bluuebunny/crossref_metadata_2025 huggingface dataset:
Shape factors for constant‐head double‐packer permeameters
Authors: Mathias S. A. , Butler A. P.
Published: June 2007

Local estimates of hydraulic conductivity are often obtained from constant‐head double‐packer tests. The tests yield ratios of flow against head difference. These are converted to values of hydraulic conductivity by dividing by a shape factor. Previously used shape factors have generally assumed that the packers are infinitely large. Such shape factors lead to overestimates of hydraulic conductivity as they ignore the effect of water flow around the packers. A semianalytical solution is derived for a constant‐head double‐packer permeameter test. The solution is compared with existing, approximate techniques, including the spheroidal equipotential solution, the narrow packer approximation of Rehbinder (1996), and the long‐packer approximation of Rehbinder (2005). Generally, it is shown that ignoring the length of the packer and assuming a fully cased well lead to an overestimate of hydraulic conductivity. The significance of this reduces with increasing well‐screen aspect ratio. Evaluation of the solution is nontrivial. For this reason, a polynomial relationship between shape factor, well‐screen aspect ratio, and packer length (obtained by curve fitting with the semianalytical solution) is provided.
Streamflow variations of the Yellow River over the past 593 years in western China reconstructed from tree rings
Authors: Gou Xiaohua , Chen Fahu , Cook Edward , Jacoby Gordon , Yang Meixue , Li Jinbao
Published: June 2007

Annual streamflow of the Yellow River has decreased in recent years (1980 to 2000) because of climate change and human activity. This decrease affects the environment and the lives of the people in the drainage area. Tree ring width chronologies from six sites in the headwaters of the Yellow River were developed to provide estimates of past Yellow River streamflow in order to place the recent flow reduction in a long‐term context. The ring width indices of the six local Juniperus przewalski chronologies correlate significantly with the observed streamflow of the Yellow River recorded at the Tangnaihai hydrological station. Principal components analysis shows that the first principal component (PC) of the tree ring indices explains 49% of the streamflow variance. On the basis of this result, Yellow River streamflow was reconstructed for the past 593 years. Several severe droughts and low‐flow events are recognized in the decades 1920–1930, 1820–1830, 1700–1710, 1590–1600, and 1480–1490. The most severe droughts in 1480–1490 were also recorded in other studies on the Tibetan Plateau. Regional historical climate archives further support the validity of our streamflow reconstruction. The reconstructed increase in streamflow during much of the twentieth century also coincides with generally wetter conditions in the Tienshan and Qilianshan Mountains of China, as well as in northern Pakistan and Mongolia. After the 1980s, our reconstruction indicates a decreasing trend in streamflow, which is cause for concern. Presently, Yellow River streamflow is relatively low but not yet outside the range of streamflow fluctuations that occurred during the past six centuries.
Molecular Static and Dynamic Analyses reveal Flaw in Murine Model used by US FDA to Detect Drug Carcinogenicity
Authors: Marshall Trevor
Published: June 2007

The US FDA currently accepts carcinogenicity studies of pharmaceutical drugs based on murine models. In addition to 6 month studies with p53(+/-) and ras.H2 transgenic mice, lifetime studies (typically 2 years) in WT mice or rats are also considered as evidence that a drug lacks carcinogenic activity. This model is not always exhaustive. For example, during the acceptance testing of the ARB Olmesartan[1], possible carcinogenicity observed in hamsters was not able to be duplicated in rats, or in transgenic mice. We have previously used the static molecular modeling of AutoDock to demonstrate that Olmesartan has agonostic activity in the PDB:1DB1 model of the human VDR Nuclear Receptor[2], while it has antagonistic activity in the PDB:1RK3 model of the rat VDR. This agonism has now been confirmed with Molecular Dynamics, using GROMACS. The murine VDR indeed lost its ability to bind the DRIP-205 co-activator when Olmesartan was the ligand, while the human VDR was activated by Olmesartan similarly to its native ligand (1,25-dihydroxyvitamin-D). Since the VDR is believed to express 913 genes[3], many of which are known to be associated with cancer pathogenesis, good homology between human VDR, and the animal model VDR, is exceedingly important.

CONCLUSION: The murine environment is inadequate to accurately model drug carcinogenic activity in humans. A species should be chosen which has a VDR LBP homology closer to that of man. AutoDock and GROMACS molecular analyses are useful in resolving any remaining anomalies in the observed data.
Role of the C-terminal domains of rice (Oryza sativa L.) bZIP proteins RF2a and RF2b in regulating transcription
Authors: Liu Yi , Dai Shunhong , Beachy Roger N.
Published: June 2007

Rice (Oryza sativa L.) transcription factors RF2a and RF2b are bZIP (basic leucine zipper) proteins that interact with, and activate transcription from the RTBV (rice tungro bacilliform virus) promoter. Here we characterize the C-terminal domains of RF2a and RF2b: these domains are rich in glutamine and proline/glutamine, respectively. Affinity pull-down assays demonstrated that the C-terminal domains of RF2a and RF2b can associate to form either homodimers or heterodimers; however, they do not interact with other domains of RF2a or RF2b. Results of in vitro transcription assays using a rice whole-cell extract demonstrate that the C-terminal domains of both RF2a and RF2b activate transcription from the RTBV promoter. In addition, dimerization of the RF2a C-terminal domain is involved in regulating the transcription activation function of RF2a. The predicted helical region within the RF2a C-terminal glutamine-rich domain was determined to be involved in inter-molecular dimerization, and contributed to the regulatory functions of RF2a in these assays.
The peptide-binding activity of GRP94 is regulated by calcium
Authors: Biswas Chhanda , Ostrovsky Olga , Makarewich Catherine A. , Wanderling Sherry , Gidalevitz Tali , Argon Yair
Published: June 2007

GRP94 (glucose-regulated protein of 94 kDa) is a major luminal constituent of the endoplasmic reticulum with known high capacity for calcium in vivo and a peptide-binding activity in vitro. In the present study, we show that Ca2+ regulates the ability of GRP94 to bind peptides. This effect is due to a Ca2+-binding site located in the charged linker domain of GRP94, which, when occupied, enhances the association of peptides with the peptide-binding site in the N-terminal domain of the protein. We further show that grp94−/− cells are hypersensitive to perturbation of intracellular calcium and thus GRP94 is important for cellular Ca2+ storage.
A novel aminopeptidase in the fat body of the moth Achaea janata as a receptor for Bacillus thuringiensis Cry toxins and its comparison with midgut aminopeptidase
Authors: Budatha Madhusudhan , Meur Gargi , Dutta-Gupta Aparna
Published: June 2007

Bacillus thuringiensis insecticidal crystal proteins bind to cell-surface receptors which represent a family of aminopeptidases [APN (aminopeptidase N)] present on the brush border membrane of insect midgut cells of susceptible insects leading to pore formation and death of the insect. We report here for the first time the presence of a novel APN in the fat body of the moth Achaea janata. Northern blotting detected at least one APN-specific transcript in the fat body, whereas two transcripts of different sizes were detected in the midgut. We have cloned two full-length APN cDNAs of 3015 bp and 2850 bp from fat body and midgut respectively, which encode proteins of 1004 and 950 amino acids. These two APNs share only 33% amino acid sequence identity, but both display the typical APN features, such as the N-terminal signal peptide, several putative glycosylation sites, C-terminal glycosylphosphatidylinositol anchor signal, the APN-specific zinc-binding/gluzincin motif HEXXHX18E and gluzincin motif GAMENWG. The fat body APN manifested a variation in its expression with respect to tissue and developmental stage. In spite of the abundance of the APN transcript in the fat body, fairly low APN activity was detected in this tissue. The fat-body- and midgut-specific APNs showed differential interaction with various Cry1A toxins. Besides, the level of toxicity of different Cry subtypes varied enormously with mode/site of delivery, such as intrahaemocoelic injections and feeding bioassays. These data indicate that the fat body might be a potential alternative Cry toxin target site in the moth.
LRRK2 phosphorylates moesin at threonine-558: characterization of how Parkinson's disease mutants affect kinase activity
Authors: Jaleel Mahaboobi , Nichols R. Jeremy , Deak Maria , Campbell David G. , Gillardon Frank , Knebel Axel , Alessi Dario R.
Published: June 2007

Mutations in the LRRK2 (leucine-rich repeat kinase-2) gene cause late-onset PD (Parkinson's disease). LRRK2 contains leucine-rich repeats, a GTPase domain, a COR [C-terminal of Roc (Ras of complex)] domain, a kinase and a WD40 (Trp-Asp 40) motif. Little is known about how LRRK2 is regulated, what its physiological substrates are or how mutations affect LRRK2 function. Thus far LRRK2 activity has only been assessed by autophosphorylation and phosphorylation of MBP (myelin basic protein), which is catalysed rather slowly. We undertook a KESTREL (kinase substrate tracking and elucidation) screen in rat brain extracts to identify proteins that were phosphorylated by an activated PD mutant of LRRK2 (G2019S). This led to the discovery that moesin, a protein which anchors the actin cytoskeleton to the plasma membrane, is efficiently phosphorylated by LRRK2, at Thr558, a previously identified in-vivo-phosphorylation site that regulates the ability of moesin to bind actin. LRRK2 also phosphorylated ezrin and radixin, which are related to moesin, at the residue equivalent to Thr558, as well as a peptide (LRRKtide: RLGRDKYKTLRQIRQ) encompassing Thr558. We exploited these findings to determine how nine previously reported PD mutations of LRRK2 affected kinase activity. Only one of the mutations analysed, namely G2019S, stimulated kinase activity. Four mutations inhibited LRRK2 kinase activity (R1941H, I2012T, I2020T and G2385R), whereas the remainder (R1441C, R1441G, Y1699C and T2356I) did not influence activity. Therefore the manner in which LRRK2 mutations induce PD is more complex than previously imagined and is not only caused by an increase in LRRK2 kinase activity. Finally, we show that the minimum catalytically active fragment of LRRK2 requires an intact GTPase, COR and kinase domain, as well as a WD40 motif and a C-terminal tail. The results of the present study suggest that moesin, ezrin and radixin may be LRRK2 substrates, findings that have been exploited to develop the first robust quantitative assay to measure LRRK2 kinase activity.
Mechanism of membrane redistribution of protein kinase C by its ATP-competitive inhibitors
Authors: Takahashi Hideyuki , Namiki Hideo
Published: June 2007

ATP-competitive inhibitors of PKC (protein kinase C) such as the bisindolylmaleimide GF 109203X, which interact with the ATP-binding site in the PKC molecule, have also been shown to affect several redistribution events of PKC. However, the reason why these inhibitors affect the redistribution is still controversial. In the present study, using immunoblot analysis and GFP (green fluorescent protein)-tagged PKC, we showed that, at commonly used concentrations, these ATP-competitive inhibitors alone induced redistribution of DAG (diacylglycerol)-sensitive PKCα, PKCβII, PKCδ and PKCϵ, but not atypical PKCζ, to the endomembrane or the plasma membrane. Studies with deletion and point mutants showed that the DAG-sensitive C1 domain of PKC was required for membrane redistribution by these inhibitors. Furthermore, membrane redistribution was prevented by the aminosteroid PLC (phospholipase C) inhibitor U-73122, although an ATP-competitive inhibitor had no significant effect on acute DAG generation. Immunoblot analysis showed that an ATP-competitive inhibitor enhanced cell-permeable DAG analogue- or phorbol-ester-induced translocation of endogenous PKC. Furthermore, these inhibitors also enhanced [3H]phorbol 12,13-dibutyrate binding to the cytosolic fractions from PKCα–GFP-overexpressing cells. These results clearly demonstrate that ATP-competitive inhibitors cause redistribution of DAG-sensitive PKCs to membranes containing endogenous DAG by altering the DAG sensitivity of PKC and support the idea that the inhibitors destabilize the closed conformation of PKC and make the C1 domain accessible to DAG. Most importantly, our findings provide novel insights for the interpretation of studies using ATP-competitive inhibitors, and, especially, suggest caution about the interpretation of the relationship between the redistribution and kinase activity of PKC.
Evidence for oxygen as the master regulator of the responsiveness of soluble guanylate cyclase and cytochrome c oxidase to nitric oxide
Authors: Landar Aimee , Darley-Usmar Victor M.
Published: June 2007

Haem is used as a versatile receptor for redox active molecules; most notably NO (nitric oxide) and oxygen. Three haem-containing proteins, myoglobin, haemoglobin and cytochrome c oxidase, are now known to bind NO, and in all these cases competition with oxygen plays an important role in the biological outcome. NO also binds to the haem group of sGC (soluble guanylate cyclase) and initiates signal transduction through the formation of cGMP in a process that is oxygen-independent. From biochemical studies, it has been shown that sGC is substantially more sensitive to NO than is cytochrome c oxidase, but a direct comparison in a cellular setting under various oxygen levels has not been reported previously. In this issue of the Biochemical Journal, Cadenas and co-workers reveal how oxygen can act as the master regulator of the relative sensitivity of the cytochrome c oxidase and sGC signalling pathways to NO. These findings have important implications for our understanding of the interplay between NO and oxygen in both physiology and the pathology of diseases associated with hypoxia.
Demographic and risk analyses applied to management and conservation of the blue shark (Prionace glauca) in the North Atlantic Ocean
Authors: Aires-da-Silva Alexandre M. , Gallucci Vincent F.
Published: June 2007

Management and conservation of the North Atlantic blue shark are handicapped by a fishery-data-limited stock-assessment process. The status of this stock is ambiguous at a time when catch appears to be on the rise and new trade and exploitation patterns are emerging. This research provides fishery-independent demographic and risk analysis results for use in conservation and management. An age-structured matrix population model in which the vital rates are stochastic was constructed. A mean finite rate of population increase (λ) of 1.23 year–1 and a mean population doubling time (t2) of 3.08 years suggests that the blue shark is one of the most productive shark species. However, this concept of high productivity could be misleading because an elasticity analysis shows a strong dependence of the population growth rate on the survival of juveniles (0–4 years). An analysis of the risk that the harvested population will decline to levels below an assumed threshold of 50% of pre-exploited levels was conducted. The risk analysis is proposed as a supplement to the data-limited stock assessment, to better evaluate the probability that a given management strategy will put the population at risk of decline.
Context-sensitive data integration and prediction of biological networks
Authors: Myers Chad L. , Troyanskaya Olga G.
Published: June 2007

Motivation: Several recent methods have addressed the problem of heterogeneous data integration and network prediction by modeling the noise inherent in high-throughput genomic datasets, which can dramatically improve specificity and sensitivity and allow the robust integration of datasets with heterogeneous properties.

However, experimental technologies capture different biological processes with varying degrees of success, and thus, each source of genomic data can vary in relevance depending on the biological process one is interested in predicting. Accounting for this variation can significantly improve network prediction, but to our knowledge, no previous approaches have explicitly leveraged this critical information about biological context.

Results: We confirm the presence of context-dependent variation in functional genomic data and propose a Bayesian approach for context-sensitive integration and query-based recovery of biological process-specific networks. By applying this method to Saccharomyces cerevisiae, we demonstrate that leveraging contextual information can significantly improve the precision of network predictions, including assignment for uncharacterized genes. We expect that this general context-sensitive approach can be applied to other organisms and prediction scenarios.

Availability: A software implementation of our approach is available on request from the authors.

Contact:  ogt@genomics.princeton.edu

Supplementary information: Supplementary data are available at http://avis.princeton.edu/contextPIXIE/
Different packing of external residues can explain differences in the thermostability of proteins from thermophilic and mesophilic organisms
Authors: Glyakina Anna V. , Garbuzynskiy Sergiy O. , Lobanov Michail Yu. , Galzitskaya Oxana V.
Published: June 2007

Motivation: Understanding the basis of protein stability in thermophilic organisms raises a general question: what structural properties of proteins are responsible for the higher thermostability of proteins from thermophilic organisms compared to proteins from mesophilic organisms?

Results: A unique database of 373 structurally well-aligned protein pairs from thermophilic and mesophilic organisms is constructed. Comparison of proteins from thermophilic and mesophilic organisms has shown that the external, water-accessible residues of the first group are more closely packed than those of the second. Packing of interior parts of proteins (residues inaccessible to water molecules) is the same in both cases. The analysis of amino acid composition of external residues of proteins from thermophilic organisms revealed an increased fraction of such amino acids as Lys, Arg and Glu, and a decreased fraction of Ala, Asp, Asn, Gln, Thr, Ser and His. Our theoretical investigation of folding/unfolding behavior confirms the experimental observations that the interactions that differ in thermophilic and mesophilic proteins form only after the passing of the transition state during folding. Thus, different packing of external residues can explain differences in thermostability of proteins from thermophilic and mesophilic organisms.

Availability: The database of 373 structurally well-aligned protein pairs is available at http://phys.protres.ru/resources/termo_meso_base.html

Contact:  ogalzit@vega.protres.ru

Supplementary information: Supplementary data are available at Bioinformatics online.
Acupuncture Treatment for Low Back Pain and Lower Limb Symptoms—The Relation between Acupuncture or Electroacupuncture Stimulation and Sciatic Nerve Blood Flow
Authors: Inoue Motohiro , Kitakoji Hiroshi , Yano Tadashi , Ishizaki Naoto , Itoi Megumi , Katsumi Yasukazu
Published: June 2007

To investigate the clinical efficacy of acupuncture treatment for lumbar spinal canal stenosis and herniated lumbar disc and to clarify the mechanisms in an animal experiment that evaluated acupuncture on sciatic nerve blood flow. In the clinical trial, patients with lumbar spinal canal stenosis or herniated lumbar disc were divided into three treatment groups; (i) Ex‐B2 (at the disordered level), (ii) electrical acupuncture (EA) on the pudendal nerve and (iii) EA at the nerve root. Primary outcome measurements were pain and dysesthesia [evaluated with a visual analogue scale (VAS)] and continuous walking distance. In the animal study, sciatic nerve blood flow was measured with laser‐Doppler flowmetry at, before and during three kinds of stimulation (manual acupuncture on lumber muscle, electrical stimulation on the pudendal nerve and electrical stimulation on the sciatic nerve) in anesthetized rats. For the clinical trial, approximately half of the patients who received Ex‐B2 revealed amelioration of the symptoms. EA on the pudendal nerve was effective for the symptoms which had not improved by Ex‐B2. Considerable immediate and sustained relief was observed in patients who received EA at the nerve root. For the animal study, increase in sciatic nerve blood flow was observed in 56.9% of the trial with lumber muscle acupuncture, 100% with pudendal nerve stimulation and 100% with sciatic nerve stimulation. Sciatic nerve stimulation sustained the increase longer than pudendal nerve stimulation. One mechanism of action of acupuncture and electrical acupuncture stimulation could be that, in addition to its influence on the pain inhibitory system, it participates in causing a transient change in sciatic nerve blood blow, including circulation to the cauda equine and nerve root.
D-Cycloserine enhances memory consolidation of hippocampus-dependent latent extinction
Authors: Gabriele Amanda , Packard Mark G.
Published: June 2007

Adult male Long-Evans rats were trained to run in a straight-alley maze for food reward and subsequently received hippocampus-dependent latent extinction training. Immediately following latent extinction, rats received peripheral injections of the NMDA receptor partial agonist D-cycloserine (DCS, 15 mg/kg), or saline. Twenty-four hours later, rats received four extinction “probe” trials. Relative to saline controls, latencies to reach the goal box on probe trials were significantly higher in rats that had received DCS. These findings indicate that memory consolidation underlying hippocampus-dependent latent extinction, a cognitive form of learning in which the previously rewarded response is not made during extinction training, can be enhanced by NMDA-receptor agonism.
Orphan nuclear receptor NGFI‐B forms dimers with nonclassical interface
Authors: Calgaro Marcos R. , Neto Mario de Oliveira , Figueira Ana Carolina M. , Santos Maria A.M. , Portugal Rodrigo V. , Guzzi Carolina A. , Saidemberg Daniel M. , Bleicher Lucas , Vernal Javier , Fernandez Pablo , Terenzi Hernán , Palma Mario Sergio , Polikarpov Igor
Published: June 2007

The orphan receptor nerve growth factor‐induced B (NGFI‐B) is a member of the nuclear receptor's subfamily 4A (Nr4a). NGFI‐B was shown to be capable of binding both as a monomer to an extended half‐site containing a single AAAGGTCA motif and also as a homodimer to a widely separated everted repeat, as opposed to a large number of nuclear receptors that recognize and bind specific DNA sequences predominantly as homo‐ and/or heterodimers. To unveil the structural organization of NGFI‐B in solution, we determined the quaternary structure of the NGFI‐B LBD by a combination of ab initio procedures from small‐angle X‐ray scattering (SAXS) data and hydrogen–deuterium exchange followed by mass spectrometry. Here we report that the protein forms dimers in solution with a radius of gyration of 2.9 nm and maximum dimension of 9.0 nm. We also show that the NGFI‐B LBD dimer is V‐shaped, with the opening angle significantly larger than that of classical dimer's exemplified by estrogen receptor (ER) or retinoid X receptor (RXR). Surprisingly, NGFI‐B dimers formation does not occur via the classical nuclear receptor dimerization interface exemplified by ER and RXR, but instead, involves an extended surface area composed of the loop between helices 3 and 4 and C‐terminal fraction of the helix 3. Remarkably, the NGFI‐B dimer interface is similar to the dimerization interface earlier revealed for glucocorticoid nuclear receptor (GR), which might be relevant to the recognition of cognate DNA response elements by NGFI‐B and to antagonism of NGFI‐B–dependent transcription exercised by GR in cells.
Probing mechanisms of resistance to the tuberculosis drug isoniazid: Conformational changes caused by inhibition of InhA, the enoyl reductase from Mycobacterium tuberculosis
Authors: Kruh Nicole A. , Rawat Richa , Ruzsicska Béla P. , Tonge Peter J.
Published: June 2007

The frontline tuberculosis drug isoniazid (INH) inhibits InhA, the NADH‐dependent fatty acid biosynthesis (FAS‐II) enoyl reductase from Mycobacterium tuberculosis (MTB), via formation of a covalent adduct with NAD+ (the INH‐NAD adduct). Resistance to INH can be correlated with many mutations in MTB, some of which are localized in the InhA cofactor binding site. While the InhA mutations cause a substantial decrease in the affinity of InhA for NADH, surprisingly the same mutations result in only a small impact on binding of the INH‐NAD adduct. Based on the knowledge that InhA interacts in vivo with other components of the FAS‐II pathway, we have initiated experiments to determine whether enzyme inhibition results in structural changes that could affect protein–protein interactions involving InhA and how these ligand‐induced conformational changes are modulated in the InhA mutants. Significantly, while NADH binding to wild‐type InhA is hyperbolic, the InhA mutants bind the cofactor with positive cooperativity, suggesting that the mutations permit access to a second conformational state of the protein. While cross‐linking studies indicate that enzyme inhibition causes dissociation of the InhA tetramer into dimers, analytical ultracentrifugation and size exclusion chromatography reveal that ligand binding causes a conformational change in the protein that prevents cross‐linking across one of the dimer–dimer interfaces in the InhA tetramer. Interestingly, a similar ligand‐induced conformational change is also observed for the InhA mutants, indicating that the mutations modulate communication between the subunits without affecting the two conformational states of the protein that are present.
The role of protein homochirality in shaping the energy landscape of folding
Authors: Nanda Vikas , Andrianarijaona Aina , Narayanan Chitra
Published: June 2007

The homochirality, or isotacticity, of the natural amino acids facilitates the formation of regular secondary structures such as α‐helices and β‐sheets. However, many examples exist in nature where novel polypeptide topologies use both l‐ and d‐amino acids. In this study, we explore how stereochemistry of the polypeptide backbone influences basic properties such as compactness and the size of fold space by simulating both lattice and all‐atom polypeptide chains. We formulate a rectangular lattice chain model in both two and three dimensions, where monomers are chiral, having the effect of restricting local conformation. Syndiotactic chains with alternating chirality of adjacent monomers have a very large ensemble of accessible conformations characterized predominantly by extended structures. Isotactic chains on the other hand, have far fewer possible conformations and a significant fraction of these are compact. Syndiotactic chains are often unable to access maximally compact states available to their isotactic counterparts of the same length. Similar features are observed in all‐atom models of isotactic versus syndiotactic polyalanine. Our results suggest that protein isotacticity has evolved to increase the enthalpy of chain collapse by facilitating compact helical states and to reduce the entropic cost of folding by restricting the size of the unfolded ensemble of competing states.
Effect of Asp69 and Arg310 on the pK of His68, a key catalytic residue of adenylosuccinate lyase
Authors: Sivendran Sharmila , Segall Mark L. , Rancy Pumtiwitt C. , Colman Roberta F.
Published: June 2007

Adenylosuccinate lyase (ASL) of Bacillus subtilis contains three conserved histidines, His68, His89, and His141, identified by affinity labeling and site‐directed mutagenesis as critical to the intersubunit catalytic site. The pH‐Vmax profile for wild‐type ASL is bell‐shaped (pK1 = 6.74 and pK2 = 8.28). Only the alkaline side changes with temperature, characteristic of histidine pKs. To identify determinants of pK2 in the enzyme‐substrate complex, we replaced residues at two positions close to His68 (but not to His89 or His141) in the structure. Compared with the specific activity of 1.75 μmol adenylosuccinate reacting/min/mg of wild‐type enzyme at pH 7.0, mutant enzymes D69E, D69N, R310Q, and R310K exhibit specific activities of 0.40, 0.04, 0.00083, and 0.10, respectively. While D69E has a Km for adenylosuccinate similar to that of wild‐type ASL, D69N and R310K exhibit modest increases in Km, and R310Q has an 11‐fold increase in Km. The mutant enzymes show no significant change in molecular weight or secondary structure. The major change is in the pH‐Vmax profile: pK2 is 8.48 for the D69E mutant and is decreased to 7.83 in D69N, suggesting a proximal negative charge is needed to maintain the high pK of 8.28 observed for wild‐type enzyme and attributed to His68. Similarly, R310Q exhibits a decrease in its pK2 (7.33), whereas R310K shows little change in pK2 (8.24). These results suggest that Asp69 interacts with His68, that Arg310 interacts with and orients the β‐carboxylate of Asp69, and that His68 must be protonated for ASL to be active.
Clinical experience with a highly purified factor IX concentrate in patients undergoing surgical operations
Authors: Thomas D. P. , Lee C. A. , Colvin B. T. , Dasani H. , Dolan G. , Giangrande P. L. F. , Jones P. , Lucas G. , Cantwell O. , Harman C. T.
Published: June 2007

The haemostatic efficacy of a new highly purified factor IX concentrate, prepared by metal chelate affinity chromatography, was assessed in 13 patients with haemophilia B undergoing a variety of surgical operations. Four of the patients had developed post‐operative thromboembolic complications following previous operations, when treated with a prothrombin complex concentrate. None of the patients in the present series developed any evidence of post‐operative thrombotic complications. Effective haemostasis was achieved in all patients, with the exception of a surgical bleed in one case, and late post‐operative bleeding in a second patient when the factor IX activity fell below 20iu/dl. The product is treated with a solvent‐detergent process that destroys lipid‐enveloped viruses, while the affinity chromatography process during manufacture removes in excess of 4 log10 of a non‐lipid‐enveloped virus. In follow‐up studies, none of the patients has shown evidence of fresh infection from the concentrate, when assessed by virological markers. It is concluded that this high‐purity concentrate (tradenane ‘Replenine’) is effective for the treatment of patients with haemophilia B who undergo surgical operations.
Prophylaxis for Pneumocystis carinii pneumonia: its impact on the natural history of HIV infection in men with haemophilia
Authors: Sabin Caroline A. , Elford Jonathan , Phillips Andrew N. , Janossy George , Lee Christine A.
Published: June 2007

It has been suggested that the range of AIDS‐defining conditions witnessed in patients with HIV infection has changed since the early years of the HIV epidemic. In this paper we consider the range of AIDS‐defining conditions in a cohort of 111 HIV‐positive men with haemophilia registered at the Royal Free Hospital Haemophilia Centre. In particular we assess whether the incidence of Pneumocystis carinii pneumonia (PCP) has changed over time. The men were all infected between 1979 and 1985 after treatment with infected blood products and have now been followed prospectively for up to 13 years from HIV seroconversion. By the end of 1992, 44/111 patients had developed AIDS. Of the 44 men, 18 (41%) presented with PCP as their first AIDS‐defining condition (ADC), mainly before the initiation of primary prophylaxis in 1989. The remaining 26 patients presented with a range of conditions as their first ADC, but there were no more than four cases in any one disease category. It is estimated that patients suffer from 0.7 further ADCs per year after being diagnosed with AIDS. After taking account of the increased levels of immuno‐suppression in the cohort with time, it appears that the incidence of PCP, both as the first ADC or as any ADC, has declined since the introduction of primary prophylaxis for the disease in 1989. However, non‐compliance with prophylaxis for PCP appears to have played a major role in the continuing occurrence of PCP since 1988. Improvements in compliance with therapy should result in a further reduction in the incidence of PCP both as a first ADC and as any ADC.
Long‐term surveillance for human anti‐murine antibodies of a group of haemophiliacs treated only with immunoaffinity‐purified FVIII concentrates
Authors: BRACKMANN H. H. , GOMPERTS E. D. , COURTER S. C. , VIDOR A. , O'BRIEN S. , OLDENBURG J.
Published: June 2007

An immunoaffinity purified, solvent/detergent virally inactivated factor VIII (FVIII) concentrate (Hemofil® M) has been in clinical use in persons with haemophilia A since March 1987 and under clinical trial prior to licencing in the USA, Europe and Japan.

The specification set and consistently met for the monoclonal antibody (mAb) content of the final product is 0.1 ng/IU FVIII. This level was considered non‐immunogenic, based upon animal studies, recommendations of the European PHarmacopoeia with ovalbumin contamination of influenza vaccine and also the experience during clinical research studies using aAb for diagnostic or other clinical purpose as reported in the medical literature.

This research communication documents the surveillance for human anti‐murine antibodies (HAMA) in 13 patients with serve haemophilia A. These patients have been treated with a large amount of product (mean 14,026 IU), numerous different lots of Hemofil M (total 45; mean 17) over a substantial period of time (mean 28 months).

The data generated from this group of 13 patients before and during therapeutic Hemofil M administration failed to show the development of any HAMA response at any timepoint. Therefore Hemofil M prepared by mAb resin immunoaffinity column chromatography under the currently set specifications for mAb contaminant of 0.1 ng/IU FVIII or less is a safe therapeutic agent in the management and prevention of bleeding episodes in persons with haemophilia A.
Spontaneous infection of soft tissue haematomas in two HIV seropositive haemophilia patients
Authors: MERCHAN E. C. RODRIGUEZ , VILLAR ANA , MAGALLON MANUEL , DE ORBE ARANZAZU
Published: June 2007

Two HIV seropostiive haemophilic patients with spontaneous infected soft tissue haematomas are described. In one patient the prepatellar bursa was infected, and in the other the posterior aspect of the shoulder. Treatmetn included evacuation of the suppurative focus, antibiotic therapy and prompt rehabilitation. The results were good in both cases and therefore operative management is recommended.
Polymorphisms associated with the FVIII and FIX genes in the Turkish population
Authors: CÇAǦLAYAN S. HANDE , GÖKMEN YESIM , KIRDAR BETUL
Published: June 2007

Summary In order to determine the gene frequencies of nine polymorphic sites associated with FVIII and FIX genes in the Turkish population a sample of 50‐235 unrelated X chromosomes from healthy individuals were analysed by using PCR‐based assays. The Turkish population was found to be as polymorphic as Europeans in the FVII and FIX genes. Analysis of FIX haplotypes revealed that the most frequent haplotype observed in European populations and Anglo‐Americans was also very common among Turks. The present population‐based study indicates that two marker loci, namely HindIII and St14 in the factor VIII gene and DdeI and HhaI in the factor IX gene, are highly informative and useful markers that can be used in DNA linkage analysis for the assessment of haemophilia carriers and affected fetuses in the Turkish population.
A description of two cases of factor V deficiency
Authors: CERNECA FEDERICO , PARCO ERGIO , SIMEONE ROBERT , BEMBI BRUNO , GIORGI RITA
Published: June 2007

Summary We report two cases of factor V deficiency. A 12‐year‐old girl with thalassemia major was admitted for bone marrow transplant (mismatched). She was found to have a heterozygous deficiency of factor V (21%), but this was considered compatible with the transplant, which was performed without the support of suppernatant cryoprecipitate. A 14‐year‐old girl (factor V 2%) with a negative history (menarche at age 12, menstrual cycle regular and normal in quantity and length). One year previously she had complained of menorrhagia (length of period, 9 days); tests revealed server anaemia (Hb 4.9 g/dl). We show how even severe cases of factor V deficiency may often be silent, and not require transfusion in surgical procedures, thus avoiding the risks associated with such therapy.
PCP pneumonia with bilateral pneumothoraces in a haemophiliac with factor VIII inhibitors
Authors: DASANI H. , DAVIES V. , WYNN R.
Published: June 2007

Summary Pneumocystis carinii pneumonia (PCP) infection is a well‐recognized feature of advanced HIV infection in all risk groups. This serious opportunistic parasitic infection is almost invariably fatal if left untreated. When associated with bilateral pneumo‐thoraces in a patient with severe haemophilia A and factor VIII inhibitors, it poses a critical management problem and can be life‐threatening. We report the case of a young man with severe haemophilia A with factor VIII inhibitors presenting with bilateral pneumothoraces which was successfully managed with conservative therapy only.
Carrier detection and prenatal diagnosis by intron 22 inversion analysis of the factor VIII gene
Authors: ONONYE C. , JENKINS P.V. , GOLDMAN E. , PASI K. J.
Published: June 2007

Summary In approximately 50% of severe haemophilia A patients the mutation is present in the form of a large chromosomal disruption in the factor VIII gene; this disruption is described as an inversion. It results in the physical breakage and separation of exons 1‐2 and exons 23‐26 of the factor VIII gene.
Clinical experience of factor XI deficiency: the role of fresh frozen plasma and factor XI concentrate
Authors: COLLINS P.W. , GOLDMAN E. , LILLEY P. , PASI K. J. , LEE C. A.
Published: June 2007

Summary. Factor XI deficiency is a rare autosomally transmitted coagulopathy that is associated with a variable bleeding tendency. Recently there have been reports of thrombotic events following the administration of a virally inactivated factor XI concentrate (BPL) to factor XI deficient patients. We have therefore reviewed a single centre's experience of the use of factor XI concentrate over a 6‐year period and compared this to our previous experience of either no treatment or treatment with fresh frozen plasma (FFP) in 103 patients.

There were 156 procedures performed without haemo‐ static cover. The incidence of bleeding was greatest following tonsillectomy (71%) and dental extraction (Sl'h). There was a trend for bleeding complications to be associated with lower levels of factor XI but patients with all levels of factor XI suffered bleeding complica‐ tions. There were 38 procedures carried out under FFP cover, with only one patient suffering excessive bleeding and no serious complications.

Factor XI concentrate was given to 25 patients to cover 45 episodes. There were no bleeding complications. Three patients suffered serious complications. One patient, with a previous history of cardiovascular disease, died of a myocardial infarction and a second had an ischaemic episode resulting in a %day hospital admission. These episodes both occurred on the same day as the factor XI infusion. A third patient suffered bilateral pulmonary emboli 7 weeks after a prolonged course of factor XI concentrate.

These finding suggest that factor XI concentrate should be contraindicated in patients with a history of cardiovascular disease, when FFP should be used. Guide‐ lines for the use of factor XI concentrates should be revised, and work performed to establish the mechanism of these thrombotic events.
Resection of the radial head and partial open synovectomy of the elbow in the young adult with haemophilia: long‐term results
Authors: MERCHAN E. C. RODRIGUEZ , GALINDO E. , MAGALLON M. , GAGO J. , VILLAR A. , SANJURJO M. J.
Published: June 2007

Resection of the radial head and partial open synovectomy of the elbow have been used in the treatment of repeated haemarthroses of the elbows in 15 patients with haemophilia. The indications for the index operation were: severe pain and bleeding in the elbow that occurred in spite of appropriate, episodic, replacement therapy over a period of more than 6 months, associated with hypertrophy of the radial head and a significant loss of pronation‐supination.

The long‐term results were assessed retrospectively according to the classification recommended by the Orthopaedic Advisory Committee of the World Federation of Hemophilia. In the operated group, three patients had a good result, seven were fair, and five poor. It is concluded that this procedure appears to reduce the incidence of haemarthrosis but did not slow the rate of evolution of radiographic changes.
Counselling challenges in haemophilia and HIV infection/AIDS
Authors: PITTADAKI JANE
Published: June 2007

Summary. The advent of AIDS has had such a deep‐reaching effect on the international haemophilia community that one can make a reasonable distinction between a pre‐AIDS and a post‐AIDS era in haemophilia management. In the context of counselling, however, talking about a ‘before’ and an ‘after’ in haemophilia does not (and, in our opinion, should not) necessarily imply a separation of the past from the present. Dealing with the psycho‐social implications of haemophilia and HIV infection does not mean focusing exclusively on HIV‐generated problems at the expense of haemophilia‐related issues.

Since the HIV crisis, counselling has posed the multiple challenge of: (a) assessing and alleviating the more immediate emotional effects of HIV infection; (b) paying due attention to the underlying influence of haemophilia on reaction, defence and coping; (c) formulating a flexible approach that is based on close cooperation with the medical staff and effective interpersonal communication with the counsellees.*

In practice, the flexibility and effectiveness of the counselling model are promoted by means of: (a) ongoing counselling, (b) multiple counselling sites (i.e. the Haemophilia Centre and other appropriate locations), (c) interdisciplinary team‐work, (d) respect for individual/ ethnic values, (e) maintenance of exo‐empathy (i.e. neutrality), and (f) transfer of coping skills.

The above framework can help maximize the effectiveness of counselling sessions through a personalized rapport of mutual trust and confidence between the counselling team and the counsellees.
A post‐marketing safety and efficacy assessment of a monoclonal antibody purified high‐purity factor VIII concentrate
Authors: HAY C. R. M. , LEE C. A. , SAVIDGE G.
Published: June 2007

Summary. The identification of infrequent side‐effects of clotting factor concentrates, undetected by clinical trials, is facilitated by post‐marketing surveillance. We present a post‐marketing surveillance study in which 97 patients with haemophilia A, attending three haemophilia centres, were treated over a median follow‐up period of 284 days (range 1–1074), and a total follow‐up period of 30,080 days, with a pasteurized immunoaffinity purified factor VIII concentrate (Monoclate‐P, Armour, Collegeville, USA). 5216 infusions, using 10,527,000 units of Monoclate‐P, were carried out, mostly for routine haemarthroses or prophylaxis.

No new inhibitors were observed during the study. At the start of the study 60/97 were HIV seropositive, 67/97 HBs antibody positive, 12 HbsAb negative and the remainder HBsAb positive before the study period. 13/14 tested were HAV seropositive at the beginning of the study. One patient became HAV seropositive during the study period, an infection thought to be community acquired. No other seroconversions were observed. Only one mild transfusion reaction was observed. This study confirms the safety and efficacy of Monoclate‐P. Post‐marketing surveillance or nationally organized pharmaco‐vigilance should be practiced more widely to enable identification of low‐frequency side‐effects of treatment.
The impact of a very high‐purity factor VIII concentrate on the immune system of HIV‐infected haemophiliacs: a randomized, two‐year comparison with a high‐purity concentrate
Authors: de BIASI R. , ROCINO A. , QUIRINO A. A. , MIRAGLIA E. , ZIELLO L.
Published: June 2007

Summary. Randomized and cohort studies have provided evidence confirming the hypothesis, based on in‐vitro observations, that the use of very high‐purity factor VIII (FVIII) concentrates, either immuoaffinity chromatography purified or produced by recombinant DNA technology, may slow immunological deterioration in human immunodeficiency virus (HIV)‐infected haemophiliacs, while high‐purity concentrates, produced by ion‐exchange chromatography, did not produce a benefit. Even though these data clearly indicate that very high‐purity concentrates should be preferred for the replacement therapy of HIV‐positive haemophiliacs, there are little data, based on direct comparison, supporting the use of very high‐purity concentrates rather than high‐purity preparations, which are less expensive. In an attempt to address this issue, we prospectively compared CD4 cell counts and changes of clinical status in 18 HIV‐positive haemophiliacs, randomly assigned either to receive the treatment with a very high‐purity FVIII concentrate, purified by immunoaffinity chromatography, or a high‐purity product, produced by ion‐exchange chromatography. All patients had CD4 lymphocyte counts below 300 μL‐1, were negative for the hepatitis B surface antigen and the HIV p24 antigen, and were receiving antiretroviral treatment with Zidovudine for at least 6 months. There were no significant changes of CD4 cell counts over the 96‐week follow‐up period or between the two groups. No signficant differences between the two groups were detected in the occurrence of AIDS‐defining diagnoses (one in each group). On the whole, no striking benefit is conferred to the immune status of asymptomatic HIV‐positive haemophiliacs by using either of these high‐purity and very high‐purity FVIII concentrates for 96 weeks. Larger prospective randomized trials are needed to establish definitely whether it is necessary to resort to very high‐purity concentrates or it is sufficient to use high‐purity concentrates to slow the fall of CD4 cell counts that occurs in HIV‐positive haemophiliacs. Randomized trials, based on clinical end‐points, are also needed to demonstrate whether slowing the fall in CD4 cells results in clinical benefits, delaying the occurrence of AIDS.