bluuebunny/crossref_metadata_2025 huggingface dataset:
High‐titre factor VIII inhibitor in two children with mild haemophilia A
Authors: Puetz J. J. , Bouhasin J. D.
Published: March 2003

A frequently encountered complication of therapy given to patients with severe haemophilia A is the development of antibodies to infused factor VIII. While much less common, inhibitors also occur in patients with mild or moderate severity haemophilia A. Often thought to be of low titre and transient, several cases of high‐titre inhibitors have been described in patients with mild or moderate haemophilia A. Generally these occur in adults or adolescents following significant infused factor VIII exposure. A review of reported cases revealed only two cases of high‐titre inhibitor formation in mild haemophilia A patients younger than 10 years of age. We wish to report our experience with an additional two children with mild haemophilia A and high titre inhibitors, and offer suggestions for the management of these children.
Access to medical information in Spain
Authors: GonzÁlez QuinzÁ A.
Published: March 2003

Summary.  The 21st century is marked by advances in medical science and the rise of the ‘information society’. It is possible to identify and systematize the intersections between both of these areas and point out a matrix of access to medical information from a legal perspective. There are four connecting levels: (1) informed consent; (2) medical histories; (3) healthcare administrative information; and (4) information on the impact of environmental factors such as food and nutrition or contamination on health. This four‐way scenario acquires new importance with the generalization of the information society and the advances in biomedicine. Globalization is challenged by the ‘here and now’ mentioned before. The large number of medical data and their new uses and consequences is a subject requiring an interdisciplinary approach that emerges from ethics as much as from science.
Orthopaedic surgery of haemophilia in the 21st century: an overview
Authors: RODRIGUEZ‐MERCHAN E. C.
Published: March 2003

Close co‐operation between haematologists, orthopaedic surgeons, rehabilitation physicians and physiotherapists is essential for obtaining satisfactory results after orthopaedic procedures that are performed on haemophilic patients. Although continuous prophylaxis could avoid the development of the orthopaedic complications of haemophilia that we still see in the 21st century, such a goal has not been achieved so far, not even in developed countries. Therefore, orthopaedic surgeons are still required to carry out many different surgical procedures, such as arthrocentesis, synoviorthesis, synovectomies, tendon lengthening, articular debridements, alignment osteotomies, joint arthroplasties, nerve releases, opening of compartment syndromes, removal of pseudotumours and osteosynthesis of fractures. Furthermore, the emergence of human immunodeficiency virus has meant that immunosuppressed patients in developed countries sometimes require an arthrotomy for the treatment of spontaneous septic arthritis, or the surgical drainage of a spontaneously infected haematoma (abscess). In addition, they have a high risk of postoperative infection after any surgical procedure, particularly a joint arthroplasty.
Acquired anti‐FVIII inhibitors in children
Authors: MORACA R. J. , RAGNI M. V.
Published: March 2003

Acquired inhibitors to FVIII (anti‐FVIII) are uncommon in children. An acquired anti‐FVIII developed in a previously healthy 4‐year‐old boy treated with penicillin for streptococcal pharyngitis. Aspirin prophylaxis begun for suspected rheumatic fever led to compartment syndromes of all four extremities, which resolved with high‐dose FVIII and surgical decompression. Anti‐FVIII in this patient, and the five additional cases identified in a survey of 160 haemophilia treatment centres, occurred at a median age of 8 years, with median initial and peak titres of 4.6 and 6.9 Bethesda Units (BU), respectively. All six presented with bleeding, including haematomas (three intramuscular, one intracranial), and ecchymoses in three. The median baseline FVIII was 0.05 U mL−1, and the median baseline activated partial thromboplastin time (APTT) was 79.8 s. The inhibitor resolved completely in five patients (83%) within a median 5 months, after treatment with FVIII concentrate, steroids, cytoxan, methotrexate, and no treatment. The inhibitor persisted in the patient with Goodpasture's disease, despite steroids, cytoxan, cyclosporin, and intravenous gamma globulin. Aspirin therapy, in two, worsened ongoing bleeding. The association of penicillin‐like drugs in this and three other cases in the literature suggest that to avoid potential catastrophic bleeding, it is prudent to obtain an APTT prior to initiating aspirin for suspected rheumatic fever. In conclusion, acquired anti‐FVIII inhibitors in children may cause severe bleeding, and remit in the majority after FVIII and/or immunosuppressive therapy.
Postoperative hepatic laceration in a child with Type 3 von Willebrand disease
Authors: BUYUKAVCI M. , AKDAG R. , OLGUN H.
Published: March 2003

We report an unusual postoperative complication (hepatic laceration) in a child with type 3 vWD, who has not received any prophylaxis.
Massive retroperitoneal pseudotumour in a patient with type 3 von Willebrand disease
Authors: EBY C. S. , CARACIONI A. A. , BADAR S. , JOIST J. H.
Published: March 2003

Formation of destructive haemorrhagic pseudocysts or pseudotumours thought to arise from unresolved, encapsulated haematomas is a well‐recognized, rare complication of severe haemophilia A or B, and has been reported in a single patient with von Willebrand disease (vWD). We report a 41‐year‐old patient with type 3 vWD who underwent incomplete resection of a large retroperitoneal pseudocyst in 1995 and presented with a recurrent, extensive right abdominal and flank mass and signs and symptoms of large bowel obstruction. He required emergency partial colectomy for bowel ischaemia and removal of his right kidney, which was hydronephrotic due to prolonged ureteral obstruction by the pseudocyst. Following repeat partial resection of the pseudotumour, he developed persistent bleeding into the operative site despite aggressive administration of von Willebrand factor (vWF)‐rich factor VIII concentrates, resulting in retroperitoneal haematomas and abscesses, which resolved after 13 months of percutaneous drainage, extended supplementation of vWF and antibiotic therapy.
Ethical issues in the genetic aspects of haemophilia
Authors: SHENFIELD F.
Published: March 2003

The aim of this paper is to put some of the ethical considerations concerning the genetic aspects of haemophilia not only in the national, but also in the international context, especially from the point of view of human rights. Particular attention is given to the issues pertaining to consent from children, and the respect for their autonomy within family relationships. This will be analysed within the context of genetic screening and testing, as well as codes of practice and guidelines. The specific issues around prenatal diagnosis in general and pre‐implantation genetic diagnosis (PGD) are also discussed.
Arthroscopic synovectomy: state of the art
Authors: WIEDEL J. D.
Published: March 2003

Arthroscopic synovectomy for first performed for haemophiic arthropathy in 1980 and was considered a major improvement over open surgical synovectomy, causing less postoperative morbidity and fewer hospitalizations, and preserving range of motion. Long‐term results have shown that recurrent haemarthroses are decreased and joint function is preserved. Radiographic changes, however, suggest continued progression of the disease process. The procedure is technically demanding and requires considerable resources including skilled personnel, surgical equipment and an adequate supply of factor concentrate, all of which limit the availability of the procedure worldwide. Where this procedure fits into the treatment of chronic haemophilic synovitis depends very much on location and availability of resources. Consequently, the procedure should not be considered as the first‐line approach for recurrent haemarthroses, but rather as a secondary procedure if alternative measures have failed.
Patient autonomy: evolution of the doctor–patient relationship
Authors: STEINHART B.
Published: March 2003

In recent years, much has been said about the shifting locus of control in clinical practice. This article discusses the influence on the physician‐patient relationship of recent social and political history, patient culture and experience, and advances in medical practice. I suggest that effective partnerships between physicians and patients are possible in a supportive institutional environment in which the participants acknowledge and fulfil their roles and responsibilities in shared decision‐making. While I recognize that there is considerable variability in practices across cultures and in individual behaviour, the scope of this article is based on literature and my knowledge, which is representative primarily of the North American experience.
Efficacy and safety of a factor VIII–von Willebrand factor concentrate 8Y: stability, bacteriological safety, pharmacokinetic analysis and clinical experience
Authors: Lubetsky A. , Martinowitz U. , Luboshitz J. , Kenet G. , Keller N. , Tamarin I. , Inbal A.
Published: March 2003

Summary.  The present study was undertaken to evaluate stability, pharmacokinetic profile and efficacy of continuous infusion of 8Y in patients with different types of von Willebrand disease (vWD). Following reconstitution, 8Y levels of von Willebrand factor ristocetin cofactor (vWF:Rco), vWF antigen and factor VIII coagulant activity (FVIII:C) decreased to about 80% of the baseline levels; addition of low molecular weight heparin decreased the level of FVIII:C even further. Reconstituted 8Y was found to be sterile for up to 6 days postreconstitution. Ten vWD patients (four with type 2A, three with type 3, two with type 1 and one with 2N) underwent pharmacokinetic analysis. The recovery of vWF: RCo was significantly lower in patients with type 3 vWD (1.4 ± 0.05% U−1 kg−1) compared withthat of the patients with types 1 (2.3 ± 0.52% U−1 kg−1) or 2A (2.0 ± 0.06% U−1 kg−1) vWD (P = 0.015). Type 3 vWD patients exhibited significantly higher vWF:RCo clearance (5.1 ± 1.1 mL kg−1 h−1) compared with that of patients with type 2A (2.8 ± 0.7 mL kg−1 h−1) and type 1 (2.6 ± 1.0 mL kg−1 h−1) vWD (P = 0.028). Accordingly, terminal half‐life was lower in patients with type 3 vWD (8.0 ± 0.6 h−1) compared with type 2A (12.7 ± 5.9 h−1) or type 1 (14 ± 1.2 h−1) vWD patients. Multimeric pattern of vWF from patients' plasma was similar to that of 8Y. In two patients treated with 8Y by continuous infusion for prevention or treatment of bleeding haemostasis was achieved. Thus, 8Y is suitable and haemostatically effective for continuous infusion treatment in patients with vWD.
Orthopaedic surgery in severe bleeding disorders: a low‐volume, high‐cost procedure
Authors: Mishra V. , Tjønnfjord G. E. , Paus A. C. , Vaaler S.
Published: March 2003

Summary. As more and more nations are scrutinizing their health care costs, attention has been focused on high‐cost low‐density disease. Assessment of actual total cost of care for haemophilia and its positive outcome becomes essential to justify support for these patients. In this study, we assessed hospital cost and diagnosis‐related group (DRG) reimbursement for patients undergoing elective orthopaedic surgical procedures from May 1999 to December 1999. Hospital cost was assessed by a prospective microcost‐analysis method. To identify real hospital costs, we performed registration of preoperative phase, operative phase and 1‐year follow‐up costs. Hospital cost included personnel costs and costs for clinical and laboratory procedures, blood products, prosthetic implants, coagulation factor concentrates and drugs. These data were compared with hospital DRG reimbursement. We included nine consecutive patients, with a mean age 38 years (19–54 years) who had had 10 major orthopaedic surgical procedures performed during the study period. Six patients had haemophilia A, two had haemophilia B and one had factor VII deficiency. Data analysis showed a mean cost of US$ 54 201 (range US$ 25 795–105 479; 1US$ = 8.5 NOK). The average actual hospital revenue (50% DRG reimbursement + income related to length of stay) was $4730 (range $ 1 308–13 601). Our study confirms that orthopaedic surgery in patients with severe bleeding disorders puts the hospital to a considerable expense. Activity‐based financing, as used in Norway, does not provide a proper reimbursement for this part of the haemophilia care.
Carrier testing and prenatal diagnosis of haemophilia B by SSCP in an Iranian family
Authors: Karimipoor M. , Zeinali S. , Lak M. , Safaee R.
Published: March 2003

Summary. Haemophilia B is an X‐linked recessive bleeding disorder caused by multiple molecular defects in factor IX gene. During our genotyping programme of haemophilia B patients, a family from the west of Iran was referred to us. We first detected the mutation in the index case of the family by single‐strand conformation polymorphism (SSCP) technique. This technique was then applied in carrier detection and prenatal diagnosis for females of this family. Sequencing later revealed the mutation as being G to A at 20519.
Historical and political implications of haemophilia in the Spanish royal family
Authors: Ojeda‐Thies C. , Rodriguez‐Merchan E. C.
Published: March 2003

Summary.  The political implications of haemophilia in the marriage of King Alfonso XIII of Spain and Princess Victoria Eugenie Battenberg of England have been reviewed in recent books on history. However, the fact that they had haemophilic sons also affected their personal relationship. In this article, we review the consequences haemophilia bore on their lives. We feel great compassion for families who suffer the illness, be it ordinary people or members of royalty; however, in this case, it can be said that when the disease affected a royal couple, the political consequences were great.
Weak links: ‘Rapoport's rule’ and large‐scale species richness patterns
Authors: Kerr Jeremy T.
Published: March 2003

Many hypotheses have been proposed to explain regional species richness patterns. Among these, ‘Rapoport's rule’ has sparked considerable controversy by stating that the latitudinal gradient in species richness can be explained indirectly as a function of narrower geographic ranges for species at low latitudes. Annual climatic variability, or deviation from mean climatic conditions, has been hypothesized to moderate this phenomenon. Furthermore, taxa that avoid much of this seasonality, such as temperate zone insects that enter diapause or species that migrate, were predicted to show reduced latitudinal gradients in richness. I test the suggested link between ‘Rapoport's rule’ and species richness for two higher level insect taxa as well as for the class Mammalia. Although these taxa exhibit the well‐known latitudinal gradient in species richness, simple annual climatic variability and deviation from mean annual climatic conditions provide very poor predictions of species richness in each of them. Potential evapotranspiration, a measurement of ambient climatic energy, explains most of the observed variance in regional species richness patterns for all three taxa, consistent with the species richness‐energy hypothesis. I find no support for an indirect link between ‘Rapoport's rule’ and terrestrial species richness patterns in North America.
Mammals in South American drylands: faunal similarity and trophic structure
Authors: Ojeda Ricardo A. , Blendinger Pedro G. , Brandl Roland
Published: March 2003

We compared the fauna of small mammals (less than 500 g body weight) among five major South American drylands (Atacama, Altiplano, Monte, Patagonia and Caatinga) and found considerable heterogeneity and distinctiveness in species richness and composition between these biomes. From a total of 89 recorded species, 76 of them are restricted to only one of these drylands. The highland desert, or Altiplano, is the biome with the highest number of species. Despite the marked differences in the composition of the mammalian fauna, the trophic structure shows a rather consistent pattern: herbivores are the most important trophic group in all drylands. This consistency seems to be more the result of phylogenetic inertia than of similar ecological processes. Our results are compared with recent studies on desert small mammals across continents.
Species as units of analysis in ecology and biogeography: are the blind leading the blind?
Authors: Kelt Douglas A. , Brown James H.
Published: March 2003

Riddle & Hafner (1999) suggest that genetically differentiated lineages should be formally recognized as evolutionary significant units (ESUs) and that ESUs should replace currently recognized species as units in quantitative ecological and biogeographic analyses. Riddle & Hafner imply that if comparisons of desert rodent communities across many localities in south‐western North America by Brown & Kurzius (1987) and Kelt et al. (1996) had used ESUs rather than species, substantially different results and conclusions would have been obtained. Here we defend our use of species in these studies, and question the wisdom of formalizing the concept of ESU and applying it in most biogeographic, ecological and behavioural studies. Studies of genetic variation and description of ESUs are uneven across taxa of mammals and geographical regions, and those ESUs that have been described are often difficult or impossible to identify on the basis of the morphological traits used to distinguish currently recognized species. Consequently, most of the specimens in museum drawers, nearly all of the fossil remains, and most of the animals studied in the field or laboratory by ecologists, behaviourists and other biologists cannot be assigned to ESUs. The currently recognized species, usually described originally on the basis of morphological and biological species concepts, provide the only relatively consistent, operational taxonomic units. Results of our studies on the biogeography of desert small mammals are not seriously altered by recent subdivisions of some species into multiple ESUs.
Risk assessment for nonindigenous pests: 2. Accounting for interyear climate variability
Authors: Jarvis Claire H. , Baker Richard H. A.
Published: March 2003

The paper firstly discusses the importance of accounting for interyear variability when assessing the likelihood of establishment of an alien pest. The potential establishment of Colorado beetle (Leptinotarsa decemlineata) is used as an illustration within the geographical context of England and Wales. An aggregate risk index is introduced as a probabilistic representation of the likelihood that a pest might complete a single generation over a 30‐year period (1961–90). Data for individual years were used to compute, objectively, the interyear distribution of risk across the landscape. The standard deviation in area at risk (26 800 km2) was high relative to the average proportion of the landscape potentially at risk (95 700 km2). In 40% of years, the area at risk was estimated to be higher than ‘average’.

Secondly, the paper demonstrates multiple indices of risk that reflect different aspects of pest risk assessment. Viewing risk from a variety of perspectives provides a means of gauging the consistency and therefore reliability of the results. This contrasts with current practice, where a single mapped output is commonly presented to decision makers. Modelling using a daily time step allowed the use of indices to investigate the long‐term probabilities of biotic and abiotic events of short duration. These indices include estimates of pest activity and flight potential.
Using conjoint analysis to elicit the views of health service users: an application to the patient health card
Authors: Ryan Mandy , McIntosh Emma , Shackley Phil
Published: March 2003

Objective

To demonstrate the application of conjoint analysis (CA) for eliciting the views of health service users.

Methods

A CA study was conducted alongside a randomized controlled trial evaluating the introduction of a patient health card (PHC). The PHC was evaluated with respect to three other aspects of general practice: number of days between making a non‐urgent appointment and seeing a doctor; waiting time in reception between the time of the appointment and seeing a doctor; and whether the patient is usually seen by the doctor of their choice. A postal questionnaire was sent to 100 individuals from a general practice in Inverurie, Scotland.

Results

Seventy‐five individuals returned the questionnaire, of whom 51 answered the CA section. The PHC was the least important of the attributes considered. The number of days between making a non‐urgent appointment and seeing a doctor was considered to be the most important. A 1‐day reduction in the number of days to appointment was four and a half times more important than having a PHC; a 1‐minute reduction in waiting time in the reception area was three and a half times more important than having a PHC; and seeing a doctor of choice was over three times more important than having a PHC. Satisfaction or utility scores for different ways of providing a general practice service also indicated that priority should be given to reducing waiting time to see a doctor or reducing waiting time in reception.

Conclusions

While the PHC is a significant and positive predictor of satisfaction in general practice, it is less important than the other three attributes considered. More generally, CA appears to be a potentially useful instrument for eliciting the views of health service users.
Domain organization, folding and stability of bacteriophage T4 fibritin, a segmented coiled‐coil protein
Authors: Boudko Sergei P. , Londer Yuri Y. , Letarov Andrei V. , Sernova Natalia V. , Engel Juergen , Mesyanzhinov Vadim V.
Published: March 2003

Fibritin is a segmented coiled‐coil homotrimer of the 486‐residue product of phage T4 gene wac. This protein attaches to a phage particle by the N‐terminal region and forms fibrous whiskers of 530 Å, which perform a chaperone function during virus assembly. The short C‐terminal region has a β‐annulus‐like structure. We engineered a set of fibritin deletion mutants sequentially truncated from the N‐termini, and the mutants were studied by differential scanning calorimetry (DSC) and CD measurements. The analysis of␣DSC curves indicates that full‐length fibritin exhibits three␣thermal‐heat‐absorption peaks centred at 321 K (ΔH=1390 kJ·mol trimer−1), at 336 K (ΔH=7600 kJ·mol trimer−1), and at 345 K (ΔH=515 kJ·mol trimer−1). These transitions were assigned to the N‐terminal, segmented coiled‐coil, and C‐terminal functional domains, respectively. The coiled‐coil region, containing 13 segments, melts co‐operatively as a single domain with a mean enthalpy ΔHres=21 kJ·mol residue−1. The ratio of ΔHVH/ΔHcal for the coiled‐coil part of the 120‐, 182‐, 258‐ and 281‐residue per monomer mutants, truncated from the N‐termini, and for full‐length fibritin are 0.91, 0.88, 0.42, 0.39, and 0.13, respectively. This gives an indication of the decrease of the ‘all‐or‐none’ character of the transition with increasing protein size. The deletion of the 12‐residue‐long loop in the 120‐residue fibritin increases the thermal stability of the coiled‐coil region. According to CD data, full‐length fibritin and all the mutants truncated from the N‐termini refold properly after heat denaturation. In contrast, fibritin XN, which is deleted for the C‐terminal domain, forms aggregates inside the cell. The XN protein can be partially refolded by dilution from urea and does not refold after heat denaturation. These results confirm that the C‐terminal domain is essential for correct fibritin assembly both in vivo and in vitro and acts as a foldon.
Regulation of RAS in human platelets
Authors: Tulasne David , Bori Teresa , Watson Steve P.
Published: March 2003

In this study, we show that the G protein‐coupled receptor agonist thrombin, the glycoprotein VI agonist convulxin, and the cytokine receptor Mpl agonist thrombopoietin (TPO) are able to induce activation of RAS in human platelets. Recruitment of GRB2 by tyrosine‐phosphorylated proteins in response to TPO and convulxin but not by thrombin occurred with a similar time‐course to RAS activation, consistent with a causal relationship. On the other hand, activation of ERK2 by thrombin and convulxin is delayed and also inhibited by the protein kinase C inhibitor Ro‐31 8220, whereas RAS activation is unaffected. Further evidence for differential regulation of RAS and ERK is provided by the observations that TPO, which activates RAS but not protein kinase C, does not activate ERK, and that the inhibitor of SRC kinases PP1 inhibits activation of RAS but not ERK2 in response to thrombin. Our results demonstrate that activation of RAS is not necessarily coupled to ERK in human platelets.
The role of hydrophobic active‐site residues in substrate specificity and acyl transfer activity of penicillin acylase
Authors: Alkema Wynand B. L. , Dijkhuis Anne‐Jan , de Vries Erik , Janssen Dick B.
Published: March 2003

Penicillin acylase of Escherichia coli catalyses the hydrolysis and synthesis of β‐lactam antibiotics. To study the role of hydrophobic residues in these reactions, we have mutated three active‐site phenylalanines. Mutation of αF146, βF24 and βF57 to Tyr, Trp, Ala or Leu yielded mutants that were still capable of hydrolysing the chromogenic substrate 2‐nitro‐5‐[(phenylacetyl)amino]‐benzoic acid. Mutations on positions αF146 and βF24 influenced both the hydrolytic and acyl transfer activity. This caused changes in the transferase/hydrolase ratios, ranging from a 40‐fold decrease for αF146Y and αF146W to a threefold increase for αF146L and βF24A, using 6‐aminopenicillanic acid as the nucleophile. Further analysis of the βF24A mutant showed that it had specificity constants (kcat/Km) for p‐hydroxyphenylglycine methyl ester and phenylglycine methyl ester that were similar to the wild‐type values, whereas the specificity constants for p‐hydroxyphenylglycine amide and phenylglycine amide had decreased 10‐fold, due to a decreased kcat value. A low amidase activity was also observed for the semisynthetic penicillins amoxicillin and ampicillin and the cephalosporins cefadroxil and cephalexin, for which the kcat values were fivefold to 10‐fold lower than the wild‐type values. The reduced specificity for the product and the high initial transferase/hydrolase ratio of βF24A resulted in high yields in acyl transfer reactions.
Secretion of egg envelope protein ZPC after C‐terminal proteolytic processing in quail granulosa cells
Authors: Sasanami Tomohiro , Pan Jianzhi , Doi Yukio , Hisada Miki , Kohsaka Tetsuya , Toriyama Masaru , Makoto Mori
Published: March 2003

In avian species, an egg envelope homologous to the mammalian zona pellucida is called the perivitelline membrane. We have previously reported that one of its components, a glycoprotein homologous to mammalian ZPC, is synthesized in the granulosa cells of the quail ovary. In the present study, we investigated the proteolytic cleavage of the newly synthesized ZPC and the secretion of ZPC from the granulosa cells. Western blot analysis of the cell lysates demonstrated that the 43‐kDa protein is the precursor of mature ZPC (proZPC), and is converted to the 35‐kDa protein before secretion. The accumulation of proZPC in the presence of brefeldin A, and conversion of proZPC to ZPC in the presence of monensin, indicate the possibility that the proteolytic processing of ZPC occurs in the Golgi apparatus. An analysis of amino‐acid sequence identified that the C terminus of mature ZPC protein is Phe360, and the N‐terminal amino‐acid sequence of the proZPC‐derived fragment was determined as Asp363. These results suggest that newly synthesized ZPC is cleaved at the consensus furin cleavage site, and the resulting two basic residues at the C terminus are subsequently trimmed off to generate mature ZPC prior to secretion.
When the embryonic genome flexes its muscles
Authors: Rupp Ralph A. W. , Singhal Nishant , Veenstra Gert Jan C.
Published: March 2003

During the development of multicellular organisms, both transient and stable gene expression patterns have to be established in a precisely orchestrated sequence. Evidence from diverse model organisms indicates that this epigenetic program involves not only transcription factors, but also the local structure, composition, and modification of chromatin, which define and maintain the accessibility and transcriptional competence of the nucleosomal DNA template. A paradigm for the interdependence of development and chromatin is constituted by the mechanisms controlling the specification and differentiation of the skeletal muscle cell lineage in vertebrates, which is the topic of this review.
Beyond the ABCs of CKC and SCC
Authors: Meluh Pamela B. , Strunnikov Alexander V.
Published: March 2003

The centromere–kinetochore complex is a highly specialized chromatin domain that both mediates and monitors chromosome–spindle interactions responsible for accurate partitioning of sister chromatids to daughter cells. Centromeres are distinguished from adjacent chromatin by specific patterns of histone modification and the presence of a centromere‐specific histone H3 variant (e.g. CENP‐A). Centromere‐proximal regions usually correspond to sites of avid and persistent sister chromatid cohesion mediated by the conserved cohesin complex. In budding yeast, there is a substantial body of evidence indicating centromeres direct formation and/or stabilization of centromere‐proximal cohesion. In other organisms, the dependency of cohesion on centromere function is not as clear. Indeed, it appears that pericentromeric heterochromatin recruits cohesion proteins independent of centromere function. Nonetheless, aspects of centromere function are impaired in the absence of sister chromatid cohesion, suggesting the two are interdependent. Here we review the nature of centromeric chromatin, the dynamics and regulation of sister chromatid cohesion, and the relationship between the two.
Autophosphorylation‐dependent inactivation of plant chimeric calcium/calmodulin‐dependent protein kinase
Authors: Sathyanarayanan P. V. , Poovaiah B. W.
Published: March 2003

Chimeric calcium/calmodulin dependent protein kinase (CCaMK) is characterized by the presence of a visinin‐like Ca2+‐binding domain unlike other known calmodulin‐ dependent kinases. Ca2+‐Binding to the visinin‐like domain leads to autophosphorylation and changes in the affinity for calmodulin [Sathyanarayanan P.V., Cremo C.R. & Poovaiah B.W. (2000) J. Biol. Chem. 275, 30417–30422]. Here, we report that the Ca2+‐stimulated autophosphorylation of CCaMK results in time‐dependent loss of enzyme activity. This time‐dependent loss of activity or self‐inactivation due to autophosphorylation is also dependent on reaction pH and ATP concentration. Inactivation of the enzyme resulted in the formation of a sedimentable enzyme due to self‐association. Specifically, autophosphorylation in the presence of 200 µm ATP at pH 7.5 resulted in the formation of a sedimentable enzyme with a 33% loss in enzyme activity. Under similar conditions at pH 6.5, the enzyme lost 67% of its activity and at pH 8.5, 84% enzyme activity was lost. Furthermore, autophosphorylation at either acidic or alkaline reaction pH lead to the formation of a sedimentable enzyme. Transmission electron microscopic studies on autophosphorylated kinase revealed particles that clustered into branched complexes. The autophosphorylation of wild‐type kinase in the presence of AMP‐PNP (an unhydrolyzable ATP analog) or the autophosphorylation‐site mutant, T267A, did not show formation of branched complexes under the electron microscope. Autophosphorylation‐ dependent self‐inactivation may be a mechanism of modulating the signal transduction pathway mediated by CCaMK.
A chimeric scorpion α‐toxin displays de novo electrophysiological properties similar to those of α‐like toxins
Authors: Bouhaouala‐Zahar Balkiss , Benkhalifa Rym , Srairi Najet , Zenouaki Ilhem , Ligny‐Lemaire Caroline , Drevet Pascal , Sampieri François , Pelhate Marcel , El Ayeb Mohamed , Ménez André , Karoui Habib , Ducancel Frédéric
Published: March 2003

BotXIV and LqhαIT are two structurally related long chain scorpion α‐toxins that inhibit sodium current inactivation in excitable cells. However, while LqhαIT from Leiurus quinquestriatus hebraeus is classified as a true and strong insect α‐toxin, BotXIV from Buthus occitanus tunetanus is characterized by moderate biological activities. To assess the possibility that structural differences between these two molecules could reflect the localization of particular functional topographies, we compared their sequences. Three structurally deviating segments located in three distinct and exposed loops were identified. They correspond to residues 8–10, 19–22, and 38–43. To evaluate their functional role, three BotXIV/LqhαIT chimeras were designed by transferring the corresponding LqhαIT sequences into BotXIV. Structural and antigenic characterizations of the resulting recombinant chimera show that BotXIV can accommodate the imposed modifications, confirming the structural flexibility of that particular α/β fold. Interestingly, substitution of residues 8–10 yields to a new electrophysiological profile of the corresponding variant, partially comparable to that one of α‐like scorpion toxins. Taken together, these results suggest that even limited structural deviations can reflect functional diversity, and also that the structure–function relationships between insect α‐toxins and α‐like scorpion toxins are probably more complex than expected.
Two independent, light‐sensing two‐component systems in a filamentous cyanobacterium
Authors: Jorissen Helena J. M. M. , Quest Benjamin , Remberg Anja , Coursin Thérèse , Braslavsky Silvia E. , Schaffner Kurt , Tandeau de Marsac Nicole , Gärtner Wolfgang
Published: March 2003

Two ORFs, cphA and cphB, encoding proteins CphA and CphB with strong similarities to plant phytochromes and to the cyanobacterial phytochrome Cph1 of Synechocystis sp. PCC 6803 have been identified in the filamentous cyanobacterium Calothrix sp. PCC7601. While CphA carries a cysteine within a highly conserved amino‐acid sequence motif, to which the chromophore phytochromobilin is covalently bound in plant phytochromes, in CphB this position is changed into a leucine. Both ORFs are followed by rcpA and rcpB genes encoding response regulator proteins similar to those known from the bacterial two‐component signal transduction. In Calothrix, all four genes are expressed under white light irradiation conditions, albeit in low amounts. For heterologous expression and convenient purification, the cloned genes were furnished with His‐tag encoding sequences at their 3′ end and expressed in Escherichia coli. The two recombinant apoproteins CphA and CphB bound the chromophore phycocyanobilin (PCB) in a covalent and a noncovalent manner, respectively, and underwent photochromic absorption changes reminiscent of the Pr and Pfr forms (red and far‐red absorbing forms, respectively) of the plant phytochromes and Cph1. A red shift in the absorption maxima of the CphB/PCB complex (λmax = 685 and 735 nm for Pr and Pfr, respectively) is indicative for a noncovalent incorporation of the chromophore (λmax of Pr, Pfr of CphA: 663, 700 nm). A CphB mutant generated at the chromophore‐binding position (Leu246→Cys) bound the chromophore covalently and showed absorption spectra very similar to its paralog CphA, indicating the noncovalent binding to be the only cause for the unexpected absorption properties of CphB. The kinetics of the light‐induced Pfr formation of the CphA–PCB chromoprotein, though similar to that of its ortholog from Synechocystis, showed differences in the kinetics of the Pfr formation. The kinetics were not influenced by ATP (probing for autophosphorylation) or by the response regulator. In contrast, the light‐induced kinetics of the CphB–PCB complex was markedly different, clearly due to the noncovalently bound chromophore.
Effects of ATP depletion and phosphate analogues on P‐glycoprotein conformation in live cells
Authors: Goda Katalin , Nagy Henrietta , Mechetner Eugene , Cianfriglia Maurizio , Szabó Gábor
Published: March 2003

P‐glycoprotein (Pgp), a membrane pump often responsible for the multidrug resistance of cancer cells, undergoes conformational changes in the presence of substrates/modulators, or upon ATP depletion, reflected by its enhanced reactivity with the UIC2 monoclonal antibody. When the UIC2‐shift was elicited by certain modulators (e.g. cyclosporin A or vinblastine, but not with verapamil or Tween 80), the subsequent binding of other monoclonal anti‐Pgp Ig sharing epitopes with UIC2 (e.g. MM12.10) was abolished [Nagy, H., Goda, K., Arceci, R., Cianfriglia, M., Mechetner, E. & Szabó Jr, G. (2001) Eur. J. Biochem.268, 2416–2420]. To further study the relationship between UIC2‐shift and the suppression of MM12.10 binding, we compared, on live cells, how ATP depletion and treatment of cells with phosphate analogues (sodium orthovanadate, beryllium fluoride and fluoro‐aluminate) that trap nucleotides at the catalytic site, affect the two phenomena. Similarly to modulators or ATP depleting agents, all the phosphate analogues increased daunorubicin accumulation in Pgp‐expressing cells. Prelabeling of ATP depleted cells with UIC2 completely abolished the subsequent binding of MM12.10, in accordance with the enhanced binding of the first mAb. Vanadate and beryllium fluoride, but not fluoro‐aluminate, reversed the effect of cyclosporin A, preventing UIC2 binding and allowing for labeling of cells with MM12.10. Thus, changes in UIC2 reactivity are accompanied by complementary changes in MM12.10 binding also in response to direct modulation of the ATP‐binding site, confirming that conformational changes intrinsic to the catalytic cycle are reflected by both UIC2‐related phenomena. These data also fit a model where the UIC2 epitope is available for antibody binding throughout the catalytic cycle including the step of ATP binding, to become unavailable only in the catalytic transition state.
Structural determination of the O‐chain polysaccharide from Agrobacterium tumefaciens, strain DSM 30205
Authors: De Castro Cristina , De Castro Olga , Molinaro Antonio , Parrilli Michelangelo
Published: March 2003

Agrobacterium tumefaciens is a Gram‐negative, phytopathogenic bacterium and is characterized by an unique mode of action on dicotyledonous plants: it is able to genetically modify the host, and because of this feature, it is used as a tool for transgenic plants. Many experiments have demonstrated that lipopolysaccharides (LPSs) play an important role for the disease development, as they are involved in the adhesion process of the bacterium on the plant cell wall. Despite the wealth of information on the role of LPS on phytopathogenesis, the present paper appears as the first report on the molecular primary structure of the O‐chain produced from Agrobacterium. Its repeating unit was determined by means of chemical and spectroscopical analysis, and has the following structure: (3)‐α‐d‐Araf‐(1→3)‐α‐l‐Fucp‐(1→.
Structural and biochemical characterization of calhepatin, an S100‐like calcium‐binding protein from the liver of lungfish (Lepidosiren paradoxa)
Authors: Di Pietro Santiago M. , Santomé José A.
Published: March 2003

We report the biochemical characterization of calhepatin, a calcium‐binding protein of the S100 family, isolated from lungfish (Lepidosiren paradoxa) liver. The primary structure, determined by Edman degradation and MS/MS, shows that the sequence identities with the other members of the family are lower than those between S100 proteins from different species. Calhepatin is composed of 75 residues and has a molecular mass of 8670 Da. It is smaller than calbindin D9k (78 residues), the smallest S100 described so far. Sequence analysis and molecular modelling predict the two EF‐hand motifs characteristic of the S100 family. Metal‐binding properties were studied by a direct 45Ca2+‐binding assay and by fluorescence titration. Calhepatin binds Ca2+ and Cu2+ but not Zn2+. Cu2+ binding does not change the affinity of calhepatin for Ca2+. Calhepatin undergoes a conformational change upon Ca2+ binding as shown by the increase in its intrinsic fluorescence intensity and λmax, the decrease in the apo‐calhepatin hydrodynamic volume, and the Ca2+‐dependent binding of the protein to phenyl‐Superose. Like most S100 proteins, calhepatin tends to form noncovalently associated dimers. These data suggest that calhepatin is probably involved in Ca2+‐signal transduction.
S‐Decyl‐glutathione nonspecifically stimulates the ATPase activity of the nucleotide‐binding domains of the human multidrug resistance‐associated protein, MRP1 (ABCC1)
Authors: Cool Robbert H. , Veenstra Marloes K. , van Klompenburg Wim , Heyne René I. R. , Müller Michael , de Vries Elisabeth G. E. , van Veen Hendrik W. , Konings Wil N.
Published: March 2003

Thehumanmultidrugresistance‐associatedprotein(MRP1) is an ATP‐dependent efflux pump that transports anionic conjugates, and hydrophobic compounds in a glutathione dependent manner. Similar to the other, well‐characterized multidrug transporter P‐gp, MRP1 comprises two nucleotide‐binding domains (NBDs) in addition to transmembrane domains. However, whereas the NBDs of P‐gp have been shown to be functionally equivalent, those of MRP1 differ significantly. The isolated NBDs of MRP1 have been characterized in Escherichia coli as fusions with either the glutathione‐S‐transferase (GST) or the maltose‐binding domain (MBP). The nonfused NBD1 was obtained by cleavage of the fusion protein with thrombin. The GST‐fused forms of NBD1 and NBD2 hydrolyzed ATP with an apparent Km of 340 µm and a Vmax of 6.0 nmol PI·mg−1·min−1, and a Km of 910 µm ATP and a Vmax of 7.5 nmol PI·mg−1·min−1, respectively. Remarkably, S‐decyl‐glutathione, a conjugate specifically transported by MRP1 and MRP2, was able to stimulate the ATPase activities of the isolated NBDs more than 2‐fold in a concentration‐dependent manner. However,the stimulation of the ATPase activity was found to coincide with the formation of micelles by S‐decyl‐glutathione. Equivalent stimulation of ATPase activity could be obtained by surfactants with similar critical micelle concentrations.
ISC1‐encoded inositol phosphosphingolipid phospholipase C is involved in Na+/Li+ halotolerance of Saccharomyces cerevisiae
Authors: Betz Christian , Zajonc Dirk , Moll Matthias , Schweizer Eckhart
Published: March 2003

In Saccharomyces cerevisiae, toxic concentrations of Na+ orLi+ ions induce the expression of the cation‐extrusion ATPase gene, ENA1. Several well‐studied signal transduction pathways are known correlating high salinity to the transcriptional activation of ENA1. Nevertheless, information on the actual sensing mechanism initiating these pathways is limited. Here, we report that the ISC1‐encoded phosphosphingolipid‐specific phospholipase C appears to be involved in stimulation of ENA1 expression and, consequently, in mediating Na+ and Li+ tolerance in yeast. Deletion of ISC1 distinctly decreased cellular Na+ and Li+ tolerance as growth of the Δisc1::HIS5 mutant, DZY1, was severely impaired by 0.5 m NaCl or 0.01 m LiCl. In contrast,K+ tolerance and general osmostress regulation wereunaffected. Isc1Δ mutant growth with 0.9 m KCl and glycerol accumulation in the presence of 0.9 m NaCl or 1.5 m sorbitol were comparable to that of the wild‐type. ENA1‐lacZ reporter studies suggested that the increased salt sensitivity of the isc1Δ mutant is related to a significant reduction of Na+/Li+‐stimulated ENA1 expression. Correspondingly, Ena1p‐dependent extrusion of Na+/Li+ ions was less efficient in the isc1Δ mutant than in wild‐type cells. Itis suggested that ISC1‐dependent hydrolysis of an unidentified yeast inositol phosphosphingolipid represents an early event in one of the salt‐induced signalling pathways of ENA1 transcriptional activation.
Effect of sequence polymorphism and drug resistance on two HIV‐1 Gag processing sites
Authors: Fehér Anita , Weber Irene T. , Bagossi Péter , Boross Péter , Mahalingam Bhuvaneshwari , Louis John M. , Copeland Terry D. , Torshin Ivan Y. , Harrison Robert W. , Tözsér József
Published: March 2003

The HIV‐1 proteinase (PR) has proved to be a good target for antiretroviral therapy of AIDS, and various PR inhibitors are now in clinical use. However, there is a rapid selection of viral variants bearing mutations in the proteinase that are resistant to clinical inhibitors. Drug resistance also involves mutations of the nucleocapsid/p1 and p1/p6 cleavage sites of Gag, both in vitro and in vivo. Cleavages at these sites have been shown to be rate limiting steps for polyprotein processing and viral maturation. Furthermore, these sites show significant sequence polymorphism, which also may have an impact on virion infectivity. We have studied the hydrolysis of oligopeptides representing these cleavage sites with representative mutations found as natural variations or that arise as resistant mutations. Wild‐type and five drug resistant PRs with mutations within or outside the substrate binding site were tested. While the natural variations showed either increased or decreased susceptibility of peptides toward the proteinases, the resistant mutations always had a beneficial effect on catalytic efficiency. Comparison of the specificity changes obtained for the various substrates suggested that the maximization of the van der Waals contacts between substrate and PR is the major determinant of specificity: the same effect is crucial for inhibitor potency. The natural nucleocapsid/p1 and p1/p6 sites do not appear to be optimized for rapid hydrolysis. Hence, mutation of these rate limiting cleavage sites can partly compensate for the reduced catalytic activity of drug resistant mutant HIV‐1 proteinases.
Characterization of phycoviolobilin phycoerythrocyanin‐α84‐cystein‐lyase‐(isomerizing) from Mastigocladus laminosus
Authors: Zhao Kai‐Hong , Wu Dong , Wang Lu , Zhou Ming , Storf Max , Bubenzer Claudia , Strohmann Brigitte , Scheer Hugo
Published: March 2003

Cofactor requirements and enzyme kinetics have been studied of the novel, dual‐action enzyme, the isomerizing phycoviolobilin phycoerythrocyanin‐α84‐cystein‐lyase(PVB‐PEC‐lyase) from Mastigocladus laminosus, which catalyses both the covalent attachment of phycocyanobilin to PecA, the apo‐α‐subunit of phycoerythrocyanin, and its isomerization to phycoviolobilin. Thiols and the divalent metals, Mg2+ or Mn2+, were required, and the reaction was aided by the detergent, Triton X‐100. Phosphate buffer inhibits precipitation of the proteins present in the reconstitution mixture, but at the same time binds the required metal. Kinetic constants were obtained for both substrates, the chromophore (Km = 12–16 µm, depending on [PecA], kcat ≈ 1.2 × 10−4·s−1) and the apoprotein (Km = 2.4 µm at 14 µm PCB, kcat = 0.8 × 10−4·s−1). The kinetic analysis indicated that the reconstitution reaction proceeds by a sequential mechanism. By a combination of untagged and His‐tagged subunits, evidence was obtained for a complex formation between PecE and PecF (subunits of PVB‐PEC‐lyase), and by experiments with single subunits for the prevalent function of PecE in binding and PecF in isomerizing the chromophore.