A new murine monoclonal antibody against Kx protein.

Mice immunized with a synthetic peptide located on an intracellular segment of the polytopic Kx protein (37 kDa) from human red blood cells (RBCs) produced a monoclonal antibody called C7B8. As expected, this antibody did not agglutinate common RBCs but reacted with permeabilized cells in flow cytometry. C7B8 recognizes the Kx protein on Western blots. Cross-reactivity of C7B8 with human calpain of human muscle extracts was demonstrated by Western blot analysis. This cross-reactivity precludes the use of C7B8 for Kx tissue distribution studies, but immobilized C7B8 was a convenient tool for purification of the Kell-Kx complex from RBC membrane extract by immunochromatography.

Structural characterization of the epitope recognized by the new anti-Fy6 monoclonal antibody NaM 185-2C3.

The epitope recognized by a new anti-Fy6 monoclonal antibody (MoAb) (clone name: NaM185-2C3) was characterized using peptides synthesized on pins (Epitope scanning kit). The clone was obtained from splenocytes of mice immunized with CHO cells expressing the recombinant Duffy glycoprotein. NaM185-2C3 recognized a linear epitope, the essential portion of which was pentapeptide Phe-Glu-Asp-Val-Trp comprising amino acid residues 22-26 of the main (336aa) isoform of the Duffy antigen. All the amino acid residues of the epitope, except Asp, were essential for the antibody-binding, because they could not be replaced by any or most other amino acid residues. The Asp residue could be replaced by most other amino acid residues and its replacement by some amino acid residues gave a distinct increase in the antibody-binding. The MoAb NaM185-2C3, similarly as other anti-Fy6 antibodies, inhibits interleukin (IL)-8-binding to the Duffy antigen. A part of the results was presented at ISBT meeting (Blanchard et al., 1998, Vox Sanguinis, 74, S1, Abstract no. 71).

A murine monoclonal antibody against Kx protein which reacts also with beta-spectrin.

Kx is a polytopic membrane protein of human erythrocytes carrying the Kx blood group antigen, which is deficient in rare patients with McLeod syndrome. Kx is disulphide bond linked to the Kell glycoprotein, which is a bitopic type II membrane protein carrying the Kell blood group antigen. Mice immunized with a synthetic peptide predicted to be located on the second external loop of Kx produced a monoclonal antibody called 3E12 which does not recognize red cells with common Kell phenotype by agglutination and flow cytometry. 3E12 recognizes the Kx protein and the spectrin beta-chain on western blots, the affinity for these two proteins being lowered with increasing ionic strength. Linear epitopes recognized by 3E12 are E116EIEKE121 and L484AQELEKE491 on the Kx protein and spectrin beta-chain, respectively. To quantify the relative amount of Kx in Empigen BB extracts of red cell membranes, an ELISA for Kx was set up which showed conclusively that (i) there is less Kx in membranes of K0 individuals (lacking the Kell glycoprotein) than in membranes of common individuals, and (ii) that all common individuals, typed as K+k-, K-k+ and K+k+, have the same amount of Kx on their red cell membranes. When an erythrocyte membrane detergent extract from one K0 individual was chromatographed on an immobilized 3E12 column, a minute amount of authentic Kell glycoprotein was recovered in acid eluted fractions, indicating that at least the K0 individual under study may still produce some Kell protein.

Structural changes induced by binding of the high-mobility group I protein to a mouse satellite DNA sequence.

Using spectroscopic methods, we have studied the structural changes induced in both protein and DNA upon binding of the High-Mobility Group I (HMG-I) protein to a 21-bp sequence derived from mouse satellite DNA. We show that these structural changes depend on the stoichiometry of the protein/DNA complexes formed, as determined by Job plots derived from experiments using pyrene-labeled duplexes. Circular dichroism and melting temperature experiments extended in the far ultraviolet range show that while native HMG-I is mainly random coiled in solution, it adopts a beta-turn conformation upon forming a 1:1 complex in which the protein first binds to one of two dA.dT stretches present in the duplex. HMG-I structure in the 1:1 complex is dependent on the sequence of its DNA target. A 3:1 HMG-I/DNA complex can also form and is characterized by a small increase in the DNA natural bend and/or compaction coupled to a change in the protein conformation, as determined from fluorescence resonance energy transfer (FRET) experiments. In addition, a peptide corresponding to an extended DNA-binding domain of HMG-I induces an ordered condensation of DNA duplexes. Based on the constraints derived from pyrene excimer measurements, we present a model of these nucleated structures. Our results illustrate an extreme case of protein structure induced by DNA conformation that may bear on the evolutionary conservation of the DNA-binding motifs of HMG-I. We discuss the functional relevance of the structural flexibility of HMG-I associated with the nature of its DNA targets and the implications of the binding stoichiometry for several aspects of chromatin structure and gene regulation.

Triple helix formation and homologous strand exchange in pyrene-labeled oligonucleotides.

The orientations of the symmetrical third strands (G3A4G3) and (G3T4G3) within the triplexes (C3T4C3) - (G3A4G3) x (G3A4G3) and (C3T4C3) - (G3A4G3) x (G3T4G3) were investigated by fluorescence spectroscopy and thermal denaturation using pyrene-labeled oligodeoxynucleotides. In the two triplex structures, both parallel and antiparallel orientations of the third strand with respects to the purine Watson-Crick one were identified by means of pyrene excimer formation. The pyrene labels do not modify the melting temperature of the (C3T4C3) - (G3A4G3) x (G3T4G3) triplex but somewhat stabilize the corresponding duplex against thermal denaturation. The absorption melting profiles of the (C3T4C3) - (G3A4G3) x (G3A4G3) triplex are monophasic in agreement with previous reports. In contrast, the melting of this structure, when monitored by the pyrene excimer band, reveals a biphasic behavior. These data, together with kinetics measurements, strongly suggest exchange mechanisms between the homologous oligomers (G3A4G3), Hoogsteen, and Watson-Crick strands.

A continuous transition from A-DNA to B-DNA in the 1:1 complex between nogalamycin and the hexamer dCCCGGG.

The antibiotic nogalamycin, a drug with high specificity for TG and CG steps in double-stranded DNA, has been crystallized as a 1:1 complex with the hexamer d(CCCGGG). The antibiotic is inserted at the central CG step of the duplex, with the two sugars oriented in the same direction and with strong interactions with the DNA within the grooves. The amino-glucose residue makes an integral part of a well defined major groove hydration network with van der Waals contacts and several strong hydrogen bonds to the duplex. The nogalose residue resides in the minor groove, making primarily van der Waals contacts. The single site allows an accurate molecular description of the intercalation, without perturbations from end effects observed previously. The local unwinding induced by nogalamycin is completely relaxed 2 base pairs away from the intercalation site. The two strands of the DNA show a continuous deformation from the A to the B form: 1) the cytosines toward the 5' end of the nogalomycin site in each strand have c3'-endo conformations while 5 guanosines toward the 3' ends have c2'-endo conformations; 2) within each strand, the phosphate-phosphate distances increase in a continuous manner from 5.7 A (A-form) to 7.1 A (B-form).

Synthesis of 4-Amino-1-Hydroxy-Butane-1,1-Diphosphonate (AHBDP) - Stannous Complexes for the Preparation of Ahbdp-Sn(II)-Tc and its Biodistribution in Rats.

The new potential tracer of bone imaging, AHBDP-Sn(II)-TcO.3H(2)O was synthesized by reducing the TcO(4) (-) to TcO(2) (+) in the presence of AHBDP and Sn(ll)'s reducing agent. We found that tin rapidly forms a stable complex with AHBDP, giving AHBDP-Sn(II).3H(2)O. In the excess of AHBDP-Sn(ll).3H(2)O, the AHBDP-Sn(II).3H(2)O coordinates with TcO(2) (+) to give AHBDP-Sn(II)-TcO.3H(2)O which could polymerise or oligomerise to give hydrophobic species. The overall process appears as a first-order reaction (K= 0.67 +/- 0.005s(-1)). In rats, the fixation of AHBDP-Sn(II)-(99m)TcO. 3H(2)O on bone is homogeneous and the scintigraphic images have the same quality as those of 1-hydroxymethane-1,1-diphosphonate-Technetium (HMDP-(99m)Tc). The activity in non-target organs was neglible.

[Mesentric venous thrombosis. Risk factors, treatment and outcome. An analysis of 18 cases]. / Thromboses veineuses mésentériques. Facteurs de risque, traitement et évolution. Analyse de 18 observations.

Eighteen patients with an acute thrombosis of the splanchnic veins were reviewed. Most of apparently idiopathic cases of splanchnic vein thrombosis are related to an increased coagulation related to a congenital or acquired defect of haemostasis. The aim of this study was to assess the effects of a new and effective treatment. Nine male and 9 female patients (range of age: 19 to 81 years) experienced a mesenteric venous thrombosis. There were 14 mesenteric vein thromboses with infarction, two transient mesenteric venous ischaemias without bowel infarction and two acute thromboses of the splanchnic veins without bowel ischaemia. A coagulopathy was detected in seven patients: oral contraception, protein C (PC) or antithrombin III (AT III) congenital deficiencies, acquired deficiency of AT III, PC and protein S (PS), polycythaemia in the post-partum period and primary myeloproliferative disorder. No coagulopathy was associated with thrombosis in eight cases: mesenteric haematoma, splenomegaly, cirrhosis, appendicectomy, cholescytectomy, chronic heart failure, treatment with beta-adrenergic receptor antagonist and digitalis, stenosis of the portal anastomosis after liver transplantation. Twelve patients required surgery: eight intestinal bowel resections with immediate anastomosis, four resections without immediate anastomosis. Only one patient underwent a second look for a repeat bowel resection. No death occurred in the early postoperative period and 17 out of 18 patients were alive after 12 years. An oral anticoagulant therapy was undertaken from two months to seven years. However, three patients suffered a recurrent thrombosis. Two of them required a long-term anticoagulation. Six patients experienced a portal hypertension and oral anticoagulants were discontinued in three of them because of bleeding oesophageal varices. Six patients were treated only by unfractionated heparin (UFH) or low molecular weight heparin (LMWH) followed by oral anticoagulants. After laparotomy, two were only treated with UFH without any bowel resection, as mesenteric venous ischaemia was too extensive. These observations suggest that the choice between an appropriate medical or surgical treatment is important and must be discussed. Since 1989, the therapeutic choice has been modified by ultrasonography and contrast enhanced computed tomographic scan which confirms diagnosis, allows to follow up and check the effects of anticoagulation and to choose the time for surgery. When the diagnosis is established and the patient's risk is low, the IU . kg(-1) . d(-1) to obtain an antifactor Xa activity between 0.3 and 0.6 antiXa IU mL(-1). When the diagnosis is uncertain and the patient's risk if high a laparotomy is required.(ABSTRACT TRUNCATED AT 400 WORDS)

Efficacy of 3,4,3-LIHOPO for reducing the retention of 238Pu in rat after inhalation of the tributyl phosphate complex.

The efficacy of 3,4,3-LIHOPO, a siderophore analogue, has been tested for removing 238Pu from rat after inhalation of plutonium as the tri-N-butylphosphate (TBP) complex. The amounts of Pu retained in the lung of untreated rat, 7 days after exposure ranged from 0.86 to 37 kBq. The results have been compared with DTPA, the current therapy of choice for man. The ligand 3,4,3-LIHOPO was more effective than DTPA for removing Pu from the body when repeated treatment began 1 h after inhalation. This observation was independent of the mass of Pu deposited in the lungs. The efficacy of 3,4,3-LIHOPO was mainly due to the decrease of Pu retention in lung, 1.5 times less than after DTPA administration; in liver and skeleton, retention was about four times less. Seven days after internal contamination, < 10% of the activity was found in organs other than lung when rat was treated with 3,4,3-LIHOPO. As this ligand showed an apparent lack of irreversible toxicity, it is likely to be of interest in the development of new decorporation treatments after inhalation of Pu as a TBP complex.

Comparative efficacies of 3,4,3-LIHOPO and DTPA for enhancing the excretion of plutonium and americium from the rat after simulated wound contamination as nitrates.

With DTPA as a comparison, the siderophore analogue 3,4,3-LIHOPO has been examined for its ability to remove 238Pu and 241Am from the rat after subcutaneous (s.c.) and intramuscular (i.m.) injection of about 200 Bq of each actinide (0.3 ng Pu, 1.6 ng Am). After the s.c. deposition of 238Pu and 241Am, both ligands were more effective after local administration than (in decreasing order) their repeated interperitoneal (i.p.) injection, single i.p. injection and continuous infusion. Dosages of 3 mumol kg-1 of 3,4,3-LIHOPO were at least as effective as 30 mumol kg-1 DTPA after each mode of administration. The most effective regimen of those investigated for s.c. 238Pu and 241Am involved local administration of 30 mumol kg-1 of 3,4,3-LIHOPO at 30 min followed by i.p. injections at 6 h, 1, 2 and 3 day. By day 7 after exposure, the amounts of 238Pu and 241Am retained in the body were 2 and 7% of those in controls, respectively and 10 and four times less than when DTPA was administered using the same regimen. The ligand 3,4,3-LIHOPO was more effective for 238Pu and 241Am after their i.m. injection. This was attributed to the greater retention of these actinides at the wound site (97 versus 67%) when treatment commenced. After a single local injection of 30 mumol kg-1 at 30 min, the amounts of 238Pu and 241Am retained in the body at 7 day were 0.9 and 0.8% of controls. These values were 34 and 27 times less than after local and repeated i.p. injections of DTPA at dosages of 30 mumol kg-1. It is concluded that the administration of 3,4,3-LIHOPO represents potentially a most significant advance in the treatment of wound contamination by 238Pu and 241Am by chelating agents.

The efficacies of 3,4,3-LIHOPO and DTPA for enhancing the excretion of plutonium and americium from the rat: comparison with other siderophore analogues.

With DTPA as a comparison, the siderophore analogue code named 3,4,3-LIHOPO has been tested for its ability to remove 238Pu and 241Am from rats after their inhalation or intravenous injection as nitrate. The most effective treatment regimen for inhaled Pu was the repeated administration of 30 mumol kg-1 3,4,3-LIHOPO. By 7 days after exposure, the Pu contents of the lungs and total body were reduced respectively to 2 and 4% of those in untreated animals. These values were six and three times less than when DTPA was administered using the same protocol. For inhaled Am, 3,4,3-LIHOPO and DTPA were considered equally effective, the lung and total body contents being reduced respectively to 13 and 10% of those in controls. Some animals showed slight degenerative changes in the liver and proximal tubules of the kidneys after the repeated administration of 30 mumol kg-1 of 3,4,3-LIHOPO; however these changes were less marked than after DTPA treatment. After the intravenous injection of Pu, the most effective regimen was the single administration of 3 mumol kg-1 3,4,3-LIHOPO. The body content at 7 days was reduced to 7% controls compared with 19% after the repeated administration of 30 mumol kg-1 DTPA. At a dosage of 30 mumol kg-1, 3,4,3-LIHOPO was less effective owing to the higher retention of Pu in the liver. With repeated dosages of 30 mumol kg-1 3,4,3-LIHOPO was more effective than DTPA for the decorporation of Am; the body contents were 16 and 31% of those in controls respectively. Importantly, the body content was still reduced to 28% of control after a single administration of 3 mumol kg-1. The ligand 3,4,3-LIHOPO, which is also superior to other siderophore analogues, could represent a most significant development in the decorporation of Pu and Am.

Evidence for broken minocycline by NMR and HPLC techniques: a new additional resistance mechanism mediated by tetB determinant.

As demonstrated by microbiological assays, a decrease in the active minocycline level occurs in spent media from each Escherichia coli K12 recipient containing one of 10 different plasmids bearing tetB determinants. No such decrease was detected when tetA, C, D or E determinants were tested under the same conditions. Likewise, no decrease in tetracycline or doxycycline levels was detected when 20 plasmids bearing tetA to E determinants were tested. Studies carried out by nuclear magnetic resonance and high pressure liquid chromatography proved that minocycline is broken by a mechanism mediated by the tetB determinant. This new mechanism can be considered as additional to the active efflux of minocycline.

Pharmacokinetics of metapramine (19560 RP) in man after infusion and intravenous injection.

The time course of the plasma level metapramine following infusion at three different rates (1.18, 2.36 and 4.71 mg/h) has been studied in six healthy volunteers. The correlation coefficient of drug concentration and rate of infusion was 0.974 (alpha less than 0.001, n = 18). Plasma clearance, estimated from the steady-state level, varied from 68 to 1071/h. 3 subjects also received 35 mg of drug by i.v. bolus injection and plasma concentrations were determined at set times for up to 24 h. The plasma level of metapramine decreased tri-exponentially, with a terminal half-life of about 7.4 h. The plasma clearance after i.v. injection was in good agreement with that observed during the infusion study. Apparent volumes of distribution ranged from 41.9 to 90.31.