[Spinal cord infarction and recurrent venous thrombosis in association with estrogens and the 20210A allele of the prothrombin gene]. / Mielopatía aguda isquémica y trombosis venosa recurrente asociada a estrógenos y alelo 20210A del gen de la protrombina.

The acute spinal cord infarction is a rare cause of acute-onset paraplegia. Furthermore, it is specially uncommon that the infarction occurs in patients with apparent low predisposition to vascular disease. The 20210A allele of the prothrombin gene (causing a threefold-increased risk in venous thromboembolism) was recently associated with unexplained spinal cord infarction in young women under treatment with estrogens (contraceptive pill). We report a case of anterior spinal artery syndrome resulting from an ischaemic infarction at the anterior aspect of the spinal cord in a healthy 50-year-old woman, carrying this mutation, being the first published case under treatment with transdermal estradiol. She referred the typical sudden-onset back pain associated to clinical anterior spinal artery syndrome with sphincter dysfunction and nontraumatic paraplegia. A possible multiple sclerosis was ruled out and the steroids or immunoglobulin therapy induced no clinical improvement. Cerebrospinal fluid and other investigations were all negative. Sequential MRI scans revealed development of spinal cord infarction from T10 to T11, with increased signal in T2-weighted image (T2). Because she referred a previous thrombophlebitis and suffered a deep-vein thrombosis one month after paraplegia, a complete coagulation study was performed. Antithrombin, proteins C and S, homocysteine, factor V Leiden, lupus anticoagulant and anticardiolipin antibodies were all normal or negatives. In opposite, the 20210A variation was positive (heterozygous) and the factor VIIIc level was very high (280 U/dl eight months later). We argue the relative importance of both findings. The patient had no a substantial recovery over a period of 20 months.Certainly, the prothrombin 20210A seems to be associated with unexplained ischemic myelopathy among the young women with estrogens.

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).

Increased expression of vascular endothelial growth factor in pyogenic granulomas.

The expression of vascular endothelial growth factor (VEGF) was analysed in biopsy samples from patients with pyogenic granuloma. The results disclosed the presence of a strong VEGF signal in pyogenic granulomas, which are constituted by a vast majority of cells of endothelial lineage. A marked positivity was evident in areas of proliferating endothelial cells without vessel lumen formation. In the same respect, staining for VEGF was less marked in the vessels with a well-developed lumen. The fact that VEGF production appears to be limited to endothelial cell precursors or immature endothelial cells prior to the complete development of the vessels, leads to the possibility that VEGF may act as an autocrine factor in circumstances of endothelial cell stimulation.

Molecular genetic analysis of a compound heterozygote for the glycoprotein (GP) IIb gene associated with Glanzmann's thrombasthenia: disruption of the 674-687 disulfide bridge in GPIIb prevents surface exposure of GPIIb-IIIa complexes.

This work was aimed at elucidating the molecular genetic lesion(s) responsible for the thrombasthenic phenotype of a patient whose low platelet content of glycoprotein (GP) IIb-IIIa indicated that it was a case of type II Glanzmann's thrombasthenia (GT). The parents did not admit consanguinity and showed a reduced platelet content of GPIIb-IIIa. Polymerase chain reaction (PCR)-single-stranded conformational polymorphism analysis of genomic DNA showed no mutations in the patient's GPIIIa and two novel mutations in the GPIIb gene: one of them was a heterozygous splice junction mutation, a C-->A transversion, at position +2 of the exon 5-intron 5 boundary [IVS5(+2)C-->A] inherited from the father. The predicted effect of this mutation, insertion of intron 5 (76 bp) into the GPIIb-mRNA, was confirmed by reverse transcription-PCR analysis of platelet mRNA. The almost complete absence of this mutated form of GPIIb-mRNA suggests that it is very unstable. Virtually all of the proband's GPIIb-mRNA was accounted for by the allele inherited from the mother showing a T2113-->C transition that changes Cys674-->Arg674 disrupting the 674-687 intramolecular disulfide bridge. The proband showed a platelet accumulation of proGPIIb and minute amounts of GPIIb and GPIIIa. Moreover, transfection and immunoprecipitation analysis demonstrated that [Arg674]GPIIb is capable of forming a heterodimer complex with GPIIIa, but the rate of subunit maturation and the surface exposure of GPIIb-IIIa are strongly reduced. Thus, the intramolecular 674-687 disulfide bridge in GPIIb is essential for the normal processing of GPIIb-IIIa complexes. The additive effect of these two GPIIb mutations provides the molecular basis for the thrombasthenic phenotype of the proband.

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)

[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.

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.

Interchain and intrachain disulphide bonds in human platelet glycoprotein IIb. Localization of the epitopes for several monoclonal antibodies.

The single interchain disulphide bond in platelet glycoprotein IIb (GPIIb) is accessible to extracellular reductants, and selective cleavage does not liberate GPIIb alpha from platelet plasma membrane, confirming that non-covalent interactions contribute to maintaining attachment of this subunit to the membrane. Eosin-maleimide labelling of isolated GPIIb after selective cleavage of this interchain disulphide bond, followed by full reduction and alkylation, CNBr cleavage, and analysis of the cleavage products allowed us to establish that this interchain disulphide bridge is formed between GPIIb beta (GPIIb beta-subunit) Cys-9 and GPIIb alpha Cys-826, and this conclusion was confirmed by independent routes. The other two cysteines of GPIIb beta (Cys-14 and Cys-19) form the single intrachain disulphide bond in this subunit. Last, the intrachain disulphides in GPIIb alpha (GPIIb alpha-subunit) are distributed in four main peptide domains which are not disulphide-bonded among themselves. The linear epitope for monoclonal antibody M1 is localized between Pro-4 and Met-24 (or Met-31) of GPIIb beta. The linear epitope for M3 is situated between Cys-826 and the C-terminus of GPIIb alpha. The M4 epitope is also linear and localized somewhere between residues 115 and 285 of GPIIb alpha. Finally, the epitopes for M5 and M6 are somewhere between Cys-608 and Met-704, within a 35 kDa membrane-bound chymotryptic product of digestion of GPIIb in whole platelets. The N-terminal amino acid sequences determined for eight different cleavage products of GPIIb alpha and GPIIb beta agree with the corresponding amino acid sequences predicted by cDNA sequence for human-erythroleukaemic-cell GPIIb [Poncz, Eisman, Heindenreich, Silver, Vilaire, Surrey, Schwartz & Bennett (1987) J. Biol. Chem. 262, 8476-8482].

[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.

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.