Interaction of the von Willebrand factor with platelets and thrombosis

The human von Willebrand factor (vWf) is a multimeric glycoprotein present in plasma, platelets, endothelial cells and subendothelium and synthesized in endothelial cells and megakaryocytes. vWf plays a pivotal role in the mechanisms of blood clotting and platelet thrombus formation; quantitative and qualitative abnormalities of vWf cause the most common congenital bleeding disorder in man, the von Willebrand disease. vWf stabilizes factor VIII and interacts with subendothelial components and with platelet membrane receptors. The multimeric structure of vWf provides an array of binding sites which allows multivalent interactions with its ligands, thus supporting the formation of stable platelet aggregates at the site of vascular injury, particularly under flow conditions characterized by high shear stress. In the last years, remarkable progress has been made toward understanding the structure of vWf protein and gene, and the elucidation of many structure-function relationships, which may result in improved therapeutic intervention for vWD patients, and in the development of effective strategies for antithrombotic therapy.     

Properties of the regulatory subunit of cAMP-dependent protein kinase type II from human brain

The regulatory subunit type II (RII) of cAMP-dependent protein kinase purified from human brain was represented by two proteins with apparent molecular masses of 51-52 kD and 54 kD. Dephosphorylation of human RII containing 3 mol phosphate/mol protein did not change the electrophoretic pattern. One-dimensional peptide mapping of 51-52 kD and 54 kD proteins after digestion with St. aureus V8 protease evidenced to their being distinct proteins. The data obtained permit to assume that human RII of neural type is represented by two isoforms.     

Homocamptothecins: synthesis and antitumor activity of novel E-ring-modified camptothecin analogues

Homocamptothecin (hCPT), a camptothecin (CPT) analogue with a seven membered beta-hydroxylactone which combines enhanced plasma stability and potent topoisomerase I (Topo I)-mediated activity, is an attractive template for the elaboration of new anticancer agents. Like CPT, hCPT carries an asymmetric tertiary alcohol and displays stereoselective inhibition of Topo I. The preparation and biological screening of racemic hCPT analogues are described. The 10 hCPTs tested were better Topo I inhibitors than CPT. Fluorinated hCPTs 23c, d,f,g were found to have potent cytotoxic activity on A427 and PC-3 tumor cell lines. Their cytotoxicity remained high on the K562adr and MCF7mdr cell lines, which overexpress a functionally active P-glycoprotein. Fluorinated hCPTs were more efficacious in vivo than CPT on HT-29 xenografts. In this model, a tumor growth delay of 25 days was reached with hCPT 23g at a daily dose of 0.32 mg/kg, compared to 4 days with CPT at 0.625 mg/kg. Thus difluorinated hCPT 23g warrants further investigation as a novel Topo I inhibitor with high cytotoxicity toward tumor cells and promising in vivo efficacy.     

Effect of propofol and thiopentone on free radical mediated oxidative stress of the erythrocyte

Propofol has free radical scavenging properties similar to those of recognized phenol-based antioxidants. We have examined these properties in an in vitro model of radical-induced cellular injury, comparing its activity with that of thiopentone (which has also been shown to have radical scavenging activity). Haemolysis of human erythrocytes was induced using the azo compound 2,2'-azo-bis(2-amidinopropane) dihydrochloride (ABAP). This was achieved by incubating a 10% suspension of erythrocytes with ABAP 100 mmol litre-1 at 37 degrees C. For propofol, at concentrations of 12.5, 25 and 50 mumol litre-1, the times to achieve 50% haemolysis were mean 126 (SEM 7) min (95% confidence interval 108-144 min), 150 (8) (129-170) min and 182 (12) (160-180) min, respectively (Intralipid control 107 (7) (90-125) min, ANOVA P < 0.0001). For thiopentone, at concentrations of 62.5, 125 and 250 mumol litre-1, the values were 117 (2) (112-121) min, 126 (3) (119-133) min and 138 (2) (132-144) min, respectively (saline control 109 (2) (104-113) min, ANOVA P < 0.0001). Spectroscopic analysis in the visible and ultraviolet spectra demonstrated a steady increase in the proportion of methaemoglobin during haemolysis, with the highest concentrations in the propofol-containing flasks. The formation of methaemoglobin was preceded by the generation of ferrylhaemoglobin (a Fe4+ haemoglobin species). Further experiments examining oxidation of purified methaemoglobin to ferrylhaemoglobin by hydrogen peroxide suggested that propofol, but not Intralipid or thiopentone, reduced ferrylhaemoglobin back to the met- state, and thereby explained the higher concentrations of methaemoglobin in the propofol-containing erythrocyte suspensions. We conclude that propofol is a more potent free radical scavenger in this model of oxidant stress than thiopentone, and that reduction of high oxidation states of haemoglobin may contribute to such activity.     

A longitudinal study of maternal serum inhibin-A, inhibin-B, activin-A, activin-AB, pro-alphaC and follistatin during pregnancy

Maternal serum concentrations of inhibin-A, inhibin-B, activin-A, activin-AB, pro-alphaC-related inhibin forms, total follistatin, steroids and gonadotrophins were measured longitudinally in six normal singleton pregnancies. Maternal venous blood was collected randomly during a spontaneous follicular phase prior to donor insemination, at 5, 7, 9, 11, 16, 20, 24, 28, 32 and 36 weeks after the first missed menses and in the early puerperium. Steroid and gonadotrophin profiles conformed to previous reports. While at week 5 of gestation inhibin-A, activin-A and follistatin concentrations were similar to those at the follicular phase, all three increased progressively (P < 0.001) to maximal concentrations in week 36: approximately 48-fold (3740 +/- 1349 ng inhibin-A/ml), approximately 22-fold (6109 +/- 1443 ng activin-A/ml) and approximately 10-fold (3563 +/- 418 ng follistatin/ml) higher. Pro-alphaC concentrations reached a maximum in weeks 5 (approximately 5-fold, P < 0.001) and 36 (1027 +/- 174 pg/ml, P < 0.01). Inhibin-B (71 +/- 23 pg/ml prior to pregnancy) was undetectable (<12 pg/ml) between week 5-16 of gestation but increased slightly in the third trimester (26 +/- 7 pg/ml in week 36). Activin-AB was undetectable throughout pregnancy. Post-partum concentrations of inhibin-A (41 +/- 12 ng/ml), inhibin-B (<12 pg/ml), activin-A (950 +/- 149 pg/ml), pro-alphaC (128 +/- 22 pg/ml) and follistatin (990 +/- 79 ng/ml) were substantially lower than at week 36 of gestation. The activin-A:follistatin ratio increased from 0.5 in week 5 to 1.8 in week 36, suggesting that more free activin-A is available in the maternal circulation during late pregnancy.     

Brachial plexus injury during cardiac catheterisation in children. Report of two cases

The arm is particularly vulnerable to neurological injury, due to the intimate anatomical relationship between the brachial plexus and the traction zones. Severe injuries of the brachial plexus can be caused by compression, traction or laceration. Fortunately, many deficits are superficial and permanent neurovascular deficits are rare. Nevertheless, it is important to identify the probable cause of the injury since the prognosis for recovery directly depends on the underlying nature of the neurological deficit. Two anaesthetised children who suffered brachial plexus injury during cardiac catheterisation are reported. The first, with Ebstein's anomaly and significant pulmonary valve stenosis, presented, after the procedure, with a right arm motor deficit with proximal predominance. The second patient, with tetralogy of Fallot and pulmonary atresia, presented difficulty in left arm abduction and external rotation on awakening. The risk factors for brachial plexus lesions during anaesthesia are discussed. These include improper positioning, anaesthetic agents, extreme variations of body mass index and anatomical anomalies. Prevention, evolution and treatment of the brachial plexus injury are also considered. With proper care by the cardio-radiologist and anaesthesiologist the frequency of this injury can be reduced.     

Shigella and the fluorinated quinolones

An 8-year-old girl presented with marked shortening of the right forearm due to destruction of both the radius and ulna secondary to neonatal osteomyelitis. A one-bone forearm operation was performed to achieve a stable forearm. Two years later, the one-bone forearm was lengthened for 6 months by callus distraction (callotasis) achieving 12 cm of extra length. The patient was last followed up at the age of 16. The appearance and functional outcome of the right upper limb had been improved and she was independent in all activities of daily living.     

Modulation of an early step in the secretory machinery in hippocampal nerve terminals

In hippocampal neurons, neurotransmitter release can be regulated by protein kinase A (PKA) through a direct action on the secretory machinery. To identify the site of PKA modulation, we have taken advantage of the ability of the neurotoxin Botulinum A to cleave the synaptic protein SNAP-25. Cleavage of this protein decreases the Ca2+ responsiveness of the secretory machinery by partially uncoupling Ca2+-sensing from fusion per se. This is expressed as a shift toward higher Ca2+ levels of the Ca2+ to neurotransmitter release relationship and as a perturbation of synaptic delay under conditions where secretion induced by the Ca2+-independent secretagogue ruthenium red is unimpaired. We find that SNAP-25 cleavage also perturbs PKA-dependent modulation of secretion; facilitation of ruthenium red-evoked neurotransmitter release by the adenylyl cyclase activator forskolin is blocked completely after Botulinum toxin A action. Together with our observation that forskolin modifies the Ca2+ to neurotransmitter release relationship, our results suggest that SNAP-25 acts as a functional linker between Ca2+ detection and fusion and that PKA modulates an early step in the secretory machinery related to calcium sensing to facilitate synaptic transmission.     

Formulary review of the carbapenems: comparison of imipenem/cilastatin and meropenem

The duplicative mutation of an Ala-Val-Arg sequence at positions 208 to 210 in the loop structure of Enterobacter cloacae class C beta-lactamase caused substrate specificity extension to oxyimino beta-lactam antibiotics and this chromosomal mutation provided bacterial cells with high resistance to the beta-lactams (M. Nukaga et al, 1995, J. Biol. Chem. 270, 5729-5735). In order to confirm the universality of this phenomenon among other class C beta-lactamases, the duplicative mutation was applied to a class C beta-lactamase of Citrobacter freundii, which has 74% homology to the E. cloacae beta-lactamase amino acid sequence. The counterpart sequence to the Ala-Val-Arg of the E. cloacae enzyme in C. freundii beta-lactamase was identified to be Pro-Val-His. A Pro-Val-His sequence was inserted just after the native Pro-Val-His sequence at positions 208 to 210 in the C. freundii beta-lactamase. The resulting mutant of C. freundii beta-lactamase obtained a striking characteristic that we expected, showing substrate specificity extension to oxyimino beta-lactams. Nearly the same result was obtained with the insertion of an Ala-Val-Arg sequence after the native Pro-Val-His sequence. These results indicate that structural modification of this locus commonly induces modification of the substrate specificity to unfavorable substrates for many chromosomal class C beta-lactamases produced by gram-negative bacteria.