bluuebunny/crossref_metadata_2025 huggingface dataset:
Evaluation of the de‐selection of men who have had sex with men from blood donation in England
Authors: Soldan K. , Sinka K.
Published: May 2003

Background and Objectives The Blood Services of the UK permanently de‐select men who have had sex with men (MSM) from donating blood. The rationale for this has been questioned. This article attempts to evaluate whether this selection criterion does contribute to blood safety.

Materials and Methods Data about transfusion‐transmissible infections, in particular about human immunodeficiency virus (HIV) infection, were used to evaluate whether de‐selection of MSM meets the aims of donor selection. Models were constructed to estimate the risk of HIV‐infectious donations entering the blood supply should this criterion be changed.

Results Many assumptions were required to generate estimates of the risk of HIV infection entering the blood supply. The accuracy of the estimates is therefore uncertain and the probable ranges around the estimates were wide. However, by using the most probable assumptions, our models suggested that de‐selection of MSM for 12 months since the last sexual contact, or complete removal of this selection criterion, would be expected to increase the risk of HIV‐infectious donations entering the blood supply in England by approximately 60% (from the current risk of 0·45 per year to 0·75 per year) and 500% (to 2·5 per year), respectively. The increase in numbers of non‐infected donations would be relatively small – less than 2% of donations. The probability of a relatively high frequency of other sexually transmissible blood‐borne infections also currently favours maintaining permanent de‐selection of MSM, irrespective of the risk of HIV‐infectious donations. Current compliance with this selection criteria was estimated to be 95%.

Conclusions Based on current knowledge, accepting blood donations from MSM would probably increase the risk of transfusion–transmission of HIV and of other blood‐borne infections. Good compliance with this criterion has contributed greatly to the safety of blood transfusions in England. Better communication about donor selection, to maintain and improve compliance with this and other selection criteria, is recommended. Other risk groups are gaining in relative importance for the risk of transfusion‐transmitted HIV infection, and ongoing evaluation of all donor‐selection criteria is also recommended.
Estimation of the risk of hepatitis B virus, hepatitis C virus and human immunodeficiency virus infectious donations entering the blood supply in England, 1993–2001
Authors: Soldan K. , Barbara J. A. J. , Ramsay M. E. , Hall A. J.
Published: May 2003

Background and Objectives The frequency of hepatitis B virus (HBV), hepatitis C virus (HCV) or human immunodeficiency virus (HIV) infectious donations entering the blood supply in England is too low to monitor using observational studies. The expected frequency of infectious donations can be estimated and these estimates may be used to contribute to monitoring of blood safety and used in the design of strategies to decrease the risk of transfusion‐transmitted infections.

Materials and Methods The prevalence and incidence of hepatitis B surface antigen (HBsAg), and antibodies to HCV and HIV (anti‐HCV and anti‐HIV, respectively) in donors in England, between 1993 and 2001, were used together with data about the length of negative ‘window‐periods’ of current assays for each of these markers and data about test performance, to estimate the number of infectious donations that enter the blood supply. The risks were calculated separately for donations from new donors and from repeat donors, and for the three time periods 1993–95, 1996–98 and 1999–01.

Results The estimated frequency of infectious donations entering the blood supply in England, between 1993 and 2001 was 1 in 260 000 for HBV and 1 in 8 million for HIV. For HCV, the frequency of infectious donations was 1 in 520 000 during 1993–98 and fell to 1 in 30 million during 1999–2001 when all donations were tested for HCV RNA. The frequency of HBV‐ and HCV‐infectious donations entering the blood supply fell over these 9 years: the frequency of HIV‐infectious donations remained essentially unchanged. The risk from donations from new donors was found to be approximately sevenfold higher than the risk from donations from repeat donors.

Conclusions The risks of HBV‐, HCV‐ or HIV‐infectious donations entering the blood supply in England are very low, and have decreased since 1993. Although the accuracy of these estimates is imperfect, mainly owing to uncertainty in some assumptions and to small numbers of infections, they provide some quantification of the risk of HBV, HCV or HIV transmission by transfusion, and allow comparison of the magnitude of these risks for each infection and over time. The methods we have used have been developed and improved from previously published methods.
A novel DI*A allele without the band 3‐Memphis mutation in Amazonian Indians
Authors: Baleotti W. , Rios M. , Reid M. E. , Fabron A. , Pellegrino J. , Saad S. T. O. , Castilho L.
Published: May 2003

Background and Objectives The blood‐group antigens Dia and Dib are carried on erythrocyte band 3 and are defined by a single amino acid substitution at position 854 (Leu for Dia and Pro for Dib). The Band 3‐Memphis variant has a point mutation (166A>G) in the SLC4A1 gene, which encodes the amino acid substitution Lys56Glu. Two types of Band 3‐Memphis, variants I and II, are distinguished by their susceptibility to covalent labelling with 4,4′‐diisothiocyanato‐1,2‐diphenylethane‐2,2′‐disulphonic acid (H2DIDS). Memphis II is more readily labelled than Memphis I or normal band 3. It is reported that Memphis II is associated with Dia. In a study designed to determine the frequency of the DI*A/DI*B and 166A>G polymorphisms in different populations in Brazil, we found a new DI*A allele.

Materials and Methods We studied DNA samples from 70 Amazonian Indians, 71 individuals of Japanese descent, 93 random Brazilian blood donors and 84 blacks with sickle cell disease. Polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) analyses were performed on all samples, using MspI for DI*A/DI*B (exon 19) and MnlI for 166A>G (exon 4). Exon 4 and exon 19 from four outliers were sequenced.

Results Among Amazonian Indians, DI*A and 166G mutations both had a high frequency (0·57 and 0·54, respectively). In individuals of Japanese descent, these alleles were moderately frequent (0·07 and 0·19, respectively). We identified a new allele with DI*A and 166A (56Lys) in four Amazonian Indians.

Conclusions Our results revealed that DI*A does not have a strict association with 166G. They also show the relevance of testing a cohort of different populations.
Prevalence of human erythrovirus B19 DNA in healthy Belgian blood donors and correlation with specific antibodies against structural and non‐structural viral proteins
Authors: Thomas I. , Di Giambattista M. , Gérard C. , Mathys E. , Hougardy V. , Latour B. , Branckaert T. , Laub R.
Published: May 2003

Background and Objectives Human parvovirus (erythrovirus) B19 is recognized as a major contaminant of blood and blood products. To reduce the risk of contamination, plasma‐pool screening and exclusion of highly viraemic donations are recommended. The objectives of this study were to estimate the prevalence of B19 DNA in our blood‐donor population, to determine the appropriate pool size to be tested (taking into account parameters such as prevalence, viral load, test sensitivity, and the efficacy of inactivation procedures), and to correlate viral loads with the serological status of donors as regards antibodies against different viral proteins.

Materials and Methods Pools of different sizes were tested for B19, using a sensitive nested polymerase chain reaction (PCR) as well as an simple, un‐nested, less sensitive PCR. Positive pools were resolved to the level of individual donations, and the viral load and serological markers were determined.

Results Of 16 859 donations, 27 (one of 625) were found to be B19 DNA positive, with viral loads ranging from 102 to > 107 IU/ml. Twenty‐five of the positive donations were tested for VP‐specific anti‐B19 antibodies, and eight (32%) were negative for both immunoglobulin (Ig)M and IgG. They were probably collected in the preseroconversion window period or from chronic carriers without detectable antibodies. We regarded the seven (28%) IgM‐positive donors as being in the early phase of infection. The remaining 10 (40%) IgM‐negative, IgG‐positive donors were probably carriers of persistent infection (i.e. PCR positive despite the presence of IgG antibodies), as suggested by their low viral loads (< 104 IU/ml). Fifteen out of 36 major pools contained one or more contaminated donations. Among these, 12 tested positive by nested PCR and only three by un‐nested PCR, this reflecting a viral load of > 104 IU/ml.

Conclusions By testing all donations as pools of 480 by un‐nested PCR, and resolving positive pools to identify the responsible donations, it is possible to ensure that the viral load in fractionation pools (5000 donations) remains < 103 IU/ml, compatible with the efficacy of inactivation procedures and complying with Food and Drug Administration (FDA) recommendations.
High frequency of hepatitis B virus infection in patients with β‐thalassemia receiving multiple transfusions
Authors: Singh H. , Pradhan M. , Singh R. L. , Phadke S. , Naik S. R. , Aggarwal R. , Naik S.
Published: May 2003

Background and Objectives Hepatitis B virus (HBV) may occasionally be transmitted through transfusion of blood units that are hepatitis B surface antigen (HBsAg) negative but HBV DNA positive. Children with β‐thalassemia are particularly susceptible to HBV because they receive multiple blood transfusions. These children have high infection rates despite vaccination against HBV. Post‐vaccination infections may be a result of viruses harbouring surface (S)‐gene mutations (e.g. G587A) in a region critical for reactivity to antibody to hepatitis B surface antigen (anti‐HBs). The true prevalence of HBV in individuals with β‐thalassemia has not been studied previously.

Patients and Methods Seventy patients with β‐thalassemia (median age 6 years; range 8 months to 22 years; 49 male), who had received seven to 623 (median 61) units of blood each and three doses (10/20 µg) of HBV vaccine (Engerix B) before presentation to us, were included in the study; 50 of the 70 patients had received transfusions prior to vaccination. Enzyme‐linked immunoassay for serological markers [HBsAg, antibody to hepatitis B core antigen (anti‐HBc) and quantitative anti‐HBs] and polymerase chain reaction (PCR) followed by Southern hybridization for molecular detection of hepatitis B, was performed on all samples. The PCR‐amplified product was cloned, sequenced and the nucleotide and deduced amino acid sequences for the HBV S and polymerase (P) genes were analysed for mutations.

Results Four of 70 (5·7%) individuals with β‐thalassemia were HBsAg positive and 14 (20%) were anti‐HBc positive. The prevalence of serological markers increased with number of transfusions (P < 0·01). Of 70 patients, 53 (75·7%) had an anti‐HBs titre of > 10 IU/l following vaccination and 17 (24·3%) were non‐responders (< 10 IU/l); 22 (31·4%) of the 70 were DNA positive. The frequency of HBV infection in β‐thalassemia was similar in vaccine responders and non‐responders. The virus was of subtype ayw (genotype D) in the five DNA‐positive samples in which a 388‐nucleotide region of the S gene was sequenced. Mutations occurred at 13 positions in the S gene and at 10 positions in the P gene. Hydrophobicity plots revealed differences in amino acid regions 117–165 and 195–211. Some of these amino acid substitutions coincided with the putative cytotoxic T‐lymphocyte epitopes of both S and P proteins.

Conclusions A high frequency of HBV infection was seen using molecular methods in thalassemic patients. The frequency of infection was similar in vaccine responders and non‐responders. A number of mutations were observed in the S gene, which could have implications for viral replication as well as virus–host cell interaction.
Will it ever be possible to balance the risk of intracranial haemorrhage in fetal or neonatal alloimmune thrombocytopenia against the risk of treatment strategies to prevent it?
Authors: Radder C. M. , Brand A. , Kanhai H. H. H.
Published: May 2003

Background and Objectives Intracranial haemorrhage (ICH) of the fetus or newborn is a severe complication of fetal or neonatal alloimmune thrombocytopenia (FNAIT). In order to attain management decisions to prevent ICH, the risk of ICH in successive pregnancies with thrombocytopenia, with or without a history of ICH, must be established.

Materials and Methods We performed a search of medline for ICH cases in untreated FNAIT pregnancies. After exclusion of cases with confounding factors, 24 reports, describing 62 pregnancies of 27 mothers, were eligible. In addition, two mothers with five pregnancies were included from our own case records. Observational studies were examined to estimate the risk of ICH in subsequent FNAIT pregnancies without a history of ICH. Finally, medline was searched for complication rates in the treatment of FNAIT pregnancies.

Results In 52% of the ICH cases, a previous sibling suffered from ICH. The recurrence rate of ICH in the subsequent offspring of women with a history of FNAIT with ICH was 72%[confidence interval (CI): 46–98%] without inclusion of fetal deaths and 79% (CI: 61–97%) with inclusion of fetal deaths. In 48% of the ICH cases, the previous sibling had thrombocytopenia but not ICH. Population studies revealed an overall ICH risk in thrombocytopenic infants of 11% (CI: 0·8–23%) without inclusion of fetal deaths and 15% (CI: 1·5–19%) with inclusion of fetal deaths. Assuming occurrence in 48%, the risk of ICH in a subsequent pregnancy following a history of FNAIT without ICH, was estimated to be 7% (CI: 0·5–13%). Invasive treatment strategies carry a risk of 2·8% (CI: 1·2–4·4%) on complications.

Conclusions The number of eligible publications on ICH in untreated FNAIT pregnancies is strikingly limited. The recurrence rate is high. As sufficient data on successive FNAIT cases without ICH are lacking, the occurrence of ICH in pregnancies with thrombocytopenia, but without ICH in a previous sibling, cannot be predicted. We estimate this risk to be 7%. This risk must be balanced against the risk of interventions in treatment strategies.
In vivo and in vitro comparison of platelets stored in either synthetic media or plasma
Authors: De Wildt‐Eggen J. , Gulliksson H.
Published: May 2003

Since 1980, several synthetic media have been developed for the storage of platelets for transfusion. At present, platelets suspended in approximately 70% synthetic medium and 30% plasma can be stored for at least 5 days at very stable pH levels, generally pH 6·8–7·2. Present knowledge suggests that synthetic media should contain at least acetate, citrate, phosphate, potassium and magnesium. Future studies will probably result in the inclusion of other components to this list. Glucose for platelet metabolism will generally be supplied by carryover of plasma from the original platelet preparation. In addition, improved plastic containers for the storage of platelets will probably facilitate the introduction of new synthetic media. In six studies comparing synthetic media with plasma as the storage environment, and involving patients with intensive chemotherapy for haematological malignancies, the clinical outcome in terms of corrected count increments (CCI) generally indicated similar results. Three studies suggested significant reduction of the incidence of transfusion reactions of platelets suspended in synthetic media as compared with plasma. For future comparisons of platelet storage in either plasma or new synthetic media, additional platelet survival and recovery studies, as well as patient‐transfusion studies, will be needed as in vitro data may not always reflect the clinical outcome. This will add further knowledge to data from the present few clinical studies available that compare storage of platelets in either synthetic media or plasma.
Near‐patient testing of haemostasis in the operating theatre: an approach to appropriate use of blood in surgery
Authors: Samama C. M. , Ozier Y.
Published: May 2003

Several haemostasis point‐of‐care (POC) monitors are now available in the operating theatre. Two of these are widely used; the Coaguchek® and the thromboelastograph (TEG), but they have been developed in very different ways. Bedside‐activated partial thromboplastin time and prothrombin time performed with the Coaguchek monitor seem to be reliable and have been used to build algorithms for transfusion decision making. They have been developed in close collaboration with haemostasis groups and therefore gain a benefit from these links. Conversely, TEG provides very important information except it has never been validated. The number of collaborative studies with biologists has to increase in order to implement the use of TEG in the routine practice.
Detection and characterization of a novel extracellular fungal enzyme that catalyzes the specific and hydrolytic cleavage of lignin guaiacylglycerol β‐aryl ether linkages
Authors: Otsuka Yuichiro , Sonoki Tomonori , Ikeda Seiichiro , Kajita Shinya , Nakamura Masaya , Katayama Yoshihiro
Published: May 2003

Cleavage of the arylglycerol β‐aryl ether linkage is the most important process in the biological degradation of lignin. The bacterial β‐etherase was described previously and shown to be tightly associated with the cellular membrane. In this study, we aimed to detect and isolate a new extracellular function that catalyses the β‐aryl ether linkage cleavage of high‐molecular lignin in the soil fungi. We screened and isolated 2BW‐1 cells by using a highly sensitive fluorescence assay system. The β‐aryl ether cleavage enzyme was produced by a newly isolated fungus, 2BW‐1, and is secreted into the extracellular fraction. The β‐aryl ether cleavage enzyme converts the guaiacylglycerol β‐O‐guaiacyl ether (GOG) to guaiacylglycerol and guaiacol. It requires the Cα alcohol structure and p‐hydroxyl group and specifically attacks the β‐aryl ether linkage of high‐molecular mass lignins with addition of two water molecules at the Cα and Cβ positions.
Thyroid Ca2+/NADPH‐dependent H2O2 generation is partially inhibited by propylthiouracil and methimazole
Authors: Freitas Ferreira Andrea C. , de Carvalho Cardoso Luciene , Rosenthal Doris , Pires de Carvalho Denise
Published: May 2003

H2O2 generation is a limiting step in thyroid hormone biosynthesis. Biochemical studies have confirmed that H2O2 is generated by a thyroid Ca2+/NADPH‐dependent oxidase. Decreased H2O2 availability may be another mechanism of inhibition of thyroperoxidase activity produced by thioureylene compounds, as propylthiouracil (PTU) and methimazole (MMI) are antioxidant agents. Therefore, we analyzed whether PTU or MMI could scavenge H2O2 or inhibit thyroid NADPH oxidase activity in vitro. Our results show that PTU and thiourea did not significantly scavenge H2O2. However, MMI significantly scavenged H2O2 at high concentrations. Only MMI was able to decrease the amount of H2O2 generated by the glucose–glucose oxidase system. On the other hand, both PTU and MMI were able to partially inhibit thyroid NADPH oxidase activity in vitro. As PTU did not scavenge H2O2 under the conditions used here, we presume that this drug may directly inhibit thyroid NADPH oxidase. Also, at the concentration necessary to inhibit NADPH oxidase activity, MMI did not scavenge H2O2, also suggesting a direct effect of MMI on thyroid NADPH oxidase. In conclusion, this study shows that MMI, but not PTU, is able to scavenge H2O2 in the micromolar range and that both PTU and MMI can impair thyroid H2O2 generation in addition to their potent thyroperoxidase inhibitory effects.
Investigation of the kinetics and order of tyrosine phosphorylation in the T‐cell receptor ζ chain by the protein tyrosine kinase Lck
Authors: Housden Hazel R. , Skipp Paul J. S. , Crump Matthew P. , Broadbridge Robert J. , Crabbe Tom , Perry Martin J. , Gore Michael G.
Published: May 2003

We report experiments to investigate the role of the physiologically relevant protein tyrosine kinase Lck in the ordered phosphorylation of the T‐cell receptor ζ chain. Six synthetic peptides were designed based on the sequences of the immunoreceptor tyrosine‐based activation motifs (ITAMs) of the ζ chain. Preliminary 1H‐NMR studies of recombinant ζ chain suggested that it is essentially unstructured and therefore that peptide mimics would serve as useful models for investigating individual ITAM tyrosines. Phosphorylation kinetics were determined for each tyrosine by assaying the transfer of 32P by recombinant Lck on to each of the peptides. The rates of phosphorylation were found to depend on the location of the tyrosine, leading to the proposal that Lck phosphorylates the six ζ chain ITAM tyrosines in the order 1N (first) > 3N > 3C > 2N > 1C > 2C (last) as a result of differences in the amino‐acid sequence surrounding each tyrosine. This proposal was then tested on cytosolic, recombinant T‐cell receptor ζ chain. After in vitro phosphorylation by Lck, the partially phosphorylated ζ chain was digested with trypsin. Separation and identification of the ζ chain fragments using LC–MS showed, as predicted by the peptide phosphorylation studies, that tyrosine 1N is indeed the first to be phosphorylated by Lck. We conclude that differences in the amino‐acid context of the six ζ chain ITAM tyrosines affect the efficiency of their phosphorylation by the kinase Lck, which probably contributes to the distinct patterns of phosphorylation observed in vivo.
Selectivity of pyruvate kinase for Na+ and K+ in water/dimethylsulfoxide mixtures
Authors: Ramírez‐Silva Leticia , Oria‐Hernández Jesús
Published: May 2003

In aqueous media, muscle pyruvate kinase is highly selective for K+ over Na+. We now studied the selectivity of pyruvate kinase in water/dimethylsulfoxide mixtures by measuring the activation and inhibition constants of K+ and Na+, i.e. their binding to the monovalent and divalent cation binding sites of pyruvate kinase, respectively [Melchoir J.B. (1965) Biochemistry4, 1518–1525]. In 40% dimethylsulfoxide the K0.5 app for K+ and Na+ were 190 and 64‐fold lower than in water. Ki app for K+ and Na+ decreased 116 and 135‐fold between 20 and 40% dimethylsulfoxide. The ratios of Ki app/K0.5 app for K+ and Na+ were 34–3.5 and 3.3–0.2, respectively. Therefore, dimethylsulfoxide favored the partition of K+ and Na+ into the monovalent and divalent cation binding sites of the enzyme. The kinetics of the enzyme at subsaturating concentrations of activators show that K+ and Mg2+ exhibit high selectivity for their respective cation binding sites, whereas when Na+ substitutes K+, Na+ and Mg2+ bind with high affinity to their incorrect sites. This is evident by the ratio of the affinities of Mg2+ and K+ for the monovalent cation binding site, which is close to 200. For Na+ and Mg2+ this ratio is approximately 20. Therefore, the data suggest that K+ induces conformational changes that prevent the binding of Mg2+ to the monovalent cation binding site. Circular dichroism spectra of the enzyme and the magnitude of the transfer and apparent binding energies of K+ and Na+ indicate that structural arrangements of the enzyme induced by dimethylsulfoxide determine the affinities of pyruvate kinase for K+ and Na+.
Tumor necrosis factor‐α converting enzyme is processed by proprotein‐convertases to its mature form which is degraded upon phorbol ester stimulation
Authors: Endres Kristina , Anders Andreas , Kojro Elzbieta , Gilbert Sandra , Fahrenholz Falk , Postina Rolf
Published: May 2003

Tumor necrosis factor‐α converting enzyme (TACE or ADAM17) is a member of the ADAM (a disintegrin and metalloproteinase) family of type I membrane proteins and mediates the ectodomain shedding of various membrane‐anchored signaling and adhesion proteins. TACE is synthesized as an inactive zymogen, which is subsequently proteolytically processed to the catalytically active form. We have identified the proprotein‐convertases PC7 and furin to be involved in maturation of TACE. This maturation is negatively influenced by the phorbol ester phorbol‐12‐myristate‐13‐acetate (PMA), which decreases the cellular amount of the mature form of TACE in PMA‐treated HEK293 and SH‐SY5Y cells. Furthermore, we found that stimulation of protein kinase C or protein kinase A signaling pathways did not influence long‐term degradation of mature TACE. Interestingly, PMA treatment of furin‐deficient LoVo cells did not affect the degradation of mature TACE. By examination of furin reconstituted LoVo cells we were able to exclude the possibility that PMA modulates furin activity. Moreover, the PMA dependent decrease of the mature enzyme form is specific for TACE, as the amount of mature ADAM10 was unaffected in PMA‐treated HEK293 and SH‐SY5Y cells. Our results indicate that the activation of TACE by the proprotein‐convertases PC7 and furin is very similar to the maturation of ADAM10 although there is a significant difference in the cellular stability of the mature enzyme forms after phorbol ester treatment.
Selective inhibition of ADAMTS‐1, ‐4 and ‐5 by catechin gallate esters
Authors: Vankemmelbeke Mireille N. , Jones Gavin C. , Fowles Cyprianne , Ilic Mirna Z. , Handley Christopher J. , Day Anthony J. , Knight C. Graham , Mort John S. , Buttle David J.
Published: May 2003

Three mammalian ADAMTS enzymes, ADAMTS‐1, ‐4 and ‐5, are known to cleave aggrecan at certain glutamyl bonds and are considered to be largely responsible for cartilage aggrecan catabolism observed during the development of arthritis. We have previously reported that certain catechins, polyphenolic compounds found in highest concentration in green tea (Camellia sinensis), are capable of inhibiting cartilage aggrecan breakdown in an in vitro model of cartilage degradation. We have now cloned and expressed recombinant human ADAMTS‐1, ‐4 and ‐5 and report here that the catechin gallate esters found in green tea potently inhibit the aggrecan‐degrading activity of these enzymes, with submicromolar IC50 values. Moreover, the concentration needed for total inhibition of these members of the ADAMTS group is approximately two orders of magnitude lower than that which is needed to partially inhibit collagenase or ADAM‐10 activity. Catechin gallate esters therefore provide selective inhibition of certain members of the ADAMTS group of enzymes and could constitute an important nutritional aid in the prevention of arthritis as well as being part of an effective therapy in the treatment of joint disease and other pathologies involving the action of these enzymes.
Sulfation of hydroxychlorobiphenyls
Authors: Sugahara Takuya , Liu Chau‐Ching , Pai T. Govind , Collodi Paul , Suiko Masahito , Sakakibara Yoichi , Nishiyama Kazuo , Liu Ming‐Cheh
Published: May 2003

As a first step toward developing a zebrafish model for investigating the role of sulfation in counteracting environmental estrogenic chemicals, we have embarked on the identification and characterization of cytosolic sulfotransferases (STs) in zebrafish. By searching the zebrafish expressed sequence tag database, we have identified two cDNA clones encoding putative cytosolic STs. These two zebrafish ST cDNAs were isolated and subjected to nucleotide sequencing. Sequence data revealed that the two zebrafish STs are highly homologous, being ≈ 82% identical in their amino acid sequences. Both of them display ≈ 50% amino acid sequence identity to human SULT1A1, rat SULT1A1, and mouse SULT1C1 ST. These two zebrafish STs therefore appear to belong to the SULT1 cytosolic ST gene family. Recombinant zebrafish STs (designated SULT1 STs 1 and 2), expressed using the pGEX‐2TK prokaryotic expression system and purified from transformed Escherichia coli cells, migrated as ≈ 35 kDa proteins on SDS/PAGE. Purified zebrafish SULT1 STs 1 and 2 displayed differential sulfating activities toward a number of endogenous compounds and xenobiotics including hydroxychlorobiphenyls. Kinetic constants of the two enzymes toward two representative hydroxychlorobiphenyls, 3‐chloro‐4‐biphenylol and 3,3′,5,5′‐tetrachloro‐4,4′‐biphenyldiol, and 3,3′,5‐triiodo‐l‐thyronine were determined. A thermostability experiment revealed the two enzymes to be relatively stable over the range 20–43 °C. Among 10 different divalent metal cations tested, Co2+, Zn2+, Cd2+, and Pb2+ exhibited considerable inhibitory effects, while Hg2+ and Cu2+ rendered both enzymes virtually inactive.
Differential post‐translational modification of CD63 molecules during maturation of human dendritic cells
Authors: Engering Anneke , Kuhn Lotte , Fluitsma Donna , Hoefsmit Elisabeth , Pieters Jean
Published: May 2003

The capacity of dendritic cells to initiate T cell responses is related to their ability to redistribute MHC class II molecules from the intracellular MHC class II compartments to the cell surface. This redistribution occurs during dendritic cell development as they are converted from an antigen capturing, immature dendritic cell into an MHC class II‐peptide presenting mature dendritic cell. During this maturation, antigen uptake and processing are down‐regulated and peptide‐loaded class II complexes become expressed in a stable manner on the cell surface. Here we report that the tetraspanin CD63, that associates with intracellularly localized MHC class II molecules in immature dendritic cells, was modified post‐translationally by poly N‐acetyl lactosamine addition during maturation. This modification of CD63 was accompanied by a change in morphology of MHC class II compartments from typical multivesicular organelles to structures containing densely packed lipid moieties. Post‐translational modification of CD63 may be involved in the functional and morphological changes of MHC class II compartments that occur during dendritic cell maturation.
Comparative analysis of the ATP‐binding sites of Hsp90 by nucleotide affinity cleavage: a distinct nucleotide specificity of the C‐terminal ATP‐binding site
Authors: So˝ti Csaba , Vermes Ákos , Haystead Timothy A. J. , Csermely Péter
Published: May 2003

The 90‐kDa heat shock protein (Hsp90) is a molecular chaperone that assists both in ATP‐independent sequestration of damaged proteins, and in ATP‐dependent folding of numerous targets, such as nuclear hormone receptors and protein kinases. Recent work from our lab and others has established the existence of a second, C‐terminal nucleotide binding site besides the well characterized N‐terminal, geldanamycin‐sensitive ATP‐binding site. The cryptic C‐terminal site becomes open only after the occupancy of the N‐terminal site. Our present work demonstrates the applicability of the oxidative nucleotide affinity cleavage in the site‐specific characterization of nucleotide binding proteins. We performed a systematic analysis of the nucleotide binding specificity of the Hsp90 nucleotide binding sites. N‐terminal binding is specific to adenosine nucleotides with an intact adenine ring. Nicotinamide adenine dinucleotides and diadenosine polyphosphate alarmones are specific N‐terminal nucleotides. The C‐terminal binding site is much more unspecific—it interacts with both purine and pirimidine nucleotides. Efficient binding to the C‐terminal site requires both charged residues and a larger hydrophobic moiety. GTP and UTP are specific C‐terminal nucleotides. 2′,3′‐O‐(2,4,6‐trinitrophenyl)‐nucleotides (TNP‐ATP, TNP‐GTP) and pyrophosphate access the C‐terminal binding site without the need for an occupied N‐terminal site. Our data provide additional evidence for the dynamic domain–domain interactions of Hsp90, give hints for the design of novel types of specific Hsp90 inhibitors, and raise the possibility that besides ATP, other small molecules might also interact with the C‐terminal nucleotide binding site in vivo.
Characterization of a high‐affinity binding site for the pea albumin 1b entomotoxin in the weevil Sitophilus
Authors: Gressent Frédéric , Rahioui Isabelle , Rahbé Yvan
Published: May 2003

The toxicity of the pea albumin 1b (PA1b), a 37 amino‐acid peptide extracted from pea seeds, for cereal weevils (Sitophilus oryzae, Sitophilus granarius and Sitophilus zeamais) was recently discovered. The mechanism of action of this new entomotoxin is still unknown and potentially involves a target protein in the insect tissues. This work describes the characterization of a high‐affinity binding site for PA1b in a microsomal fraction of Sitophilus spp. extracts. Purified PA1b was labeled to a high specific radioactivity (c. 900 Ci·mmol−1) using 125I, and the iodinated ligand was found to be biologically active. Binding of this ligand to the microsomal fraction of S. oryzae extract was found to be saturable and reversible, with an affinity (Kd) of 2.6 nm, and a high maximal binding capacity (Bmax) of 40 pmol·mg−1 of protein. A binding site displaying similar characteristics was detectable in the five susceptible weevils strains tested, as well as in the pea aphid or in the fruit fly. However, no binding activity was detectable in extracts from four S. oryzae strains previously shown to be resistant to the toxin through a recessive monogenic mechanism. Therefore, we suggest that this binding site might be involved in the mechanism of action of PA1b.
A novel retinol‐binding protein in the retina of the swallowtail butterfly, Papilio xuthus
Authors: Wakakuwa Motohiro , Arikawa Kentaro , Ozaki Koichi
Published: May 2003

Retinoid‐binding proteins are indispensable for visual cycles in both vertebrate and invertebrate retinas. These proteins stabilize and transport hydrophobic retinoids in the hydrophilic environment of plasma and cytoplasm, and allow regeneration of visual pigments. Here, we identified a novel retinol‐binding protein in the eye of a butterfly, Papilio xuthus. The protein that we term Papilio retinol‐binding protein (Papilio RBP) is a major component of retinal soluble proteins and exclusively binds 3‐hydroxyretinol, and emits fluorescence peaking at 480 nm under ultraviolet (UV) illumination. The primary structure, deduced from the nucleotide sequence of the cDNA, shows no similarity to any other lipophilic ligand‐binding proteins. The molecular mass and isoelectric point of the protein estimated from the amino‐acid sequence are 26.4 kDa and 4.92, respectively. The absence of any signal sequence for secretion in the N‐terminus suggests that the protein exists in the cytoplasmic matrix. All‐trans 3‐hydroxyretinol is the major ligand of the Papilio RBP in dark‐adapted eyes. Light illumination of the eyes increases the 11‐cis isomer of the ligand and induces redistribution of the Papilio RBP from the proximal to the distal part of the photoreceptor layer. These results suggest that the Papilio RBP is involved in visual pigment turnover.
Investigating signal transduction with genetically encoded fluorescent probes
Authors: Pozzan Tullio , Mongillo Marco , Rudolf Rüdiger
Published: May 2003

Ca2+ and cAMP are ubiquitous second messengers in eukaryotes and control numerous physiological responses ranging from fertilization to cell death induction. To distinguish between these different responses, their subtle regulation in time, space and amplitude is needed. Therefore, the characterization of the signalling process requires measurement of second messengers with tools of precise localization, high dynamic range and as little disturbance of cell physiology as possible. Recently, fluorescent proteins of marine jellyfish have given rise to a set of genetically encoded biosensors which fulfil these criteria and which have already led to important new insights into the subcellular handling of Ca2+ and cAMP. The use of these probes in combination with new microscopical methods such as two‐photon microscopy now enables researchers to study second messenger signalling in intact tissues. In this review, the genetically encoded measurement probes and their origin are briefly introduced and some recent insights into the spatio‐temporal complexity of both Ca2+ and cAMP signalling obtained with these tools are discussed.
Reversed‐phase HPLC determination of chlorophyll a′ and phylloquinone in Photosystem I of oxygenic photosynthetic organisms
Authors: Nakamura Akimasa , Akai Masahiko , Yoshida Emi , Taki Takashi , Watanabe Tadashi
Published: May 2003

Chlorophyll (Chl) a′, the C132‐epimer of Chl a, is a constituent of the primary electron donor (P700) of Photosystem (PS) I of a thermophilic cyanobacterium Synechococcus (Thermosynechococcus) elongatus, as was recently demonstrated by X‐ray crystallography. To determine whether PS I of oxygenic photosynthetic organisms universally contains one molecule of Chl a′, pigment compositions of thylakoid membranes and PS I complexes isolated from the cyanobacteria T. elongatus and Synechocystis sp. PCC 6803, the green alga Chlamydomonas reinhardtii, and the green plant spinach, were examined by simultaneous detection of phylloquinone (the secondary electron acceptor of PS I) and Chl a′ by reversed‐phase HPLC. The results were compared with the Chl a/P700 ratio determined spectrophotometrically. The Chl a′/PS I ratios of thylakoid membranes and PS I were about 1 for all the organisms examined, and one Chl a′ molecule was found in PS I even after most of the peripheral subunits were removed. Chl a′ showed a characteristic extraction behaviour significantly different from the bulk Chl a in acetone/methanol extraction upon varying the mixing ratio. These findings confirm that a single Chl a′ molecule in P700 is the universal feature of PS I of the Chl a‐based oxygenic photosynthetic organisms.
Emerin interacts in vitro with the splicing‐associated factor, YT521‐B
Authors: Wilkinson Fiona L. , Holaska James M. , Zhang Zhayi , Sharma Aarti , Manilal Sushila , Holt Ian , Stamm Stefan , Wilson Katherine L. , Morris Glenn E.
Published: May 2003

Emerin is a nuclear membrane protein that interacts with lamin A/C at the nuclear envelope. Mutations in either emerin or lamin A/C cause Emery–Dreifuss muscular dystrophy (EDMD). The functions of emerin are poorly understood, but EDMD affects mainly skeletal and cardiac muscle. We used a high‐stringency yeast two‐hybrid method to screen a human heart cDNA library, with full‐length emerin as bait. Four out of five candidate interactors identified were nuclear proteins: lamin A, splicing factor YT521‐B, proteasome subunit PA28γ and transcription factor vav‐1. Specific binding between emerin and the functional C‐terminal domain of YT521‐B was confirmed by pull‐down assays and biomolecular interaction analysis (BIAcore). Inhibition by emerin of YT521‐B‐dependent splice site selection in vivo suggests that the interaction is physiologically significant. A ‘bipartite’ binding site for YT521‐B in emerin was identified using alanine substitution or disease‐associated mutations in emerin. The transcription factor GCL (germ cell‐less) has previously been shown to bind to the same site. The results are consistent with an emerging view that lamins and lamina‐associated proteins, like emerin, have a regulatory role, as well as a structural role in the nucleus. YT521‐B joins a growing list of candidates for a role in a gene expression model of the pathogenesis of EDMD.
The Hansenula polymorpha MOX gene presents two alternative transcription start points differentially utilized and sensitive to respiratory activity
Authors: Genu Victor , Gödecke Stefanie , Hollenberg Cornelis P. , Pereira Gonçalo G.
Published: May 2003

The peroxisomal methanol metabolism of Hansenula polymorpha depends on a group of genes that are coordinately regulated. Methanol oxidase (Mox) plays a key role in this pathway and its synthesis has been shown to be regulated at the transcriptional level. MOX expression is strongly repressed on glucose and activated on glycerol or methanol. In this study we have identified two MOX transcripts that are differentially expressed along MOX derepression. The first one, named l‐MOX (for longer MOX), starts at position −425, is only weakly and transiently transcribed and is not translated into the Mox protein. The other is the true MOX mRNA, which initiates around position −25. Using a strain bearing multiple copies of MOX(Q1N) and a reporter gene fused to the MOX promoter, regulation of the two transcripts was investigated. Initiation of the true MOX correlates with repression of l‐MOX and conditions that are repressive for MOX transcription, such as the inhibition of mitochondrial activity, lead to higher levels of l‐MOX expression. This effect was first observed in a mox mutant (Q1N‐M8) unable to grow on nonfermentable carbon sources. No function was detected for l‐MOX, but its regulation follows a pattern similar to that of catalase, which is essential for methanol metabolism. This suggests that, l‐MOX, although precisely regulated, seems to be a remnant of the evolution of the methanol metabolism network.
Two W‐containing formate dehydrogenases (CO2‐reductases) involved in syntrophic propionate oxidation by Syntrophobacter fumaroxidans
Authors: de Bok Frank A. M. , Hagedoorn Peter‐Leon , Silva Pedro J. , Hagen Wilfred R. , Schiltz Emile , Fritsche Kathrin , Stams Alfons J. M.
Published: May 2003

Two formate dehydrogenases (CO2‐reductases) (FDH‐1 and FDH‐2) were isolated from the syntrophic propionate‐oxidizing bacterium Syntrophobacter fumaroxidans. Both enzymes were produced in axenic fumarate‐grown cells as well as in cells which were grown syntrophically on propionate with Methanospirillum hungatei as the H2 and formate scavenger. The purified enzymes exhibited extremely high formate‐oxidation and CO2‐reduction rates, and low Km values for formate. For the enzyme designated FDH‐1, a specific formate oxidation rate of 700 U·mg−1 and a Km for formate of 0.04 mm were measured when benzyl viologen was used as an artificial electron acceptor. The enzyme designated FDH‐2 oxidized formate with a specific activity of 2700 U·mg−1 and a Km of 0.01 mm for formate with benzyl viologen as electron acceptor. The specific CO2‐reduction (to formate) rates measured for FDH‐1 and FDH‐2, using dithionite‐reduced methyl viologen as the electron donor, were 900 U·mg−1 and 89 U·mg−1, respectively. From gel filtration and polyacrylamide gel electrophoresis it was concluded that FDH‐1 is composed of three subunits (89 ± 3, 56 ± 2 and 19 ± 1 kDa) and has a native molecular mass of approximately 350 kDa. FDH‐2 appeared to be a heterodimer composed of a 92 ± 3 kDa and a 33 ± 2 kDa subunit. Both enzymes contained tungsten and selenium, while molybdenum was not detected. EPR spectroscopy suggested that FDH‐1 contains at least four [2Fe‐2S] clusters per molecule and additionally paramagnetically coupled [4Fe‐4S] clusters. FDH‐2 contains at least two [4Fe‐4S] clusters per molecule. As both enzymes are produced under all growth conditions tested, but with differences in levels, expression may depend on unknown parameters.
Ycf1p‐dependent Hg(II) detoxification in Saccharomyces cerevisiae
Authors: Gueldry Olivier , Lazard Myriam , Delort Florence , Dauplais Marc , Grigoras Ioana , Blanquet Sylvain , Plateau Pierre
Published: May 2003

In Saccharomyces cerevisiae, disruption of the YCF1 gene increases the sensitivity of cell growth to mercury. Transformation of the resulting ycf1 null mutant with a plasmid harbouring YCF1 under the control of the GAL promoter largely restores the wild‐type resistance to the metal ion. The protective effect of Ycf1p against the toxicity of mercury is especially pronounced when yeast cells are grown in rich medium or in minimal medium supplemented with glutathione. Secretory vesicles from S. cerevisiae cells overproducing Ycf1p are shown to exhibit ATP‐dependent transport of bis(glutathionato)mercury. Moreover, using β‐galactosidase as a reporter protein, a relationship between mercury addition and the activity of the YCF1 promoter can be shown. Altogether, these observations indicate a defence mechanism involving an induction of the expression of Ycf1p and transport by this protein of mercury–glutathione adducts into the vacuole. Finally, possible coparticipation in mercury tolerance of other ABC proteins sharing close homology with Ycf1p was investigated. Gene disruption experiments enable us to conclude that neither Bpt1p, Yor1p, Ybt1p nor YHL035p plays a major role in the detoxification of mercury.
Trehalose influence on beta‐lactoglobulin stability and hydration by time resolved fluorescence
Authors: D'Alfonso Laura , Collini Maddalena , Baldini Giancarlo
Published: May 2003

The stabilizing role of the disaccharide trehalose on beta‐lactoglobulin (BLG) against its chemical denaturation both at native and acidic pH has been explored by means of time‐resolved fluorescence of the probe acrylodan covalently bound to the unique free cysteine of BLG. The changes in acrylodan fluorescence lifetime with guanidinium chloride concentration reveal BLG sigmoidal denaturation profiles which depend upon the amount of trehalose in solution. When adding trehalose the transition midpoint shifts towards higher denaturant concentration. This effect has been measured by fitting the data with a two‐state model whose parameters indicate that an almost 60% increase in the denaturation free energy is induced independently of trehalose concentrations and pH values. Fluorescence anisotropy measurements performed in the same conditions reveal that the internal dynamics are largely affected by the sugar, which makes the acrylodan environment more rigid, and by the denaturant that acts in the opposite way. The overall rotational diffusion of BLG suggests that trehalose affects the hydrodynamic properties of the solution in the proximity of the protein; tentative mechanisms are discussed.
NMR solution structure of calerythrin, an EF‐hand calcium‐binding protein fromSaccharopolyspora erythraea
Authors: Tossavainen Helena , Permi Perttu , Annila Arto , Kilpeläinen Ilkka , Drakenberg Torbjörn
Published: May 2003

The structure of calerythrin, a prokaryotic 20 kDa calcium‐binding protein has been determined by solution NMR spectroscopy. Distance, dihedral angle,Jcoupling, secondary chemical shift, residual dipolar coupling and radius of gyration restraints reveal four EF‐hand motifs arranged in a compact globular structure. A tight turn in the middle of the amino acid sequence brings the two halves, each comprising a pair of EF‐hands, close together. The structural similarity between calerythrin and the eukaryotic sarcoplasmic calcium‐binding proteins is notable.
A novel type of family 19 chitinase from Aeromonas sp. No.10S‐24
Authors: Ueda Mitsuhiro , Kojima Miki , Yoshikawa Tomoye , Mitsuda Norio , Araki Keiichi , Kawaguchi Takashi , Miyatake Kazutaka , Arai Motoo , Fukamizo Tamo
Published: May 2003

A family 19 chitinase gene from Aeromonas sp. No.10S‐24 was cloned, sequenced, and expressed in Escherichia coli. From the deduced amino acid sequence, the enzyme was found to possess two repeated N‐terminal chitin‐binding domains, which are separated by two proline‐threonine rich linkers. The calculated molecular mass was 70 391 Da. The catalytic domain is homologous to those of plant family 19 chitinases by about 47%. The enzyme produced α‐anomer by hydrolyzing β‐1,4‐glycosidic linkage of the substrate, indicating that the enzyme catalyzes the hydrolysis through an inverting mechanism. When N‐acetylglucosamine hexasaccharide [(GlcNAc)6] was hydrolyzed by the chitinase, the second glycosidic linkage from the nonreducing end was predominantly split producing (GlcNAc)2 and (GlcNAc)4. The evidence from this work suggested that the subsite structure of the enzyme was (−2)(−1)(+1)(+2)(+3)(+4), whereas most of plant family 19 chitinases have a subsite structure (−3)(−2)(−1)(+1)(+2)(+3). Thus, the Aeromonas enzyme was found to be a novel type of family 19 chitinase in its structural and functional properties.