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J Saldanha

Publications and source records attributed to J Saldanha.

At least 19 recordsLinked to original sources
PubMed2001

The conformational switch in muscarinic acetylcholine receptors.

The recently-determined structure of rhodopsin has provided a suitable basis for modeling the three-dimensional structure of the M1 muscarinic acetylcholine receptor. Using this as a framework for interpreting mutagenesis studies, we have been able to suggest most of the contacts which the receptor makes with acetylcholine and many of the intramolecular contacts which are important for the ground-state structure of the receptor. It is possible to outline a mechanism of G-protein interaction.

Animals↗
PubMed2001

Multiple allosteric sites on muscarinic receptors.

Proteins and small molecules are capable of regulating the agonist binding and function of G-protein coupled receptors by multiple allosteric mechanisms. In the case of muscarinic receptors, there is the well-characterised allosteric site that binds, for example, gallamine and brucine. The protein kinase inhibitor, KT5720, has now been shown to bind to a second allosteric site and to regulate agonist and antagonist binding. The binding of brucine and gallamine does not affect KT5720 binding nor its effects on the dissociation of [3H]-N-methylscopolamine from M1 receptors. Therefore it is possible to have a muscarinic receptor with three small ligands bound simultaneously. A model of the M1 receptor, based on the recently determined structure of rhodopsin, has the residues that have been shown to be important for gallamine binding clustered within and to one side of a cleft in the extracellular face of the receptor. This cleft may represent the access route of acetylcholine to its binding site.

Allosteric Site↗
PubMed2001

Validation and standardisation of nucleic acid amplification technology (NAT) assays for the detection of viral contamination of blood and blood products.

Standardisation of NAT assays is necessary before the introduction of such assays for routine screening of blood and blood products for viral contaminants such as HBV, HCV, and HIV-1. Standardisation can be achieved by the use of well-characterised reference materials (working reagents) to validate each assay run. Working reagents for HCV, HIV-1, HBV, HAV, and human parvovirus B19 have been established by the NIBSC. Such reagents and reference panels are also available from other official medicinal control laboratories and commercial organisations. However, the nucleic acid content of these reagents are expressed in many different units, e. g. genome equivalents/ml, copies/ml, PCR detectable units/ml, making comparisons of results from laboratories using different reagents difficult. The establishment of internationally accepted standards against which all working reagents could be calibrated, using a common standard unit of measurement, IU, would overcome this major problem. The first International Standard for HCV RNA assays was established in 1997. This reagent, 96/790, is a lyophilised preparation of a genotype 1 isolate and the concentration of the standard is 10(5) IU/ml. Two further International Standards have since been established; for HIV-1 and HBV, containing 10(5) IU/ml and 10(6) IU/ml respectively. The establishment of the HCV International Standard has been critical in the introduction of mandatory testing. Since 1st July 1999, all batches of blood products marketed in Europe have to be prepared from plasma pools tested and found non-reactive for HCV RNA using a validated assay which can detect a sample containing 100 IU/ml of HCV RNA. In Germany, screening of blood donations for HCV RNA by NAT has been mandatory since 1st April 1999. The minimum sensitivity of assays should be 5000 IU/ml for a single donation.

DNA, Viral↗
PubMed2001

An international collaborative study to establish a World Health Organization international standard for hepatitis B virus DNA nucleic acid amplification techniques.

BACKGROUND AND OBJECTIVES: Twenty-two laboratories from nine countries participated in an international collaborative study to establish a World Health Organization (WHO) international standard for hepatitis B virus (HBV) DNA nucleic acid amplification techniques (NAT). MATERIALS AND METHODS: Three samples, AA, BB (both of which were lyophilized) and CC (which was a liquid preparation), were analysed using several different NAT assays. The mean HBV DNA content of each sample was determined from the study. RESULTS: Despite the range of assays (commercial and in-house) used by participants, there was good agreement among the overall mean 'equivalents'/ml obtained by the different assays, except for one laboratory (laboratory 4). The variation in estimates of log10 'equivalents'/ml was 1.75-1.25 for the three samples if results from laboratory 4 were excluded. The mean log10 'equivalents'/ml for all laboratories were 6.42 for sample AA, 6.30 for sample BB and 5.03 for sample CC (exclusion of results from laboratory 4 made little difference). The difference in titres between the two lyophilized samples (AA and BB) was not statistically significant but the titre of the frozen sample (CC) was significantly lower. Material AA (code 97/746) was accepted as the first WHO international standard for HBV DNA NAT assays and assigned a potency of 10(6) international units (IU)/ml. CONCLUSIONS: The titres (genome equivalents/ml) of three HBV preparations were determined by several laboratories using different NAT assays. This study enabled the establishment of an international standard, 97/746, for HBV DNA NAT assays.

DNA, Viral↗
PubMed2001

Peroxide sensors for the fission yeast stress-activated mitogen-activated protein kinase pathway.

The Schizosaccharomyces pombe stress-activated Sty1p/Spc1p mitogen-activated protein (MAP) kinase regulates gene expression through the Atf1p and Pap1p transcription factors, homologs of human ATF2 and c-Jun, respectively. Mcs4p, a response regulator protein, acts upstream of Sty1p by binding the Wak1p/Wis4p MAP kinase kinase kinase. We show that phosphorylation of Mcs4p on a conserved aspartic acid residue is required for activation of Sty1p only in response to peroxide stress. Mcs4p acts in a conserved phospho-relay system initiated by two PAS/PAC domain-containing histidine kinases, Mak2p and Mak3p. In the absence of Mak2p or Mak3p, Sty1p fails to phosphorylate the Atf1p transcription factor or induce Atf1p-dependent gene expression. As a consequence, cells lacking Mak2p and Mak3p are sensitive to peroxide attack in the absence of Prr1p, a distinct response regulator protein that functions in association with Pap1p. The Mak1p histidine kinase, which also contains PAS/PAC repeats, does not regulate Sty1p or Atf1p but is partially required for Pap1p- and Prr1p-dependent transcription. We conclude that the transcriptional response to free radical attack is initiated by at least two distinct phospho-relay pathways in fission yeast.

Activating Transcription Factor 1↗
PubMed2000

Bacterial orthologues indicate the malarial plastid gene ycf24 is essential.

ycf24 is a well conserved gene found in all major groups of bacteria, as well as on red algal plastid genomes and the vestigal plastid genome of apicomplexan pathogens like the malaria parasite Plasmodium falciparum (ORF470). Some database annotations describe Ycf24 as an ABC transporter subunit, but we find the level of significance is low. To investigate ycf24's function we disrupted it in the cyanobacterium Synechocystis sp., strain PCC6803 which has a multi-copy genome. This showed ycf24 is essential, partial loss producing a terminal phenotype of chlorosis, reduced cell size, loss of DNA, and a striking arrest in cytokinesis. Attempts to disrupt the single copy of ycf24 in E. coli failed to give stable transformants. When Ycf24 was over-expressed in E. coli as a soluble fusion protein, it localized mostly as a band on either side of the nucleoid and nucleoid partitioning was aberrant. We propose the relict plastid organelle of apicomplexans retains its capacity for protein synthesis because Ycf24 is essential.

ATP-Binding Cassette Transporters↗
PubMed2000

Genetic diversity and clonal patterns among antibiotic-susceptible and -resistant Streptococcus pneumoniae colonizing children: day care centers as autonomous epidemiological units.

Characterization by antibiotype of the 1,096 Streptococcus pneumoniae recovered from 2,111 nasopharyngeal samples of children attending 16 day care centers (DCCs) in Lisbon, Portugal, and molecular typing of 413 drug-resistant pneumococci (DRPn) and 89 fully drug-susceptible pneumococci (DSPn) has allowed several conclusions. (i) There was an increase in the frequency of DRPn colonizing children in DCCs from 40% in 1996 to 45% in 1997 to 50% in 1998. (ii) Drug resistance spread by cross-transmission of DRPn clones. A few (8 out of 57) DRPn clones were repeatedly isolated from a large number of children in several DCCs and during each period of surveillance, suggesting the epidemic nature of these clones, which included lineages representing internationally spread S. pneumoniae clones. (iii) Dissemination of resistance determinants among pneumococci colonizing the nasopharynx occurred. Association of identical pulsed-field gel electrophoresis patterns with diverse antibiotypes among pneumococci colonizing children suggests that the high prevalence of DRPn involves not only cross-transmission of resistant strains but also dispersal of resistance genes through recombinational mechanisms. (iv) DCCs are autonomous epidemiological units. Among the 413 DRPn, 57 different lineages were detected; these lineages were dispersed among the 16 DCCs to produce unique microbiological profiles for each of the DCCs. Higher genetic diversity and less sharing of clonal types were observed among the DSPn.

Anti-Bacterial Agents↗
PubMed2000

Calibration of HCV working reagents for NAT assays against the HCV international standard. The Collaborative Study Group.

BACKGROUND AND OBJECTIVES: Five HCV RNA reference reagents, the Paul Ehrlich Institut (PEI) reference 75, the National Institute for Biological Standards and Control (NIBSC) reagent 96/586, the Central Laboratory of the Netherlands Red Cross Blood Transfusion Service (CLB) Pelispy HCV RNA run control S2001, the Istituto Superiore di Sanità (ISS) reagent 0498 and the CBER panel member No. 1, were calibrated against the WHO International Standard, 96/790. MATERIALS AND METHODS: The reference materials were calibrated in a collaborative study organised by NIBSC. Nineteen laboratories, using a range of qualitative and quantitative assays returned results. RESULTS: The concentrations of the reagents were: 25,000 IU/ml for the PEI material, 710 IU/ml for the NIBSC material 96/586, 1,000 IU/ml for the CLB material, 1,700 IU/ml for the ISS material 0498 and 250 IU/ml for the CBER panel member No. 1. CONCLUSIONS: The calibration of these five reference reagents for HCV RNA nucleic acid amplification technology (NAT) assays enables them to be used for standardisation and validation of assays. Such calibrants are essential for meeting the requirements of the European Medicinal Evaluation Agency (EMEA) for the testing of plasma pools and donations for HCV RNA for the release of blood products and the PEI requirements for the release testing of erythrocyte and thrombocyte concentrates.

Calibration↗
PubMed1999

Standardization: a progress report.

The introduction of nucleic acid amplification technology (NAT) assays for the detection of viral contamination of blood and blood products requires the availability of well-characterized reference reagents. Working reagents for hepatitis C virus RNA, hepatitis B virus DNA, HIV-1 RNA and human parvovirus B19 DNA have been established at NIBSC and at many other laboratories (both official medicinal control laboratories and commercial laboratories). However, as these reagents have been characterised independently, it is difficult to compare results from assays using different working reagents. Recently, a WHO International Standard was established for HCV RNA NAT assays. This standard has been calibrated in International Units (IU) and provides a common standard against which all working reagents can be calibrated. Collaborative studies to characterise two further candidate International Standards for HBV DNA and HIV-1 RNA NAT assays have been completed.

Blood Banks↗
PubMed1999

Carriage of respiratory tract pathogens and molecular epidemiology of Streptococcus pneumoniae colonization in healthy children attending day care centers in Lisbon, Portugal.

In an effort to establish the rate of carriage of antibiotic resistant respiratory pathogens in children attending urban day care centers (DCC) in Portugal, seven DCC in Lisbon were selected for determining the rate of nasopharyngeal colonization of children between the ages of 6 months to 6 years by Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis. Of the 586 children studied between January and March 1996, 47% carried S. pneumoniae, 72% H. influenzae, and 54% M. catarrhalis. Twenty-four percent of the pneumococci had reduced susceptibility to penicillin, and most of these belonged to serogroups 19, 23, 14, and 6. An additional 19% were fully susceptible to penicillin but showed decreased susceptibility to other antimicrobials. These isolates expressed serogroups 6, 11, 14, 18, 19, and 34. The majority (96%) of M. catarrhalis and 20% of H. influenzae were penicillin resistant due to the production of beta-lactamases. Recent antimicrobial use was associated with carriage of penicillin non-susceptible pneumococci and beta-lactamase producing H. influenzae (p < 0.05). Individual DCC differed substantially from one another in their rates of carriage of antibiotic resistant H. influenzae and S. pneumoniae. Characterization of antibiotic resistant S. pneumoniae isolates by molecular fingerprinting techniques showed that each DCC had a unique microbiological profile, suggesting little, if any, exchange of the resistant microbial flora among them. An exception to this was the presence of isolates belonging to two internationally spread epidemic clones: the multiresistant Spanish/USA clone expressing serotype 23F, and the penicillin and sulfamethoxazole-trimethoprim resistant French/Spanish clone (serotype 14) which were detected in four and three DCC, respectively.

Anti-Bacterial Agents↗
PubMed1999

Establishment of the first international standard for nucleic acid amplification technology (NAT) assays for HCV RNA. WHO Collaborative Study Group.

BACKGROUND AND OBJECTIVES: The aims of this study were the establishment of a WHO International standard for HCV RNA for nucleic acid amplification technology (NAT) assays and the determination of the HCV RNA content of the candidate standard. MATERIALS AND METHODS: Twenty-two laboratories evaluated three candidate materials (two lyophilised, AA and BB, which were derived from the same source and one a liquid preparation, CC). All samples were HCV genotype 1 with a concentration of approximately 10(5) genome equivalents/ml. The methods used included the Roche Amplicor assay (version 1), Chiron Quantiplex (bDNA) assay, Organon Teknika NASBA assay, Transcription Mediated assay and various in-house assays, using single or nested primers. RESULTS: There was reasonable agreement between the overall mean NAT detectable units/ml obtained by the different assays except for some of the in-house assays using single primers which gave substantially lower estimates. These titres were 5.0 log10 for samples AA and BB and 4.6 log10 for sample CC. CONCLUSIONS: Sample AA was accepted as the candidate standard and assigned a titre of 10(5) international units (IU)/ml. The International Standard consists of a batch of vials each containing 50,000 IU/vial. Preliminary studies indicated that the material is stable at +4 degrees C and +20 degrees C for up to 200 days.

Gene Amplification↗
PubMed1999

Sensitivity of PCR assays for the determination of hepatitis A virus RNA in plasma pools. A collaborative study.

BACKGROUND AND OBJECTIVES: A collaborative study was organised to establish a hepatitis A virus (HAV) RNA standard for genomic amplification assays (GAT). MATERIALS AND METHODS: A panel of 10 samples consisting of a 10-fold serial dilution of wild-type HAV diluted in HAV-negative cryosupernatant and samples of the diluent were sent to 14 laboratories in duplicate for testing for HAV RNA by the polymerase chain reaction. RESULTS: Data returned by 12 laboratories indicated that the sensitivities of the assays performed by different laboratories were fairly close: there was a 100-fold difference in sensitivity between the majority of laboratories. Only one laboratory reported false-positive results. CONCLUSION: A 10-5 dilution of the virus in cryosupernatant could be an appropriate working reagent for GAT assays for HAV RNA in plasma pools.

Hepatovirus↗
PubMed1998

Identification of a Frizzled-like cysteine rich domain in the extracellular region of developmental receptor tyrosine kinases.

In Drosophila, members of the Frizzled family of tissue-polarity genes encode proteins that appear to function as cell-surface receptors for Wnts. The Frizzled genes belong to the seven transmembrane class of receptors (7TMR) and have on their extracellular region a cysteine-rich domain that has been implicated as the Wnt binding domain. This region has a characteristic spacing of ten cysteines, which has also been identified in FrzB (a secreted antagonist of Wnt signaling) and Smoothened (another 7TMR, which is involved in suppression of the hedgehog pathway). We have identified, using BLAST, sequence similarity between the cysteine-rich domain of Frizzled and several receptor tyrosine kinases, which have roles in development. These include the muscle-specific receptor tyrosine kinase (MuSK), the neuronal specific kinase (NSK2), and ROR1 and ROR2. At present, the ligands for these developmental tyrosine kinases are unknown. Our results suggest that Wnt-like ligands may bind to these developmental tyrosine kinases.

Amino Acid Sequence↗
PubMed1998

Identification of a Frizzled-like cysteine rich domain in the extracellular region of developmental receptor tyrosine kinases.

In Drosophila, members of the Frizzled family of tissue-polarity genes encode proteins that appear to function as cell-surface receptors for Wnts. The Frizzled genes belong to the seven transmembrane class of receptors (7TMR) and have on their extracellular region a cysteine-rich domain that has been implicated as the Wnt binding domain. This region has a characteristic spacing of ten cysteines, which has also been identified in FrzB (a secreted antagonist of Wnt signaling) and Smoothened (another 7TMR, which is involved in the hedgehog signalling pathway). We have identified, using BLAST, sequence similarity between the cysteine-rich domain of Frizzled and several receptor tyrosine kinases, which have roles in development. These include the muscle-specific receptor tyrosine kinase (MuSK), the neuronal specific kinase (NSK2), and ROR1 and ROR2. At present, the ligands for these developmental tyrosine kinases are unknown. Our results suggest that Wnt-like ligands may bind to these developmental tyrosine kinases

Amino Acid Sequence↗
PubMed1997

Report of EPFA/NIBSC workshop 'nucleic acid amplification tests (NAT) for the detection of blood-borne viruses' held on 31 October 1996 in Amsterdam, The Netherlands.

The 3rd annual European Plasma Fractionators Association/National Institut of Biological Standard and Control (EPFA/NIBSC) meeting provided a forum for regulators, blood product and test kit manufacturers and organisations developing standards to present and discuss their latest data. The main conclusions were as follows. There has been substantial progress during the last year in the development of NAT technology specifically for improving the safety of blood products though there is an urgent need for the development of international reference materials. The technology is not yet sufficiently developed to be used as a routine screening test though testing of plasma pools for hepatitis C virus may be achieved within a year. Introduction of testing should not result in the creation of dual standards for plasma derived and cellular products. Once the technology is fully developed it could significantly improve the safety of all blood products, particularly those derived from starting materials with a high incidence of viral markers.

Blood↗
PubMed1997

Collaborative study to assess the suitability of a proposed working reagent for human parvovirus B19 DNA detection in plasma pools by gene amplification techniques. B19 Collaborative Study Group.

BACKGROUND AND OBJECTIVES: A collaborative study was done to examine the sensitivity and specificity of assays for the detection of human parvovirus B19 DNA in plasma pools by PCR techniques and to establish a working reagent for B19 DNA testing of plasma pools. MATERIALS AND METHODS: Duplicate samples consisting of a tenfold dilution series of a positive cryosupernatant diluted in B19 DNA-negative cryosupernatant were sent to 17 laboratories. RESULTS: The sensitivity of the assays varied: 2 laboratories were able to detect the 10(-7) dilution while 1 laboratory failed to detect B19 DNA in any samples. In addition, 5 laboratories obtained false-positive results. CONCLUSIONS: In general, laboratories using assays optimised for rapid detection of B19 DNA in serum samples did not perform well, indicating that such rapid methods are not adequate for examination of plasma pools. The 10(-6) dilution was detected by approximately half the laboratories and could be used as the working reagent.

DNA, Viral↗
PubMed1997

Humanization of a mouse antibody against human alpha-4 integrin: a potential therapeutic for the treatment of multiple sclerosis.

alpha 4 beta 1 integrin (VLA-4) is crucial for the adhesion of leukocytes to human vascular cell adhesion molecule-1 (VCAM-1) on inflamed endothelium. This cell adhesion event is the first step in leukocyte extravasation across the blood-brain barrier in inflammatory diseases of the central nervous system (CNS) such as experimental autoimmune encephalomyelitis (EAE). Prevention of leukocyte infiltration by antibodies against the alpha 4 integrin, which block the alpha 4 beta 1 integrin/VCAM-1 interaction, have been shown to suppress clinical and pathological features of EAE. In this study, two mouse monoclonal antibodies (MAb) directed against human alpha 4 integrin were analyzed in vitro for their ability to block the interaction of leukocytes with VCAM-1 under different assay conditions. The best blocking MAb, AN100226m, was humanized by complementarily-determining region grafting, associated with human C regions and expressed. We found that modification of two structural determinants (H27 and H29) for the heavy chain CDR1 loop in one hand, and modification of framework amino acid H38, H40 and H44 in the other hand, had no effect on antigen binding. In contrast, modification of a structural determinant (H71) for the heavy chain CDR2 loop resulted in loss of binding. The humanized antibody. AN100226, was equivalent to the murine antibody. AN100226m, in binding to alpha 4 beta 1 integrin and in blocking cell adhesion. More importantly, AN100226 was as effective as AN100226m in the reversal of active EAE in guinea pigs and thus may be useful in the treatment of autoimmune diseases such as multiple sclerosis. AN100226 is currently in phase II clinical trials in the UK for the treatment of multiple sclerosis exacerbations.

Amino Acid Sequence↗