Direct conjugation of antibodies to the ZnS shell of quantum dots for FRET immunoassays with low picomolar detection limits.

Compact and functional nanoparticle-antibody conjugates are of paramount importance for the development of quantum dot (QD)-based immunoassays. Here, we present a simple strategy to directly conjugate IgG, F(ab')2, and Fab antibodies via their endogenous disulfide groups directly to the inorganic ZnS shell of compact penicillamine-coated QDs. The functionality of the conjugates was demonstrated by terbium (Tb)-to-QD FRET immunoassays against prostate specific antigen in serum samples. Detection limits of 2.5 pM (0.080 ng mL-1) were 10 and 25 times lower compared to conjugation via maleimide-terminated ligands and polymer chains, respectively. These more compact, simple, and sensitive QD-antibody conjugates will be highly advantageous for nanocrystal-based biosensing applications.

Reentrant surface anisotropy in the antiferromagnetic/ferromagnetic bilayer Mn/Co/Cu(001).

We investigated the magnetic anisotropy energy of monatomic surface-step atoms in antiferromagnetic/ferromagnetic (AF/FM) epitaxial Mn/Co bilayers grown on vicinal Cu(001) surfaces. The step-induced anisotropy of the Co/Cu(001) films was quenched upon submonolayer Mn deposition, but a reentrant uniaxial surface anisotropy was observed for Mn thickness (tMn) between 1 and 2 monolayers, which disappears for Mn thickness above 2 monolayers. In the Mn/Co/Cu(001) system, Mn films undergo a tMn-dependent transition from FM to AF in the 1-2 Mn monolayer thickness range, which entails the coexistence of FM and AF Mn phases in the film. The observation of a sizeable uniaxial anisotropy exclusively in the Mn-thickness range of coexistence of the FM and AF phases points out the crucial role of the boundaries between FM and AF regions within the Mn film. A symmetry-breaking mechanism of a magnetic type, rather than a purely geometric one, is therefore proposed as the origin of the reentrant anisotropy.

Tuning the magneto-optical response of iron oxide nanocrystals in Au- and Ag-based plasmonic media.

We investigated the magneto-optical response of chemically synthesized iron oxide magnetic nanocrystals, optically coupled with ordered planar arrays of plasmonic nanoparticles. We compare the signals from two classes of systems, featuring either Au or Ag as the plasmonic counterpart. The localized surface plasmon resonance of the Ag and Au nanoparticles arrays were superimposed or detuned, respectively, with respect to the dominant magneto-optical transitions of the magnetic material. Under resonance, a significant enhancement of the magneto-optical signal was observed. In both cases, we could separate the purely plasmonic and the magnetic contributions in the magneto-optical spectrum of the optically coupled composite based on their different magnetic-field dependence.

NF-κB subunits are differentially distributed in cells of lumbar dorsal root ganglia in naïve and diabetic rats.

Previous studies demonstrated that nuclear factor κB (NF-κB) activation is decreased in dorsal root ganglia (DRG) of rats having streptozotocin (STZ)-induced diabetes. DRG contain cell bodies of neurons that convey sensory signals from the periphery. To determine the relationship between diabetes-induced neuropathy and NF-κB expression in DRG, behavioral, immunohistochemical, and biochemical studies were performed on naïve and 3-month diabetic rats. Behavioral studies confirmed that many diabetic rats develop tactile allodynia, or increased sensitivity to light touch, in the hind paws. Immunohistochemical studies on lumbar DRG that receive input from the affected regions revealed that p50 and p65, frequent NF-κB subunit partners, are differentially localized. Intense p65 immunostaining was detected in the cytoplasm of small- and medium-sized neurons as well as in satellite cells. In contrast, p50 was localized in the cytoplasm of virtually all neurons. In many cases, prominent staining was also present in nuclei, a location consistent with transcription factor activation. Immunohistochemical and biochemical studies found that the nuclear to cytoplasmic ratio of p50 expression was significantly reduced in diabetic rats compared to that in naïve animals. Our findings raise the possibility that changes in NF-κB activation in a subset of DRG neurons participates in mediating diabetes-induced sensory neuropathy.

The p400/Tip60 ratio is critical for colorectal cancer cell proliferation through DNA damage response pathways.

The Tip60 histone acetyltransferase belongs to a multimolecular complex that contains many chromatin remodeling enzymes including the ATPase p400, a protein involved in nucleosomal incorporation of specific histone variants and that can directly or indirectly repress some Tip60-dependent pathways. Tip60 activity is critical for the cellular response to DNA damage and is affected during cancer progression. Here, we found that the ratio between Tip60 and p400 mRNAs is affected in most colorectal carcinoma. Strikingly, reversing the p400/Tip60 imbalance by Tip60 overexpression or the use of siRNAs resulted in increased apoptosis and decreased proliferation of colon-cancer-derived cells, suggesting that this ratio defect is important for cancer progression. Furthermore, we demonstrate that the p400/Tip60 ratio controls the oncogene-induced DNA damage response, a known anticancer barrier. Finally, we found that it is also critical for the response to 5-fluorouracil, a first-line treatment against colon cancer. Together, our data indicate that the p400/Tip60 ratio is critical for colon cancer cells proliferation and response to therapeutic drugs through the control of stress-response pathways.

Growth dynamics of L-cysteine SAMs on single-crystal gold surfaces: a metastable deexcitation spectroscopy study.

We report on a metastable deexcitation spectroscopy investigation of the growth of L-cysteine layers deposited under UHV conditions on well-defined Au(110)- (1 × 2) and Au(111) surfaces. The interaction of He(*) with molecular orbitals gave rise to well-defined UPS-like Penning spectra which provided information on the SAM assembly dynamics and adsorption configurations. Penning spectra have been interpreted through comparison with molecular orbital DFT calculations of the free molecule and have been compared with XPS results of previous works. Regarding adsorption of first-layer molecules at room temperature (RT), two different growth regimes were observed. On Au(110), the absence of spectral features related to orbitals associated with SH groups indicated the formation of a compact SAM of thiolate molecules. On Au(111), the data demonstrated the simultaneous presence, since the early stages of growth, of strongly and weakly bound molecules, the latter showing intact SH groups. The different growth mode was tentatively assigned to the added rows of the reconstructed Au(110) surface which behave as extended defects effectively promoting the formation of the S-Au bond. The growth of the second molecular layer was instead observed to proceed similarly for both substrates. Second-layer molecules preferably adopt an adsorption configuration in which the SH group protrudes into the vacuum side.

Isolating the step contribution to the uniaxial magnetic anisotropy in nanostructured Fe/Ag(001) films.

We have investigated the possibility of isolating the step-induced in-plane uniaxial magnetic anisotropy in Fe/Ag(001) films on which nanoscale surface ripples were fabricated by the ion sculpting technique. For rippled Fe films deposited on flat Ag(001), the steps created along the ripple sidewalls are shown to be the only source of uniaxial anisotropy. Ion sculpting of ultrathin magnetic films allows one to selectively study the step-induced anisotropy and to investigate the correlation between local atomic environment and magnetic properties.

Temperature driven reversible breakdown of pseudomorphism in ultrathin Fe/Cu3Au films.

We observe that ultrathin Fe/Cu(3)Au(001) films in the 6-13 A thickness range, beyond the thickness of pseudomorphism breakdown at room temperature, exhibit a temperature dependent structural phase transition in the range T(c) approximately 345-380 K. In the high temperature state the Fe film becomes pseudomorphic, while breakdown of pseudomorphism reversibly occurs as the system is cooled below the transition temperature. The difference between substrate and overlayer thermal expansion coefficient is highlighted as the driving force for the observed transition.

Uniaxial magnetic anisotropy in nanostructured Co/Cu(001): from surface ripples to nanowires.

We have investigated the correlation between morphology and magnetic anisotropy in nanostructured Co films on Cu(001). The formation of nanoscale ripples by ion erosion is found to deeply affect the magnetic properties of the Co film. A surface-type uniaxial magnetic anisotropy with easy axis parallel to the ripples is observed. The origin of the magnetic anisotropy has been identified with the modification of thermodynamic-step distribution induced by ripple formation. At higher ion doses, when Co ripples detach and crystalline nanowires form, a strong enhancement of the magnetic anisotropy due to magnetostatic contributions is observed.

Proliferative and morphological effects of endothelins in Schwann cells: roles of p38 mitogen-activated protein kinase and Ca(2+)-independent phospholipase A2.

The regulation of Schwann cell (SC) proliferation and morphology is critical to nerve homeostasis. We have previously reported that endothelins (ETs) regulate the activity of different effectors in SC including adenylyl cyclase, phospholipases C and A2 and mitogen-activated protein kinases (MAPKs). These effects imply a possible participation of ETs in the regulation of SC phenotype. We have now investigated the effects of endothelins on the proliferation and morphology of SC, and compared them with the responses to platelet-derived growth factor (PDGF), a known mitogen in these cells. Both endothelin-1 (ET-1) and PDGF increased the incorporation of [3H]thymidine and the proportion of SC in S and G2/M, with a concomitant decrease in the G0/G1 stage cells. Treatment with ET-1 produced rapid changes in the morphology of the SC, characterized by the appearance of cell spreading with shorter processes. The response to ET-1 was considered to represent a proliferative phenotype, in contrast to the effects of forskolin, which decreased [3H]thymidine incorporation in immortalized SC (iSC) and lead to a differentiated morphology with longer extensions. While both ET-1 and PDGF displayed a proliferative effect on SC, treatment with PDGF did not affect the morphology of these cells to a significant extent. A role for p38 MAPK and Ca(2+)-independent phospholipase A2 in the changes in morphology and proliferation of iSC driven by ET-1 was suggested by the effects of selective inhibitors of these pathways [SB202190 and HELSS, respectively]. The unique pattern of signaling pathways recruited by ET-1 and its combined effects on regulation of phenotype and proliferation of SC suggest an important role for this peptide during nerve degeneration/regeneration.

Role of the human V1 vasopressin receptor COOH terminus in internalization and mitogenic signal transduction.

We studied the role played by the intracellular COOH-terminal region of the human arginine vasopressin (AVP) V1-vascular receptor (V1R) in ligand binding, trafficking, and mitogenic signal transduction in Chinese hamster ovary cells stably transfected with the human AVP receptor cDNA clones that we had isolated previously. Truncations, mutations, or chimeric alterations of the V1R COOH terminus did not alter ligand binding, but agonist-induced V1R internalization and recycling were reduced in the absence of the proximal region of the V(1)R COOH terminus. Coupling to phospholipase C was altered as a function of the COOH-terminal length. Deletion of the proximal portion of the V1R COOH terminus or its replacement by the V2-renal receptor COOH terminus prevented AVP stimulation of DNA synthesis and progression through the cell cycle. Mutation of a kinase consensus motif in the proximal region of the V1R COOH terminus also abolished the mitogenic response. Thus the V1R cytoplasmic COOH terminus is not involved in ligand specificity but is instrumental in receptor trafficking and facilitates the interaction between the intracellular loops of the receptor, G protein, and phospholipase C. It is absolutely required for transmission of the mitogenic action of AVP, probably via a specific kinase phosphorylation site.

Endothelins regulate arachidonic acid release and mitogen-activated protein kinase activity in Schwann cells.

Immortalized rat Schwann cells (iSC) express endothelin (ET) receptors coupled to inhibition of adenylyl cyclase and stimulation of phospholipase C (PLC). These effects precede phenotypic changes and increased DNA synthesis. We have investigated the role of ETs in the regulation of arachidonic acid (AA) release and mitogen-activated protein kinases (MAPKs). Both ET-1 and ET-3 increased AA release in iSC. This effect was sensitive to the phospholipase A(2) (PLA(2)) inhibitors E:-6-(bromomethylene)tetrahydro-3-(1-naphthalenyl)-2H:-pyran-2-one and arachidonyl-trifluoromethyl ketone but was insensitive to inhibitors of PLC or phospholipase D-dependent diacylglycerol generation. ET-1-dependent AA release was also unaffected by removal of extracellular Ca(2+) and blocking the concomitant elevation in [Ca(2+)](i), consistent with participation of a Ca(2+)-independent PLA(2). Treatment of iSC with ETs also resulted in activation of extracellular signal-regulated kinase, c-Jun-NH(2)-terminal kinase (JNK), and p38 MAPK. A cause-effect relationship between agonist-dependent AA release and stimulation of MAPKs, but not the opposite, was suggested by activation of JNK by exogenous AA and by the observation that inhibition of MAPK kinase or p38 MAPK was inconsequential to ET-1-induced AA release. Similar effects of ETs on AA release and MAPK activity were observed in cultures expanded from primary SC and in iSC. Regulation of these effectors may mediate the control of proliferation and differentiation of SC by ETs during peripheral nerve development and regeneration.

Signal transduction pathways of the human V1-vascular, V2-renal, V3-pituitary vasopressin and oxytocin receptors.

Vasopressin (VP) and oxytocin (OT) are cyclic nonapeptides whose actions are mediated by stimulation of specific G protein-coupled receptors (GPCRs) currently classified into V1-vascular (V1R), V2-renal (V2R) and V3-pituitary (V3R) VP receptors and OT receptors (OTR). The recent cloning of the different members of the VP/OT family of receptors now allows the extensive characterization of the molecular determinants involved in ligand binding and signal transduction pathways coupled to a given VP/OT receptor subtype in stably transfected mammalian cell lines. In this article, we review the present knowledge of the signal transduction pathways coupled to the different VP/OT receptor subtypes and we present new observations derived from the study of each human VP or OT receptor subtype stably expressed in CHO cells.

Molecular pharmacology of human vasopressin receptors.

Vasopressin (AVP) and oxytocin (OT) are cyclic nonapeptides whose actions are mediated by activation of specific G protein-coupled receptors (GPCRs) currently classified into V1-vascular (V1R), V2-renal (V2R) and V3-pituitary (V3R) AVP receptors and OT receptors (OTR). The cloning of the different members of the AVP/OT family of receptors now allows the extensive molecular pharmacological characterization of a single AVP/OT receptor subtype in stably transfected mammalian cell lines. The human V1-vascular (CHO-V1), V2-renal (CHO-V2), V3-pituitary (CHO-V3) and oxytocin (CHO-OT) receptors stably expressed in CHO cells display distinct binding profiles for 18 peptide and 5 nonpeptide AVP/OT analogs. Several peptide and nonpeptide compounds have a greater affinity for the V1R than AVP itself. V2R peptide agonists and antagonists tend to be non-selective ligands whereas nonpeptide V2R antagonists are potent and subtype-selective. None of the 22 AVP/OT analogs tested has a better affinity for the human V3R than AVP itself. Several peptide antagonists do not select well between V1R and OTR. These results underscore the need for developing specific and potent analogs interacting specifically with a given human AVP/OT receptor subtype.

The human V3 pituitary vasopressin receptor: ligand binding profile and density-dependent signaling pathways.

The vasopressin (AVP) V3 pituitary receptor (V3R) is a G protein-coupled corticotropic phenotypic marker that is overexpressed in ACTH-hypersecreting tumors. Studies of the agonist/antagonist binding profile and signal transduction pathways linked to the human V3R have been limited because of the scarcity of this protein. To define the signals activated by V3Rs and the eventual changes triggered by developmental or pathological receptor regulation, we developed Chinese hamster ovary (CHO)-V3 cells stably expressing low, medium, or high levels of human V3Rs (binding capacity, <10, 10-25, and 25-100 pmol/mg, respectively). The affinity of the V3R for 21 peptide and nonpeptide AVP analogs was clearly distinct from that exhibited by the human V1R and V2R. AVP triggered stimulation of phospholipase C in CHO-V3 cells (partially sensitive to treatment with pertussis toxin) with a potency directly proportional to receptor density. V3R-mediated arachidonic acid release also was also sensitive to pertussis toxin and more efficacious in cells exhibiting medium than in those with high receptor density. AVP also stimulated the pertussis toxin-insensitive uptake of [3H]thymidine in CHO-V3 cells. The concentration-response curves for this effect were monophasic in cells expressing low and medium levels of V3Rs; on the contrary, a biphasic curve was observed in cells with high V3R density. Coupling of V3R to increased production of cAMP was only observed in CHOV3 high cells, suggesting a negative relationship between increased cAMP production and DNA synthesis. Activation of mitogen-activated protein kinases by V3R was pertussis toxin insensitive, but was dependent on activation of phospholipase C and protein kinase C; both the level and duration of activation were a function of the receptor density. Thus, the human V3R has a pharmacological profile clearly distinct from that of the human V1R and V2R and activates several signaling pathways via different G proteins, depending on the level of receptor expression. The increased synthesis of DNA and cAMP levels observed in cells expressing medium and high levels of V3Rs, respectively, may represent important events in the tumorigenesis of corticotroph cells.

Immortalized schwann cells express endothelin receptors coupled to adenylyl cyclase and phospholipase C.

Endothelins (ETs) are potent regulators of renal, cardiovascular and endocrine functions and act as neurotransmitters in the CNS. Here we report that immortalized Schwann cells express receptors for ETs and characterize some of the cellular events triggered by their activation. Specific binding of [125I]-ET-1 to Schwann cell membranes was inhibited by ET-1 and ETB-selective agonists ET-3, sarafotoxin 6c and [Ala1,3,11,15]-ET-1 with IC50cor values ranging between 2 and 20 nM. No competition was observed with the ETA receptor-selective antagonist BQ123. Incubation of [3H]-inositol pre-labeled Schwann cells with ET-1, ET-3 or sarafotoxin 6c elicited a concentration-dependent increase in the release of [P1 that reached a plateau at approximately 100 nM. The efficacy of [Ala1,3,11,15]-ET-1 (a linear peptide analog of ET-1) was half of that corresponding to ET-1. These stimulatory effects were partially blocked by pre-incubation with pertussis toxin. When Schwann cells were incubated in the presence of 100 nM ET-1 or ET-3 there was a significant inhibition of basal and isoproterenol-stimulated cAMP levels. The inhibitory effects of sarafotoxin 6c and [Ala1,3,11,15]-ET-1 on isoproterenol-stimulated cAMP levels were similar to that observed with ET-1. Pre-incubation with pertussis toxin completely prevented this effect. These observations indicate that immortalized Schwann cells express receptors for ET peptides (predominantly ETB) coupled to modulation of phospholipase C and adenylyl cyclase activities. The actions of ETs on Schwann cells provide a novel example of the influence of vascular factors on nerve function.

P2-purigenic receptors regulate phospholipase C and adenylate cyclase activities in immortalized Schwann cells.

Schwann cells play an important role in both the development and regeneration of peripheral nerves. Proliferation and differentiation of Schwann cells are critically dependent on changes in the levels of cAMP. ATP is a fast excitatory transmitter in the peripheral nervous system, inducing depolarization of the vagus nerve through occupancy of P2-purinergic receptors. In the present study we demonstrate that extracellular ATP stimulates phospholipase C and inhibits adenylate cyclase activities in cultured Schwann cells. Addition of ATP inhibited, in a concentration-dependent manner, forskolin- or isoprenaline-stimulated adenylate cyclase activity. The rank order of potency corresponding to different purinergic receptor agonists was 2-methylthio-ATP > ATP = ADP > or = adenosine 5'-[gamma-thio]triphosphate (ATP[S]) > UTP, consistent with the involvement of a P2y subtype. Adenosine and adenosine 5'-[alpha,beta-methylene]-triphosphate (pp[CH2pA) were ineffective. Preincubation with pertussis toxin completely blocked this inhibitory effect. When Schwann cells were pre-labelled with myo-[3H]inositol and incubated in Hanks' balanced salt solution containing Ca2+ and Mg2+, addition of ATP[S] resulted in a concentration-dependent increase in the release of InsP with a concomitant increase in intracellular free [Ca2+] ([Ca2+]i). Under these conditions, the effects of both ATP and UTP were of lower magnitude. Removal of Ca2+ and Mg2+ from the assay medium resulted in a significant increase in the effects of ATP[S], ATP and UTP. The decreased response observed in the presence of both bivalent cations (1.2 mM Ca2+ and 1 mM Mg2+) could not be explained either by increased degradation of ATP by Ca2+/Mg2+-dependent nucleotidases or by cation influx. The rank order of potency for the effects of agonists on phospholipase C activity was ATP[S] = adenosine 5'[gamma-imido]triphosphate > ATP -UTP > ADP, indicating the involvement of a P(2U) receptor subtype in this response. Adenosine, AMP and pp[CH2]pA were ineffective. These results demonstrate that immortalized Schwann cells express P(2U) and P(2Y) purinoceptors, which are coupled to stimulation of phospholipase C and inhibition of adenylate cyclase, respectively. Our observations unveil signal-transduction pathways that may be used by ATP to regulate proliferation and differentiation of Schwann cells, and ultimately to influence nerve homeostasis.

An aldose reductase inhibitor but not myo-inositol blocks enhanced polyphosphoinositide turnover in peripheral nerve from diabetic rats.

Experimental diabetic neuropathy, whether chemically induced or present in several spontaneously diabetic animal models, is characterized by sorbitol accumulation and myo-inositol depletion and usually also by enhanced turnover of the monoesterified moieties of polyphosphoinositides, particularly phosphatidylinositol-4,5-bisphosphate (PIP2). This study examined the relationship of these alterations by assessing the effects of myo-inositol and the aldose reductase inhibitor, sorbinil, supplied as dietary supplements, on sorbitol and myo-inositol concentrations and incorporation of 32P into polyphosphoinositides in sciatic nerve from rats killed 8 weeks after induction of diabetes with streptozotocin. Nerves from diabetic rats killed after 8 weeks of disease exhibited 52% to 76% greater PIP2 labeling, markedly elevated sorbitol levels, and 30% less myo-inositol when compared with age-matched normal rats. Incorporation of isotope into PIP2 in nerves from animals fed a myo-inositol supplement, added to either a high-sucrose diet or standard rat chow beginning immediately after induction of diabetes, remained substantially elevated, whereas myo-inositol levels were corrected to normal. Essentially the same results were obtained when rats were fed the myo-inositol-containing diet beginning 4 weeks after streptozotocin injection. In contrast, PIP2 labeling in nerves from diabetic rats that received the sorbinil-supplemented diet for either 4 or 8 weeks was not different from that in controls. myo-Inositol levels in these animals were also restored to normal, whereas sorbitol levels remained elevated, albeit reduced by approximately 30%. These results indicate that myo-inositol administration is unable to completely counteract the impact of diabetes on the turnover of monoesterified phosphate groups in PIP2. In contrast, sorbinil can correct this abnormality, but this beneficial effect is not dependent on the presence of normal sorbitol concentrations.