIIIa, GPIIbH is highly susceptible to proteolysis all along its sequence. Equally susceptible are the extracellular end of the transmembrane segment of both GPIIIa and the light (L) chain of GPIIb (GPIIbL), and the N-terminal end of GPIIbL. Three sequence stretches along the C-terminal half of GPIIbH, one sequence stretch in GPIIbL and three sequence stretches within the GPIIIa-(217-421) region were putatively involved in the subunit interface of the heterodimer. Most likely, the N-terminal end of GPIIbL is folded over the N- and C-terminal regions of GPIIbH, and the N-terminal end of GPIIbH is folded against the GPIIbH-(600-700) domain. This map of GPIIb/IIIa does not fit the current accommodation of the amino acid sequence of GPIIb and GPIIIa in the head/two-tails image of the heterodimer obtained by metal-rotary-shadowing electron microscopy.

[Synthesis of 1-substituted nitroimidazoles and its evaluation as radiosensitizing agents]. / Síntesis de nitroimidazoles 1-sustituidos y su evaluación como radiosensibilizantes.

The synthesis of various substituted nitroimidazoles with lipophilic and hydrophilic side chains as potential radiosensitizing agents is described. The starting material employed was 4(5)-nitroimidazole, which was alkylated via the sodium salt with various chloro-methylated, substituted alcohols and esters, in order to obtain analogues of misonidazole, metronidazole and desmethylmisonidazole of known radiosensitizing and bactericidal activity. Some final products were assayed for their radiosensitizing properties giving negative results under the testing conditions used.

Rotational mobility of the fibrinogen receptor glycoprotein IIb/IIIa or integrin alpha IIb beta 3 in the plasma membrane of human platelets.

Integrin alpha IIb beta 3 or glycoprotein IIb/IIIa (GPIIb/IIIa, 228 kDa) is a Ca(2+)-dependent, noncovalent heterodimer of glycoproteins IIb (GPIIb or alpha IIb, 136 kDa) and IIIa (GPIIIa or beta 3, 92 kDa), which serves as the receptor for fibrinogen and other adhesive proteins at the surface of activated platelets. We have determined the microsecond-range rotational motions of alpha IIb beta 3 in resting platelets, in isolated plasma membranes, and reconstituted in 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) bilayers. The measurements were based on the time-resolved phosphorescence anisotropy [r(t)] of erythrosin-labeled F(ab) fragments [Er-F(ab)] of monoclonal antibodies bound to alpha IIb beta 3. In general, the r(t) decays were satisfactorily fitted to the sum of the two exponential terms and a constant, from which the initial anisotropy (r(in) approximately 0.05-0.11), the short (phi 1 approximately 1-14 microseconds) and the long (phi 2 approximately 15-60 microseconds) rotational correlation times, and the limiting anisotropy (r infinity approximately 0.02-0.07) were obtained. The observed values depended on the platelet preparation, temperature, Ca2+ concentration, and the antibody used. In accordance with data on the order parameter and the viscosity of the lipid bilayer of the platelet plasma membrane, phi 2 and r infinity of the alpha IIb beta 3-Er-F(ab) complexes in the three preparations decreased with the increase of temperature, the r(t) curves being fully reversible within the interval from 5 to 35 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)

Twofold symmetry of human fibrinogen proved at the beta chain distal domains by monoclonal-immunoelectron microscopy and image analysis.

Using a murine antibody (F7) specific for the C-terminal domain of the beta chain of human fibrinogen combined with electron microscopy and image analysis, we show unequivocally that the epitopes for F7 are at the distal nodules of fibrinogen, equidistant from the center of the molecule and arranged not colinearly with the long axis of the molecule but at opposite sides of it, i.e., following twofold symmetry. Thus, given the monoclonality of the immunochemical probe used and the dimeric nature of the fibrinogen molecule, we can conclude that the distal domains of the two beta chains are arranged in the same manner as these epitopes and, therefore, that the fibrinogen molecule has twofold symmetry. This symmetry pattern found here for F7 is the same as that found recently for the platelet fibrinogen receptor binding sites [Weisel, J. W., Nagaswami, C., Vilaire, G., & Bennett, J. S. (1992) J. Biol. Chem. 23, 16637-16643], located almost certainly at the C-terminal end of the gamma chains, and gives further support to the most accurate model of fibrinogen available so far. We discuss the consequences of this symmetry pattern and of the molecular rigidity of fibrinogen in the actual models of fibrin polymerization and platelet aggregation and adhesion.

Cardiac troponin I (cTn I) and the postmortem diagnosis of myocardial infarction.

In clinical practice several biochemical markers are used for the diagnosis of myocardial infarction. Because of its extremely high specificity for myocardial damage, cardiac troponin I (cTn I) is frequently used. The aim of this study was to evaluate the diagnostic efficacy of postmortem cTn I determinations in pericardial fluid and serum and to compare these results with other biochemical markers and with structural findings used to diagnose acute myocardial ischaemia. We studied 89 cadavers with a mean age of 51.38 +/- 2.04 (SD 19.27 years). Cases were allocated to 1 of 4 diagnostic groups depending on the probable intensity of myocardial damage and cause of death. In pericardial fluid we obtained statistically significant differences for the four biochemical parameters, while in serum myosin heavy chains and myoglobin showed statistically significant differences. The highest levels of biochemical markers in pericardial fluid were observed in subjects who had died from definite myocardial infarction.

Studies on cis-dichlorodiammineplatinum (II) as a radiosensitizer.

Cis-dichlorodiammineplatinum (II) (cisDDP) has been extensively studied as an antitumour agent; its binding to DNA has been proved but the radiosensitizing action has scarcely been tested. We report here that on TC.SV -40 mammalian cells cisDPP acts as a dose-modifying factor for ionizing radiation. The radiosensitizing action can be induced by two different mechanisms: reaction with non protein-SH groups and inhibition of repair processes. The cisDDP-DNA complex was studied against increasing radiation doses by analytical centrifugation and by spectrophotometrical measurements. The native complex seems to be more radiation resistant than the denatured one.

Theoretical approach for radiosensitizers. Correlation between calculated electroaffinity and sensitization factors of nitro-compounds.

The electron affinity of eight well-known nitroaromatic and nitroheterocyclic radiosensitizers is computed resorting to a quantum mechanical approach. Four of the compounds studied were nitroimidazoles; but p-nitroacetophenone and some 5-nitrofuran derivatives were also investigated for comparison. A good correlation between the theoretical electroaffinity values and the radiosensitization efficiencies is found in the case of the nitroimidazoles, in which the pi-electron system appears to be limited to the aromatic ring, except for small side groups. In the case of molecules where the pi-electron system spreads out on the side-chain the theoretical procedure seems to fail.

[Platinum complexes as radiosensitizers in radiotherapy]. / Complejos de platino como radiosensibilizadores en radioteriapia.

The authors study the radiosensitizing effect of cisplatinum, cis-dichloroethylenediamine platinum (II), and cis-dichlorobis-cyclohexylamine platinum (II) by the of TC-SV40 cells radiated under aerobic and hypoxic conditions. Cis-platinum shows the highest radiosensitizing activity but is very toxic. Perhaps cis-dischloro-bis-cyclohexylamine platinum (II) could be assayed as a less toxic radiosensitizing agent. On the other hand, DNA synthesis is studied in mouse spleen and TC-SV40 cells labeled with tritiated thymidine. Cis-platinum inhibitory activity is related to the preincubation time, cis-dichloroethylenediamine platinum(II) does not inhibit the semiconservative synthesis and stimulates the repair synthesis of DNA, and cis-dichloro-bis-cyclohexylamine platinum (II) produces a 60 per 100 inhibition of the semiconservative synthesis and a smaller inhibition of the repair synthesis.

Further studies on the topography of the N-terminal region of human platelet glycoprotein IIIa. Localization of monoclonal antibody epitopes and the putative fibrinogen-binding sites.

The precise localization of the epitopes for six monoclonal antibodies specific for the N-terminal region of human platelet glycoprotein IIIa (GPIIIa) was determined. The epitope for P37, a monoclonal antibody that inhibits platelet aggregation, was found at GPIIIa 101-109, flanked by the epitopes for P23-3 (GPIIIa 16-28), P23-4 (GPIIIa 83-91), P23-5 (GPIIIa 67-73), P23-7 (GPIIIa 114-122) and P40 (GPIIIa 262-302), and very close to the early chymotryptic cleavage site of GPIIIa in whole platelets (Phe-100). When the amino acid sequence of GPIIIa was searched for peptide sequences hydropathically complementary to the fibrinogen gamma-chain C-terminal (gamma 400-411) and A alpha-chain RGD-containing peptides, none was found for the gamma 400-411, two (GPIIIa 128-132 and 380-384) were found complementary to fibrinogen A alpha 571-575 and two (GPIIIa 109-113 and 129-133) were found for A alpha 94-99. Two of these putative fibrinogen-binding sites overlap with each other, and a third one overlaps with the epitope for P37. These findings reinforce the earlier suggestion that the N-terminal region of GPIIIa is involved in fibrinogen binding, and suggest the existence in GPIIIa of either multiple or alternative RGD-binding sites or one RGD-binding domain with several moieties. Finally, early chymotryptic cleavage of GPIIIa in whole platelets liberates to the soluble fraction the peptide stretch Ser-101-Tyr-348, which carries the epitope for P37 and the putative binding sites for fibrinogen. The rest of the molecule, together with the GPIIb-resistant moiety, remains membrane-bound. This leads us to propose that the fibrinogen-binding domain of GPIIIa is not involved in the binding to GPIIb to form the Ca2(+)-dependent GPIIb-GPIIIa complex.

Further studies on the topography of human platelet glycoprotein IIb. Localization of monoclonal antibody epitopes and the putative glycoprotein IIa- and fibrinogen-binding regions.

Glycoprotein IIb (GPIIb) is a major glycoprotein of the human platelet plasma membrane, which together with glycoprotein IIIa (GPIIIa) forms a Ca2(+)-dependent heterodimer, GPIIb/IIIa, which serves as the major fibrinogen receptor in activated platelets. The precise localization of the epitopes for six anti-GPIIb monoclonal antibodies (M1-M6) has been determined by a combination of enzymic and chemical cleavage procedures, peptide isolation, N-terminal sequence analysis, peptide synthesis and enzyme immunoassay. The following localizations were found: M1, beta 1-16-36, beta 2-4-24; M2, alpha 747-755; M alpha 2, alpha 837-843; M3, alpha 849-857; M4, alpha 143-151; M5, alpha 550-558; M6, alpha 657-665. Besides considerations of the degree of exposure of these epitopes, several remarkable features are readily apparent. The earliest and main chymotryptic cleavage site of GPIIb in whole platelets is between alpha cysteine-545 and alpha phenylalanine-551. The epitope for M3 was located within the same sequence (alpha 842-857) as is the epitope for PMI-1 [Loftus, Plow, Frelinger, D'Souza, Dixon, Lacy, Sorge & Ginsberg (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 7114-7118] in spite of the fact that the exposure of the latter in whole platelets is EDTA-dependent whereas that in the former is not. The epitope for M5 shares full homology with the 540-548 peptide stretch of the alpha-subunit of the vitronectin receptor, and this antibody cross-reacts with endothelial cells. The M6 epitope is located in the 25 kDa membrane-bound fragment of GPIIb, which is most epitope is destroyed at an early stage of chymotrypic digestion. This suggests that this region of GPIIb, somewhere between the epitope for M5 (alpha 550-558) and the epitope for M2 (alpha 747-755), may carry the surface of interaction of GPIIb with GPIIIa in the GPIIb/IIIa heterodimer. Finally, the sequence where the epitope for M6 has been located (alpha 657-667) was the only one found to be hydropathically complementary to the gamma 402-411 peptide of fibrinogen within the amino acid sequence of both GPIIb and GPIIIa. This complementariness, the EDTA- or thrombin-dependence of the exposure of the alpha 657-665 stretch in whole platelets to M6 and the ability of this antibody to inhibit platelet aggregation led us to postulate that this peptide stretch is a putative binding site for fibrinogen in the platelet receptor.(ABSTRACT TRUNCATED AT 400 WORDS)