Search

The myristoylated alanine-rich protein kinase C substrate (MARCKS) is a major protein kinase C (PKC) substrate in many different cell types. MARCKS is bound to the plasma membrane, and several recent studies suggest that this binding requires both hydrophobic insertion of its myristate chain into the bilayer and electrostatic interaction of its cluster of basic residues with acidic lipids. Phosphorylation of MARCKS by PKC introduces negative charges into the basic cluster, reducing its electrostatic interaction with acidic lipids and producing translocation of MARCKS from membrane to cytoplasm. The present study shows that physiological concentrations of MARCKS (

Using an affinity resin and photoaffinity label based on phospholipid analogs of inositol 1,3,4,5-tetrakisphosphate (InsP4), we have isolated, characterized, and cloned a 46-kDa protein from rat brain, which we have named centaurin-alpha. Binding specificity was determined using displacement of 1-O-[3H](3-[4-benzoyldihydrocinnamidyl]propyl)-InsP4 photoaffinity labeling. Centaurin-alpha displayed highest affinity for phosphatidylinositol 3,4,5-trisphosphate (PtdInsP3) (IC50 = 120 nM), whereas InsP4, PtdInsP2, and InsP3 bound with 5-, 12-, and >50-fold lower affinity, respectively. Screening a rat brain cDNA library with a polymerase chain reaction product, generated using partial amino acid sequence from tryptic peptides, yielded a full-length clone. The 2,450-base pair cDNA contained an open reading frame (ORF) encoding a novel protein of 419 amino acids. Northern analysis revealed a 2.5-kilobase transcript that is highly expressed in brain. The deduced sequence contains a novel putative zinc finger motif, 10 ankyrin-like repeats, and shows homology to recently identified yeast and mammalian Arf GTPase-activating proteins. Given the specificity of binding and enrichment in brain, centaurin-alpha is a candidate PtdInsP3 receptor that may link the activation of phosphoinositide 3-kinase to downstream responses in the brain.

Diphosphoinositol pentakisphosphate (PP-IP5) and bis(diphospho)inositol tetrakisphosphate (bis-PP-IP4) are recently identified inositol phosphates that possess pyrophosphate bonds. We have purified an inositol hexakisphosphate (IP6) kinase from rat brain supernatants. The pure protein, a monomer of 54 kDa, displays high affinity (Km = 0.7 microM) and selectivity for inositol hexakisphosphate as substrate. It can be dissociated from bis(diphospho)inositol tetrakisphosphate synthetic activity. The purified enzyme transfers a phosphate from PP-IP5 to ADP to form ATP. This ATP synthase activity indicates the high phosphate group transfer potential of PP-IP5 and may represent a physiological role for PP-IP5.

Photoactivatable analogues of 1-L-phosphatidyl-D-myo-inositol 4,5-bisphosphate (PtdIns(4,5)P(2) or PtdInsP(2)) and the corresponding 3,4,5-trisphosphate (PtdIns(3,4,5)P(3) or PtdInsP(3)) were prepared from the two chiral precursors, methyl alpha-D-glucopyranoside and 1,2-isopropylidene-sn-glycerol. Two key synthetic transformations included the Ferrier rearrangement reaction to construct the optically-pure inositol skeleton and the sequential acylation of the primary and secondary hydroxyl groups on the glycerol derivatives. The sn-1-O-(6-aminohexanoyl) PtdInsP(2) and PtdInsP(3) derivatives were further modified to contain benzophenone photophores in unlabeled and high specific activity tritium-labeled forms.

A synthetic route was developed for the preparation of novel O-(3-aminopropyl) tethered phosphotriester analogs (5) of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5,)P2, or PIP2) and phosphatid...

A photoreactive dihydropyridine (DHP), BZDC-DHP (2,6-dimethyl-4-(2-(trifluoromethyl)-phenyl)-1,4-dihydropyridine-3,5- dicarboxylic acid (2-[3-(4-benzoylphenyl)propionylamino]ethyl) ester ethyl ester), and its tritiated derivative were synthesized as novel probes for human p-glycoprotein (p-gp). (-)-[3H]BZDC-DHP specifically photolabeled p-gp in membranes of multidrug-resistant CCRF-ADR5000 cells. In reversible labeling experiments a saturable, vinblastine-sensitive and high-affinity (Kd = 16.3 nM, Bmax = 58 pmol/mg of protein, k(+1) = 0.031 nM-1 min-1, k(-1) = 0.172 min-1) binding component was present in CCRF-ADR5000 membranes but absent in the sensitive parent cell line. Binding was inhibited by cytotoxics and known chemosensitizers with a p-gp characteristic pharmacological profile. For eight chemosensitizers tested, the potency for binding inhibition correlated (r > 0.94) with the potency for drug transport inhibition (measured using rhodamine 123 accumulation). The DHP niguldipine and a structurally related pyrimidine stereoselectively stimulated reversible (-)-[3H]BZDC-DHP binding, suggesting that more than one DHP molecule can bind to p-gp at the same time. Our data demonstrate that DHPs label multiple chemosensitizer domains on p-gp, distinct from the vinblastine interaction site. (-)-[3H]BZDC-DHP represents a valuable tool to characterize the molecular organization of chemosensitizer binding domains on p-gp by both reversible binding and photoinduced covalent modification. It provides a novel simple screening assay for p-gp active drugs.

A diverse group of proteins has evolved to bind and metabolize insect juvenile hormones (JHs). Synthetic radiolabeled JHs and their photoaffinity analogs have enabled us to isolate and characterize JH binding proteins (JHBPs), a putative nuclear JH receptor, JH esterases (JHEs), JH epoxide hydrolases (JHEHs), and methyl farnesoate binding proteins (MFBPs). Highlights of recent progress on structural characterization of JHBPs and JHEHs of two lepidopterans will be described. Efforts to identify MFBPs of penaeid shrimp will be discussed, and the discovery of a possible vertebrate JHBP will be presented. © 1996 Wiley-Liss, Inc.

d - myo -P-1-( O -3-Aminopropyl)-Ins(l,4,5)P 3 has been synthesized from methyl α- d -glucopyranoside. This optically-pure tethered IP 3 derivative has been converted to a selective photoaffinity label for modification of the ligand binding site of IP 3 receptor proteins.

The ligand specificities and binding affinities of three recombinant pheromone binding proteins (PBPs) of two saturniid moths (genus Antheraea) were determined by using a novel binding assay in conjunction with two tritium-labeled constituents of the pheromone blend, [3H]-6E,11Z-hexadecadienyl acetate and [3H]-4E,9Z-tetradecadienyl acetate. The new binding assay, in which nonspecific adsorption to a plastic vessel is suppressed by presaturation of the surface with a 1-alkanol, allows measurement of dissociation constants (KD) for lipophilic ligands for their carrier proteins. The three PBPs showed KD values for [3H]-6E,11Z-16:Ac and [3H]-4E,9Z-14:Ac between 0.6 and 30 microM, as determined by Scatchard analysis. Importantly, two PBPs (Aper-1 and Aper-2) from one species showed opposite binding specificities for these two ligands. Aper-1, like Apol-3, showed 15-fold higher affinity for 6E,11Z-16:Ac than for 4E,9Z-14:Ac, while Aper-2 showed a 3.5-fold preference for binding the shorter chain compound. In addition, for the Apol-3 PBP, displacement of [3H]-6E,11Z-16:Ac binding by other pheromone components or analogs showed a clear trend in relative binding affinity: 6E,11Z-16:Ac > 4E,9Z-14:Ac > 6E,11Z-16:Al approximately 16:Ac > 6E,11Z-16:OH > 4E,9Z-14:OH. These data clearly demonstrate a > 1000-fold range of binding affinities among these very similar structures and unambiguously demonstrate the specificity of the PBP-pheromone interaction. Moreover, this assay offers the potential for determining ligand specificities for odorant binding proteins and other proteins in the vertebrate lipocalin superfamily.

A new radiolabeled, bifunctional photoaffinity cross-linking reagent, N-succinimidyl p-benzoyl-[2,3-3H2]dihydrocinnamate, has been synthesized in high yield and with high specific activity. This reagent can be used to append the benzophenone photophore to amino groups of small molecules, such as O-aminoalkylinositol polyphosphates and polypeptides. The resulting tritiated photoaffinity labels can be purified and manipulated in ambient light and can be activated at 360 nm.

A decline in juvenile hormone (JH) titer early in the last larval instar leads to the metamorphosis of lepidopterous larvae into pupae. Clearance of JH is associated with an increase in the level of JH-specific esterase (JHE) that hydrolyzes JH into the biologically inactive JH-acid. Experiments with purified recombinant JHBP (rJHBP) and with both natural and recombinant JHE (rJHE) were designed to test the hypothesis that JHBP “protects” JH from JHE degradation in insect hemolymph. First, at low JHE concentrations, the rate of hydrolysis of JHBP-bound JH by JHE was determined by the rate of dissociation of JH from JHBP. Second, the rate of hydrolysis of JH increased with increasing JHE concentrations even in the presence of excess JHBP. In contrast, JH was not hydrolyzed by a general carboxylesterase in the presence of JHBP. Third, the rate of JH hydrolysis when the JHBP-JH complex was added to JHBP-depleted hemolymph was essentially identical to that in undiluted hemolymph. Taken together, these results suggest that JHBP protects JH from nonspecific hydrolytic processes, while still allowing highly specific JHE to hydrolyze JH by recognizing the JH-JHBP complex. Finally, JHBP efficiently extracted JH from lipophilic depots (the so-called “refractory pool”, resulting in more rapid degradation of JH by JHE. Thus, JHBP is crucial for maintaining and distributing JH in hemolymph in the absence of JHE, and may assist in the rapid clearance of JH in the presence of JHE.

The last enzyme in the biosynthetic pathway to juvenile hormone III in the corpora allata of hemimetabolous insects is methyl farnesoate epoxidase, a cytochrome P450 monooxygenase. Assays with intact glands incubated in vitro and with gland homogenates have identified a series of 1,5-disubstituted imidazoles as potent inhibitors of the enzyme. We have designed, synthesized and tested two imidazoles, diazirine-Ice T and benzophenone-Ice T, in which a radiolabeled and photoactivatable diazirine or benzophenone group was introduced to label the hydrophobic substrate binding site of the enzyme. Our results show that these bifunctional compounds inhibit JH III synthesis by intact glands as well as methyl farnesoate epoxidation by gland homogenates. Moreover both compounds selectively label a protein of ca. 55 kDa in corpora allata of the cockroach, Diploptera punctata. These photoaffinity labels, which use an imidazole to coordinate to the heme iron and a photoreactive group to modify the hydrophobic substrate binding pocket, are specific and effective probes for the molecular analysis of methyl farnesoate epoxidase.

Background Periodontitis is characterized by microbial infection, inflammation, tissue breakdown, and accelerated loss of alveolar bone matrix. Treatment targeting these multiple stages of the disease provides ways to treat or prevent periodontitis. Certain glycosaminoglycans (GAGs) block multiple inflammatory mediators as well as suppress bacterial growth, suggesting that these GAGs may be exploited as a therapeutic for periodontitis. Methods We investigated the effects of a synthetic GAG, GM-0111, on various molecular events associated with periodontitis: growth of Porphyromonas gingivalis (P. gingivalis) and Aggregatibacter actinomycetemcomitans (A. actinomycetemcomitans) pathogenic bacteria associated with periodontitis; activation of pro-inflammatory signaling through TLR2 and TLR4 in mouse macrophage RAW 264.7 cells and heterologously expressed HEK 293 cells; osteoclast formation and bone matrix resorption in cultured mouse pre-osteoclasts. Results (1) GM-0111 suppressed the growth of P. gingivalis and A. actinomycetemcomitans even at 1% (w/v) solution. The antibacterial effects of GM-0111 were stronger than hyaluronic acid (HA) or xylitol in P. gingivalis at all concentrations and comparable to xylitol in A. actinomycetemcomitans at ≥2% (w/v) solution. We also observed that GM-0111 suppressed biofilm formation of P. gingivalis and these effects were much stronger than HA. (2) GM-0111 inhibited TLR-mediated pro-inflammatory cellular signaling both in macrophage and HEK 293 cells with higher selectivity for TLR2 than TLR4 (IC50 of 1–10 ng/mL vs. > 100 μg/mL, respectively). (3) GM-0111 blocked RANKL-induced osteoclast formation (as low as 300 ng/mL) and bone matrix resorption. While GM-0111 showed high affinity binding to RANKL, it did not interfere with RANKL/RANK/NF-κB signaling, suggesting that GM-0111 inhibits osteoclast formation by a RANKL-RANK-independent mechanism. Conclusions We report that GM-0111 inhibits multiple molecular events involved in periodontitis, spanning from the early pro-inflammatory TLR signaling, to pathways activated at the later stage component of bone loss.

The hemolymph juvenile hormone binding protein (JHBP) from the early fifth instar larvae of Heliothis virescens (Lepidoptera, Noctuidae) has been purified, and three cDNA clones for this protein have been isolated from a fat body cDNA library constructed in bacteriophage λZAP XR. The deduced amino acid sequence of the full-length clone predicts a mature protein consisting of 224 residues, a molecular mass of 24 976 Da, and a p/ of 5.29. Comparison of the amino acid sequence to that of the previously described JHBP from Manduca sexta shows 51 % overall identity with highly conserved N- and C-terminal regions. One of the three clones bound photoactivatable analogs of juvenile hormones with much lower affinity than the other two. This clone had Phe150 in place of the expected Cys150 conserved in other JHBP clones. The F150C mutant of this clone regained native binding affinity. For native Hvir-JHBP, the affinity for [3H]JH I was lower under reducing conditions (87 nM) relative to a 40 nM affinity under nonreducing conditions. The importance of pairs of Cys residues was addressed by preparing Cys to Ala mutants at each site. Expressed proteins were tested for binding affinity by photoaffinity labeling with tritium-labeled JH analogs and by binding assays using (10R,11S)-[3H]JH I. Curiously, the C150A mutant retained full activity, implying that the aberrant C150F was dysfunctional due to steric hindrance rather than to a missing disulfide linkage. Likewise, C29A and C194A had binding affinities unchanged from that of the full-length wild-type clone. Two constructs, C9A and C16A, showed reduced binding affinity, suggesting that they form a disulfide bond important for ligand recognition.

The haemolymph of Procambrus clarkii does not contain detectable levels of methyl farnesoate esterase activity. Homogenates of the hepatopancreas and the vas deferens contain significant methyl farnesoate esterase activity which is sensitive to inhibition by diisopropylfluorophosphate. This activity may not be specific to methyl farnesoate since nonspecific esterase activity is also inhibited by diisopropylfluorophosphate. A methyl farnesoate binding protein, 93 ± 3 kDa MW, has been identified in the haemolymph using the photoaffinity analog [3H]FDK and both native and SDS-PAGE. The binding kinetics of this protein were characterized with [3H] methyl farnesoate and the Kd for methyl farnesoate was found to be 320 ± 150 nM. Competition experiments demonstrated that this protein has a higher affinity toward methyl farnesoate than farnesoic acid or juvenile hormone III and is therefore a methyl farnesoate binding protein.

Membrane preparations from vitellogenic follicles of Locusta migratoria incubated with tritium-labeled photoaffinity analogs of juvenile hormones and methoprene in the presence or absence of excess unlabeled hormone or methoprene were irradiated, subsequently solubilized, the peptides separated by SDS-PAGE and the radioactive proteins visualized by fluorography. Both [3H] epoxyhomofarnesyl diazoacetate ([3H]EHDA), the photoaffinity analog of JH II, and [3H] epoxyfarnesyl diazoacetate ([3H]EFDA), the photoaffinity analog of JH III, covalently modified a 35 kDa peptide, and this labeling was displaced by an excess of unlabeled JH II and JH III respectively. No JH 1-displaceable labeling by [3H] epoxybishomofarnesyl diazoacetate ([3H]EBDA), the photoaffinity analog of JH I, was detected. [3H] methoprene diazoacetate ([3H]MDK), the photoaffinity analog of methoprene, exhibited specific binding to a 17 kDa protein. Using (10R)-[3H]JH III as a ligand, membrane preparations were demonstrated to possess specific and saturable binding: Scatchard analysis revealed a single binding site with a Kp of 3.68 ± 0.46 nM and a total binding capacity (Bmax) of 0.375 ± 0.07 pmol per mg protein. The ability of the various JHs to bind reflects previous findings concerning their biological activity on follicle cells in vitro, suggesting that the binding site is a membrane receptor for JH III.

Juvenile hormone epoxide hydrolase (JHEH) and juvenile hormone diol phosphotransferase (JHDPT) were characterized from the Malpighian tubules of day 1 fifth instar Manduca sexta. An improved RP-HPLC assay is described for the major metabolites of (lOR,lIS) juvenile hormone I: diol, acid, aciddiol, and diol-phosphate. JHEH is strictly associated with membrane fractions, while JHDPT is cytosolic. JHEH may be solubilized in active form by the nonionic detergents Thesit or MEGA-8. Separation of Malpighian tubule cytosol proteins using preparative isoelectric focusing yields two zones which contain JHDPT activity, at pi 4.8-5.1 and 6.8-8.2. The partially purified JHDPT from either zone requires both ATP and Mg2+ for activity, so this enzyme may be formally called either ATP:juveni/e hormone diol phosphotransferase or juvenile hormone diol kinase (EC 2.1.7.3). Metabolites more polar than JH I aciddiol and JH I diol-phosphate are generated in vivo from either [3H]JH I or 13H]JH I diol. 0 1995 Wiley-Liss, inc.

Leaders in the field comment on what they perceive to be the greatest barriers to biomaterial translation.

The current practice of cell therapy, in which multipotent or terminally differentiated cells are injected into tissues or intravenously, is inefficient. Few therapeutic cells are retained at the site of administration and engraftment is low. An injectable and biologically appropriate vehicle for delivery, retention, growth and differentiation of therapeutic cells is needed to improve the efficacy of cell therapy. We focus on a hyaluronan-based semi-synthetic extracellular matrix (sECM), HyStem®, which is a manufacturable, approvable and affordable clinical product. The composition of this sECM can be customized for use with mesenchymal stem cells as well as cells derived from embryonic or induced pluripotent sources. In addition, it can support therapeutic uses of progenitor and mature cell populations obtained from skin, fat, liver, heart, muscle, bone, cartilage, nerves and other tissues. This overview presents four pre-clinical uses of HyStem® for cell therapy to repair injured vocal folds, improve post-myocardial infarct heart function, regenerate damaged liver tissue and restore brain function following ischemic stroke. Finally, we address the real-world limitations - manufacture, regulation, market acceptance and financing - surrounding cell therapy and the development of clinical combination products.

To determine the resorption rate and biocompatibility characteristics of novel cross-linked hydrogel ventilation tubes and varied formulations of polyester ventilation tubes in a Chinchilla model.Animal Study.Three cross-linked glycosaminoglycan hydrogel ventilation tubes fabricated by cross-linking thiol-modified chondroitin sulfate or thiol-modified carboxymethylated hyaluronic acid, four different polyester ventilation tubes (poly L-lactide [PLA], 50/50 poly D,L-lactide-co-glycolide [PLGA], and silver-impregnated versions of PLA and PLGA tubes) were placed into the tympanic membranes of chinchillas. Commercially available fluoroplastic ventilation tubes were placed in the contralateral ear of each animal to serve as a control. Integrity of the tubes was assessed by weekly otoscopy. Biocompatibility was assessed by auditory brainstem response, by otoscopic and histologic examination of the tympanic membrane at the tube site.The hydrogel tubes had very short resorption times that expanded and enlarged the myringotomy site. PLGA and silver-coated PLGA tubes maintained their integrity in the tympanic membrane for similar durations of 18.9 ± 6.4 days and 21.0 ± 6.0 days, respectively. The silver-coated PLGA tubes had lower neutrophil and fibrosis scores than PLGA tubes. PLA tubes demonstrated equivalent findings to commercially available nonresorbable tubes with respect to otoscopic findings, auditory brainstem response thresholds, and histologic inflammatory scores.Resorbable polyester pressure equalization tubes demonstrate predictable resorption behavior and similar biocompatibility characteristics when compared with nonresorbable tubes. Silver modification may confer some stability to PLGA tubes. Hydrogel tubes have very short resorption times, tend to enlarge the myringotomy site, and show greater inflammatory changes.

The vast majority of cells delivered into the heart by conventional means are lost within the first 24 hours. Methods are needed to enhance cell retention, so as to minimize loss of precious material and maximize effectiveness of the therapy. We tested a cell-hydrogel delivery strategy. Cardiosphere-derived cells (CDCs) were grown from adult human cardiac biopsy specimens. In situ polymerizable hydrogels made of hyaluronan and porcine gelatin (Hystem®-C™) were formulated as a liquid at room temperature so as to gel within 20 minutes at 37°C. CDC viability and migration were not compromised in Hystem-C™. Myocardial infarction was created in SCID mice and CDCs were injected intramyocardially in the infarct border zone. Real time PCR revealed engraftment of CDCs delivered in Hystem-C™ was increased by nearly an order of magnitude. LVEF (left ventricular ejection fraction) deteriorated in the control (PBS only) group over the 3-week time course. Hystem-C™ alone or CDCs alone preserved LVEF relative to baseline, while CDCs delivered in Hystem-C™ resulted in a sizable boost in LVEF. Heart morphometry revealed the greatest attenuation of LV remodeling in the CDC + Hystem-C™ group. Histological analysis suggested cardiovascular differentiation of the CDCs in Hystem-C™. However, the majority of functional benefit is likely from paracrine mechanisms such as tissue preservation and neovascularization. A CDC/hydrogel formulation suitable for catheter-based intramyocardial injection exhibits superior engraftment and functional benefits relative to naked CDCs.

The synthesis of D- myo -P-5-( O -aminopropyl)-Ins(1,2,5,6)P 4 , a phosphodiester analog of Ins(1,2,6)P 3 tethered at the C-5 position, has been achieved and a photoaffinity label has been prepared.

Specific affinity labeling of the squalene binding site of squalene epoxidase remains an elusive goal. We wish to report here the design and synthesis of six novel, non-squalenoid, photoaffinity labels for squalene epoxidase, based on the potent inhibitor NB-598 ( 3 ).

Arachidonoyl ethanolamide (anandamide) is a naturally occurring brain constituent that binds to a specific brain cannabinoid receptor (CBR1). An amidase activity (anandamide amidase) in membrane fractions of brain and in cultured neuroblastoma cells rapidly degrades anandamide to arachidonic acid (Deutsch, D. G., and Chin, S. (1993) Biochem. Pharmacol. 46, 791-796). In the current study, analogs of anandamide representing three classes of putative transition-state inhibitor (trifluoromethyl ketones, alpha-keto esters, and alpha-keto amides) were synthesized and tested as inhibitors of anandamide hydrolysis in vitro and as ligands for CBR1. The trifluoromethyl ketones and alpha-keto esters showed nearly 100% inhibition of anandamide hydrolysis in vitro at 7.5 microM inhibitor and 27.7 microM anandamide. Arachidonyl trifluoromethyl ketone was the only synthetic compound in the series of fatty acid derivatives able to displace [3H]CP-55940 binding to CBR1 with a Ki of 0.65 microM. It was also the most effective inhibitor in intact neuroblastoma cells, leading to a 12-fold increase of cellular anandamide levels at 12 microM. From the action of these inhibitors on this hydrolytic enzyme, it seems likely that anandamide is cleaved by a mechanism that involves an active-site serine hydroxyl group. These inhibitors may serve as useful tools to elucidate the role anandamide plays in vivo.

Oligosaccharides derived from hyaluronic acid (HA), a naturally occurring linear polysaccharide composed of repeating disaccharide units of N-acetyl-D-glucosamine and D-glucuronic acid, can be chemically modified to introduce a pendant amine-like functionality (patent application pending). Covalent attachment of steroidal and nonsteroidal antiinflammatory drugs to functionalized HA oligosaccharides was accomplished with the incorporation of hydrolytically labile bonds. Further derivatization of the pendant group with homobifunctional crosslinkers allowed the introduction of covalent crosslinks. Chemically-modified HA oligosaccharides were unambiguously characterized in solution by high-resolution 1H NMR spectroscopy.

A 29-kDa nuclear juvenile hormone (JH)-binding protein from the epidermis of Manduca sexta larvae was purified by using the photoaffinity analog for JH II ([3H]epoxyhomofarnesyldiazoacetate) and partially sequenced. A 1.1-kb cDNA was isolated by using degenerate oligonucleotide primers for PCR based on these sequences. The cDNA encoded a 262-amino acid protein that showed no similarity with other known proteins, except for short stretches of the interphotoreceptor retinoid-binding protein, rhodopsin, and human nuclear protein p68. Recombinant baculovirus containing this cDNA made a 29-kDa protein that was covalently modified by [3H]epoxyhomofarnesyldiazoacetate and specifically bound the natural enantiomer of JH I (Kd = 10.7 nM). This binding was inhibited by the natural JHs but not by methoprene. Immunocytochemical analysis showed localization of this 29-kDa protein to epidermal nuclei. Both mRNA and protein are present during the intermolt periods; during the larval molt, the mRNA disappears but the protein persists. Later when cells become pupally committed, both the mRNA and protein disappear with a transient reappearance near pupal ecdysis. The properties of this protein are consistent with its being the receptor necessary for the antimetamorphic effects of JH.

Developmental profiles of a membrane-associated 50 kDa juvenile hormone epoxide hydrolase (JHEH) and a cytosolic 32 kDa juvenile hormone binding protein (JHBP) in the ovaries and eggs of Manduca sexta were obtained using photoaffinity analogs of JHs. The 50 kDa JHEH was present at a constant level in adult female ovaries, in eggs, and in first instar larvae. In contrast, the cytosolic 32 kDa JHBP was absent in pupae and increased significantly in the adult. The cytosolic 32 kDa JHBP in eggs had properties similar to the hemolymph 32 kDa JHBP in terms of size, isoelectric point, ligand specificity, and immunoreactivity. The level of hemolymph 32 kDa JHBP, however, was present throughout pupal and adult stages. The JH-specific esterase (JHE) activity in ovaries also increased in the adult, remained unchanged until oviposition, and decreased in eggs between days 1 and 2 after oviposition. These results show an increase of cytosolic JHBP and JHE at the termination phase of vitellogenesis and oogenesis. Moreover, it appears that high JHE and JHEH activity in the embryo helps maintain a sufficiently low JH titer to circumvent inhibition of embryonic development. Finally, a hemolymph 80 kDa protein, which was present in larvae and pupae but was absent in the newly eclosed adult female, was also specifically labeled by the JH photoaffinity analogs.

A series of analogues of disparlure, the gypsy moth (Lymantria dispar) sex attractant, was synthesized, and the potency of these inhibitors in suppressing the metabolism of disparlure by the L. dispar epoxide hydrolase (EH) was determined. The analogues substituted at the 6-position (6-hydroxy-, 6-oxo-, and 6,6-difluorodisparlure; (±)-threo,cis-11, (±)-13, and (±)-17, respectively), along with 9,9-difluorodisparlure [(±)-26], were the most potent inhibitors (IC 50 values of 4-9 μM). Two other 9-substituted analogues, 9-hydroxydisparlure [(±)-threo,cis-21] and 9-oxodisparlure [(±)-22], were slightly less potent (IC 50 values of 18 and 30 μM, respectively). Analogues substituted at both the 6- and 9-positions (threo,erythro-6,9-dihydroxy-, threo,threo-6,9-dihydroxy-, and 6,9-dioxodisparlure; (±)-threo,erythro-32, (±)-33, respectively) were generally the least potent inhibitors (IC 50 values of 27-200 μM). On the basis of a model of the EH active site, a hypothesis is advanced to rationalize the higher potencies of the 6-substituted analogues.Pheromone metabolism plays a key role in pheromone perception, and the potential consequences of inhibition of pheromone metabolism are discussed

Juvenile hormone (JH) is catabolized primarily by two enzymes, JH esterase (JHE) and JH epoxide hydrolase (JHEH). A JH binding protein (JHBP) may modulate this degradation by ligand sequestration. In this study, the effects of JHBP on the metabolism of JH by JHEH were examined. 1. (1) Purified JHEH from Manduca sexta (Lepidoptera) eggs converted JH to JH-diol four to five times faster than JH-acid was converted to JH-acid-diol in the absence of JHBP. 2. (2) The presence of JHBP dramatically decreased the degradation of JH, but not that of JH-acid. Similar results were obtained by using crude unsolubilized microsomes. 3. (3) The inhibition of JHEH activity by JHBP was due to the formation of a JH-JHBP complex that could be detected at the origin of a silica gel TLC plate. 4. (4) A crude homogenate of newly oviposited M. sexta eggs was incubated with a physiological concentration of JH. JH-acid-diol and JH-acid were found as the major metabolites, and the rate of hydration of JH-acid by JHEH was faster than that of JH. These results suggested that JH was protected from JHEH by cytosolic JHBP as a JH-JHBP complex; in contrast, JH-acid, a poor ligand for JHBP, was efficiently metabolized into JH-acid-diol by JHEH in vivo. Thus, while JH was not efficiently degraded by JHEH in the presence of JHBP, JHEH could nevertheless act as the ultimate scavenger for JH by hydrating JH-acid generated by JHE during embryogenesis in vivo.

The photoactivatable aryl ketone derivatives have been rediscovered as biochemical probes in the last 5 years. The expanding use of benzophenone (BP) photoprobes can be attributed to three distinct chemical and biochemical advantages. First, BPs are chemically more stable than diazo esters, aryl azides, and diazirines. Second, BPs can be manipulated in ambient light and can be activated at 350-360 nm, avoiding protein-damaging wavelengths. Third, BPs react preferentially with unreactive C-H bonds, even in the presence of solvent water and bulk nucleophiles. These three properties combine to produce highly efficient covalent modifications of macromolecules, frequently with remarkable site specificity. This Perspectives includes a brief review of BP photochemistry and a selection of specific applications of these photoprobes, which address questions in protein, nucleic acid, and lipid biochemistry.

The bacterially expressed recombinant pheromone binding protein (PBP) of Antheraea polyphemus was photoaffinity labeled with (6E,11Z)-[3H]hexadecadienyl diazoacetate, a photoactivatable analog of the naturally occurring acetate pheromone. Radiolabeled peptides were separated from an endoproteinase Lys-C digestion by HPLC and characterized by Edman degradation. The label was exclusively found in the Asp39-Lys58 fragment. Cleavage of this peptide (DDYVMTDRLAGCAINCLATK) with Arg-C gave a single radiolabeled peptide (DDYVMTDR), which was predicted to be alpha-helical. The adjoining LAGCAINCLATK fragment, which is highly conserved in PBP sequences, was predicted to be a hydrophobic beta-strand and has been proposed to be important in recognition of the alkadienyl chain. Edman degradation confirmed the location of the covalently attached ligand at Thr44 of the smaller hydrophilic peptide. In addition, the synthesis of the newly identified pheromone component (4E,9Z)-tetradecadienyl acetate and its photoaffinity analog, (4E,9Z)-[3H]tetradecadienyl diazoacetate, is also described. Mapping of PBP photoaffinity labeled by (4E,9Z)-[3H]14:Dza revealed that the hydrophobic region Asp21-Lys38 adjacent to the primary binding domain Asp39-Lys58 contained a second modification site. The 14-carbon odorant molecule thus had two binding positions within the recognition site, while only a single binding position was available to the 16-carbon pheromone.

The 32 kD juvenile hormone binding protein (JHBP) and two 80 kD proteins in larval Manduca sexta hemolymph were labeled with [3H]FDK, a photoaffinity analog of methyl farnesoate (MF). The labeling could be completely displaced by a 30-fold excess of either MF or JH II, demonstrating that [3H]FDK binds specifically to the JH binding sites of the 32 kD JHBP and the 80 kD proteins. In addition, a high molecular-mass protein was labeled with [3H]FDK; labeling could be displaced by excess MF but not by JH II, demonstrating the selectivity in binding MF. The 32 kD JHBP also appeared to weakly bind the potent juvenoid, methoprene, at the JH binding site. Covalent modification by [3H]FDK induced a change in the apparent size and the isoelectric point of the JHBP. These changes were not induced by substrate alone, nor by UV irradiation alone. The same effect was also observed during labeling with [3H]MDK, an analog of methoprene. These data provide an important caveat for anticipating artifactual changes of protein properties during chemical or photochemical affinity labeling experiments. The molecular dimensions of [3H]FDK more closely resemble those of JH II than those of [3H]EHDA, a photoactivatable analog of JH II. We suggest that covalent modification by a diazoketone photolabel involves a hydrophilic amino acid important in the recognition of the ester group of JH. © 1994 Wiley-Liss, Inc.

Synthesis of (10R)-juvenile hormone III (JH III) outside the corpora allata (CA) was investigated in female Aedes aegypti. Intact females or ligated abdomens of blood-fed and sugar-fed females synthesized in vivo [12-3H]JH III-like molecules from [12-3H]-methyl farnesoate, indicating that an organ(s) in the female abdomen, other than the CA, converted methyl farnesoate into JH III. To find out the organ(s) that synthesized JH III-like molecules, ovaries, fat bodies, and midguts were incubated in vitro with [12-3H]methyl farnesoate and the synthesis of JH III-like molecules was compared with JH III synthesized by CA. To identify tissue(s) having both farnesoic acid methyl transferase and farnesoate epoxidase, enzymes that convert farnesoic acid into JH III, ovaries, and fat bodies were removed from sugar and blood-fed females and incubated with [12-3H]farnesoic acid. Chemical derivatization by methoxyhydrin formation followed by esterification with (+)-α-methoxy- α-trifluoromethyl phenylacetic (MTPA) acid chloride and reversed phase liquid chromatography identified (10R)-JH III methoxyhydrin (+)-MTPA ester as the sole JH III-like molecule produced in tissue culture incubation of ovaries. Since only (10R)-JH III is produced and not racemic JH III, the oxidation of farnesoic acid must be enzymatically mediated. Ovaries and corpora allata of female A. aegypti also synthesized [3H,14C]JH III from L-[methyl-3H]methionine and [14C]acetate which was characterized by HPLC and gas chromatography. These results suggest that mosquito ovary can synthesize (10R)-JH III from farnesoic acid, and that this tissue synthesizes JH III-like molecules from L-methionine and acetate. © 1994 Wiley-Liss, Inc.

The cellular microenvironment is recognized to play a key role in stabilizing cell differentiation states and phenotypes in culture. This study addresses the hypothesis that preservation of in vivo-like tissue architecture in vitro produces a cell culture more capable of responding to environmental stimuli with clinically relevant toxicity biomarkers. This was achieved using kidney proximal tubules in three-dimensional organoid hydrogel culture, with comparisons to conventional monolayer kidney cell cultures on plastic. Kidney proximal tubule cultures and two immortalized kidney cell line monolayer cultures exposed to known nephrotoxic drugs were evaluated for inflammatory cytokines, nephrotoxicity-associated genes, Kim-1 protein, cytochrome enzymes, and characteristic cellular enzyme shedding. Significant similarities are shown for these traditional biomarkers of kidney toxicity between in vivo and 3-D organoid endpoints of drug toxicity, and significantly, a consistent lack of clinically relevant endpoints produced by traditional 2-D kidney cell cultures. These findings impact both in vitro bioreactor-based kidney functional and regenerative medicine models, as well as high-throughput cell-based drug screening validations.

The incidence of melanoma continues to rise, in particular among adult males residing in the U.S. (Jemal, Siegel et al. 2010). Although this disease is treatable in its early stages, diagnosis of or progression to an advanced, metastatic disease stage drastically reduces the prognosis, with few patients surviving 5 years. It is especially disturbing that there is a lack of FDA-approved therapeutics that can adequately treat the disease and very low percentages (5-15%) of patients who will respond to these traditional chemotherapeutics (Thompson, Morton et al. 2004). Even among responders, the response to therapy is usually unsustainable over the long-term, with approximately 10.8% of patients achieving long-term survival (Kim, Lee et al. 2010). Dacarbazine or DTIC is considered the most appropriate cytotoxic chemotherapy, but response rates are poor. Many other single-agents have been tried with response rates ≤18%, these include temozolomide, lomustine, carmustine, cisplatin, carboplatin, vincristine, vinblastine, vindesine, paclitaxel, docetaxel, gemcitabine and topotecan. (Thompson, Morton et al. 2004) Without patient selection to decipher who belongs to the small portion of responders, the more common approach to therapy is to enroll patients into a clinical trial. Recent trials examining more targeted and selective agents like PLX4032 (Flaherty, Puzanov et al. 2010) and ipilimumab (Hodi, O'Day et al. 2010) have further reinforced this clinical ideology due to the successes observed among some patients. Although the development of chemoresistance is an impediment to achieving cure using these novel agents, they represent the current ideal and a hopeful future for promising targeted therapeutics. Thus, more specific drugs are desirable to increase the options for therapy and improve overall outcomes. A recently discovered and highly “druggable” method to target melanoma cells is through inhibition of a G protein-coupled receptor (GPCR) that is responsible for viability among melanomas that predominantly express it (Altman, Gopal et al. 2010). This GPCR is called the Lysophosphatidic Acid (LPA) receptor 3 or the LPA3 receptor (Bandoh, Aoki et al. 1999) and is found along the cell surface of specific types of melanomas. In addition, the high

Metastasis is a spread of cancer cells to a distant location from the primary tumor. This process involves a complex series of events similar to those that occur during inflammation. The events of metastasis can be divided into four major steps. First, cancer cells proliferate and transform to acquire motility and ability to invade basement membrane to reach blood vessels. Second, cancer cells penetrate blood vessels due to increased vascular permeability facilitated by inflammatory mediators and enter the circulation. Third, in the circulation cancer cells are shielded by host cells (platelets and neutrophils) to escape surveillance by immune cells and survive shear forces of the bloodstream. Next, integrin-mediated arrest of circulating cancer cells occurs on the endothelial surface before extravasation. Once cancer cells extravasate, heparanase from cancer cells degrades heparan sulfates of the extra cellular matrix. The cleavage of heparan sulfates release growth factors that stimulate cancer cell growth as well as angiogenesis. The Receptor for Advanced Glycation Endproducts (RAGE) belongs to the immunoglobulin superfamily of cell surface molecules. The receptor’s name, RAGE, was coined for its ability to bind its first described ligand, advanced glycation end products (AGEs), which accumulate in physiological (aging) and pathological disorders such as diabetes (Schmidt et al., 1992). RAGE is a pattern recognition receptor for its ligation to structurally unrelated ligands that include Mac-1, HMGB1 and S100 /calgranulins. Similar to immunoglobulin, RAGE contains an extracellular structure with a V-type binding region and two C-type regions. Immediately following the C-type region is a transmembrane region and a short cytoplasmic domain (Fig. 1). The important roles played by RAGE in inflammation, diabetes, Alzhiemer’s disease and cancer have been discussed in detail (Ellerman et al., 2007; Logsdon et al., 2007; Schmidt et al., 2001; Sims et al., 2010; Tang et al., 2010). RAGE is ubiquitously expressed in tissues and inflammatory cells at low levels in homeostasis and its expression is increased in stress conditions. RAGE expression is observed in many tumors, including brain, breast, colon, lung, prostate, pancreatic, ovarian cancers, lymphoma and melanoma (Hsieh et al., 2003; Logsdon et al., 2007) and elevated levels of RAGE have been reported in colon (Sasahira et al., 2005), prostate (Ishiguro et al., 2005) and gastric cancers

Human livers have maturational lineages of cells within liver acini, beginning periportally in stem cell niches, the canals of Hering, and ending in polyploid hepatocytes pericentrally and cholangiocytes in bile ducts. Hepatic stem cells (hHpSCs) in vivo are partnered with mesenchymal precursors to endothelia (angioblasts) and stellate cells, and reside in regulated microenvironments, stem cell niches, containing hyaluronans (HA). The in vivo hHpSC niche is modeled in vitro by growing hHpSC in two-dimensional (2D) cultures on plastic. We investigated effects of 3D microenvironments, mimicking the liver’s stem cell niche, on these hHpSCs by embedding them in HA-based hydrogels prepared with Kubota’s Medium (KM), a serum-free medium tailored for endodermal stem/progenitors. The KM-HA hydrogels mimicked the niches, matched diffusivity of culture medium, exhibited shear thinning and perfect elasticity under mechanical loading, and had predictable stiffness depending on their chemistry. KM-HA hydrogels, which supported cell attachment, survival and expansion of hHpSC colonies, induced transition of hHpSC colonies towards stable heterogeneous populations of hepatic progenitors depending on KM-HA hydrogel stiffness, as shown by both their gene and protein expression profile. These acquired phenotypes did not show morphological evidence of fibrotic responses. In conclusion, this study shows that the mechanical properties of the microenvironment can regulate differentiation in endodermal stem cell populations.

Phosphatidylinositol-4-phosphate (PtdIns(4)P) is an essential component of eukaryotic membranes and a marker of the Golgi complex. The pivotal role of PtdIns(4)P in signaling and trafficking necessitates the development of synthetic derivatives of this lipid that are resistant to hydrolysis and therefore can be used in biochemical, structural and functional assays to monitor time-dependent processes at the Golgi membranes. Here, we developed novel metabolically stabilized (ms) analogues of the PtdIns(4)P lipid and the inositol 1,4-bisphosphate (IP2) head group derivative suitable for outfitting with a reporter tag and demonstrated that these compounds can substitute the natural lipid in peripheral protein recruitment and membrane deformation. The methylenephosphonate (MP) and phosphorothioate (PT) analogues of PtdIns(4)P and the aminohexyl (AH)-IP2 probe are recognized by the PtdIns(4)P-specific PH domain of four phosphate adaptor protein 1 (FAPP1). Binding of FAPP1 to the PtdIns(4)P derivatives stimulates insertion of the PH domain into the lipid layers and induces tubulation of membranes. Both ms-analogues and IP2-probes could be invaluable for identifying novel protein effectors and characterizing and monitoring PtdIns(4)P-dependent signaling cascades within the trans-Golgi network (TGN).

The development of injectable and biocompatible vehicles for delivery, retention, growth, and differentiation of stem cells is of paramount importance for regenerative medicine. For cell therapy and the development of clinical combination products, we created a hyaluronan (HA)-based synthetic extracellular matrix (sECM) that provides highly reproducible, manufacturable, approvable, and affordable biomaterials. The composition of the sECM can be customized for use with progenitor and mature cell populations obtained from skin, fat, liver, heart, muscle, bone, cartilage, nerves, and other tissues. This overview describes the design criteria for “living” HA derivatives, and the many uses of this in situ crosslinkable HA-based sECM hydrogel for three-dimensional (3-D) culture of cells in vitro and translational use in vivo. Recent advances allow rapid expansion and recovery of cells in 3-D, and the bioprinting of engineered tissue constructs. The uses of HA-derived sECMs for cell and molecule delivery in vivo will be reviewed, including applications in cancer biology and tumor imaging.

Publisher Summary With the rapidly increasing demand for stem cells, the problems related to their storage and preservation, maintenance, and expansion become ever more acute. The requirements for stem cell banking would place the need for preserving quiescence and stemness at the top of the list, whereas the requirements for transplantation of stem and progenitor cells would give preference to the possibility of increasing the mass of stem cells without losing their properties. The constellation of different requirements should orbit around the physiological ways of preserving stem cells, i.e. stem cell niches. The niches are defined as the umbrella microenvironment supporting cell attachment and quiescence by sheltering cells from proliferation and differentiation signals, enhancing cell survival, regulating stem cell division and renewal, and coordinating the population of resident stem cells to meet the actual requirements of an organ. The way in which these diverse functions are accomplished by the niches may vary from one type of stem cells and their niche to another, yet the general outline is preserved. It includes specific components of the extracellular matrix (ECM) that create a scaffold and a boundary to the niche, cell-cell and cell-matrix adhesion molecules, locally stored growth and/or quiescence factors, and a set of instructions for proliferation and/or migration that can be triggered on demand. The vicinity of vascular beds, often covered with sinusoidal endothelial cells, provides a portal for entry into the circulation.

A significant stumbling block in the creation of functional three-dimensional (3D) engineered tissues is the proper vascularization of the constructs. Furthermore, in the context of electrospinning, the development of 3D constructs using this technique has been hindered by the limited infiltration of cells into their structure. In an attempt to address these issues, a hybrid mesh of poly (ɛ-caprolactone)-collagen blend (PCL/Col) and hyaluronic acid (HA) hydrogel, Heprasil™ was created via a dual electrodeposition system. Simultaneous deposition of HA and PCL/Col allowed the dual loading and controlled release of two potent angiogenic growth factors VEGF 165 and PDGF-BB over a period of five weeks in vitro. Furthermore, this manner of loading sustained the bioactivity of the two growth factors. Utilizing an in-house developed 3D co-culture assay model of human umbilical vein endothelial cells and lung fibroblasts, the growth factor-loaded hybrid meshes was shown to not only support cellular attachment, but also their infiltration and the recapitulation of primitive capillary network in the scaffold’s architecture. Thus, the creation of a PCL/Col-Heprasil hybrid scaffold is a step forward toward the attainment of a 3D bio-functionalized, vascularized tissue engineering construct.