Factor Xa and VIIa inhibition by tissue factor pathway inhibitor is prevented by a monoclonal antibody to its Kunitz-1 domain.

Essentials Activated FVII (FVIIa) and FX (FXa) are inhibited by tissue factor pathway inhibitor (TFPI). A monoclonal antibody, mAb2F22, was raised against the N-terminal fragment of TFPI (1-79). mAb2F22 bound exclusively to the K1 domain of TFPI (KD ∼1 nm) and not to the K2 domain. mAb2F22 interfered with inhibition of both FVIIa and FXa activities and restored clot formation. SUMMARY: Background Initiation of coagulation is induced by binding of activated factor VII (FVIIa) to tissue factor (TF) and activation of factor X (FX) in a process regulated by tissue factor pathway inhibitor (TFPI). TFPI contains three Kunitz-type protease inhibitor domains (K1-K3), of which K1 and K2 block the active sites of FVIIa and FXa, respectively. Objective To produce a monoclonal antibody (mAb) directed towards K1, to characterize the binding epitope, and to study its effect on TFPI inhibition. Methods A monoclonal antibody, mAb2F22, was raised against the N-terminal TFPI(1-79) fragment. Binding data were obtained by surface plasmon resonance analysis. The Fab-fragment of mAb2F22, Fab2F22, was expressed and the structure of its complex with TFPI(1-79) determined by X-ray crystallography. Effects of mAb2F22 on TFPI inhibition were measured in buffer- and plasma-based systems. Results mAb2F22 bound exclusively to K1 of TFPI (KD ~1 nm) and not to K2. The crystal structure of Fab2F22/TFPI (1-79) mapped an epitope on K1 including seven residues upstream of the domain. TFPI inhibition of TF/FVIIa amidolytic activity was neutralized by mAb2F22, although the binding epitope on K1 did not include the P1 residue. Binding of mAb2F22 to K1 blocked TFPI inhibition of the FXa amidolytic activity and normalized hemostasis in hemophilia human A-like plasma and whole blood. Conclusion mAb2F22 blocked TFPI inhibition of both FVIIa and FXa activities and mapped a FXa exosite for binding to K1. It reversed TFPI feedback inhibition of TF/FVIIa-induced coagulation and restored clot formation in FVIII-neutralized human plasma and blood.

Oxytocin use during Caesarean sections in Denmark - are we getting the dose right?

BACKGROUND: In Denmark, an iv bolus of 10 IU oxytocin was traditionally given after delivery to prevent atony during caesarean sections. Randomized controlled trials have shown that lower iv bolus doses have same efficacy with fewer side effects and many countries now recommend a 5 IU maximum dose. The aims of this study were to investigate whether patients referred for allergy testing after oxytocin exposure had dose-related side effects to oxytocin rather than true allergic reactions and to investigate whether updated international recommendations on lower bolus doses had been implemented in practice. METHODS: Medical notes of patients tested with oxytocin as part of investigations in the Danish Anaesthesia Allergy Centre from May 2004 to January 2014 were reviewed retrospectively. A telephone survey of on-duty obstetricians at all Danish obstetric departments was performed and most recent online recommendations from the Danish societies of obstetrics and anaesthesia about the use of oxytocin were identified. RESULTS: In total 30 women were tested with oxytocin as part of investigations. None were allergic to oxytocin but 19 had symptoms consistent with dose-related side effects on iv provocation. The telephone survey revealed that iv doses of 10 IU oxytocin were still used and recommendations on the websites were not updated. CONCLUSION: Too high oxytocin doses are still used in Denmark leading to dose-related side effects mimicking allergic reactions. Coordination between obstetricians and anaesthesiologists on producing common updated guidelines on the administration of oxytocin and dissemination of this information to obstetric and anaesthetic departments in Denmark is needed.

Impaired postural stability after laparoscopic surgery.

BACKGROUND: Early postoperative mobilisation may reduce patient morbidity and improve hospital efficiency by accelerated discharge. The aim of this study was to measure postural stability early after laparoscopic surgery in order to assess how early it is safe to mobilise and discharge patients. METHODS: We included 25 women undergoing outpatient gynaecological laparoscopic surgery in the study. Patients received standardised anaesthesia with propofol, remifentanil and rocuronium. Postural stability was assessed preoperatively, at 30 min after tracheal extubation, and at discharge from the post-anaesthesia care unit using a force platform where sway area, mean sway and sway velocity were determined. The assessments were done with eyes closed and with eyes open. The primary outcome was the change in sway area with eyes closed 30 min after extubation. Data are reported as median (25-75% range). RESULTS: Three patients could not perform all the test's 30 min after extubation. Thirty minutes after extubation, sway area with eyes closed had increased significantly with 84 mm(2) (9-172, P = 0.011) and 108 mm(2) with eyes open (25-295, P = 0.0017). Median mean sway had also increased significantly 30 min postoperatively. No significant changes were found for sway velocity. We found no significant changes in mean sway, sway area or sway velocity at discharge from the post-anaesthesia care unit approximately 2 h after surgery. CONCLUSION: Postural stability was significantly impaired 30 min after outpatient gynaecological laparoscopic surgery. However, the postural stability was normalised at discharge from the post-anaesthesia care unit 2 h after surgery.

Synthesis and anti-angiogenic effect of conjugates between serum albumin and non-steroidal anti-inflammatory drugs.

Non-steroidal anti-inflammatory drugs (NSAIDs) inhibit tumor growth and angiogenesis. Covalent linkage of naproxen to human serum albumin (HSA) has been shown to target it efficiently to the liver and this may potentially be exploited for liver-selective inhibition of angiogenesis. With the aim of investigating the anti-angiogenic efficiency of NSAID-HSA conjugates in vitro, three NSAIDs, aspirin, ibuprofen, and naproxen were conjugated to HSA using different concentrations of their N-hydroxysuccinimide esters. Conjugation ratios from 10 to 50 were achieved and the conjugates retained a growth inhibitory effect on endothelial cells at or above the level of the non-conjugated NSAIDs in an in vitro angiogenesis assay.

Photoinduced transient absorbance spectra of P840/P840(+) and the FMO protein in reaction centers of Chlorobium vibrioforme.

The kinetics of photoinduced absorbance changes in the 400-ns to 100-ms time range were studied between 770 and 1025 nm in reaction center core (RCC) complexes isolated from the green sulfur bacterium Chlorobium vibrioforme. A global, multiple stretched-exponential analysis shows the presence of two distinct but strongly overlapping spectra. The spectrum of the 70-micros component consists of a broad bleaching with two minima at 810 and 825 nm and a broad positive band at wavelengths greater than 865 nm and is assigned to the decay of (3)Bchl a of the Fenna-Matthews-Olson (FMO) protein. The contribution of the 70-micros component correlates with the amount of FMO protein in the isolated RCC complex. The spectrum of the 1.6-micros component has a sharp bleaching at 835 nm, a maximum at 805 nm, a broad positive band at wavelengths higher than 865 nm, and a broad negative band at wavelengths higher than 960 nm. When the RCC is incubated with inorganic iron and sulfur, the 1.6-micros component is replaced by a component with a lifetime of approximately 40 micros, consistent with the reconstruction of the F(X) cluster. We propose that the 1.6-micros component results from charge recombination between P840(+) and an intermediate electron acceptor operating between A(0) and F(X). Our studies in Chlorobium RCCs show that approaches that employ a single wavelength in the measurement of absorption changes have inherent limitations and that a global kinetic analysis at multiple wavelengths in the near-infrared is required to reliably separate absorption changes due to P840/P840(+) from the decay of (3)Bchl a in the FMO protein.

[Sentinel node biopsy in cutaneous malignant melanoma of the lower extremities]. / "Sentinel node"-biopsi ved kutant malignt melanom på underekstremiteterne.

Although a substantial number of patients with intermediate thickness cutaneous malignant melanoma (> 1.5-4 mm) have non-detectable regional node metastases, elective regional node dissection still remains controversial. One-three specific lymph node(s)--sentinel node(s)--in the first drained regional lymphatic basin can be visualised peroperatively by applying Patent V Blue intradermally at the site of the previous melanoma. Histological examination of the sentinel node can reveal metastases and therefore presumably give a more accurate oncological staging, thus enabling selection of patients who may benefit from elective regional node dissection. The aim of the present study was to describe our experience with this technique in 23 patients treated for cutaneous malignant melanoma of the lower extremity with a thickness > 1.5 mm. We found that sentinel node dissection, through a minimal surgical procedure, was efficient in detecting micrometastases in the regional lymph node(s).

Epidemiology of respiratory syncytial virus infection requiring hospitalization in East Denmark.

BACKGROUND: Prophylaxis against infection caused by respiratory syncytial virus (RSV) with high titered RSV immunoglobulin or humanized antibody may soon be available in Europe. OBJECTIVE: To study the epidemiology of RSV infections requiring hospitalization in infants <6 months in East Denmark to provide a rational basis for decisions concerning prophylaxis against RSV. METHOD: Populat ion-based retrospective review of case records of infants <6 months admitted to pediatric departments with RSV infection in East Denmark from November 1, 1995, to April 30, 1996. RESULTS: Data were obtained from 459 infants. Seventy-three had predisposing conditions: prematurity, 49; pulmonary disease, 2; congenital heart disease, 7; neurologic disease, 6; others, 9. One preterm infant had bronchopulmonary dysplasia. The incidence of RSV infection requiring hospitalization in East Denmark among infants <6 months was estimated to be 34/1000/season. It was 32/1000/season among term infants and 66/ 1000/season among preterm infants (P<0.001). Infants with predisposing conditions and/or nosocomial infection (n = 24) had significantly more severe courses than otherwise healthy infants (P<0.01). One-hundred thirty infants received respiratory support by nasal continuous positive airway pressure, but only six required mechanical ventilation. No infants died. CONCLUSION: The course of RSV disease in East Denmark was milder than reported elsewhere, possibly as a result of the low prevalence of bronchopulmonary dysplasia in Denmark. However, RSV constitutes a considerable burden to the Danish pediatric health care system, and therefore prophylaxis against RSV is desirable.

Menaquinone-7 in the reaction center complex of the green sulfur bacterium Chlorobium vibrioforme functions as the electron acceptor A1.

Photosynthetically active reaction center complexes were prepared from the green sulfur bacterium Chlorobium vibrioforme NCIMB 8327, and the content of quinones was determined by extraction and high-performance liquid chromatography. The analysis showed a stoichiometry of 1.7 molecules of menaquinone-7/reaction center. No other quinones were detected in the isolated reaction centers, whereas membrane preparations also contained chlorobiumquinone. The possible involvement of quinones in electron transport was investigated by electron paramagnetic resonance (EPR) spectroscopy. A highly anisotropic radical was detected by Q-band EPR spectroscopy in both membranes and isolated reaction centers following dark reduction with sodium dithionite and photoaccumulation at 205 K. At 34 GHz, the EPR spectrum is characterized by a g tensor with gxx = 2.0063, gyy = 2.0052, gzz = 2.0020 and delta B of 0.7 mT, consistent with its identification as a quinone. This spectrum is highly similar in terms of g values and line widths to photoaccumulated A1- in photosystem I of Synechococcus sp. PCC 7002. The results indicate that menaquinone-7 in the green sulfur bacterial reaction center is analogous to phylloquinone in photosystem I.

Dopaminergic regulation of BDNF content in the pituitary intermediate lobe.

Previous studies in adult rats have demonstrated that the neurotrophin, brain-derived neurotrophic factor (BDNF), is present in virtually all cells of the pituitary intermediate lobe. In the present study, we demonstrate that cells cultured from adult intermediate lobe pituitary (ILP) rapidly lose their BDNF immunoreactivity (IR). Furthermore, a similar loss of immunostaining occurs in whole (undissociated) ILP within 30 min after removal from the rat. However, when the dopamine agonist apomorphine is present throughout the dissociation procedure and during cultivation, BDNF-IR is preserved. Supplying apomorphine only during either dissociation or cultivation did not prevent the loss of BDNF-IR in the 24 h cultures. These results suggest that a tonic dopaminergic stimulus is required to maintain BDNF-IR in ILP cells.

An isolated reaction center complex from the green sulfur bacterium Chlorobium vibrioforme can photoreduce ferredoxin at high rates.

Chlorosome-depleted membranes and a reaction center complex with well-defined subunit composition were prepared from the green sulfur bacterium Chlorobium vibrioforme under anaerobic conditions. The reaction center complex contains a 15-kDa polypeptide with the N-terminal amino acid sequence MEPQLSRPETASNQVR/. This sequence is nearly identical to the N-terminus of the pscD gene product from Chlorobium limicola (Hager-Braun et al. (1995) Biochemistry 34: 9617-9624). In the presence of ferredoxin and ferredoxin:NADP(+) oxidoreductase, the membranes and the isolated reaction center complex photoreduced NADP(+) at rates of 333 and 110 µmol (mg bacteriochlorophyll a)(-1) h(-1), respectively. This shows that the isolated reaction center complex contains all the components essential for steady state electron transport. Midpoint potentials at pH 7.0 of 160 mV for cytochrome c 551 and of 245 mV for P840 were determined by redox titration. Antibodies against cytochrome c 551 inhibit NADP(+) reduction while antibodies against the bacteriochlorophyll a-binding Fenna-Matthews-Olson protein do not.

Quantitative trait loci for heading date and straw characters in barley.

Quantitative trait loci (QTLs) for heading date and straw characters were examined in 79 chromosome-doubled haploid lines derived from the F1 generation of a cross between a six-rowed winter barley and a two-rowed spring barley. A genetic map covering 1100 cM containing 85 markers, including isozyme, morphological, RFLP, and RAPD markers, was constructed. All traits examined had two QTLs with large effects on chromosome 2. In addition, a QTL for length of the top internode was found on chromosome 6. The QTL in the chromosome segment around locus v (two row/six row) on chromosome 2 may be caused by pleiotropic effects of this locus. The same QTLs for heading date and straw length were found in both 1989 and 1991. The results indicate that two QTLs on chromosome 2 affect a group of correlated traits.

Distribution of RAPD markers on a linkage map of barley.

The RAPD technique was found to provide reliable genetic markers in barley. A linkage study of 23 RAPDs, 28 RFLPs, and 29 gene loci was conducted on 72 chromosome-doubled haploid progeny lines from a barley cross. The resulting linkage map covered 680 cM, about half of the barley genome. RAPD markers were distributed throughout the map, but a higher than expected frequency of tightly linked RAPDs was observed. Several cases of skewed segregation ratios were observed, but the RAPD markers segregated in ratios similar to their linked loci, confirming that they were reliably scored. In separate crosses, two amplified RAPD products, generated by different primers, were shown to reside in corresponding chromosomal positions. The RAPD markers seem a realistic alternative to RFLP markers in linkage analysis of barley.

Iron-sulfur centers in the photosynthetic reaction center complex fromChlorobium vibrioforme. Differences from and similarities to the iron-sulfur centers in Photosystem I.

The photosynthetic reaction center complex from the green sulfur bacteriumChlorobium vibrioforme has been isolated under anaerobic conditions. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis reveals polypeptides with apparent molecular masses of 80, 40, 30, 18, 15, and 9 kDa. The 80- and 18-kDa polypeptides are identified as the reaction center polypeptide and the secondary donor cytochromec 551 encoded by thepscA andpscC genes, respectively. N-terminal amino acid sequences identify the 40-kDa polypeptide as the bacteriochlorophylla-protein of the baseplate (the Fenna-Matthews-Olson protein) and the 30-kDa polypeptide as the putative 2[4Fe-4S] protein encoded bypscB. Electron paramagnetic resonance (EPR) analysis shows the presence of an iron-sulfur cluster which is irreversibly photoreduced at 9K. Photoaccumulation at higher temperature shows the presence of an additional photoreduced cluster. The EPR spectra of the two iron-sulfur clusters resemble those of FA and FB of Photosystem I, but also show significantly differentg-values, lineshapes, and temperature and power dependencies. We suggest that the two centers are designated Center I (with calculatedg-values of 2.085, 1.898, 1.841), and Center II (with calculatedg-values of 2.083, 1.941, 1.878). The data suggest that Centers I and II are bound to thepscB polypeptide.

A membrane-bound monoheme cytochrome c551 of a novel type is the immediate electron donor to P840 of the Chlorobium vibrioforme photosynthetic reaction center complex.

A photosynthetic reaction center complex has been isolated from the green sulfur bacterium Chlorobium vibrioforme. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis reveals polypeptides with apparent molecular masses of 80, 40, 18, 15, 9, and 6 kDa. Only the 18-kDa polypeptide is stained with 3,3',5,5'-tetramethylbenzidine, a heme-specific reagent. Oxidized minus reduced difference spectra show the presence of approximately one heme/P840 and the presence of a cytochrome c551. Flash photolysis of P840 was followed by rereduction of P840+ and oxidation of cytochrome c551, both with a biphasic kinetic with t1/2 values of 7 and 50 microseconds. Using oligonucleotide probes derived from an N-terminal amino acid sequence of the 18-kDa polypeptide, a genomic clone was isolated. The sequence of the gene, which we designate cycA, predicts a single heme binding site (Cys-Asn-Lys-Cys-His). The 621-base pair open reading frame encodes an apoprotein of 22,858 Da with three predicted membrane-spanning alpha-helices. No extensive sequence similarity is found to other cytochromes. Northern blotting indicates that the cycA gene is transcribed as a monocistronic mRNA. Southern blotting shows the presence of only one cycA gene in the C. vibrioforme and Chlorobium tepidum genomes. The unique membrane-bound monoheme cytochrome c551 of C. vibriforme is assigned to a new class of c-type cytochromes. The implications for the current view of evolution of photosynthetic reaction center complexes are discussed.