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S Searle

Publications and source records attributed to S Searle.

At least 37 records · Page 2Linked to original sources
PubMed1999

Molecular modeling and preclinical evaluation of the humanized NR-LU-13 antibody.

A mouse-human chimeric monoclonal antibody (chNR-LU-13), specific for the EGP40 pancarcinoma antigen, was humanized through three-dimensional molecular modeling. Humanization of the chNR-LU-13 antibody is expected to enhance its use for patients undergoing immunotherapy. On the basis of the observed amino acid sequence identity, chNR-LU-13 complementary determining regions (CDRs) of the V(L) and V(H) regions were grafted onto the human anti-DNA-associated idiotype immunoglobulin clone, R3.5H5G'CL. Ten amino acids residues within the humanized framework were back-mutated to their corresponding chNR-LU-13 sequence, because they were predicted to disrupt the canonical classification of the CDRs or were within 5 A of a CDR. Synthesis of the V(L) and V(H) regions was accomplished by recursive PCR, and the dual-chain expression vector p451.C4 was positioned under control of the CMV(P+E). We observed by competitive ELISA that the recombinant humanized NR-LU-13 (huNR-LU-13) IgG1 antibody exhibited an indistinguishable immunoreactivity profile when compared with the murine monoclonal antibody (muNR-LU-10). The huNR-LU-13 antibody was effective in mediating both antibody-dependent cellular cytotoxicity and complement-mediated cytotoxicity when assayed against either the breast carcinoma cell line, MCF-7, or the colon adenocarcinoma cell line, SW1222. Biodistribution studies using i.v. coinjected 131I-muNR-LU-10 and 125I-huNR-LU-13 confirmed that the huNR-LU-13 specifically targets to the tumor in athymic BALB/c mice bearing the SW1222 human tumor xenograft. Humanization of the chNR-LU-13 antibody is expected to eliminate an undesired human antimouse antibody response, allowing for repeated i.v. administration into humans.

Amino Acid Sequence↗
PubMed1999

Evidence for a functional repeat polymorphism in the promoter of the human NRAMP1 gene that correlates with autoimmune versus infectious disease susceptibility.

A polymorphism in the promoter of human NRAMP1 encodes a Z-DNA forming dinucleotide repeat with four alleles: (1) t(gt)5ac(gt)5ac(gt)11g; (2) t(gt)5ac(gt)5 ac(gt)10g; (3) t(gt)5ac(gt) ac(gt)9g; and (4) t(gt)5ac(gt)9g. Alleles 1 and 4 are rare (gene frequencies approximately 0.001); alleles 2 and 3 occur at gene frequencies approximately 0.20-0.25 and approximately 0.75-0.80, respectively. Here, luciferase reporter gene constructs are used to show that the four alleles differ in their ability to drive gene expression. In the absence of exogenous stimuli, alleles 1, 2, and 4 are poor promoters; allele 3 drives high expression, indicating that the repeat itself has endogenous enhancer activity. All four alleles show a similar percentage enhancement of reporter gene expression in the presence of interferon-gamma, consistent with the multiple interferon-gamma response elements both 5' and 3' of the Z-DNA forming repeat. However, while the addition of bacterial lipopolysaccharide (LPS) has no effect on alleles 1 and 4, it causes significant reduction in expression driven by allele 2 and enhances expression driven by allele 3, suggesting that the juxtaposition of LPS related response elements (NFkappaB, AP-1, NF-IL6) may be differentially affected by the two commonly occurring alleles. These results are consistent with the hypothesis that chronic hyperactivation of macrophages associated with allele 3 is functionally linked to autoimmune disease susceptibility, while the poor level of NRAMP1 expression promoted by allele 2 contributes to infectious disease susceptibility. Conversely, allele 3 protects against infectious disease and allele 2 against autoimmune disease. Hence, alleles that are detrimental in relation to autoimmune disease susceptibility may be maintained in the population because they improve survival to reproductive age following infectious disease challenge.

Alleles↗
PubMed1998

Localisation of Nramp1 in macrophages: modulation with activation and infection.

The murine natural resistance-associated macrophage protein, Nramp1, has multiple pleiotropic effects on macrophage activation and regulates survival of intracellular pathogens including Leishmania, Salmonella and Mycobacterium species. Nramp1 acts as an iron transporter, but precisely how this relates to macrophage activation and/or pathogen survival remains unclear. To gain insight into function, anti-Nramp1 monoclonal and polyclonal antibodies are used here to localise Nramp1 following activation and infection. Confocal microscope analysis in uninfected macrophages demonstrates that both the mutant (infection-susceptible) and wild-type (infection-resistant) forms of the protein localise to the membranes of intracellular vesicular compartments. Gold labelling and electron microscopy defines these compartments more precisely as electron-lucent late endosomal and electron-dense lysosomal compartments, with Nramp1 colocalizing with Lamp1 and cathepsins D and L in both compartments, with macrosialin in late endosomes, and with BSA-5 nm gold in pre-loaded lysosomes. Nramp1 is upregulated with interferon-(gamma) and lipopolysaccaride treatment, coinciding with an increase in labelling in lysosomes relative to late endosomes and apparent dispersion of Nramp1-positive vesicles from a perinuclear location towards the periphery of the cytoplasm along the microtubular network. In both control and activated macrophages, expression of the protein is 3- to 4-fold higher in wild-type compared to mutant macrophages. In Leishmania major-infected macrophages, Nramp1 is observed in the membrane of the pathogen-containing phagosomes, which retain a perinuclear localization in resting macrophages. In Mycobacterium avium-infected resting and activated macrophages, Nramp1-positive vesicles migrated to converge, but not always fuse, with pathogen-containing phagosomes. The Nramp1 protein is thus located where it can have a direct influence on phagosome fusion and the microenvironment of the pathogen, as well as in the more general regulation of endosomal/lysosomal function in macrophages.

Amino Acid Sequence↗
PubMed1997

Occupational physical activity and long-term risk of musculoskeletal symptoms: a national survey of post office pensioners.

The objective of this work was to address the relationship between physical activity in the workplace and subsequent musculoskeletal pain syndromes. We performed a survey of 5,042 men and women aged 70-75 years, selected from the retirement population of a large national employer (the post office). Subjects were sent a short postal questionnaire enquiring about all occupations held for at least 1 year, the physical activities performed in those jobs, and about recent rheumatic symptoms. The 1-month period prevalence of rheumatic symptoms ranged from 19.9% for hip pain or stiffness in men to 50% for knee pain or stiffness in women. Symptoms were more common in women than men at all sites and there were significant (P < 0.001) associations between symptoms at different sites. Obesity was significantly (P < 0.001) associated with the risk of pain or stiffness at the knee and hip. Prolonged occupational exposure (20+ years) to heavy lifting was associated with hip pain (RR = 1.5; 95% CI = 1.2-1.8); and prolonged exposure to working with arms elevated was associated with an increased risk of shoulder pain (RR = 1.4; 95% CI = 1.2-1.6). Tall stature (P = 0.003) and heavy lifting (P < 0.001) were both associated with increased risks of low back pain among men. This survey confirms the high prevalence of musculoskeletal symptoms observed in previous population-based studies. Associations between occupational activities and musculoskeletal symptoms were specific for activity type and skeletal site involved. Our results imply that the adverse effects of these occupational activities can be found many years after cessation of exposure.

Aged↗
PubMed1995

Genomic organization and sequence of the human NRAMP gene: identification and mapping of a promoter region polymorphism.

BACKGROUND: Murine Nramp is a candidate for the macrophage resistance gene Ity/Lsh/Bcg. Sequence analysis of human NRAMP was undertaken to determine its role in man. MATERIALS AND METHODS: A yeast artificial chromosome carrying NRAMP was subcloned and positive clones sequenced. The transcriptional start site was mapped using 5' RACE PCR. Polymorphic variants were amplified by PCR. Linkage analysis was used to map NRAMP. RESULTS: NRAMP spans 12kb and has 15 exons encoding a 550 amino acid protein showing 85% identity (92% similarity) with Nramp. Two conserved PKC sites occur in exon 2 encoding the Pro/Ser rich SH3 binding domain, and in exon 3. Striking sequence similarities (57 and 53%) were observed with yeast mitochondrial proteins, SMF1 and SMF2, especially within putative functional domains: exon 6 encoding the second transmembrane spanning domain, site of the murine susceptibility mutation; and exon 11 encoding a conserved transport motif. No mutations comparable to the murine susceptibility mutation were found. The transcriptional initiation site mapped 148 bp 5' of the translational initiation codon. 440bp of 5' flanking sequence contained putative promoter region elements: 6 interferon-gamma response elements, 3 W-elements, 3 NF kappa B binding sites and 1 AP-1 site. Nine purine-rich GGAA core motifs for the myeloid-specific PU.1 transcription factor were identified, two combining with imperfect AP1-like sites to create PEA3 motifs. TATA, GC and CCAAT boxes were absent. A possible enhancer element containing the Z-DNA forming dinucleotide repeat t(gt),ac(gt),ac(gt),g was polymorphic (4 alleles; n = 4,9,10,11), and was used to map NRAMP to 2q35. CONCLUSIONS: This analysis provides important resources to study the role of NRAMP in human disease.

Alleles↗
PubMed1994

Genetic regulation of leishmanial and mycobacterial infections: the Lsh/Ity/Bcg gene story continues.

A common basis to genetic regulation of leishmanial and mycobacterial infections is provided by the action of the murine Lsh/Ity/Bcg gene in controlling the priming/activation of macrophages for antimicrobial activity. This relies on the TNF-alpha-dependent sustained expression of the inducible nitric oxide synthase (iNOS) gene responsible for the generation of large amounts of toxic nitric oxide (NO). The Lsh/Ity/Bcg gene has many pleiotropic effects, including differential expression of the early response gene KC following stimulation of macrophages with bacterial lipopolysaccharide (LPS) and mycobacterial lipoarabinomannan (LAM). The major signal transduction pathway involved in KC induction requires the generation of low levels of NO via constitutive nitric oxide synthase (cNOS) activity, leading to activation of guanylate cyclase and the cGMP-dependent kinase pathway. NO therefore appears to provide a common link between the early influence of Lsh in regulating the expression of genes which mediate many pleiotropic effects, and the later production of NO as the final effector mechanism for kill. The recently cloned candidate for Lsh/Ity/Bcg, designated Nramp for Natural resistance associated macrophage protein, encodes a polytopic integral membrane protein that has structural features common to prokaryotic and eukaryotic transporters and includes a conserved binding-protein-dependent transport motif which may be involved in interaction with peripheral ATP-binding subunits. The N-terminal sequence also carries a proline/serine rich putative SH3 binding domain, consistent with a role for tyrosine kinases in regulating Nramp function. (ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1993

Leishmania major: characterisation and expression of a cytoplasmic stress-related protein.

The DNA sequence of a single-copy gene from Leishmania major has been determined and shown to share sequence identity with eukaryotic heat shock protein-70-related genes. Conserved features of the deduced open reading frame include amino acids implicated in ATP binding and a putative calmodulin-binding domain. Antibodies generated to the recombinant fusion protein recognise a 70-kDa molecule of pI 6.0. This molecule is constitutively expressed and localises to the cytoplasm in all stages of the parasite life cycle.

Amino Acid Sequence↗
PubMed1993

Expression of a mitochondrial stress protein in the protozoan parasite Leishmania major.

The DNA sequence has been determined of a gene from Leishmania major that shares sequence identity with members of the eukaryotic heat shock protein (hsp) 70 gene family. The deduced open reading frame for translation shares a number of features common to hsp70 stress proteins, including conserved amino acids implicated in ATP binding and a putative calmodulin-binding site. In addition, the protein has an N-terminal sequence characteristic of a mitochondrial targeting signal. Specific antibodies to this protein, generated by the use of recombinant fusion peptides, recognise a 65 kDa molecule of pI 6.7. This molecule is constitutively expressed and localises to the mitochondrion in all stages of the parasite life cycle. These features suggest a role for this protein as a molecular chaperone in Leishmania.

Amino Acid Sequence↗
PubMed1991

Levels of exposure to wood dust.

Surveys of exposure to inhalable dust have been carried out in two woodworking machine shops and five other woodworking shops where both manual and machine tool work is carried out. The Occupational Exposure Limits for wood dust were found to be frequently exceeded in some workshops. It is possible that the fraction of dust projected from woodworking machines towards the operator may have contributed both to measured dust concentrations and to exposure. Dust exposure may be reduced by careful attention to enclosure and ventilation, possibly using total enclosure at some machines. Emission of dust from portable orbital type sanding machines was considerably reduced by the use of low-volume high-velocity type extraction. It is suggested that suppliers of woodworking machines should provide data on dust emission to assist the user to manage better the introduction of new machines.

Air Pollutants, Occupational↗
PubMed1990

Fusion of Rous-sarcoma-virus-transformed rat cells to morphologically normal human or rat cells results in transcriptional suppression of the provirus that depends on its chromosomal integration site.

The fusion of a Rous sarcoma virus (RSV)-transformed rat fibroblast clone to at least 2 different human cell types reproducibly produces phenotypically normal hybrids. Analysis of such hybrids reveals that proviral silence is the result of transcriptional down-regulation, presumably by a trans-acting human molecule. Furthermore, this phenomenon seems to be strongly influenced by the proviral chromosomal integration site and its imposition may entail a mechanism that is required only transiently.

Animals↗
PubMed1989

A family of heat shock protein 70-related genes are expressed in the promastigotes of Leishmania major.

We describe the isolation and characterisation of two novel genes of the parasitic protozoan Leishmania major that are related by nucleotide sequence homology to eukaryotic genes encoding 70 Kd. heat shock proteins. The transcription of neither gene is heat-inducible but both are constituitively-expressed throughout the promastigote stage of the parasite life cycle. A third gene shows differential expression between non-infective and infective promastigote stages in the absence of any temperature change. These genes are related by sequence homology to the tandemly-repeated hsp70 genes of trypanosomatids, but are located on different, dispersed chromosomes within the genome of L. major. The open reading frame for translation derived from one of these sequences contains a putative mitochondrial signal peptide at its amino-terminus.

Animals↗
PubMed1989

A kinetoplast DNA probe diagnostic for Leishmania major: sequence homologies between regions of Leishmania minicircles.

A restriction fragment from a cloned kinetoplast minicircle DNA has been shown to be diagnostic for Leishmania major. This 402-bp TaqI fragment has been used routinely (as a radiolabelled probe) to detect 10(4) parasites in simple dot blots, both experimentally and in epidemiological surveys. It positively identified all stocks of L. major tested (including all six known zymodemes) and showed very low homology to kinetoplast DNA (kDNA) and chromosomal DNA of Leishmania infantum and Leishmania tropica, two species commonly isolated from patients and wild hosts within foci of L. major in the Old World. DNA sequence analysis of a minicircle of L. major is reported for the first time, and it is demonstrated that this species shares with Leishmania aethiopica, Sauroleishmania tarentolae and several species of Trypanosoma a region of conserved sequence that is involved in DNA replication, a process that could present targets for selective chemotherapeutic attack. Sequence and restriction fragment analyses have indicated the difficulties of selecting species-specific sequences from kDNA which, even in the same parasite clone, contains several predominant minicircle classes, not all of which contain diagnostic sequences.

Animals↗
PubMed1989

CpG island clones from a deletion encompassing the gene for adenomatous polyposis coli.

Adenomatous polyposis coli (APC), a dominantly inherited disorder, has been mapped to chromosome 5q15-q21 by family linkage studies. Cells from patients with deletions in this region, in one case associated with polyposis in a family, have been used to construct human hamster hybrid cell lines that retain either the normal or deleted chromosome 5. These lines have been used to identify markers from the region of the polyposis gene obtained by cloning the ends of 0.5- to 2-megabase BssHII fragments purified by pulsed-field gel electrophoresis. Three markers are described that map within the deletions and must therefore be close to the APC gene.

Adenoma↗
PubMed1988

Construction of a NotI linking library and isolation of new markers close to the Huntington's disease gene.

Linking clones contain sequences flanking recognition sites for enzymes cutting rarely in mammalian DNA. They can be used to obtain and correlate both physical and genetic mapping information over subregions of mammalian chromosomes. We have constructed and used a NotI linking clone library representing unmethylated NotI sites from HHW693 DNA, a hamster hybrid cell line containing 4p15-4pter and a fragment of 5p as its only human chromosome contribution. Human clones were identified by hybridisation with a cloned human repeat sequence, and localised further to subregions of human chromosome 4p15-4pter using a panel of additional hybrids. Clones from the region distal to the DNA probes (D4S10, D4S43, D4S95) linked to the Huntington's disease mutation, were further analysed. Four markers close to the HD gene: D4S111, D4S113, D4S114 and clone 417 are described here. In addition to serving as markers in physical and genetic mapping experiments, these linking clones provide probes next to cleavable NotI sites, and can therefore be used to screen NotI based chromosome jumping libraries. They also provide indications for potential gene sequences, identifiable as evolutionarily conserved sequences.

Chromosome Mapping↗
PubMed1987

Transcription of Rous sarcoma proviruses in rat cells is determined by chromosomal position effects that fluctuate and can operate over long distances.

Most Rous sarcoma proviruses integrated in rat DNA are inactive. The active minority are not necessarily a consequence of long terminal repeat mutations and their transcription can fluctuate in parallel with previously demonstrated transitions in chromatin configuration. Transcriptional alternations cannot be attributed to variations in positive or negative factors that operate independently of the site of provirus integration. Moreover, distinct proviruses in the same cell can be differentially expressed and flanking cellular elements can act over several kilobases to inhibit provirus transcription. We postulate that position-dependent fluctuations in proviral repression can be mediated by trans-acting factors, and DNA transfections indicate that initiation and maintenance of this phenomenon are separable entities. Provirus activity presumably reflects otherwise inapparent cell controls that affect chromatin structure over long distances.

Animals↗
PubMed1986

Expression of integrated Rous sarcoma viruses: DNA rearrangements 5' to the provirus are common in transformed rat cells but not seen in infected but untransformed cells.

The study of Rous sarcoma virus (RSV)-infected rat cell clones offers a novel approach to unravelling the mechanisms controlling eukaryotic gene expression. RSV-transformed rat cell clones frequently contain duplicated proviral sequences immediately upstream of an intact provirus. This category of proviral rearrangement is not seen in cells that remain untransformed after RSV infection nor in subsequently segregating transformants. These results suggest that such rearrangements occur during or soon after proviral integration, and that they may favour early proviral expression.

Animals↗
PubMed1986

Perturbed hemopoiesis and the generation of multipotential stem cell clones in src-infected bone marrow cultures is an indirect or transient effect of the oncogene.

Multipotential stem cell lines, derived specifically from long-term bone marrow cultures infected with a recombinant retrovirus carrying v-src, lack v-src. Stable consequences thus result from transient actions or indirect effects of v-src on other cells, with the latter possibility being favored by its mosaic expression in marrow cultures.

Animals↗