Biomedical subjects

L Wang

Publications and source records attributed to L Wang.

At least 307 records · Page 17Linked to original sources

PubMed2001

PKA, PKC, and AKAP localization in and around the neuromuscular junction.

BACKGROUND: One mechanism that directs the action of the second messengers, cAMP and diacylglycerol, is the compartmentalization of protein kinase A (PKA) and protein kinase C (PKC). A-kinase anchoring proteins (AKAPs) can recruit both enzymes to specific subcellular locations via interactions with the various isoforms of each family of kinases. We found previously that a new class of AKAPs, dual-specific AKAPs, denoted D-AKAP1 and D-AKAP2, bind to RIalpha in addition to the RII subunits. RESULTS: Immunohistochemistry and confocal microscopy were used here to determine that D-AKAP1 colocalizes with RIalpha at the postsynaptic membrane of the vertebrate neuromuscular junction (NMJ) and the adjacent muscle, but not in the presynaptic region. The labeling pattern for RIalpha and D-AKAP1 overlapped with mitochondrial staining in the muscle fibers, consistent with our previous work showing D-AKAP1 association with mitochondria in cultured cells. The immunoreactivity of D-AKAP2 was distinct from that of D-AKAP1. We also report here that even though the PKA type II subunits (RIIalpha and RIIbeta) are localized at the NMJ, their patterns are distinctive and differ from the other R and D-AKAP patterns examined. PKCbeta appeared to colocalize with the AKAP, gravin, at the postsynaptic membrane. CONCLUSIONS: The kinases and AKAPs investigated have distinct patterns of colocalization, which suggest a complex arrangement of signaling micro-environments. Because the labeling patterns for RIalpha and D-AKAP 1 are similar in the muscle fibers and at the postsynaptic membrane, it may be that this AKAP anchors RIalpha in these regions. Likewise, gravin may be an anchor of PKCbeta at the NMJ.

A Kinase Anchor Proteins↗

PubMed2001

Human Rad17 is phosphorylated upon DNA damage and also overexpressed in primary non-small cell lung cancer tissues.

The spRAD17 gene is an essential component of the DNA damage and replication checkpoints in the fission yeast Schizosaccharomyces pombe. Cloning of the human homologue of spRAD17, hRAD17, indicated that it exhibits structural similarity with the replication accessory protein family, which include subunits of the Replication factor C complex. We have analyzed the phosphorylation status of hRad17 in response to DNA damaging agents. Our results showed that phosphorylation of hRad17 occurred immediately after UV and ionizing radiation treatment and reached peak level at approximately 3 h, suggesting that hRad17 may be a component of the DNA damage checkpoint. When primary tumor samples were analyzed, we observed that the majority (74%) of non-small cell lung carcinoma samples exhibited a significantly higher level of hRad17 expression compared with matched normal tissue controls. In contrast, hRad17 protein levels in a panel of primary colon carcinoma samples did not show an elevated level of expression compared with normal colon tissues. This observation suggests that the function of the hRAD17 gene may be involved in lung cancer development and may serve as a potential tumor marker.

Carcinoma, Non-Small-Cell Lung↗

PubMed2001

Production of ultra highly pure H2 and higher hydrocarbons from methane in one step at mild temperatures and development of the catalyst under non-equilibrium reaction conditions.

Ultra highly pure hydrogen and more valuable hydrocarbons are produced directly from methane in one step beyond the thermodynamic equilibrium conversion by integration of the dehydrogenation reaction and hydrogen separation with a Pd-Ag based membrane reactor at mild temperatures, and a highly active catalyst is developed under the non-equilibrium reaction conditions.

Journal Article↗

PubMed2001

Modulation of recombinant GABA receptor/channel subunits by domain-specific antibodies in Xenopus oocytes.

To study interaction of specific antibodies with the GABA receptor/channel, antisera were raised against the extracellular domains of the GABAA receptor/channel beta2 subunit, gamma2 subunit and the GABAC receptor/channel rho1 subunit. The specificity of the antibodies was characterized by immunocytochemistry and by Western blotting of transfected FDC-P1 cells expressing recombinant GABA receptor/channel subunits. The effects of the antibodies on whole-cell currents in Xenopus laevis oocytes expressing homomeric recombinant GABA receptor/channel beta2, gamma2, and rho1 were studied using two-microelectrode voltage clamp. In the absence of GABA, anti-alpha2, anti-gamma2, and anti-rho1 antisera elicited whole-cell currents in oocytes expressing beta2, gamma2, and rho1 subunits, respectively. The effect of antibody on channel activation was concentration-dependent. The whole-cell currents induced by anti-beta2 and anti-gamma2 were several-fold greater than those induced by application of 100 microm GABA. In Xenopus oocytes expressing recombinant rho1 subunits, GABA-induced whole-cell currents were inhibited by the anti-rho1 antibody. In contrast, the GABA-induced whole-cell currents were potentiated several-fold by anti-beta2 and anti-gamma2 antibodies in Xenopus oocytes expressing homomeric beta2 and gamma2 subunits. Our studies indicate that antibodies specific to the N-terminal domain of GABA receptor/channel subunits can modulate the neurotransmitter receptor function.

Animals↗

PubMed2001

Polycomblike PHD fingers mediate conserved interaction with enhancer of zeste protein.

The products of Polycomb group (PcG) genes are required for the epigenetic repression of a number of important developmental regulatory genes, including homeotic genes. Enhancer of zeste (E(Z)) is a Drosophila PcG protein that previously has been shown to bind directly to another PcG protein, Extra Sex Combs (ESC), and is present along with ESC in a 600-kDa complex in Drosophila embryos. Using yeast two-hybrid and in vitro binding assays, we show that E(Z) binds directly to another PcG protein, Polycomblike (PCL). PCL.E(Z) interaction is shown to be mediated by the plant homeodomain (PHD) fingers domain of PCL, providing evidence that this motif can act as an independent protein interaction domain. An association was also observed between PHF1 and EZH2, human homologs of PCL and E(Z), respectively, demonstrating the evolutionary conservation of this interaction. E(Z) was found to not interact with the PHD domains of three Drosophila trithorax group (trxG) proteins, which function to maintain the transcriptional activity of homeotic genes, providing evidence for the specificity of the interaction of E(Z) with the PCL PHD domain. Coimmunoprecipitation and gel filtration experiments demonstrate in vivo association of PCL with E(Z) and ESC in Drosophila embryos. We discuss the implications of PCL association with ESC.E(Z) complexes and the possibility that PCL may either be a subunit of a subset of ESC.E(Z) complexes or a subunit of a separate complex that interacts with ESC.E(Z) complexes.

Amino Acid Motifs↗

PubMed2001

Analyses of p53 target genes in the human genome by bioinformatic and microarray approaches.

The completion of the human genome sequence (International Human Genome Sequence Consortium (2001) Nature 409, 860-921; Venter, J. C., et al. (2001) Science 291, 1304-1351) allows for new ways to analyze global cellular regulatory mechanisms. Here we present a strategy to identify genes regulated by specific transcription factors in the human genome, and apply it to p53. We first collected promoters or introns of all genes available using two methods: GenBank(TM) annotation and a computationally derived transcript map. 4,852 genes analyzed in this way contained at least one p53 consensus binding sequence. Of 13 genes randomly selected for mRNA analysis, 11 were shown to respond to p53 expression. Five promoters were analyzed by chromatin immunoprecipitation, which revealed that all were bound by p53 in vivo. We then analyzed 33,615 unique human genes on cDNA microarrays, identifying 1,501 genes that respond to p53 expression. A parameter was derived that demonstrates that in silico prediction greatly enriches for genes that are activated and repressed by p53 and assists us to suggest other signaling pathways that may be connected to p53. The methods shown here illustrate a novel approach to analysis of global gene regulatory network through the integration of human genomic sequence information and genome-wide gene expression analysis.

Computational Biology↗

PubMed2001

Intrahepatic expression and release of vascular endothelial growth factor following orthotopic liver transplantation in the rat.

BACKGROUND: Morphological and functional changes to sinusoidal endothelial cells mediated by soluble factors released from activated Kupffer cells, including cytokines, are considered pivotal events in ischemia/reperfusion injury (IRI) to liver grafts. Vascular endothelial growth factor (VEGF) is an endothelial cell-specific cytokine with potent pro-inflammatory and mitogenic effects. We investigated the possible role of VEGF in IRI to liver grafts using a syngeneic rat orthotopic liver transplantation model. METHODS: Transplantation was performed in Lewis rats using livers preserved for various periods of time (24-48 hr) in University of Wisconsin solution at 4 degrees C. Systemic VEGF levels were measured by enzyme-linked immunosorbent assay (ELISA). Intrahepatic VEGF expression was analyzed by Northern blotting and in situ hybridization. The effects of anti-VEGF neutralizing antibody treatment on the extent of IRI were assessed by measuring liver function tests, lipid peroxidation, and metalloproteinase activity. RESULTS/CONCLUSION: VEGF is expressed and released in a biphasic pattern during the early postoperative period after liver transplantation. Anti-VEGF antibody treatment, administered during reperfusion, decreased the degree of damage, suggesting that VEGF may have a role in IRI to liver grafts.

Animals↗

PubMed2001

Sequence analysis of the Spodoptera litura multicapsid nucleopolyhedrovirus genome.

The complete Spodoptera litura multicapsid nucleopolyhedrovirus (SpltMNPV) genome contained 139,342 bp with a G+C content of 42.7%, and 141 putative open reading frames (ORFs) or genes of 150 nucleotides or greater that showed minimal overlap. Ninety-six ORFs had homologues in Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV), 16 had homologues in other baculoviruses, and 29 were unique to SpltMNPV. The homologues of ubiquitin and gp37 are fused in SpltMNPV. The genome lacked a homologue of the major budded virus glycoprotein gene gp64, but it contained a homologue of ORF130 of Lymantria dispar multicapsid nucleopolyhedrovirus (LdMNPV). There were two homologues of AcMNPV ORF2 (bro gene), and a DnaJ protein gene (SpltORF39) in which the N-terminus showed homologies with the J domain of DnaJ family proteins. Seventeen homologous regions (hrs) were identified, each containing 2-29 palindromic repeats, with an average length of 534 bp and base content (G+C%) of 33.0.

Amino Acid Sequence↗

PubMed2001

Vgamma2Vdelta2 T-cell receptor-mediated recognition of aminobisphosphonates.

Aminobisphosphonates, potent derivatives of bisphosphonates, are frequently used for the treatment of conditions such as osteoporosis and bone metastases that are characterized by excessive osteoclastic bone resorption. Using T-cell receptor (TCR) transfer studies, we show that recognition of antigenic aminobisphosphonates that are known to stimulate human gammadelta T cells in vitro and in vivo (potency: risedronate > alendronate > pamidronate) requires expression of the Vgamma2Vdelta2 TCR and is thus Vgamma2Vdelta2 TCR-dependent. Myeloma cells or monocytes pulsed with risedronate and then washed rendered these target cells sensitive to lysis by a Vgamma2Vdelta2 T-cell clone or cell line. These results suggest that Vgamma2Vdelta2 TCR-dependent recognition leading to direct cytolysis of aminobisphosphonate-sensitized osteoclast or tumor targets may be a mechanism whereby aminobisphosphonate treatment of cancers metastatic to bone decreases osteoclastic activity and tumor burden and also may account for the decreased osteoclastic activity associated with successful treatment of osteoporosis.

Alendronate↗

PubMed2001

Plasmid vaccination with insulin B chain prevents autoimmune diabetes in nonobese diabetic mice.

The insulin B (InsB) chain bears major type 1 diabetes-associated epitopes of significance for disease in humans and nonobese diabetic (NOD) mice. Somatic expression of InsB chain initiated early in life by plasmid inoculation resulted in substantial protection of female NOD mice against disease. This was associated with a T2 shift in spleen, expansion of IL-4-producing and, to a lesser extent, of IFN-gamma-secreting T cells in pancreatic lymph nodes, as well as intermolecular Th2 epitope spreading to glutamic acid decarboxylase determinants. A critical role of IL-4 for the Ag-specific protective effect triggered by plasmid administration was revealed in female IL-4(-/-) NOD mice that developed diabetes and higher Th1 responses. Coadministration of IL-4-expressing plasmid or extension of the vaccination schedule corrected the unfavorable response of male NOD mice to DNA vaccination with InsB chain. Thus, plasmid-mediated expression of the InsB chain early in diabetes-prone mice has the potential to prevent transition to full-blown disease depending on the presence of IL-4.

Animals↗

PubMed2001

Role of HPC2/ELAC2 in hereditary prostate cancer.

The HPC2/ELAC2 gene on chromosome 17p was recently identified as a candidate gene for hereditary prostate cancer (HPC). To confirm these findings, we screened 300 prostate cancer patients (2 affected members/family) from 150 families with HPC for potential germ-line mutations using conformation-sensitive gel electrophoresis, followed by direct sequence analysis. The minimum criteria for our families with HPC was the presence of 3 affected men with prostate cancer. A total of 23 variants were identified, including 13 intronic and 10 exonic changes. Of the 10 exonic changes, 1 truncating mutation was identified, a Glu216Stop nonsense mutation. This nonsense variant was found in 2 of 3 affected men in a single family. The remaining nine alterations included five missense, three silent, and one variant in the 3' untranslated region. To additionally test for potential associations of polymorphic variants and increased risk for disease, we genotyped two common polymorphisms, Ser217Leu and Ala541Thr, in 446 prostate cancer patients from 164 families with HPC and 502 population-based controls. The frequency of the Leu217 variant was similar for patients (32.3%) and controls (31.8%), as was the frequency of the Thr541 variant (5.4% among patients versus 5.2% among controls). In contrast to previous reports, we found no association of the joint effects of Leu271 and Thr541 (odds ratio, 1.04; 95% confidence interval, 0.57-1.89). Overall, our results did not reveal any association between these two common polymorphisms and the risk for HPC. The finding of a nonsense mutation in the HPC2/ELAC2 gene confirms its potential role in genetic susceptibility to prostate cancer. However, our data also suggest that germ-line mutations of the HPC2/ELAC2 are rare in HPC and that the variants Leu217 and Thr541 do not appear to influence the risk for HPC. Cumulatively, these results suggest that alterations within the HPC2/ELAC2 gene play a limited role in genetic susceptibility to HPC.

Adult↗

PubMed2001

Recognition of ATGA sequences by the unfused aromatic dication DB293 forming stacked dimers in the DNA minor groove.

Furamidine and related diamidines represent a promising series of drugs active against widespread parasites, in particular the Pneumocystic carinii pathogen. In this series, the phenylfuranbenzimidazole diamidine derivative DB293 was recently identified as the first unfused aromatic dication capable of forming stacked dimers in the DNA minor groove of GC-containing sequences. Here we present a detailed biochemical and biophysical characterization of the DNA sequence recognition properties of DB293. Three complementary footprinting techniques using DNase I, Fe(II)-EDTA, and an anthraquinone photonuclease were employed to locate binding sites for DB293 in different DNA restriction fragments. Two categories of sites were identified by DNase I footprinting: (i) 4/5 bp sequences containing contiguous A.T pairs, such as 5'-AAAA and 5'-ATTA; and (ii) sequences including the motif 5'-ATGA.5'-TCAT. In particular, a 13-bp sequence including two contiguous ATGA motifs provided a highly preferential recognition site for DB293. Quantitative footprinting analysis revealed better occupancy of the 5'-ATGA site compared to the AT-rich sites. Preferential binding of DB293 to ATGA sites was also observed with other DNA fragments and was confirmed independently by means of hydroxyl radical footprinting generated by the Fe(II)-EDTA system, as well as by a photofootprinting approach using the probe anthraquinone-2-sulfonate (AQS). In addition, this photosensitive reagent revealed the presence of sites of enhanced cutting specific to DB293. This molecule, but not other minor groove binders such as netropsin, induces specific local structural changes in DNA near certain binding sites, as independently shown by DNase I and the AQS probe. Recognition of the ATGA sequence by DB293 was investigated further using melting temperature experiments and surface plasmon resonance (SPR). The use of different hairpin oligonucleotides showed that DB293 can interact with AT sites via the formation of 1:1 drug-DNA complexes but binds much more strongly, and cooperatively, to ATGA-containing sequences to form 2:1 drug-DNA complexes. DB293 binds strongly to ATGA sequences with no significant context dependence but is highly sensitive to the orientation of the target sequence. The formation of 2:1 DB293/DNA complexes is abolished by reversing the sequence 5'-ATGA-->3'-ATGA, indicating that directionality plays an important role in the drug-DNA recognition process. Similarly, a single mutation in the A[T-->G]GA sequence is very detrimental to the dimer interactions of DB293. From the complementary footprinting and SPR data, the 5'-ATGA sequence is identified as being a highly favored dimer binding site for DB293. The data provide clues for delineating a recognition code for diamidine-type minor groove binding agents, and ultimately to guide the rational design of gene regulatory molecules targeted to specific sites of the genetic material.

Antifungal Agents↗

PubMed2001

Identification and characterization of a novel androgen receptor coregulator ARA267-alpha in prostate cancer cells.

The androgen receptor (AR) is a member of the steroid receptor superfamily that binds to the androgen response element to regulate target gene transcription. AR may need to interact with some selected coregulators for maximal or proper androgen function. Here we report the isolation of a new AR coregulator with a calculated molecular mass of 267 kDa named the androgen receptor-associated protein 267-alpha (ARA267-alpha). ARA267-alpha contains 2427 amino acids, including one Su(var)3-9, Enhancer-of-zeste, and Trithorax (SET) domain, two LXXLL motifs, three nuclear translocation signal (NLS) sequences, and four plant homeodomain (PHD) finger domains. Northern blot analyses reveal that ARA267-alpha is expressed predominantly in the lymph node as 13- and 10-kilobase transcripts. HepG2 is the only cell line tested that does not express ARA267-alpha. Yeast two-hybrid and glutathione S-transferase pull-down assays show that both the N and C terminus of ARA267-alpha interact with the AR DNA- and ligand-binding domains. Unlike other coregulators, such as CBP, which enhance the interaction between the N and C terminus of AR, we found that ARA267-alpha had little influence on the interaction between the N and C terminus of AR. Luciferase and chloramphenicol acetyltransferase assays show that ARA267-alpha can enhance AR transactivation in a dihydrotestosterone-dependent manner in PC-3 and H1299 cells. ARA267-alpha can also enhance AR transactivation with other coregulators, such as ARA24 or PCAF, a histone acetylase, in an additive manner. Together, our data demonstrate that ARA267-alpha is a new AR coregulator containing the SET domain with an exceptionally large molecular mass that can enhance AR transactivation in prostate cancer cells.

Amino Acid Sequence↗

PubMed2001

Therapeutic benefit of intracerebral transplantation of bone marrow stromal cells after cerebral ischemia in rats.

We tested the hypothesis that bone marrow stromal cells (MSCs) transplanted into the ischemic boundary zone, survive, differentiate and improve functional recovery after middle cerebral artery occlusion (MCAo). MSCs were harvested from adult rats and cultured with or without nerve growth factor (NGF). For cellular identification, MSCs were prelabeled with bromodeoxyuridine (BrdU). Rats (n=24) were subjected to 2 h of MCAo, received grafts at 24 h and were euthanized at 14 days after MCAo. Test groups consisted of: (1) control-MCAo alone (n=8); (2) intracerebral transplantation of MSCs (n=8); (3) intracerebral transplantation of MSCs cultured with NGF (n=8). Immunohistochemistry was used to identify cells from MSCs. Behavioral tests (rotarod, adhesive-removal and modified neurological severity score [NSS]) were performed before and after MCAo. The data demonstrate that MSCs survive, migrate and differentiate into phenotypic neural cells. Significant recovery of somatosensory behavior (p<0.05) and NSS (p<0.05) were found in animals transplanted with MSCs compared with control animals. Animals that received MSCs cultured with NGF displayed significant recovery in motor (p<0.05), somatosensory (p<0.05) and NSS (p<0.05) behavioral tests compared with control animals. Our data suggest that intracerebral transplantation of MSCs may provide a powerful autoplastic therapy for stroke.

Animals↗

PubMed2001

UV Induces phosphorylation of protein kinase B (Akt) at Ser-473 and Thr-308 in mouse epidermal Cl 41 cells through hydrogen peroxide.

The exposure of mammalian cells to UV irradiation leads to the activation of transcription factors and protein kinases, which are believed to be responsible for the carcinogenic effects of excessive sun exposure. The present study investigated the effect of UV exposure on reactive oxygen species (ROS) generation and protein kinase B (Akt) phosphorylation in epidermal cells and determined if a relationship exists between these UV responses. Exposure of mouse epidermal JB6 Cl 41 cells to UV radiation led to specific phosphorylation of Akt at Ser-473 and Thr-308 in a time-dependent manner. This phosphorylation was confirmed by the observation that overexpression of Akt mutant, Akt-T308/S473A, attenuated phosphorylation of Akt at Ser-473 and Thr-308. UV radiation also generated ROS as measured by electron spin resonance (ESR) in JB6 Cl 41 cells. The generation of ROS by UV radiation was measured further by H(2)O(2) and O(-.2) fluorescence staining assays. The mechanism of ROS generation involved reduction of molecular oxygen to O(-.2), which generated H(2)O(2) through dismutation. H(2)O(2) produced .OH via a metal-independent pathway. The scavenging of UV-generated H(2)O(2) by N-acety-l-cyteine (NAC, a general antioxidant) or catalase (a specific H(2)O(2) inhibitor) inhibited Akt phosphorylation at Ser-473 and Thr-308, whereas the pretreatment of cells with sodium formate (an .OH radical scavenger) or superoxide dismutase (an O(-.2) radical scavenger) did not show any inhibitory effects. Furthermore, treatment of cells with H(2)O(2) increased UV-induced phosphorylation of Akt at Ser-473 and Thr-308. These results demonstrate that UV radiation generates a whole spectrum of ROS including O(-.2), .OH, and H(2)O(2) and induces phosphorylation of Akt at Ser-473. Among the various ROS, H(2)O(2) seems most potent in mediating UV-induced phosphorylation of Akt at Ser-473 and Thr-308. It is possible that Akt may play a role in the carcinogenesis effects by UV radiation.

Animals↗

PubMed2001

Effects of N-(4-hydroxyphenyl)retinamide on hTERT expression in the bronchial epithelium of cigarette smokers.

BACKGROUND: Telomerase activation plays a critical role in tumorigenesis. To determine the role of telomerase in early lung carcinogenesis and as a potential biomarker in chemoprevention trials, we analyzed the expression of the human telomerase reverse transcriptase catalytic subunit (hTERT) in bronchial biopsy specimens from cigarette smokers who were enrolled in a randomized, double-blinded, placebo-controlled chemoprevention trial of N-(4-hydroxyphenyl)retinamide (4-HPR). METHODS: We obtained biopsy specimens from six predetermined sites in the bronchial tree from the 57 participants, before treatment and 6 months after treatment with 4-HPR or placebo. We used in situ hybridization to examine hTERT messenger RNA (mRNA) expression in 266 pretreatment (baseline) and post-treatment site-paired biopsy specimens from 27 patients in the 4-HPR-treated group and from 30 patients in the placebo-treated group. All statistical tests were two-sided. RESULTS: At baseline, 62.4% (95% confidence interval [CI] = 53.9% to 71%) of the biopsy specimens obtained from the group treated with 4-HPR and 65.2% (95% CI = 57.4% to 73.1%) of the biopsy specimens obtained from the placebo-treated group expressed hTERT mRNA. After 6 months, 45.6% (95% CI = 36.9% to 54.3%) of the biopsy specimens obtained from the 4-HPR-treated group and 68.1% (95% CI = 60.4% to 75.8%) of the biopsy specimens obtained from the placebo-treated group expressed hTERT mRNA. The reduction in hTERT expression observed between the two treatment groups over time was statistically significant (P =.01) when we used the biopsy site as the unit of analysis, but not when we used the individual as the unit of analysis (P =.37). CONCLUSIONS: Telomerase is frequently reactivated in the lungs of cigarette smokers. The modulation of hTERT expression in 4-HPR-treated smokers suggests that a novel molecular mechanism underlies the potential chemopreventive properties of 4-HPR. hTERT expression is a promising potential biomarker for risk assessment and for the evaluation of the efficacy of chemopreventive agents in lung carcinogenesis.

Adult↗

PubMed2001

Analysis of single nucleotide polymorphisms with solid phase invasive cleavage reactions.

Using microparticles as the capture surface and fluorescence resonance energy transfer as the detection technology, we have demonstrated the feasibility of performing the invasive cleavage reaction on a solid phase. An effective tool for many genomic applications, the solution phase invasive cleavage assay is a signal amplification method capable of distinguishing nucleic acids that differ by only a single base mutation. The method positions two overlapping oligonucleotides, the probe and upstream oligonucleotides, on the target nucleic acid to create a complex recognized and cleaved by a structure-specific 5'-nuclease. For microarray and other multiplex applications, however, the method must be adapted to a solid phase platform. Effective cleavage of the probe oligonucleotide occurred when either of the two required overlapping oligonucleotides was configured as the particle-bound reagent and also when both oligonucleotides were attached to the solid phase. Positioning probe oligonucleotides away from the particle surface via long tethers improved both the signal and the reaction rates. The particle-based invasive cleavage reaction was capable of distinguishing the ApoE Cys158 and Arg158 alleles at target concentrations as low as 100 amol/assay (0.5 pM).

Alleles↗

PubMed2001

Design, synthesis, and structure-activity relationships of macrocyclic hydroxamic acids that inhibit tumor necrosis factor alpha release in vitro and in vivo.

To search for TNF-alpha (tumor necrosis factor alpha) converting enzyme (TACE) inhibitors, we designed a new class of macrocyclic hydroxamic acids by linking the P1 and P2' residues of acyclic anti-succinate-based hydroxamic acids. A variety of residues including amide, carbamate, alkyl, sulfonamido, Boc-amino, and amino were found to be suitable P1-P2' linkers. With an N-methylamide at P3', the 13-16-membered macrocycles prepared exhibited low micromolar activities in the inhibition of TNF-alpha release from LPS-stimulated human whole blood. Further elaboration in the P3'-P4' area using the cyclophane and cyclic carbamate templates led to the identification of a number of potent analogues with IC(50) values of </=0.2 microM in whole blood assay (WBA). Although the P3' area can accommodate a broad array of structurally diversified functional groups including polar residues, hydrophobic residues, and amino and carboxylic acid moieties, in both the cyclophane series and the cyclic carbamate series, a glycine residue at P3' was identified as a critical structural component to achieve both good in vitro potency and good oral activity. With a glycine residue at P3', an N-methylamide at P4' provided the best cyclophane analogue, SL422 (WBA IC(50) = 0.22 microM, LPS-mouse ED(50) = 15 mg/kg, po), whereas a morpholinylamide at P4' afforded the most potent and most orally active cyclic carbamate analogue, SP057 (WBA IC(50) = 0.067 microM, LPS-mouse ED(50) = 2.3 mg/kg, po). Further profiling for SL422 and SP057 showed that these macrocyclic compounds are potent TACE inhibitors, with K(i) values of 12 and 4.2 nM in the porcine TACE assay, and are broad-spectrum MMP inhibitors. Pharmacokinetic studies in beagle dogs revealed that SL422 and SP057 are orally bioavailable, with oral bioavailabilities of 11% and 23%, respectively.

ADAM Proteins↗