Novel genetic tools reveal Cdk5's major role in Golgi fragmentation in Alzheimer's disease.

Golgi fragmentation is a common feature in multiple neurodegenerative diseases; however, the precise mechanism that causes fragmentation remains obscure. A potential link between Cdk5 and Golgi fragmentation in Alzheimer's disease (AD) was investigated in this study. Because Golgi is physiologically fragmented during mitosis by Cdc2 kinase and current Cdk5-specific chemical inhibitors target Cdc2 as well, development of novel tools to modulate Cdk5 activity was essential.

Carboxyl-coated magnetic nanoparticles for mRNA isolation and extraction of supercoiled plasmid DNA.

Carboxyl-coated magnetic nanoparticles (MNPs) were used to demonstrate dual functionality: isolation of messenger RNA (mRNA) from mammalian cells and extraction of the supercoiled (sc) form of plasmid DNA (pDNA) from agarose gel. These MNPs were attached with 5'-NH(2)-tagged oligo-(dT)(25) primer and were used to isolate mRNA from breast cancer cells. The isolated mRNA was used for amplification of beta-actin to confirm the compatibility.

Stereoselective synthesis of 3,3-diarylacrylonitriles as tubulin polymerization inhibitors.

A series of 3,3-diarylacrylonitriles were synthesized stereoselectively as tubulin polymerization inhibitors for potential use in cancer chemotherapy. This synthetic route features stannylcupration and palladium-catalyzed Stille cross-coupling chemistry, allowing both E and Z isomers of 3,3-diarylacrylonitriles to be prepared in a very short sequence of reactions.

Regulation of FoxP3 regulatory T cells and Th17 cells by retinoids.

Vitamin A has both positive and negative regulatory functions in the immune system. While vitamin A is required for normal formation of immune cells and epithelial cell barriers, vitamin A deficiency can lead to increased inflammatory responses and tissue damage. The mechanism with which vitamin A and its metabolites such as retinoids negatively regulate inflammatory responses has not been clearly defined.

Hydrolysis of thioesters in an ion trap.

The alkenyldiarylmethanes are a class of non-nucleoside reverse transcriptase inhibitors that are currently being developed as potential antivirals for the treatment of HIV infection and AIDS. As part of our continuing investigations on the alkenyldiarylmethanes, a series of thioester analogues were prepared in an effort to improve upon the metabolic stability of the parent lead compound. Hydrolysis of the thioester moieties was consistently observed during ion trap electrospray ionization (ESI) mass spectrometry to the extent that the parent molecular ion was weak in intensity or simply could not be detected.

A new series of N-[2,4-dioxo-6-d-ribitylamino-1,2,3,4-tetrahydropyrimidin-5-yl]oxalamic acid derivatives as inhibitors of lumazine synthase and riboflavin synthase: design, synthesis, biochemical evaluation, crystallography, and mechanistic implications.

The penultimate step in the biosynthesis of riboflavin is catalyzed by lumazine synthase. Three metabolically stable analogues of the hypothetical intermediate proposed to arise after phosphate elimination in the lumazine synthase-catalyzed reaction were synthesized and evaluated as lumazine synthase inhibitors. All three intermediate analogues were inhibitors of Mycobacterium tuberculosis lumazine synthase, Bacillus subtilis lumazine synthase, and Schizosaccharomyces pombe lumazine synthase, while one of them proved to be an extremely potent inhibitor of Escherichia coli riboflavin synthase with a Ki of 1.

Contribution of charged groups to the enthalpic stabilization of the folded states of globular proteins.

In this paper we use the results from all-atom molecular dynamics (MD) simulations of proteins and peptides to assess the individual contribution of charged atomic groups to the enthalpic stability of the native state of globular proteins and investigate how the distribution of charged atomic groups in terms of solvent accessibility relates to protein enthalpic stability. The contributions of charged groups is calculated using a comparison of nonbonded interaction energy terms from equilibrium simulations of charged amino acid dipeptides in water (the "unfolded state") and charged amino acids in globular proteins (the "folded state"). Contrary to expectation, the analysis shows that many buried, charged atomic groups contribute favorably to protein enthalpic stability.

Cdc28 and Cdc14 control stability of the anaphase-promoting complex inhibitor Acm1.

The anaphase-promoting complex (APC) regulates the eukaryotic cell cycle by targeting specific proteins for proteasomal degradation. Its activity must be strictly controlled to ensure proper cell cycle progression. The co-activator proteins Cdc20 and Cdh1 are required for APC activity and are important regulatory targets.

Investigation of the alkenyldiarylmethane non-nucleoside reverse transcriptase inhibitors as potential cAMP phosphodiesterase-4B2 inhibitors.

The alkenyldiarylmethanes (ADAMs) are currently being investigated as non-nucleoside HIV-1 reverse transcriptase inhibitors (NNRTIs) of potential value in the treatment of HIV infection and AIDS. During the course of these studies, a number of ADAM analogues have been identified that protect HIV-infected cells from the cytopathic effects of the virus by an unknown, HIV-1 RT-independent mechanism. Since the phosphodiesterase 4 family is required for HIV infection, the effect of various ADAMs on the activity of PDE4B2 was investigated in an effort to determine if the ADAMs could possibly be targeting phosphodiesterases.

Analysis of self-associating proteins by singular value decomposition of solution scattering data.

We describe a method by which a single experiment can reveal both association model (pathway and constants) and low-resolution structures of a self-associating system. Small-angle scattering data are collected from solutions at a range of concentrations. These scattering data curves are mass-weighted linear combinations of the scattering from each oligomer.

Visualization of AP-1 NF-kappaB ternary complexes in living cells by using a BiFC-based FRET.

Protein-protein interactions are essential for maintaining cell structure and for executing almost all cellular processes. Determination of where and how each protein interacts with its partners provides significant insight into proteins' cellular roles. Although several assays, such as FRET and bimolecular fluorescence complementation (BiFC), have been developed and widely used for visualization and identification of protein interactions in living cells, there is no simple and convenient assay to visualize and identify multiple protein complexes in living cells.

In vitro histone methyltransferase assay.

INTRODUCTIONHistone methyltransferases catalyze the addition of one or more methyl groups to a specific lysine or arginine residue within histones. Currently, there is a great deal of interest in histone methyltransferases, because mutations and misregulation of the genes encoding these proteins have been linked to various cancers and other diseases. Many genes encoding putative histone methyltransferases have been identified in eukaryotes, but the proteins they encode have not been functionally characterized.

Optimizing Bayes error for protein structure model selection by stability mutagenesis.

Site-directed mutagenesis affects protein stability in a manner dependent on the local structural environment of the mutated residue; e.g., a hydrophobic to polar substitution would behave differently in the core vs.

Human Th17 cells share major trafficking receptors with both polarized effector T cells and FOXP3+ regulatory T cells.

It is a question of interest whether Th17 cells express trafficking receptors unique to this Th cell lineage and migrate specifically to certain tissue sites. We found several Th17 cell subsets at different developing stages in a human secondary lymphoid organ (tonsils) and adult, but not in neonatal, blood. These Th17 cell subsets include a novel in vivo-stimulated tonsil IL17+ T cell subset detected without any artificial stimulation in vitro.

Synthesis and in vivo antitumor evaluation of 2-methoxyestradiol 3-phosphate, 17-phosphate, and 3,17-diphosphate.

A prodrug strategy was investigated to address the problem of limited aqueous solubility and the resulting limited bioavailability of the antitumor agent 2-methoxyestradiol. The 3-phosphate, 17-phosphate, and 3,17-diphosphate of 2-methoxyestradiol were synthesized. 2-methoxyestradiol 3-phosphate was metabolized more efficiently to the parent compound in vivo than 2-methoxyestradiol 17-phosphate, and it was also more cytotoxic in cancer cell cultures than either the 17-phosphate or the 3,17-diphosphate.

Loss of IL-7 receptor alpha on CD4+ T cells defines terminally differentiated B cell-helping effector T cells in a B cell-rich lymphoid tissue.

IL-7 plays important roles in development and homeostatic proliferation of lymphocytes. IL-7 uses a receptor composed of IL-7Ralpha (CD127) and the common gamma-chain (CD132) to transmit its signal. It has been unknown how CD127 is regulated during Th cell differentiation to the B cell-helping T cell lineage.

ATM-NF-kappaB connection as a target for tumor radiosensitization.

Ionizing radiation (IR) plays a key role in both areas of carcinogenesis and anticancer radiotherapy. The ATM (ataxia-telangiectasia mutated) protein, a sensor to IR and other DNA-damaging agents, activates a wide variety of effectors involved in multiple signaling pathways, cell cycle checkpoints, DNA repair and apoptosis. Accumulated evidence also indicates that the transcription factor NF-kappaB (nuclear factor-kappaB) plays a critical role in cellular protection against a variety of genotoxic agents including IR, and inhibition of NF-kappaB leads to radiosensitization in radioresistant cancer cells.

Soluble polymer-based isotopic labeling (SoPIL): a new strategy to discover protein biomarkers?

Much attention has been given to protein biomarker discovery in the field of proteomics in the past few years. Proteomic strategies for biomarker discovery normally include the identification of proteins that alter during the progression of a particular disease state in high throughput. To perform these studies requires the ability to measure changes of low-abundance proteins in highly complex mixtures from different biological states.

The molecular basis for inhibition of BphD, a C-C bond hydrolase involved in polychlorinated biphenyls degradation: large 3-substituents prevent tautomerization.

The microbial degradation of polychlorinated biphenyls (PCBs) by the biphenyl catabolic (Bph) pathway is limited in part by the pathway's fourth enzyme, BphD. BphD catalyzes an unusual carbon-carbon bond hydrolysis of 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid (HOPDA), in which the substrate is subject to histidine-mediated enol-keto tautomerization prior to hydrolysis. Chlorinated HOPDAs such as 3-Cl HOPDA inhibit BphD.

Synthesis and characterization of bioactive tamoxifen-conjugated polymers.

Macromolecular conjugates of tamoxifen could perhaps be used to circumvent some of the limitations of the extensively used breast cancer drug. To test the feasibility of these conjugates, a 4-hydroxytamoxifen analogue was conjugated to a diaminoalkyl linker and then conjugated to activated esters of a poly(methacrylic acid) polymer synthesized by atom transfer radical polymerization. A polymer conjugated to the 4-hydroxytamoxifen analogue with a six-carbon linker showed high affinity for both estrogen receptor alpha and estrogen receptor beta and potent antagonism of the estrogen receptor in cell-based transcriptional reporter assays.

ABC transporters: how small machines do a big job.

Transporters from the ATP-binding cassette (ABC) superfamily operate in all organisms, from bacteria to humans, to pump substances across biological membranes. Recent high-resolution views of ABC transporters in different conformational states provide clues as to how ATP might be used to drive the structural reorganizations that accompany membrane transport. Importantly, it now appears that a putative translocation pathway running through the center of the transporter might be gated alternately, either at the inside or the outside of the cytoplasmic membrane, coupling substrate translocation to a cycle of ATP-dependent conformational changes.

Generation of an analog-sensitive Syk tyrosine kinase for the study of signaling dynamics from the B cell antigen receptor.

The Syk protein-tyrosine kinase is an essential component of the signaling machinery that couples the B cell receptor for antigen to multiple downstream signal transduction pathways. Syk is phosphorylated and activated rapidly and transiently following receptor engagement, but many signaling events, such as the activation of transcription factors occur over the course of several minutes or hours. To investigate a role for the continued activation of Syk in these processes, we generated an analog-sensitive mutant with an engineered ATP-binding pocket to render the kinase uniquely sensitive to an orthogonal inhibitor.

Phosphoinositol phosphatase SHIP2 promotes cancer development and metastasis coupled with alterations in EGF receptor turnover.

Phosphoinositol phosphatases are important regulators of signaling pathways relevant to both diabetes and cancer. A 3'-phosphoinositol phosphatase, phosphatase homologous to tensin (PTEN), is both a tumor suppressor and a negative regulator of insulin action. A 5'-phosphoinositol phosphatase, SH2-containing 5'-inositol phosphatase (SHIP2), regulates insulin signaling and its genetic knockout prevents high-fat diet-induced obesity in mice.

Vitamin A metabolites induce gut-homing FoxP3+ regulatory T cells.

In this study, we report a novel biological function of vitamin A metabolites in conversion of naive FoxP3- CD4+ T cells into a unique FoxP3+ regulatory T cell subset (termed "retinoid-induced FoxP3+ T cells") in both human and mouse T cells. We found that the major vitamin A metabolite all-trans-retinoic acid induces histone acetylation at the FoxP3 gene promoter and expression of the FoxP3 protein in CD4+ T cells. The induction of retinoid-induced FoxP3+ T cells is mediated by the nuclear retinoic acid receptor alpha and involves T cell activation driven by mucosal dendritic cells and costimulation through CD28.