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Biomedical subjects

B A Lynch

Publications and source records attributed to B A Lynch.

15 recordsLinked to original sources
PubMed2003

Effect of in vivo lymphocyte-depleting strategies on development of lymphoproliferative disorders in children post allogeneic bone marrow transplantation.

T cell depletion (TCD) of marrow is a proven method of graft-versus-host disease (GVHD) prophylaxis in allogeneic bone marrow transplantation (BMT). Nonetheless, TCD is associated with an increased risk of developing post transplant lymphoproliferative disorder (PTLD). Between 1986 and 1998, 241 pediatric patients at the University of Iowa underwent BMT using ex vivo TCD of marrow from mismatched related or matched unrelated donors. Additional GVHD prophylaxis included antithymocyte globulin (ATG) or anti lymphocyte globulin (ALG) post transplant (in vivo TCD). A total of 30 cases of PTLD were identified based upon a combination of clinical, histological, and immunological features. Nearly all cases occurred within 3 months post BMT. A statistically significant increase in PTLD incidence was noted for patients treated with ATG vs ALG (33 vs 9%). While grade I-II acute GVHD was more common in patients receiving ATG vs ALG, no difference in grade III-IV GVHD or overall survival was noted between the two groups. Assessment of immune recovery at various times post BMT revealed significantly fewer T cells in the ATG-treated group, suggesting the deleterious effect of ATG may be due to excessive depletion of donor-derived Epstein-Barr virus-specific cytotoxic T cells. Thus, caution should be exercised in the use of anti-T-cell antibody therapy for additional GVHD prophylaxis in the setting of TCD BMT.

Antilymphocyte Serum↗
PubMed2001

Bone-targeted Src SH2 inhibitors block Src cellular activity and osteoclast-mediated resorption.

Src, a nonreceptor tyrosine kinase, is an important regulator of osteoclast-mediated resorption. We have investigated whether compounds that bind to the Src SH2 domain inhibit Src activity in cells and decrease osteoclast-mediated resorption. Compounds were examined for binding to the Src SH2 domain in vitro using a fluorescence polarization binding assay. Experiments were carried out with compounds demonstrating in vitro binding activity (nmol/L range) to determine if they inhibit Src SH2 binding and Src function in cells, demonstrate blockade of Src signaling, and lack cellular toxicity. Cell-based assays included: (1) a mammalian two-hybrid assay; (2) morphological reversion and growth inhibition of cSrcY527F-transformed cells; and (3) inhibition of cortactin phosphorylation in csk-/- cells. The Src SH2 binding compounds inhibit Src activity in all three of these mechanism-based assays. The compounds described were synthesized to contain nonhydrolyzable phosphotyrosine mimics that bind to bone. These compounds were further tested and found to inhibit rabbit osteoclast-mediated resorption of dentine. These results indicate that compounds that bind to the Src SH2 domain can inhibit Src activity in cells and inhibit osteoclast-mediated resorption.

Actins↗
PubMed2001

Expression and isolation of antimicrobial small molecules from soil DNA libraries.

Natural products have been a critically important source of clinically relevant small molecule therapeutics. However, the discovery rate of novel structural classes of antimicrobial molecules has declined. Recently, increasing evidence has shown that the number of species cultivated from soil represents less than 1% of the total population, opening up the exciting possibility that these uncultured species may provide a large untapped pool from which novel natural products can be discovered. We have constructed and expressed in E. coli a BAC (bacterial artificial chromosome) library containing genomic fragments of DNA (5-120kb) isolated directly from soil organisms (S-DNA). Screening of the library resulted in the identification of several antimicrobial activities expressed by different recombinant clones. One clone (mg1.1) has been partially characterized and found to express several small molecules related to and including indirubin. These results show that genes involved in natural product synthesis can be cloned directly from S-DNA and expressed in a heterologous host, supporting the idea that this technology has the potential to provide novel natural products from the wealth of environmental microbial diversity and is a potentially important new tool for drug discovery.

Amino Acid Sequence↗
PubMed2000

Cloning the soil metagenome: a strategy for accessing the genetic and functional diversity of uncultured microorganisms.

Recent progress in molecular microbial ecology has revealed that traditional culturing methods fail to represent the scope of microbial diversity in nature, since only a small proportion of viable microorganisms in a sample are recovered by culturing techniques. To develop methods to investigate the full extent of microbial diversity, we used a bacterial artificial chromosome (BAC) vector to construct libraries of genomic DNA isolated directly from soil (termed metagenomic libraries). To date, we have constructed two such libraries, which contain more than 1 Gbp of DNA. Phylogenetic analysis of 16S rRNA gene sequences recovered from one of the libraries indicates that the BAC libraries contain DNA from a wide diversity of microbial phyla, including sequences from diverse taxa such as the low-G+C, gram-positive Acidobacterium, Cytophagales, and Proteobacteria. Initial screening of the libraries in Escherichia coli identified several clones that express heterologous genes from the inserts, confirming that the BAC vector can be used to maintain, express, and analyze environmental DNA. The phenotypes expressed by these clones include antibacterial, lipase, amylase, nuclease, and hemolytic activities. Metagenomic libraries are a powerful tool for exploring soil microbial diversity, providing access to the genetic information of uncultured soil microorganisms. Such libraries will be the basis of new initiatives to conduct genomic studies that link phylogenetic and functional information about the microbiota of environments dominated by microorganisms that are refractory to cultivation.

Amino Acid Sequence↗
PubMed1999

Simultaneous assay of Src SH3 and SH2 domain binding using different wavelength fluorescence polarization probes.

pp60(c-src) is a prototypical nonreceptor tyrosine kinase and may play a role in diseases as diverse as cancer and osteoporosis. In Src, the SH3 domain (Src homology 3) binds proteins at specific, proline-rich sequences, while the SH2 domain (Src homology 2) binds phosphotyrosine-containing sequences. Inhibition of Src SH3 and SH2 domain function is of potential therapeutic value because of their importance in signaling pathways involved in disease states. We have developed dual-wavelength fluorescent peptide probes for both the Src SH3 and the Src SH2 domains, which allow the simultaneous measurement of compounds binding to each domain in assays based on the technique of fluorescence polarization. We demonstrate the utility of these probes in a dual-binding assay (suitable for high-throughput screening) to study the interactions of various peptides with these domains, including a sequence from the rat protein p130(CAS) which has been reported to bind simultaneously to both Src SH3 and SH2 domains. Utilizing this dual-binding assay, we confirm that sequences from p130(CAS) can simultaneously bind Src via both its SH3 and its SH2 domains. We also use the dual-binding assay as an internal control to identify substances which inhibit SH3 and SH2 binding via nonspecific mechanisms.

Animals↗
PubMed1999

Discovery of potent and selective SH2 inhibitors of the tyrosine kinase ZAP-70.

A series of 1,2,4-oxadiazole analogues has been shown to be potent and selective SH2 inhibitors of the tyrosine kinase ZAP-70, a potential therapeutic target for immune suppression. These compounds typically are 200-400-fold more potent than the native, monophosphorylated tetrapeptide sequences. When compared with the high-affinity zeta-1-ITAM peptide (Ac-NQL-pYNELNLGRREE-pYDVLD-NH(2), wherein pY refers to phosphotyrosine) some of the best 1,2, 4-oxadiazole analogues are approximately 1 order of magnitude less active. This series of compounds displays an unprecedented level of selectivity over the closely related tyrosine kinase Syk, as well as other SH2-containing proteins such as Src and Grb2. Gel shift studies using a protein construct consisting only of C-terminal ZAP-70 SH2 demonstrate that these compounds can effectively engage this particular SH2 domain.

Enzyme Inhibitors↗
PubMed1999

Structure-activity relationships of a novel class of Src SH2 inhibitors.

The structure-activity relationships (SAR) of a novel class of Src SH2 inhibitors are described. Variation at the pY+1 and pY+3 side chain positions using 2,4- and 2,5-substituted thiazoles and 1,2,4-oxadiazoles as scaffolds resulted in inhibitors that bound as well as the standard tetrapeptide Ac-pYEEI-NH2.

Structure-Activity Relationship↗
PubMed1997

A fluorescence polarization based Src-SH2 binding assay.

The tyrosine kinase pp60c.src has been implicated as being a potential therapeutic target in several human diseases including cancer and osteoporosis. An important region within this kinase is the SH2 domain (Src homology 2) which binds to phosphorylated tyrosine residues contained within specific peptide sequences. Homologous domains are found in a variety of cytoplasmic proteins and have been shown to be essential for controlling many important signaling pathways. Developing specific inhibitors of SH2 interactions would therefore be extremely useful for modulating a variety of signaling pathways and potentially be useful for the treatment of human disease. Current methodology for the development of organic molecules as drug leads requires the ability to test thousands of individual compounds or natural product extracts in biochemical assays. Such tests must be reproducible, simple, and versatile. This paper describes an assay based on fluorescence polarization for measuring the binding of compounds to the Src-SH2 domain. The assay is insensitive to changes in fluorescence intensity working even in solutions with moderate optical density and functions in the presence of up to 20% dimethyl sulfoxide. These features make it especially useful for high-throughput screening of both natural and synthetic compound libraries.

Amino Acid Sequence↗
PubMed1992

The fifth Datta Lecture. Structural similarities between the aspartate receptor of bacterial chemotaxis and the trp repressor of E. coli. Implications for transmembrane signaling.

A high resolution structure of the N-terminal ligand-binding domain of the aspartate receptor which mediates aspartate chemotaxis in Salmonella typhimurium has recently been reported. A least-squares superposition of the alpha-amino nitrogen, alpha-carbon, beta-carbon, and alpha-carboxylate carbon of the aspartate bound to the aspartate receptor onto the equivalent atoms in the tryptophan bound to the trp repressor provides evidence for similarity between key parts of the active sites that bind to the alpha-amino and alpha-carboxylates of the respective ligands. Because the N-terminal domain of the aspartate receptor and the trp repressor also share other structural similarities, we hypothesize that the similarity between the aspartate receptor and the trp repressor derives from a similarity in ligand-induced conformational changes at the active sites of these proteins. This hypothesis also implies that an important signaling event in the aspartate receptor occurs through tertiary conformational changes within a single subunit.

Aspartic Acid↗
PubMed1991

Disulfide cross-linking studies of the transmembrane regions of the aspartate sensory receptor of Escherichia coli.

The Escherichia coli aspartate receptor, a dimer of identical subunits, has two transmembrane regions (TM1, residues 7-30; TM2, residues 189-212) of 24 residues each. To study the relative placement and orientation of the regions, cysteine residues were introduced individually into the center of each: at positions 17, 18, and 19 in TM1; and at positions 198, 199, 200, and 201 in TM2. Based on the patterns of disulfide cross-linking observed between subunits in the mutant receptors, there appears to be close contact between the TM1 and TM1' regions at the dimer interface but no such direct interaction between the TM2 and TM2' regions. The cross-linking results are consistent with an alpha-helical structure extending across the transmembrane region up through at least residue 36, which lies on the periplasmic side of TM1. The ability of an 18-18' cross-linked dimer to transmit an aspartate-induced transmembrane signal is also supportive of such an extended helix. The changes in relative rates of disulfide cross-linking provide experimental evidence of a conformational change transmitted through the transmembrane domain during signaling. Once formed, disulfides between the transmembrane regions are unusually resistant to reduction by low molecular weight thiols in the presence of denaturants like SDS. These targeted disulfide cross-links can be used to reveal structural and dynamic aspects of protein function.

Amino Acid Sequence↗
PubMed1985

Effects of low protein diets or feed restriction on rat lung glutathione and oxygen toxicity.

Male rats weighing 200-250 g were fed a 25% casein diet in restricted amounts or ad libitum or one of two low protein diets (3 and 0% casein) ad libitum. Decreased tolerance to hyperoxic stress was observed only in the rats fed low protein diets. These animals had a median death time of 49-50 h compared to 58-69 h for feed-restricted or normal control groups. Death was due to accelerated development of lung edema. Changes in total lung levels of glutathione reductase, glucose-6-phosphate dehydrogenase or catalase did not correlate with oxygen sensitivity. Lung glutathione levels were related to the amount of sulfur-containing amino acids in the diet and were depressed in the feed-restricted as well as the protein-restricted groups. However, feed restriction alone did not enhance oxygen toxicity. We conclude that a decrease in lung glutathione may be partially responsible for the increased oxygen sensitivity in the protein-deficient rats, but that other factors are necessary for explanation of the relative oxygen tolerance of the feed-restricted animals with reduced levels of glutathione in the lung.

Amino Acids↗
PubMed1985

Transient depletion of lung glutathione by diethylmaleate enhances oxygen toxicity.

Diethylmaleate (DEM) decreases glutathione (GSH) levels in various organs by enzymatic conjugation with reduced GSH catalyzed by GSH transferase. We have examined levels of GSH, glutathione reductase (GR), and glucose-6-phosphate dehydrogenase (G6PD) in lungs of 200-250-g rats after intraperitoneal injection of 0.5 or 1 g DEM/kg body wt. The GSH levels are severely depressed at 2 and 4 h but have essentially recovered by 12 and 24 h after either dose of DEM. The GR and G6PD activities in the 1 g/kg group are depressed at 4 h to a lesser extent than the GSH levels and also return to normal by 12 and 24 h. These enzymes are not affected in the 0.5 g/kg group. To determine whether these transient decreases in GSH and related enzymes affected O2 tolerance, we exposed rats injected with DEM to greater than 98% O2 and found that halftime (t1/2) for survival was decreased in rats receiving both 0.5 and 1 g DEM/kg body wt when compared with untreated or saline-injected controls (t1/2 control, 74 h; 0.5 g DEM, 59 h; 1 g DEM, 53 h). No deaths occurred in air controls at 1 mg/kg DEM for up to 5 days. DEM, in itself, caused no morphological alteration of the lung. Thus a decrease in lung GSH and related enzymes, occurring by 4 h and reversed by 12 h, has a significant effect on the subsequent progression of lung pathology and indicates that early biochemical events occurring in lungs exposed to hyperoxia may be very important in determining the degree of longer-term damage to rat lungs.

Animals↗
PubMedDate not supplied

Structure-based design and solid-phase parallel synthesis of phosphorylated nonpeptides to explore hydrophobic binding at the Src SH2 domain.

Using a novel, solid-phase parallel synthetic route and a computational docking program, a series of phosphorylated nonpeptides were generated to determine their structure-activity relationships (SAR) for binding at the SH2 domain of pp60src (Src). A functionalized benzoic acid intermediate was attached to solid support via Rink amide linkage, which upon acid cleavage generated the desired benzamide template-based nonpeptides in a facile manner. Compounds were synthesized using a combination of solid- and solution-phase techniques. Purification using reversed-phase, semipreparative HPLC allowed for quantitative SAR studies. Specifically, this work focused on functional group modifications, in a parallel fashion, designed to explore hydrophobic binding at the pY+3 pocket of the Src SH2 domain.

Benzamides↗