Showing papers by "Torrey Pines Institute for Molecular Studies published in 2007"

Functional interrogation of the kinome using nucleotide acyl phosphates.

Abstract: The central role of protein kinases in signal transduction pathways has generated intense interest in targeting these enzymes for a wide range of therapeutic indications. Here we report a method for identifying and quantifying protein kinases in any biological sample or tissue from any species. The procedure relies on acyl phosphate-containing nucleotides, prepared from a biotin derivative and ATP or ADP. The acyl phosphate probes react selectively and covalently at the ATP binding sites of at least 75% of the known human protein kinases. Biotinylated peptide fragments from labeled proteomes are captured and then sequenced and identified using a mass spectrometry-based analysis platform to determine the kinases present and their relative levels. Further, direct competition between the probes and inhibitors can be assessed to determine inhibitor potency and selectivity against native protein kinases, as well as hundreds of other ATPases. The ability to broadly profile kinase activities in native proteomes offers an exciting prospect for both target discovery and inhibitor selectivity profiling.

From exogenous to endogenous: the inevitable imprint of mass spectrometry in metabolomics.

Abstract: Mass spectrometry (MS) is an established technology in drug metabolite analysis and is now expanding into endogenous metabolite research. Its utility derives from its wide dynamic range, reproducible quantitative analysis, and the ability to analyze biofluids with extreme molecular complexity. The aims of developing mass spectrometry for metabolomics range from understanding basic biochemistry to biomarker discovery and the structural characterization of physiologically important metabolites. In this review, we will discuss the techniques involved in this exciting area and the current and future applications of this field.

Ligand docking and structure-based virtual screening in drug discovery.

Abstract: Ligand-docking-based methods are starting to play a critical role in lead discovery and optimization, thus resulting in new 'drug-candidates'. They offer the possibility to go beyond the pool of existing active compounds, and thus find novel chemotypes. A brief tutorial on ligand docking and structure-based virtual screening is presented highlighting current problems and limitations, together with the most recent methodological and algorithmic developments in the field. Recent successful applications of docking-based tools for hit discovery, lead optimization and target-biased library design are also presented. Special consideration is devoted to ongoing efforts to account for protein flexibility in structure-based virtual screening.

Towards a consensus on datasets and evaluation metrics for developing B-cell epitope prediction tools

Abstract: A B-cell epitope is the three-dimensional structure within an antigen that can be bound to the variable region of an antibody. The prediction of B-cell epitopes is highly desirable for various immunological applications, but has presented a set of unique challenges to the bioinformatics and immunology communities. Improving the accuracy of B-cell epitope prediction methods depends on a community consensus on the data and metrics utilized to develop and evaluate such tools. A workshop, sponsored by the National Institute of Allergy and Infectious Disease (NIAID), was recently held in Washington, DC to discuss the current state of the B-cell epitope prediction field. Many of the currently available tools were surveyed and a set of recommendations was devised to facilitate improvements in the currently existing tools and to expedite future tool development. An underlying theme of the recommendations put forth by the panel is increased collaboration among research groups. By developing common datasets, standardized data formats, and the means with which to consolidate information, we hope to greatly enhance the development of B-cell epitope prediction tools.

Tandem orthogonal proteolysis-activity-based protein profiling (TOP-ABPP)—a general method for mapping sites of probe modification in proteomes

Abstract: Activity-based protein profiling (ABPP) utilizes active site-directed chemical probes to monitor the functional state of enzymes directly in native biological systems. Identification of the specific sites of probe labeling on enzymes remains a major challenge in ABPP experiments. In this protocol, we describe an advanced ABPP platform that utilizes a tandem orthogonal proteolysis (TOP) strategy coupled with mass spectrometric analysis to simultaneously identify probe-labeled proteins together with their exact sites of probe modification. Elucidation of probe modification sites reveals fundamental insights into the molecular basis of specific probe-protein interactions. The TOP-ABPP method can be applied to any type of proteomic sample, including those derived from in vitro or in vivo labeling experiments, and is compatible with a variety of chemical probe structures. Completion of the entire protocol, including chemical synthesis of key reagents, requires approximately 8-10 days.

Type II NKT cell–mediated anergy induction in type I NKT cells prevents inflammatory liver disease

Abstract: Because of the paucity of known self lipid-reactive ligands for NKT cells, interactions among distinct NKT cell subsets as well as immune consequences following recognition of self glycolipids have not previously been investigated. Here we examined cellular interactions and subsequent immune regulatory mechanism following recognition of sulfatide, a self-glycolipid ligand for a subset of CD1d-restricted type II NKT cells. Using glycolipid/CD1d tetramers and cytokine responses, we showed that activation of sulfatide-reactive type II NKT cells and plasmacytoid DCs caused IL-12- and MIP-2-dependent recruitment of type I, or invariant, NKT (iNKT) cells into mouse livers. These recruited iNKT cells were anergic and prevented concanavalin A-induced (ConA-induced) hepatitis by specifically blocking effector pathways, including the cytokine burst and neutrophil recruitment that follow ConA injection. Hepatic DCs from IL-12(+/+) mice, but not IL-12(-/-) mice, adoptively transferred anergy in recipients; thus, IL-12 secretion by DCs enables them to induce anergy in iNKT cells. Our data reveal what we believe to be a novel mechanism in which interactions among type II NKT cells and hepatic DCs result in regulation of iNKT cell activity that can be exploited for intervention in inflammatory diseases, including autoimmunity and asthma.

Deciphering the kinetic mechanism of spontaneous self-assembly of icosahedral capsids.

Abstract: Self-assembly of viral proteins into icosahedral capsids is an interesting yet poorly understood phenomenon of which elucidation may aid the exploration of beneficial applications of capsids in materials science and medicine. Using molecular dynamics simulations of coarse-grained models for capsid proteins, we show that the competition between the formation of full capsids and nonidealized structures is strongly dependent upon the protein concentration and temperature, occurring kinetically as a cascade of elementary reactions in which free monomers are added to the growing oligomers on a downhill free-energy landscape. However, the insertion of the final subunits is the rate-limiting, energetically unfavorable step in viral capsid assembly. A phase diagram has been constructed to show the regions where capsids or nonidealized structures are stable at each concentration and temperature. We anticipate that our findings will provide guidance in identifying suitable conditions required for in vitro viral capsid assembly experiments.

Cross-regulation between type I and type II NKT cells in regulating tumor immunity: a new immunoregulatory axis.

Abstract: Negative immunoregulation is a major barrier to successful cancer immunotherapy. The NKT cell is known to be one such regulator. In this study we explored the roles of and interaction between the classical type I NKT cell and the poorly understood type II NKT cell in the regulation of tumor immunity. Selective stimulation of type II NKT cells suppressed immunosurveillance, whereas stimulation of type I NKT cells protected against tumor growth even when responses were relatively skewed toward Th2 cytokines. When both were stimulated simultaneously, type II NKT cells appeared to suppress the activation in vitro and protective effect in vivo of type I NKT cells. In the absence of type I, suppression by type II NKT cells increased, suggesting that type I cells reduce the suppressive effect of type II NKT cells. Thus, in tumor immunity type I and type II NKT cells have opposite and counteractive roles and define a new immunoregulatory axis. Alteration of the balance between the protective type I and the suppressive type II NKT cell may be exploited for therapeutic intervention in cancer.

Targeting thyroid hormone receptor-β agonists to the liver reduces cholesterol and triglycerides and improves the therapeutic index

Abstract: Despite efforts spanning four decades, the therapeutic potential of thyroid hormone receptor (TR) agonists as lipid-lowering and anti-obesity agents remains largely unexplored in humans because of dose-limiting cardiac effects and effects on the thyroid hormone axis (THA), muscle metabolism, and bone turnover. TR agonists selective for the TRβ isoform exhibit modest cardiac sparing in rodents and primates but are unable to lower lipids without inducing TRβ-mediated suppression of the THA. Herein, we describe a cytochrome P450-activated prodrug of a phosphonate-containing TR agonist that exhibits increased TR activation in the liver relative to extrahepatic tissues and an improved therapeutic index. Pharmacokinetic studies in rats demonstrated that the prodrug (2R,4S)-4-(3-chlorophenyl)-2-[(3,5-dimethyl-4-(4′-hydroxy-3′-isopropylbenzyl)phenoxy)methyl]-2-oxido-[1,3,2]-dioxaphosphonane (MB07811) undergoes first-pass hepatic extraction and that cleavage of the prodrug generates the negatively charged TR agonist (3,5-dimethyl-4-(4′-hydroxy-3′-isopropylbenzyl)phenoxy)methylphosphonic acid (MB07344), which distributes poorly into most tissues and is rapidly eliminated in the bile. Enhanced liver targeting was further demonstrated by comparing the effects of MB07811 with 3,5,3′-triiodo-l-thyronine (T3) and a non-liver-targeted TR agonist, 3,5-dichloro-4-(4-hydroxy-3-isopropylphenoxy)phenylacetic acid (KB-141) on the expression of TR agonist-responsive genes in the liver and six extrahepatic tissues. The pharmacologic effects of liver targeting were evident in the normal rat, where MB07811 exhibited increased cardiac sparing, and in the diet-induced obese mouse, where, unlike KB-141, MB07811 reduced cholesterol and both serum and hepatic triglycerides at doses devoid of effects on body weight, glycemia, and the THA. These results indicate that targeting TR agonists to the liver has the potential to lower both cholesterol and triglyceride levels with an acceptable safety profile.

An Affinity-Enhanced Neutralizing Antibody against the Membrane-Proximal External Region of Human Immunodeficiency Virus Type 1 gp41 Recognizes an Epitope between Those of 2F5 and 4E10

Abstract: The membrane-proximal external region (MPER) of human immunodeficiency virus type 1 (HIV-1) gp41 bears the epitopes of two broadly neutralizing antibodies (Abs), 2F5 and 4E10, making it a target for vaccine design. A third Ab, Fab Z13, had previously been mapped to an epitope that overlaps those of 2F5 and 4E10 but only weakly neutralizes a limited set of primary isolates. Here, libraries of Fab Z13 variants displayed on phage were engineered and affinity selected against an MPER peptide and recombinant gp41. A high-affinity variant, designated Z13e1, was isolated and found to be approximately 100-fold improved over the parental Fab not only in binding affinity for the MPER antigens but also in neutralization potency against sensitive HIV-1. Alanine scanning of MPER residues 664 to 680 revealed that N671 and D674 are crucial for peptide recognition as well as for the neutralization of HIV-1 by Z13e1. Ab competition studies and truncation of MPER peptides indicate that Z13e1 binds with high affinity to an epitope between and overlapping with those of 2F5 and 4E10, with the minimal peptide epitope WASLWNWFDITN. Still, Z13e1 remained about an order of magnitude less potent than 4E10 against several isolates of pseudotyped HIV-1. The sum of our molecular analyses with Z13e1 suggests that the segment on the MPER of gp41 between the 2F5 and 4E10 epitopes is exposed on the functional envelope trimer but that access to the specific Z13e1 epitope within this segment is limited. Thus, the ability of MPER-bearing immunogens to elicit potent HIV-1-neutralizing Abs may depend in part on recapitulating the particular constraints that the functional envelope trimer imposes on the segment of the MPER to which Z13e1 binds.

Waltzing transporters and 'the dance macabre' between humans and bacteria.

Abstract: Multidrug-resistance efflux pumps — in particular those belonging to the resistance-nodulation-cell-division (RND) family of transporters, with their unusually high degree of substrate promiscuity — significantly restrict the effectiveness of antibacterial therapy. Recent years have heralded remarkable insights into the structure and mechanisms of these fascinating molecular machines. Here, we review recent advances in the field and describe various approaches used in combating efflux-mediated resistance.

Genetically encoding unnatural amino acids for cellular and neuronal studies

Abstract: Proteins participate in various biological processes and can be harnessed to probe and control biological events selectively and reproducibly, but the genetic code limits the building block to 20 common amino acids for protein manipulation in living cells. The genetic encoding of unnatural amino acids will remove this restriction and enable new chemical and physical properties to be precisely introduced into proteins. Here we present new strategies for generating orthogonal tRNA-synthetase pairs, which made possible the genetic encoding of diverse unnatural amino acids in different mammalian cells and primary neurons. Using this new methodology, we incorporated unnatural amino acids with extended side chains into the K+ channel Kv1.4, and found that the bulkiness of residues in the inactivation peptide is essential for fast channel inactivation, a finding that had not been possible using conventional mutagenesis. This technique will stimulate and facilitate new molecular studies using tailored unnatural amino acids for cell biology and neurobiology.

Charcot–Marie–Tooth disease-associated mutant tRNA synthetases linked to altered dimer interface and neurite distribution defect

Abstract: Charcot–Marie–Tooth (CMT) diseases are the most common heritable peripheral neuropathy. At least 10 different mutant alleles of GARS (the gene for glycyl-tRNA synthetase) have been reported to cause a dominant axonal form of CMT (type 2D). A unifying connection between these mutations and CMT has been unclear. Here, mapping mutations onto the recently determined crystal structure of human GlyRS showed them within a band encompassing both sides of the dimer interface, with two CMT-causing mutations being at sites that are complementary partners of a “kissing” contact across the dimer interface. The CMT phenotype is shown here to not correlate with aminoacylation activity. However, most mutations affect dimer formation (to enhance or weaken). Seven CMT-causing variants and the wild-type protein were expressed in transfected neuroblastoma cells that sprout primitive neurites. Wild-type GlyRS distributed into the nascent neurites and was associated with normal neurite sprouting. In contrast, all mutant proteins were distribution-defective. Thus, CMT-causing mutations of GlyRS share a common defect in localization. This defect may be connected in some way to a change in the surfaces at the dimer interface.

Atomic property fields: generalized 3D pharmacophoric potential for automated ligand superposition, pharmacophore elucidation and 3D QSAR.

Abstract: The atomic property fields (APF) concept is introduced as a continuous, multi-component 3D potential that reflects preferences for various atomic properties at each point in space. Atomic property field-based approaches to several key problems in the field of ligand structure-based rational drug discovery and design are investigated. The superposition of ligands on one or multiple molecular templates is performed by Monte-Carlo minimization in the atomic property fields potentials combined with standard force-field energy. The approach is extended to multiple flexible ligand alignments using an iterative procedure, Self-Consistent atomic Property Fields by Optimization (SCAPFOld). The application of atomic property fields and SCAPFOld for virtual ligand screening and 3D Quantitive Structure-Activity Relationship (QSAR) is tested on published benchmarks. The new methods are shown to perform competitively in comparison to current state-of-the-art methods.

Is Membrane Transport of FFA Mediated by Lipid, Protein, or Both?

Abstract: ![Figure][1] J. Patrick Kampf Torrey Pines Institute for Molecular Studies, San Diego, California ![Figure][1] Alan M. Kleinfeld Torrey Pines Institute for Molecular Studies, San Diego, California akleinfeld{at}tpims.org The mechanism of free fatty acid (FFA)

CXCR2 Is Required for Neutrophil Recruitment to the Lung during Influenza Virus Infection, But Is Not Essential for Viral Clearance

Abstract: Neutrophils traffic to the lungs in large numbers during influenza virus infection. Although the ability of these cells to respond to numerous chemotactic stimuli has been described in other systems, the chemokine receptors mediating recruitment of neutrophils to the lungs during influenza virus infection and the role of this cell type in viral clearance are currently undefined. In the present study, we used CXCR2(/) mice to investigate the role of the chemokine receptor CXCR2 in neutrophil recruitment to the lungs during influenza virus infection and to determine the role of neutrophils in viral clearance. We infected CXCR2(/) or wild-type mice with influenza and assessed the level of inflammation, the cellular composition of the inflammatory infiltrate, and viral titers in the lungs. Absence of CXCR2 ablated neutrophil recruitment to the lungs, but had no effect on peak viral titers or on the kinetics of viral clearance. Thus, it appears that CXCR2 is the major receptor mediating neutrophil trafficking to the lung during influenza virus infection, but that neutrophils do not play an essential role in viral clearance.

Targeting the apoptotic machinery in pancreatic cancers using small-molecule antagonists of the X-linked inhibitor of apoptosis protein

Abstract: Resistance to apoptosis is a hallmark of many solid tumors, including pancreatic cancers, and may be the underlying basis for the suboptimal response to chemoradiation therapies. Overexpression of a family of inhibitor of apoptosis proteins (IAP) is commonly observed in pancreatic malignancies. We determined the therapeutic efficacy of recently described small-molecule antagonists of the X-linked IAP (XIAP) in preclinical models of pancreatic cancer. Primary pancreatic cancers were assessed for XIAP expression by immunohistochemistry, using a pancreatic cancer tissue microarray. XIAP small-molecule antagonists ("XAntag"; compounds 1396-11 and 1396-12) and the related compound 1396-28 were tested in vitro in a panel of human pancreatic cancer cell lines (Panc1, Capan1, and BxPC3) and in vivo in s.c. xenograft models for their ability to induce apoptosis and impede neoplastic growth. In addition, pancreatic cancer cell lines were treated with XAntags in conjunction with either tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) or with radiation to determine potential synergy for such dual targeting of the apoptotic machinery. XIAP was overexpressed in 14 of 18 (77%) of primary pancreatic cancers. The XAntags1396-11 and 1396-12, but not the inactive isomer 1396-28, induced profound apoptosis in multiple pancreatic cancer cell lines tested in vitro, with a IC(50) in the range of 2 to 5 mumol/L. Mechanistic specificity of the XAntags for the baculoviral IAP repeat-2 domain of XIAP was shown by preferential activation of downstream "effector" caspases (caspase-3 and caspase-7) versus the upstream "initiator" caspase-9. S.c. BxPC3 xenograft growth in athymic mice was significantly inhibited by monotherapy with XAntags; treated xenografts showed marked apoptosis and increased cleavage of caspase-3. Notably, striking synergy was demonstrable when XAntags were combined with either TRAIL or radiation therapy, as measured by growth inhibition in vitro and reduced colony formation in soft agar of pancreatic cancer cell lines, at dosages where these therapeutic modalities had minimal to modest effects when used alone. Finally, XAntags in combination with the standard-of-care agent for advanced pancreatic cancer, gemcitabine, resulted in significantly greater inhibition of in vitro growth than gemcitabine alone. Our results confirm that pharmacologic inhibition of XIAP is a potent therapeutic modality in pancreatic cancers. These antagonists are independently capable of inducing pancreatic cancer cell death and also show synergy when combined with proapoptotic ligands (TRAIL), with radiation, and with a conventional antimetabolite, gemcitabine. These preclinical results suggest that targeting of the apoptotic machinery in pancreatic cancers with XAntags is a promising therapeutic option that warrants further evaluation.

A public T cell clonotype within a heterogeneous autoreactive repertoire is dominant in driving EAE

Abstract: Experimental autoimmune encephalomyelitis (EAE) is an animal model of multiple sclerosis. Immunization of B10.PL mice with the Ac1–9 peptide, the immunodominant determinant of myelin basic protein (MBP), produced a single episode of EAE followed by recovery and resistance to reinduction of disease. Using the CDR3 length spectratyping technique, we characterized the clonal composition of the Ac1–9–specific T cell repertoire from induction through onset and resolution of disease. Two clonally restricted subsets within a heterogeneous self-reactive repertoire were found in mouse lymph nodes, spleen, and spinal cord soon after immunization, before any sign of EAE. These clonotypes, designated BV8S2/BJ2S7 and BV16/BJ2S5, were present in all mice examined and thus considered public. BV8S2/BJ2S7 was found in far greater excess; was exclusively Th1 polarized; disappeared from the spinal cord, spleen, and lymph nodes concomitantly with recovery; and transferred disease to naive recipients. In contrast, BV16/BJ2S5 and numerous private clonotypes were either Th1 or Th2 and persisted following recovery. These results are consistent with the hypothesis that the public clonotype BV8S2/BJ2S7 is a driver of disease and necessary for its propagation.

A Similarity‐based Data‐fusion Approach to the Visual Characterization and Comparison of Compound Databases

Abstract: A low-dimensional method, based on the use of multiple fusion-based similarity measures, is described for graphically depicting and characterizing relationships among molecules in compound databases. The measures are used to construct multi-fusion similarity maps that characterize the relationship of a set of 'test' molecules to a set of 'reference' molecules. The reference set is very general and can be made of molecules from, for example, the set of test molecules itself (the self-referencing case), from a small library or large compound collection, or from actives in a given assay or group of assays. The test set is any collection of compounds to be analyzed with respect to the specified reference set. Multiple fusion similarity measures tend to provide more information than single fusion-based measures, including information on the nature of the chemical-space neighborhoods surrounding reference-set molecules. A general discussion is presented on how to interpret multi-fusion similarity maps, and several examples are given that illustrate how these maps can be used to compare compound libraries or collections, to select compounds for screening or acquisition, and to identify new active molecules using ligand-based virtual screening.

Site specific incorporation of non-natural amino acids by vertebrate cells

Abstract: This invention provides compositions and methods for producing translational components that expand the number of genetically encoded amino acids in vertebrate cells. The components include orthogonal tRNA's, orthogonal aminoacyl-tRNA synthetases, orthogonal pairs of tRNA's/synthetases and unnatural amino acids. Proteins and methods of producing proteins with unnatural amino acids in vertebrate cells are also provided.

Sequence and Characterization of the Ig Heavy Chain Constant and Partial Variable Region of the Mouse Strain 129S1

Abstract: Although the entire mouse genome has been sequenced, there remain challenges concerning the elucidation of particular complex and polymorphic genomic loci. In the murine Igh locus, different haplotypes exist in different inbred mouse strains. For example, the Ighb haplotype sequence of the Mouse Genome Project strain C57BL/6 differs considerably from the Igha haplotype of BALB/c, which has been widely used in the analyses of Ab responses. We have sequenced and annotated the 3′ half of the Igha locus of 129S1/SvImJ, covering the CH region and approximately half of the VH region. This sequence comprises 128 VH genes, of which 49 are judged to be functional. The comparison of the Igha sequence with the homologous Ighb region from C57BL/6 revealed two major expansions in the germline repertoire of Igha. In addition, we found smaller haplotype-specific differences like the duplication of five VH genes in the Igha locus. We generated a VH allele table by comparing the individual VH genes of both haplotypes. Surprisingly, the number and position of DH genes in the 129S1 strain differs not only from the sequence of C57BL/6 but also from the map published for BALB/c. Taken together, the contiguous genomic sequence of the 3′ part of the Igha locus allows a detailed view of the recent evolution of this highly dynamic locus in the mouse.

Liver-Targeted Prodrugs of 2‘-C-Methyladenosine for Therapy of Hepatitis C Virus Infection

Abstract: 2'-C-Methyladenosine exhibits impressive inhibitory activity in the cell-based hepatitis C virus (HCV) subgenomic replicon assay, by virtue of intracellular conversion to the corresponding nucleoside triphosphate (NTP) and inhibition of NS5B RNA-dependent RNA polymerase (RdRp). However, rapid degradation by adenosine deaminase (ADA) limits its overall therapeutic potential. To reduce ADA-mediated deamination, we prepared cyclic 1-aryl-1,3-propanyl prodrugs of the corresponding nucleoside monophosphate (NMP), anticipating cytochrome P450 3A-mediated oxidative cleavage to the NMP in hepatocytes. Lead compounds identified in a primary rat hepatocyte screen were shown to result in liver levels of NTP predictive of efficacy after intravenous dosing to rats. The oral bioavailability of the initial lead was below 5%; therefore, additional analogues were synthesized and screened for liver NTP levels after oral administration to rats. Addition of a 2',3'-carbonate prodrug moiety proved to be a successful strategy, and the 1-(4-pyridyl)-1,3-propanyl prodrug containing a 2',3'-carbonate moiety displayed oral bioavailability of 39%.

Chemokine regulation of the inflammatory response to a low-dose influenza infection in CCR2–/– mice

Abstract: Influenza virus infections induce chemokines and cytokines, which regulate the immune response. The chemokine receptor CCR2 plays an important role in macrophage recruitment and in the development of T1 immunity. In the present study, we addressed the role of CCR2 in influenza A virus infection. CCR2 knockout (-/-) mice are protected against influenza A virus infection, despite delayed recruitment of macrophages. We show that low-dose influenza infection of CCR2-/- mice leads to increased neutrophilia between Days 5 and 10 after infection and decreased monocyte/macrophage and CD4(+) T cell recruitment to the lungs between Days 5 and 7 after infection. These changes in leukocyte recruitment did not result from or cause increased viral titers or delayed viral clearance. Neutrophilia in the lungs correlated with increased keratinocyte-derived chemokine (KC) and/or MIP-2 expression in CCR2-/- mice between Days 5 to 10 after infection, although the kinetics of neutrophil recruitment was not altered. MIP-2 mRNA and protein expression was increased three- to fivefold, and KC protein levels were increased two- to threefold in CCR2-/- compared with CCR2 wild-type mice at Day 5 after infection. This preceded the peak neutrophil influx, which occurred 7 days after infection. In vitro studies confirmed that MIP-2 and KC accounted for neutrophil chemotactic activity in the bronchoalveolar lavage. CCR2 deficiency also resulted in increased MIP-1alpha, MIP-1beta, MCP-1, and IFN-inducible protein 10 and decreased RANTES mRNA expression. Furthermore, IL-6 and TNF-alpha cytokine production were elevated after infection. These studies suggest that CCR2 plays a multifactorial role in the development of the immune response to influenza.

High-throughput five minute microwave accelerated glycosylation approach to the synthesis of nucleoside libraries.

Abstract: The Vorbruggen glycosylation reaction was adapted into a one-step 5 min/130 °C microwave assisted reaction. Triethanolamine in acetontrile containing 2% water was determined to be optimal for the neutralization of trimethylsilyl triflate allowing for direct MPLC purification of the reaction mixture. When coupled with a NH3/methanol deprotection reaction, a high-throughput method of nucleoside library synthesis was enabled. The method was demonstrated by examining the ribosylation of 48 nitrogen containing heteroaromatic bases that included 25 purines, four pyrazolopyrimidines, two 8-azapurines, one 2-azapurine, two imidazopyridines, two benzimidazoles, three imidazoles, three 1,2,4-triazoles, two pyrimidines, two 3-deazapyrimidines, one quinazolinedione, and one alloxazine. Of these, 32 yielded single regioisomer products, and six resulted in separable mixtures. Seven examples provided inseparable regioisomer mixtures of −two to three compounds (16 nucleosides), and three examples failed to yield isolable...

Free fatty acid transport across adipocytes is mediated by an unknown membrane protein pump

Abstract: The role of cell membranes in regulating the flux of long chain free fatty acids (FFA) into and out of adipocytes is intensely debated. Four different membrane proteins including, FABPpm, CD36/FAT, caveolin-1, and FATP have been identified as facilitating FFA transport. Moreover, CD36 and caveolin-1 are also reported to mediate transport in conjunction with lipid rafts. The principal evidence for these findings is a correlation of the level of FFA uptake with the expression level of these proteins and with the integrity of lipid rafts. The 3T3-L1 and 3T3-F442A cell lines in their preadipocyte states reveal little or no expression of these proteins and correspondingly low levels of uptake. Here we have microinjected the adipocyte and preadipocyte cell lines with ADIFAB, the fluorescent indicator of FFA. The ADIFAB fluorescence allowed us to monitor the intracellular unbound FFA concentration during FFA influx and efflux. We show that these measurements of transport, in contrast to FFA uptake measurements, correlate neither with expression of these proteins nor with lipid raft integrity in preadipocytes and adipocytes. Transport characteristics, including the generation of an ATP-dependent FFA concentration gradient, are virtually identical in adipocytes and preadipocytes. We suggest that the origin of the discrepancy between uptake and our measurements is that most of the FFA transported into the cells is lost during the uptake but not in the transport protocols. We conclude that long chain fatty acid transport in adipocytes is very likely mediated by an as-yet-unidentified membrane protein pump.

Protease screening methods and proteases identified thereby

Abstract: Methods for identifying modified proteases with modified substrate specificity or other properties are provided. The methods screen candidate and modified proteases by contacting them with a substrate, such as a serpin, an alpha macroglobulins or a p35 family protein or modified serpins and modified p35 family members or modified alpha macroglobulins, that, upon cleavage of the substrate, traps the protease by forming a stable complex. Also provided are modified proteases.