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Biomedical subjects

Stephen Brown

Publications and source records attributed to Stephen Brown.

At least 37 records · Page 2Linked to original sources
PubMed2004

A convenient synthesis of a selective gelatinase inhibitor as an antimetastatic agent.

Compound 1, 2-(4-phenoxyphenylsulfonylmethyl)thiirane, is a potent and selective inhibitor for human gelatinases (J. Am. Chem. Soc. 2000, 122, 6799-6800), enzymes implicated in a number of diseases, including cancer. This compound is showing excellent promise in animal trials in a number of disease models. Large quantities of this compound were necessary for these studies. A convenient four-step synthetic route for compound 1 is described herein. The synthesis is amenable to scale-up to tens of grams and gives an overall yield of 57% for this important compound.

Antineoplastic Agents↗
PubMed2004

Synthetic peptidoglycan substrates for penicillin-binding protein 5 of Gram-negative bacteria.

The major constituent of the bacterial cell wall, peptidoglycan, is comprised of repeating units of N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM) with an appended peptide. Penicillin-binding proteins (PBPs) are involved in the final stages of bacterial cell wall assembly. Two activities for PBPs are the cross-linking of the cell wall, carried out by dd-transpeptidases, and the dd-peptidase activity, that removes the terminal d-Ala residue from peptidoglycan. The dd-peptidase activity moderates the extent of the cell wall cross-linking. There exists a balance between the two activities that is critical for the well-being of bacterial cells. We have cloned and purified PBP5 of Escherichia coli. The membrane anchor of this protein was removed, and the enzyme was obtained as a soluble protein. Two fragments of the polymeric cell wall of Gram-negative bacteria (compounds 5 and 6) were synthesized. These molecules served as substrates for PBP5. The products of the reactions of PBP5 and compounds 5 and 6 were isolated and were shown to be d-Ala and the fragments of the substrates minus the terminal d-Ala. The kinetic parameters for these enzymic reactions were evaluated. PBP5 would appear to have the potential for turnover of as many as 1.4 million peptidoglycan strands within a single doubling time (i.e., generation) of E. coli.

Bacterial Proteins↗
PubMed2004

Dual role of prostratin in inhibition of infection and reactivation of human immunodeficiency virus from latency in primary blood lymphocytes and lymphoid tissue.

To design strategies to purge latent reservoirs of human immunodeficiency virus type 1 (HIV-1), we investigated mechanisms by which a non-tumor-promoting phorbol ester, prostratin, inhibits infection of CD4(+) T lymphocytes and at the same time reactivates virus from latency. CD4(+) T lymphocytes from primary blood mononuclear cells (PBMC) and in blocks of human lymphoid tissue were stimulated with prostratin and infected with HIV-1 to investigate the effects of prostratin on cellular susceptibility to the virus. The capacity of prostratin to reactivate HIV from latency was tested in CD4(+) T cells harboring preintegrated and integrated latent provirus. Prostratin stimulated CD4(+) T cells in an aberrant way. It induced expression of the activation markers CD25 and CD69 but inhibited cell cycling. HIV-1 uptake was reduced in prostratin-stimulated CD4(+) T PBMC and tissues in a manner consistent with a downregulation of CD4 and CXCR4 receptors in these systems. At the postentry level, prostratin inhibited completion of reverse transcription of the viral genome in lymphoid tissue. However, prostratin facilitated integration of the reverse-transcribed HIV-1 genome in nondividing CD4(+) T cells and facilitated expression of already integrated HIV-1, including latent forms. Thus, while stimulation with prostratin restricts susceptibility of primary resting CD4(+) T cells to HIV infection at the virus cell-entry level and at the reverse transcription level, it efficiently reactivates HIV-1 from pre- and postintegration latency in resting CD4(+) T cells.

Antigens, CD↗
PubMed2004

Quest for selectivity in inhibition of matrix metalloproteinases.

Matrix metalloproteinases (MMPs), of which at least 26 are known in humans, have been linked to a number of pathological conditions including tumor metastasis, inflammation, neurological and cardiovascular diseases. Inhibition of MMPs has been widely sought as a strategy in intervention of these disease processes. Whereas a large number of broad-spectrum MMP inhibitors have been developed over the past decade, these inhibitors have not met the promise and expectations in clinical trials. The broad-spectrum inhibition, which besides MMPs often targets other metalloproteinases, has been considered one of the potential problems that affects the therapeutic efficacy of MMPs inhibitors. Several MMP inhibitors that show selectivity for various MMPs have been reported in the past few years. This report describes the structural and inhibitory properties of these novel inhibitors, which hold considerable promise for effective targeting of these important enzymes.

Animals↗
PubMed2003

Zic2 patterns binocular vision by specifying the uncrossed retinal projection.

During CNS development, combinatorial expression of transcription factors controls neuronal subtype identity and subsequent axonal trajectory. Regulatory genes designating the routing of retinal ganglion cell (RGC) axons at the optic chiasm to the appropriate hemisphere, a pattern critical for proper binocular vision, have not been identified. Here, we show that the zinc finger transcription factor Zic2, a vertebrate homolog of the Drosophila gene odd-paired, is expressed in RGCs with an uncrossed trajectory during the period when this subpopulation grows from the ventrotemporal retina toward the optic chiasm. Loss- and gain-of-function analyses indicate that Zic2 is necessary and sufficient to regulate RGC axon repulsion by cues at the optic chiasm midline. Moreover, Zic2 expression reflects the extent of binocularity in different species, suggesting that Zic2 is an evolutionarily conserved determinant of RGCs that project ipsilaterally. These data provide evidence for transcriptional coding of axon pathfinding at the midline.

Animals↗
PubMed2003

Structural basis for potent slow binding inhibition of human matrix metalloproteinase-2 (MMP-2).

The zinc-dependent gelatinases belong to the family of matrix metalloproteinases (MMPs), enzymes that have been shown to play a key role in angiogenesis and tumor metastasis. These enzymes are capable of hydrolyzing extracellular matrix (ECM) components under physiological conditions. Specific and selective inhibitors aimed at blocking their activity are highly sought for use as potential therapeutic agents. We report herein on a novel mode of inhibition of gelatinase A (MMP-2) by the recently characterized inhibitors 4-(4-phenoxphenylsulfonyl)butane-1,2-dithiol (inhibitor 1) and 5-(4-phenoxphenylsulfonyl) pentane-1,2-dithiol (inhibitor 2). These synthetic inhibitors are selective for MMP-2 and MMP-9. We show that the dithiolate moiety of these inhibitors chelates the catalytic zinc ion of MMP-2 via two sulfur atoms. This mode of binding results in alternation of the coordination number of the metal ion and the induction of conformational changes at the microenvironment of the catalytic zinc ion; a set of events that is likely to be at the root of the potent slow binding inhibition behavior exhibited by these inhibitors. This study demonstrates a distinct approach for the understanding of the structural mechanism governing the molecular interactions between potent inhibitors and catalytic sites of MMPs, which may aid in the design of effective inhibitors.

Catalytic Domain↗
PubMed2003

Prenatal diagnosis of factor X deficiency using a combination of direct mutation detection and linkage analysis with an intragenic single nucleotide polymorphism.

OBJECTIVE: To present a family in which it was possible to perform prenatal diagnosis for recessively inherited factor X deficiency using both direct mutation detection as well as linkage analysis. METHODS: In a family where both parents were known to be carriers of factor X deficiency, fetal DNA was obtained from an ongoing pregnancy by CVS in the first trimester. Direct DNA sequencing was used to detect a previously identified factor X mutation, and linkage analysis using a single nucleotide polymorphism (SNP) was used to follow the segregation of the other parent's factor X alleles. RESULTS: Our studies predicted the fetus in question to be a heterozygote carrier of factor X deficiency, and this was demonstrated to be correct at birth. CONCLUSIONS: This is the first reported case of prenatal diagnosis of factor X deficiency. In addition, this case demonstrates the remarkable utility of SNPs in linkage analysis of rare genetic disorders.

Adult↗
PubMed2003

Immunolocalization of Zic2 expression in the developing mouse forebrain.

The Zic genes are a family of zinc finger transcription factors defined by their homology with the Drosophila gene, odd-paired (opa). Zic2 has a critical role in forebrain development, as is evidenced by the fact that, in both mice and humans, diminished expression results in the severe forebrain malformation known as holoprosencephaly. Published information indicates that Zic2 expression is most prominent in the dorsal neural tube/spinal cord and in the hindbrain; however, there is no published description of the pattern of expression of Zic2 in the developing forebrain where the main Zic2 associated phenotype occurs. Using a Zic2-specific antiserum, we present new information about the expression of Zic2 in the developing mouse forebrain. In addition, we show that in sonic hedgehog (Shh) null mice, the expression of Zic2 is expanded ventrally in some structures while absent in others, suggesting that Shh has a role in regulating the expression of Zic2.

Animals↗
PubMed2003

Novel level of signalling control in the JAK/STAT pathway revealed by in situ visualisation of protein-protein interaction during Drosophila development.

It is commonly accepted that activation of most signalling pathways is induced by ligand receptor dimerisation. This belief has been challenged for some vertebrate cytokine receptors of the JAK/STAT pathway. Here we study whether DOME, the Drosophila receptor of the JAK/STAT pathway, can dimerise and if the dimerisation is ligand-dependent. To analyse DOME homo-dimerisation, we have applied a beta-gal complementation technique that allows the detection of protein interactions in situ. This technique has been used previously in cell culture but this is the first time that it has been applied to whole embryos. We show that this technique, which we rename betalue-betalau technique, can be used to detect DOME homo-dimerisation in Drosophila developing embryos. Despite DOME being ubiquitously expressed, dimerisation is developmentally regulated. We investigate the state of DOME dimerisation in the presence or absence of ligand and show that DOME dimerisation is not ligand-induced, indicating that ligand independent cytokine receptor dimerisation is a conserved feature across phyla. We have further analysed the functional significance of ligand-independent receptor dimerisation by comparing the effects of ectopic ligand expression in cells in which the receptor is, or is not, dimerised. We show that ligand expression can only activate STAT downstream targets or affect embryo development in cells in which the receptor is dimerised. These results suggest a model in which ligand-independent dimerisation of the JAK/STAT receptor confers cells with competence to activate the pathway prior to ligand reception. Thus, competence to induce the JAK/STAT signalling pathway in Drosophila can be regulated by controlling receptor dimerisation prior to ligand binding. These results reveal a novel level of JAK/STAT signalling regulation that could also apply to vertebrates.

Alleles↗
PubMed2002

The fertile field of Drosophila Jak/STAT signalling.

The JAK/STAT pathway plays important roles in vertebrate and invertebrate development. The recent cloning and characterisation of the receptor in Drosophila shows that the pathway is conserved across phyla. In this review we describe current knowledge of the pathway and use genome data to discuss what elements are present in Drosophila. We also summarise recent work describing the involvement of the JAK/STAT pathway in oogenesis and spermatogenesis. Interestingly, the JAK/STAT pathway maintains the niche required for germline stem cell maintenance in the testis, providing the first molecular characterisation of a stem cell niche. Drosophila's streamlined pathway offers a simple model to find new elements and analyse the function of existing ones.

Animals↗
PubMed2002

Pronounced radiosensitization of cultured human cancer cells by COX inhibitor under acidic microenvironment.

PURPOSE: To demonstrate the influence of pH on the cytotoxicity and radiosensitization by COX (cyclooxygenase) -1 and -2 inhibitors using established human cancer cells in culture. METHODS AND MATERIALS: Nonselective COX inhibitor, ibuprofen (IB), and selective COX-2 inhibitor, SC-236, were used to determine the cytotoxicity and radiosensitization at varying pH of culture media. Human colon carcinoma cell line (HT-29) was exposed to the drug alone and in combination with radiation at different pH of the cell culture media. The end point was clonogenic ability of the single-plated cells after the treatment. RESULTS: Cytotoxicity and radiosensitization of IB increased with higher drug concentration and longer exposure time. The most significant radiosensitization was seen with IB (1.5 mM) for 2-h treatment at pH 6.7 before irradiation. The dose-modifying factor as defined by the ratio of radiation doses required to achieve the same effect on cell survival was 1.8 at 10% survival level. In contrast, SC-236 (50 microM for 2-8 h) showed no pH-dependent cytotoxicity. There was modest increase in the cell killing at lower doses of radiation. CONCLUSION: An acidic pH was an important factor affecting the increased cytotoxicity and radiosensitization by ibuprofen. Radiation response was enhanced at shoulder portion of the cell survival curve by selective COX-2 inhibitor.

Cell Survival↗
PubMed2002

Possible association of NTDs with a polyhistidine tract polymorphism in the ZIC2 gene.

Neural tube defects (NTDs) and brain malformations represent a common finding in chromosome 13q deletion patients. Hemizygosity for ZIC2, which is located in the 13q32 critical deletion region, results in holoprosencephaly (HPE) in humans, and diminished expression of ZIC2 results in HPE as well as lumbosacral NTDs in mice. Taken together, these observations led us to hypothesize that ZIC2 mutations may be a cause of isolated NTD. To test this, we screened 192 NTD patients for mutations in ZIC2. While we did not find ZIC2 mutations in these patients, we did find some evidence of a possible association between a histidine tract polymorphism in ZIC2 and NTDs. Our sample was too small to reach definitive conclusions, but the evidence is sufficiently intriguing to encourage further research. If this association is confirmed, subtle alterations in ZIC2 activity may confer a risk of NTD.

Alleles↗
PubMed2002

Design, synthesis, and characterization of potent, slow-binding inhibitors that are selective for gelatinases.

Gelatinases have been shown to play a key role in angiogenesis and tumor metastasis. Small molecular weight synthetic inhibitors for these enzymes are highly sought for potential use as anti-metastatic agents. Virtually all of the known inhibitors of matrix metalloproteinases (MMPs) are broad spectrum. We report herein the synthesis and kinetic characterization of two compounds, 4-(4-phenoxyphenylsulfonyl)butane-1,2-dithiol (compound 1) and 5-(4-phenoxyphenylsulfonyl)pentane-1,2-dithiol (compound 2), that are potent and selective gelatinase inhibitors. These compounds are slow, tight-binding inhibitors of gelatinases (MMP-2 and MMP-9) with K(i) values in the nanomolar range. In contrast, competitive inhibition of the catalytic domain of membrane-type 1 metalloproteinase (MMP-14(cat)) with comparable K(i) values (K(i) approximately 200 nm) was observed. Binding to stromelysin (MMP-3) was substantially weaker, with K(i) values in the micromolar range (K(i) approximately 10 microm). No binding to matrilysin (MMP-7) and collagenase 1 (MMP-1) was detected at inhibitor concentrations up to 60 microm. We have previously shown that synthetic MMP inhibitors work synergistically with TIMP-2 in the promotion of pro-MMP-2 activation by MT1-MMP in a process that depends on the affinity of the inhibitor toward MT1-MMP. It is shown herein that the dithiols are significantly less efficient (>100-fold) than marimastat, a broad-spectrum MMP inhibitor, in enhancing pro-MMP-2 activation in cells infected to express MT1-MMP, consistent with the lower affinity of the dithiols toward MT1-MMP. Thus, in contrast to broad-spectrum MMP inhibitors, the dithiols are less likely to promote MT1-MMP-dependent pro-MMP-2 activation in the presence of TIMP-2, while maintaining their ability to inhibit active MMP-2 effectively.

Catalytic Domain↗
PubMed2002

Rapid prenatal diagnosis of sickle cell diseases using oligonucleotide ligation assay coupled with laser-induced capillary fluorescence detection.

Prenatal diagnosis of sickle cell diseases has been available for several years, and our laboratory has performed over 1000 prenatal diagnoses. However, currently available techniques are labor-intensive and time-consuming, and thus the diagnosis is delayed, making the mother's decision difficult. We describe a rapid, high-throughput technique based on the ligation assay coupled with automated capillary fluorescence detection. This new approach allows the diagnosis of both Hgb S and Hgb C to be available in a few hours. We have utilized this technique in 30 prenatal diagnoses and found it to be in complete agreement with the standard diagnoses.

Anemia, Sickle Cell↗
PubMed2002

Effect of trained Seizure Alert Dogs on frequency of tonic-clonic seizures.

We have previously reported that dogs can be trained to recognize specific changes preceding an epileptic seizure in humans. Such dogs can provide an overt signal that acts as a useful warning to the human. Early observations suggested that seizure frequency might also be reduced. We report a prospective study of 10 consecutive referrals to our Seizure Alert Dogs service of people with tonic-clonic seizures. Seizure frequency was monitored over a 48 week period including 12 weeks baseline after entry, a 12 week training period, and 24 weeks follow up. Comparing baseline seizure frequency to the last 12 weeks of follow up, there was a 43% mean reduction in seizure frequency ( P= 0.002). Nine out of /10 subjects showed a 34% or greater reduction, 4 /10 showed a 50% or greater reduction, and only one showed no improvement. Although a significant drop in seizure frequency was seen during the first 4 weeks of training ( P= 0.0078) a further drop occurred between the first and last 4 week period of training (P = 0.038) and this final improvement was maintained for the whole 24 week follow up.

Adult↗
PubMed2002

Effects of prostratin on T-cell activation and human immunodeficiency virus latency.

Human immunodeficiency virus (HIV) replication is linked to cellular gene transcription and requires target cell activation. The latent reservoir of HIV-1 in quiescent T cells is thought to be a major obstacle to clearance of infection by highly active antiretroviral therapy (HAART). Thus, identification of agents that can induce expression of latent virus may, in the presence of HAART, allow elimination of the infected cells by the immune response. We previously used the SCID-hu (Thy/Liv) mouse model to establish that activation-inducible HIV can be generated at high frequency during thymopoiesis. Latently infected mature thymocytes can be exported into the periphery, providing an efficient primary cell model to determine cellular activation signals that induce renewed expression of latent virus. Here we characterized the effects of prostratin, a non-tumor-promoting phorbol ester, on primary human peripheral blood lymphocytes (PBLs) and assessed its ability to reactivate latent HIV infection from thymocytes and PBLs in the SCID-hu (Thy/Liv) model. Prostratin stimulation alone did not induce proliferation of quiescent PBLs; however, it could provide a secondary signal in the context of T-cell receptor stimulation or a primary activation signal in the presence of CD28 stimulation to induce T-cell proliferation. While prostratin alone was not sufficient to allow de novo HIV infection, it efficiently reactivated HIV expression from latently infected cells generated in the SCID-hu mouse. Our data indicate that prostratin alone is able to specifically reactivate latent virus in the absence of cellular proliferation, making it an attractive candidate for further study as an adjunctive therapy for the elimination of the latent HIV reservoir.

Animals↗
PubMed2002

Personal view: randomized controlled trials in epilepsy specialist nursing: the seduction of content by form.

Research into the effectiveness of epilepsy specialist nursing needs to take into account a number of factors, which have not been adequately addressed in previous studies. Nursing outcome measures are different to medical ones and it is inappropriate to confuse these. Specialist nurses affect the whole culture of a service, and their impact on service quality may go beyond that of their individual patient contacts. Thus randomized studies within a service that already has specialist nurses may not give valid results. Some service users will benefit more from direct contact with a specialist nurse than others, and people who give informed consent to take part in randomized controlled trials might not be representative of those who would benefit most from specialist nurse access. The stampede for level one evidence risks failing to address the issues properly by overvaluing research process (form) against its appropriateness (content), yet there remain great opportunities for good quality research in this area.

Humans↗