Naturally occurring peptides with biological actions have in most cases been detected by observing their biological activities in crude extracts and their isolation has been followed using bioassays. As a complement to the classical biological detection systems, we have proposed a chemical detection system based on fragmentation of peptides in tissue extracts followed by identification of certain of these peptide fragments having distinct chemical features. One such chemical feature is the C-terminal amide structure which is characteristic of many biologically active peptides. We have devised a chemical assay method for peptides having such a structure and have found several previously unknown peptide amides in procine upper small intestinal tissues. We report here the isolation and characterization of two of them, designated PHI and PYY. PHI is related to secretin, vasoactive intestinal polypeptide (VIP, glucagon and gastric inhibitory polypeptide (GIP); PYY is related to the pancreatic polypeptide and to neurotensin. Both peptides exhibit biological activities and appear to be present not only in the intestine but also in brain.

Using a reverse genetic approach, we have demonstrated that the product of the B5R open reading frame (ORF), which has homology with members of the family of complement control proteins, is a membrane glycoprotein present in the extracellular enveloped (EEV) form of vaccinia virus but absent from the intracellular naked (INV) form. An antibody (C'-B5R) raised to a 15-amino-acid peptide from the translated B5R ORF reacted with a 42-kDa protein (gp42) found in vaccinia virus-infected cells and cesium chloride-banded EEV but not INV. Under nonreducing conditions, an 85-kDa component, possibly representing a hetero- or homodimeric form of gp42, was detected by both immunoprecipitation and Western immunoblot analysis. Metabolic labeling with [3H]glucosamine and [3H]palmitate revealed that the B5R product is glycosylated and acylated. The C-terminal transmembrane domain of the protein was identified by constructing a recombinant vaccinia virus that overexpressed a truncated, secreted form of the B5R ORF product. By N-terminal sequence analysis of this secreted protein, the site of signal peptide cleavage of gp42 was determined. A previously described monoclonal antibody (MAb 20) raised to EEV, which immunoprecipitated a protein with biochemical characteristics similar to those of wild-type gp42, reacted with the recombinant, secreted product of the B5R ORF. Immunofluorescence of wild-type vaccinia virus-infected cells by using either MAb 20 or C'-B5R revealed that the protein is expressed on the cell surface and within the cytoplasm. Immunogold labeling of EEV and INV with MAb 20 demonstrated that the protein was found exclusively on the EEV membrane.

PlcR is a pleiotropic regulator that activates the expression of genes encoding various virulence factors, such as phospholipases C, proteases and hemolysins, in Bacillus thuringiensis and Bacillus cereus. Here we show that the activation mechanism is under the control of a small peptide: PapR. The papR gene belongs to the PlcR regulon and is located 70 bp downstream from plcR. It encodes a 48-amino-acid peptide. Disruption of the papR gene abolished expression of the PlcR regulon, resulting in a large decrease in hemolysis and virulence in insect larvae. We demonstrated that the PapR polypeptide was secreted, then reimported via the oligopeptide permease Opp. Once inside the cell, a processed form of PapR, presumably a pentapeptide, activated the PlcR regulon by allowing PlcR to bind to its DNA target. This activating mechanism was found to be strain specific, with this specificity determined by the first residue of the penta peptide.

Protein kinase C (PKC) isozymes translocate to unique subcellular sites following activation. We previously suggested that translocation of activated isozymes is required for their function and that in addition to binding to lipids, translocation involves binding of the activated isozymes to specific anchoring proteins (receptors for activated protein kinase C. Using cultured cardiomyocytes we identified inhibitors, the V1 fragment of epsilonPKC (epsilonV1), and an 8-amino acid peptide derived from it that selectively inhibited the translocation of epsilonPKC. Inhibition of epsilonPKC translocation but not inhibition of delta or betaPKC translocation specifically blocked phorbol ester- or norepinephrine-mediated regulation of contraction. These isozyme-selective translocation inhibitors provide novel tools to determine the function of individual PKC isozymes in intact cells.

There are nine known expanded CAG repeat neurological diseases, including Huntington's disease (HD), each involving the repeat expansion of polyglutamine (polyGln) in a different protein. Similar conditions can be induced in animal models by expression of the polyGln sequence alone or in other protein contexts. Besides the polyGln sequence, the cellular context of the disease protein, and the sequence context of the polyGln within the disease protein, are both likely to contribute to polyGln physical behavior and to pathology. In HD, the N-terminal, exon-1 segment of the protein huntingtin contains the polyGln sequence immediately followed by an oligoproline region. We show here that introduction of a P10 sequence C-terminal to polyGln in synthetic peptides decreases both the rate of formation and the apparent stability of the amyloid-like aggregates associated with this family of diseases. The sequence can be trimmed to P6 without altering the suppression, but a P3 sequence is ineffective. Spacers up to at least three amino acid residues in length can be inserted between polyGln and P10 without altering this effect. There is no suppression, however, when the P10 sequence is either placed on the N-terminal side of polyGln or attached to polyGln via a side-chain tether. The nucleation mechanism of a Q40 sequence is unchanged upon addition of a P10 C-terminal extension, yielding a critical nucleus of one. The effects of oligoPro length and structural context on polyGln aggregation are correlated strongly with alterations in the circular dichroism spectra of the monomeric peptides. For example, the P10 sequence eliminates the small amount of alpha helical content otherwise exhibited by the Q40 sequence. The P10 sequence may suppress aggregation by stabilizing an aggregation-incompetent conformation of the monomer. The effect is transportable: a P10 sequence fixed to the C terminus of the sequence Abeta similarly modulates amyloid fibril formation.

Nociceptive dorsal root ganglion (DRG) neurones have fibres that conduct in the C, Adelta and Aalpha/beta conduction velocity range. The properties of nociceptive compared with non-nociceptive somatic afferent dorsal root ganglion neurones appear to fall into two patterns, A and B. Pattern A properties of nociceptive neurones, the more common type, include longer action potential duration and slower maximum rate of fibre firing, as well as a greater expression of substance P and calcitonin gene-related peptide immunoreactivity. The values of pattern A properties appear to be graded according to the conduction velocity group (C, Adelta or Aalpha/beta) of the fibres. The most pronounced forms of A-type properties are expressed by nociceptive neurones with C-fibres, and these become less pronounced in nociceptive neurones with Adelta-fibres and least pronounced in those with Aalpha/beta fibres (C>> Adelta>> Aalpha/beta). Some of these properties are also expressed in a less extreme but similarly graded manner through C, Adelta and Aalpha/beta groups of non-nociceptive low threshold mechanoreceptive (LTM) neurone. The less common pattern B properties of nociceptive neurones have similar values in C-, Adelta- and Aalpha/beta-fibre nociceptive neurones but these clearly differ from LTM units with C-, Adelta- and Aalpha/beta-fibre conduction velocities. These features of nociceptive neurones include consistently larger action potential overshoots and longer after-hyperpolarisation durations in nociceptive than in LTM neurones.

The beta gamma subunits of heterotrimeric G proteins play important roles in regulating receptor-stimulated signal transduction processes. Recently appreciated among these is their role in the signaling events that lead to the phosphorylation and subsequent desensitization of muscarinic cholinergic (Haga, K., and Haga, T. (1992) J. Biol. Chem. 267, 2222-2227) and beta-adrenergic (Pitcher, J. A., Inglese, J., Higgins, J. B., Arriza, J. L., Casey, P. J., Kim, C., Benovic, J. L., Kwatra, M. M., Caron, M. G., and Lefkowitz, R. J. (1992) Science 257, 1264-1267) receptors. Beta gamma mediates the membrane targeting of the beta-adrenergic receptor kinase (beta ARK), in response to receptor activation, through a specific beta ARK-beta gamma interaction. This process utilizes the membrane-anchoring properties of the isoprenylated gamma subunit of beta gamma. In the present study, we have employed three distinct approaches to identify the region within the carboxyl terminus of beta ARK which binds beta gamma and thereby results in membrane translocation. We studied the ability of beta gamma to enhance the enzymatic activity of a series of truncated mutants of bovine beta ARK1, the ability of glutathione S-transferase fusion proteins containing various lengths of the carboxyl terminus of beta ARK to bind beta gamma subunits, and the ability of synthetic peptides comprised of beta ARK sequences to inhibit beta gamma activation of beta ARK1. We find that the minimal beta gamma binding domain of beta ARK is localized to a 125-amino acid residue stretch, the distal end of which is located 19 residues from the carboxyl terminus. A single 28-mer peptide (Trp643 to Ser670) derived from this sequence effectively inhibited beta gamma activation of beta ARK1, with an ICpeptide inhibitors provide important tools for the study of G protein-coupled receptor desensitization, as well as for the investigation of beta gamma activation of other G protein-effector systems.

Obesity has become an epidemic problem in western societies, contributing to metabolic diseases, hypertension, and cardiovascular disease. Overweight and obesity are frequently associated with increased plasma levels of aldosterone. Recent evidence suggests that human fat is a highly active endocrine tissue. Therefore, we tested the hypothesis that adipocyte secretory products directly stimulate adrenocortical aldosterone secretion. Secretory products from isolated human adipocytes strongly stimulated steroidogenesis in human adrenocortical cells (NCI-H295R) with a predominant effect on mineralocorticoid secretion. Aldosterone secretion increased 7-fold during 24 h of incubation. This stimulation was comparable to maximal stimulation of these cells with forskolin (2 x 10(-5) M). On the molecular level, there was a 10-fold increase in the expression of steroid acute regulatory peptide mRNA. This effect was independent of adipose angiotensin II as revealed by the stimulatory effect of fat cell-conditioned medium even in the presence of the angiotensin type 1 receptor antagonist, valsartan. None of the recently defined adipocytokines accounted for the effect. Mineralocorticoid-stimulating activity was heat sensitive and could be blunted by heating fat cell-conditioned medium to 99 degrees C. Centrifugal filtration based on molecular mass revealed at least two releasing factors: a heat sensitive fraction (molecular mass >50 kDa) representing 60% of total activity, and an inactive fraction (molecular mass <50 kDa). However, the recovery rate increased to 92% when combining these two fractions, indicating the interaction of at least two factors. In conclusion, human adipocytes secrete potent mineralocorticoid-releasing factors, suggesting a direct link between obesity and hypertension.

OBJECTIVE

The goal of this study was to evaluate the utility of a rapid "bedside" technique for measurement of B-type natriuretic peptide (BNP) in the diagnosis of congestive heart failure (CHF) in an urgent-care setting.

BACKGROUND

B-type natriuretic peptide is a protein secreted from the cardiac ventricles in response to pressure overload. One potential application of measurements of BNP in blood is distinguishing dyspnea due to CHF from other causes.

METHODS

B-type natriuretic peptide concentrations were measured in a convenience sample of 250 predominantly male (94%) patients presenting to urgent-care and emergency departments of an academic Veteran's Affairs hospital with dyspnea. Results were withheld from clinicians. Two cardiologists retrospectively reviewed clinical data (blinded to BNP measurements) and reached a consensus opinion on the cause of the patient's symptoms. This gold standard was used to evaluate the diagnostic performance of the BNP test.

RESULTS

The mean BNP concentration in the blood of patients with CHF (n = 97) was higher than it was in patients without (1,076 +/- 138 pg/ml vs. 38 +/- 4 pg/ml, p < 0.001). At a blood concentration of 80 pg/ml, BNP was an accurate predictor of the presence of CHF (95%); measurements less than this had a high negative predictive value (98%). The overall C-statistic was 0.97. In multivariate analysis, BNP measurements added significant, independent explanatory power to other clinical variables in models predicting which patients had CHF. The availability of BNP measurements could have potentially corrected 29 of the 30 diagnoses missed by urgent-care physicians.

CONCLUSIONS

B-type natriuretic peptide blood concentration measurement appears to be a sensitive and specific test to diagnose CHF in urgent-care settings.

Tetanus and botulinum neurotoxins are produced by Clostridia and cause the neuroparalytic syndromes of tetanus and botulism. Tetanus neurotoxin acts mainly at the CNS synapse, while the seven botulinum neurotoxins act peripherally. Clostridial neurotoxins share a similar mechanism of cell intoxication: they block the release of neurotransmitters. They are composed of two disulfide-linked polypeptide chains. The larger subunit is responsible for neurospecific binding and cell penetration. Reduction releases the smaller chain in the neuronal cytosol, where it displays its zinc-endopeptidase activity specific for protein components of the neuroexocytosis apparatus. Tetanus neurotoxin and botulinum neurotoxins B, D, F and G recognize specifically VAMP/ synaptobrevin. This integral protein of the synaptic vesicle membrane is cleaved at single peptide bonds, which differ for each neurotoxin. Botulinum A, and E neurotoxins recognize and cleave specifically SNAP-25, a protein of the presynaptic membrane, at two different sites within the carboxyl-terminus. Botulinum neurotoxin type C cleaves syntaxin, another protein of the nerve plasmalemma. These results indicate that VAMP, SNAP-25 and syntaxin play a central role in neuroexocytosis. These three proteins are conserved from yeast to humans and are essential in a variety of docking and fusion events in every cell. Tetanus and botulinum neurotoxins form a new group of zinc-endopeptidases with characteristic sequence, mode of zinc coordination, mechanism of activation and target recognition. They will be of great value in the unravelling of the mechanisms of exocytosis and endocytosis, as they are in the clinical treatment of dystonias.

Bee venom (BV) therapy (BVT), the therapeutic application of BV, has been used in traditional medicine to treat diseases, such as arthritis, rheumatism, pain, cancerous tumors, and skin diseases. BV contains a variety of peptides, including melittin, apamin, adolapin, the mast-cell-degranulating (MCD) peptide, enzymes (i.e., phospholipase [PL] A(2)), biologically active amines (i.e., histamine and epinephrine), and nonpeptide components which have a variety of pharmaceutical properties. BV has been reported to have anti-arthritis effects in several arthritis models. Melittin, a major peptide component of BV, has anti-inflammatory and anti-arthritis properties, and its inhibitory activity on nuclear factor kappaB (NF-kappaB) may be essential for the effects of BV. The anti-nociceptive effects of BV have also been demonstrated in thermal, visceral, and inflammatory pain models. Apcupoint stimulation (apipuncture) therapy into subcutaneous region may be important in the BV-induced anti-nociceptive effects. Multiple mechanisms, such as activation of the central and spinal opiod receptor, and alpha(2)-adrenergic activity, as well as activation of the descending serotonergic pathway have been suggested. The inhibition of c-Fos expression in the spinal cord by BV apipuncture in several nociceptive models is also reported to be a possible mechanism. BV also has anti-cancer activity. The cell cytotoxic effects through the activation of PLA(2) by melittin have been suggested to be the critical mechanism for the anti-cancer activity of BV. The conjugation of cell lytic peptide (melittin) with hormone receptors and gene therapy carrying melittin can be useful as a novel targeted therapy for some types of cancer, such as prostate and breast cancer.

Two functionally distinct forms of synaptic plasticity, Hebbian long-term potentiation (LTP) and homeostatic synaptic scaling, are thought to cooperate to promote information storage and circuit refinement. Both arise through changes in the synaptic accumulation of AMPA receptors (AMPARs), but whether they use similar or distinct receptor-trafficking pathways is unknown. Here, we show that TTX-induced synaptic scaling in cultured visual cortical neurons leads to the insertion of GluR2-containing AMPARs at synapses. Similarly, visual deprivation with monocular TTX injections results in synaptic accumulation of GluR2-containing AMPARs. Unlike chemical LTP, synaptic scaling is blocked by a GluR2 C-tail peptide but not by a GluR1 C-tail peptide. Knockdown of endogenous GluR2 with an short hairpin RNA (shRNA) also blocks synaptic scaling but not chemical LTP. Scaling can be rescued with expression of exogenous GluR2 resistant to the shRNA, but a chimeric GluR2 subunit with the C-terminal domain swapped with the GluR1 C-terminal domain (GluR2/CT1) does not rescue synaptic scaling, indicating that regulatory sequences on the GluR2 C-tail are required for the accumulation of synaptic AMPARs during scaling. Together, our results suggest that synaptic scaling and LTP use different trafficking pathways, making these two forms of plasticity both functionally and molecularly distinct.

Purified cell walls representing a wide variety in teichoic acid and peptidoglycan structure prepared from eight different gram-positive bacterial species induced the production of tumor necrosis factor alpha (TNF-alpha) and interleukin-6 from human monocytes in the presence of 10% plasma or serum. Significant amounts of cytokines began to be produced at concentrations above 100 ng to 1 microgram of cell walls per ml, with maximal production requiring 10 to 100 micrograms of cell wall material per ml. In the absence of plasma, the cytokine-inducing capacity of cell wall preparations was lower by at least an order of magnitude. The serum-derived cofactor was inactivated by heating at 90 degrees C for 30 min, suggesting that the activity is associated with a protein. On the other hand, replacement of normal with hypogammaglobulinemic plasma, inactivation of complement (at 56 degrees C), and blockade by the monoclonal antibody MY4 of the CD14 receptors on monocytes did not inhibit the production of TNF-alpha induced by whole cell walls. Cell walls also stimulated production of TNF-alpha induced by whole cell walls. Cell walls also stimulated production of TNF-alpha in the presence of polymyxin B, and macrophages derived from the lipopolysaccharide-insensitive cell line of Cpeptides and disacharride peptides had only poor, if any, activity. Cell walls may contribute to the septic shock induced by gram-positive bacteria.

We are able to make reliable predictions of the efficiency with which peptides of arbitrary lengths will be transported by TAP. The pressure exerted by TAP on Ag presentation thus can be assessed by checking to what extent MHC class I (MHC-I)-presented epitopes can be discriminated from random peptides on the basis of predicted TAP transport efficiencies alone. Best discriminations were obtained when N-terminally prolonged epitope precursor peptides were included and the contribution of the N-terminal residues to the score were down-weighted in comparison with the contribution of the C terminus. We provide evidence that two factors may account for this N-terminal down-weighting: 1) the uncertainty as to which precursors are used in vivo and 2) the coevolution in the C-terminal sequence specificities of TAP and other agents in the pathway, which may vary among the various MHC-I alleles. Combining predictions of MHC-I binding affinities with predictions of TAP transport efficiency led to an improved identification of epitopes, which was not the case when predictions of MHC-I binding affinities were combined with predictions of C-terminal cleavages made by the proteasome.

In Arabidopsis thaliana, the final step of floral organ abscission is regulated by INFLORESCENCE DEFICIENT IN ABSCISSION (IDA): ida mutants fail to abscise floral organs, and plants overexpressing IDA display earlier abscission. We show that five IDA-LIKE (IDL) genes are expressed in different tissues, but plants overexpressing these genes have phenotypes similar to IDA-overexpressing plants, suggesting functional redundancy. IDA/IDL proteins have N-terminal signal peptides and a C-terminal conserved motif (extended PIP [EPIP]) at the C terminus (EPIP-C). IDA can, similar to CLAVATA3, be processed by an activity from cauliflower meristems. The EPIP-C of IDA and IDL1 replaced IDA function in vivo, when the signal peptide was present. In addition, synthetic IDA and IDL1 EPIP peptides rescued ida and induced early floral abscission in wild-type flowers. The EPIP-C of the other IDL proteins could partially substitute for IDA function. Similarly to ida, a double mutant between the receptor-like kinases (RLKs) HAESA (HAE) and HAESA-LIKE2 (HSL2) displays nonabscising flowers. Neither overexpression of IDA nor synthetic EPIP or EPIP-C peptides could rescue the hae hsl2 abscission deficiency. We propose that IDA and the IDL proteins constitute a family of putative ligands that act through RLKs to regulate different events during plant development.

Human Tsg101 plays key roles in HIV budding and in cellular vacuolar protein sorting (VPS). In performing these functions, Tsg101 binds both ubiquitin (Ub) and the PTAP tetrapeptide 'late domain' motif located within the viral Gag protein. These interactions are mediated by the N-terminal domain of Tsg101, which belongs to the catalytically inactive ubiquitin E2 variant (UEV) family. We now report the structure of Tsg101 UEV and chemical shift mapping of the Ub and PTAP binding sites. Tsg101 UEV resembles canonical E2 ubiquitin conjugating enzymes, but has an additional N-terminal helix, an extended beta-hairpin that links strands 1 and 2, and lacks the two C-terminal helices normally found in E2 enzymes. PTAP-containing peptides bind in a hydrophobic cleft exposed by the absence of the C-terminal helices, whereas ubiquitin binds in a novel site surrounding the beta-hairpin. These studies provide a structural framework for understanding how Tsg101 mediates the protein-protein interactions required for HIV budding and VPS.

The insulinotropic hormone GLP-1 (glucagon-like peptide-1) is a new therapeutic agent that preserves or restores pancreatic beta cell mass. We report that GLP-1 and its agonist, exendin-4 (Exd4), induce Wnt signaling in pancreatic beta cells, both isolated islets, and in INS-1 cells. Basal and GLP-1 agonist-induced proliferation of beta cells requires active Wnt signaling. Cyclin D1 and c-Myc, determinants of cell proliferation, are up-regulated by Exd4. Basal endogenous Wnt signaling activity depends on Wnt frizzled receptors and the protein kinases Akt and GSK3beta but not cAMP-dependent protein kinase. In contrast, GLP-1 agonists enhance Wnt signaling via GLP-1 receptor-mediated activation of Akt and beta cell independent of GSK3beta. Inhibition of Wnt signaling by small interfering RNAs to beta-catenin or a dominant-negative TCF7L2 decreases both basal and Exd4-induced beta cell proliferation. Wnt signaling appears to mediate GLP-1-induced beta cell proliferation raising possibilities for novel treatments of diabetes.

Exons of three genes were identified within the 85-kilobase tandem triplication unit of the slow Wallerian degeneration mutant mouse, CCole1e, span the proximal and distal boundaries of the repeat unit, respectively. They have the same chromosomal orientation and form a chimeric gene when brought together at the boundaries between adjacent repeat units in Wld(S). The chimeric mRNA is abundantly expressed in the nervous system and encodes an in-frame fusion protein consisting of the N-terminal 70 amino acids of Ufd2, the C-terminal 302 amino acids of D4Cole1e, and an aspartic acid formed at the junction. Antisera raised against synthetic peptides detect the expected 43-kDa protein specifically in Wld(S) brain. This expression pattern, together with the previously established role of ubiquitination in axon degeneration, makes the chimeric gene a promising candidate for Wld. The third gene altered by the triplication, Rbp7, is a novel member of the cellular retinoid-binding protein family and is highly expressed in white adipose tissue and mammary gland. The whole gene lies within the repeat unit leading to overexpression of the normal transcript in Wld(S) mice. However, it is undetectable on Northern blots of Wld(S) brain and seems unlikely to be the Wld gene. These data reveal both a candidate gene for Wld and the potential of the Wld(S) mutant for studies of ubiquitin and retinoid metabolism.

Recognition of major histocompatibility complex class I molecules by natural killer (NR) cells leads to inhibition of target cell lysis. Based on the capacity of different human histocompatibility leukocyte antigen (HLA)-C and HLA-B molecules to inhibit target cell lysis by NK lines and clones, three NK allospecificities have been defined: NK1 and NK2 cells are inhibited by different HLA-C allotypes and NK3 cells by some HLA-B allotypes. The NK1 and NK2 inhibitory ligands on target cells correspond to a dimorphism of HLA-C at residues 77 and 80 in the alpha 1 helix: Asn77-Lys80 in NK1 and Ser77-Asn80 in NK2 inhibitory ligands. It has been reported that protection from NK1 killers depended on the presence of the Lys residue at position 80, an upward pointing residue near the end of the alpha 1 helix (and not on Asn77), whereas inhibition of NK2 effector cells required Ser77, a residue deep in the F pocket and interacting with the peptide (and not Asn80). As part of ongoing experiments to investigate the structural requirements for NK cell inhibition by HLA-C locus alleles, we also examined the effects of mutations at residues 77 and 80 on the ability of HLA-C alleles to confer protection from NK lysis. We present data confirming that the NK1 specificity depended on Lys80 (and not on Asn77); however recognition of NK2 ligands by NK cells was also controlled by the amino acid at position 80 (Asn), and mutation of Ser77 had no effect. Furthermore, bound peptide was shown to be unnecessary for the inhibition of NK cell-mediated lysis since HLA-C molecules assembled in the absence of peptide in RMA-S cells at 26 degrees C were fully competent to inhibit NK cells specifically. The implications of these data for peptide-independent recognition of HLA-C by NK receptors are discussed.

OBJECTIVE

To determine the effect of botulinum toxin type A on calcitonin gene-related peptide secretion from cultured trigeminal ganglia neurons.

BACKGROUND

The ability of botulinum toxins to cause muscle paralysis by blocking acetylcholine release at the neuromuscular junction is well known. Previous studies and clinical observations have failed to demonstrate sensory changes related to botulinum toxins or the disease of botulism. Recent studies, however, have suggested that botulinum toxin type A injected into pericranial muscles may have a prophylactic benefit in migraine. This observation has renewed the debate of a mechanism of sensory inhibition mediated by botulinum toxin type A.

METHODS

Primary cultures of rat trigeminal ganglia were utilized to determine whether botulinum toxin type A could directly decrease the release of calcitonin gene-related peptide, a neuropeptide involved in the underlying pathophysiology of migraine. Untreated cultures or cultures stimulated with a depolarizing stimulus (potassium chloride) or capsaicin, an agent known to activate sensory C fibers, were treated for 3, 6, or 24 hours with clinically effective doses of botulinum toxin type A or a control vehicle. The amount of calcitonin gene-related peptide secreted into the culture media following the various treatments was determined using a specific radioimmunoassay.

RESULTS

A high percentage (greater than 90%) of the trigeminal ganglia neurons present in 1- to 3-day-old cultures was shown to express calcitonin gene-related peptide. Treatment with depolarizing stimuli (potassium chloride), a mixture of inflammatory agents, or capsaicin caused a marked increase (4- to 5-fold) in calcitonin gene-related peptide released from the trigeminal neurons. Interestingly, overnight treatment of trigeminal ganglia cultures with therapeutic concentrations of botulinum toxin type A (1.6 or 3.1 units) did not affect the amount of calcitonin gene-related peptide released from these neurons. The stimulated release of calcitonin gene-related peptide following chemical depolarization with potassium chloride or activation with capsaicin, however, was greatly repressed by the botulinum toxin, but not by the control vehicle. A similar inhibitory effect of overnight treatment with botulinum toxin type A was observed with 1.6 and 3.1 units. These concentrations of botulinum toxin type A are well within or below the range of tissue concentration easily achieved with a local injection. Incubation of the cultures with toxin for 24, 6, or even 3 hours was very effective at repressing stimulated calcitonin gene-related peptide secretion when compared to control values.

CONCLUSIONS

These data provide the first evidence that botulinum toxin type A can directly decrease the amount of calcitonin gene-related peptide released from trigeminal neurons. The results suggest that the effectiveness of botulinum toxin type A in the treatment of migraine may be due, in part, to its ability to repress calcitonin gene-related peptide release from activated sensory neurons.

OBJECTIVE

The aim of this study was to determine if individual or multiple biomarkers are associated with cardiotoxicity in patients with breast cancer undergoing cancer therapy.

BACKGROUND

Current methods to identify patients at risk for cardiotoxicity from cancer therapy are inadequate.

METHODS

We measured 8 biomarkers in a multicenter cohort of 78 patients with breast cancer undergoing doxorubicin and trastuzumab therapy: ultrasensitive troponin I (TnI), high-sensitivity C-reactive protein (CRP), N-terminal pro-B-type natriuretic peptide (NT-proBNP), growth differentiation factor (GDF)-15, myeloperoxidase (MPO), placental growth factor (PlGF), soluble fms-like tyrosine kinase receptor (sFlt)-1, and galectin (gal)-3. Cardiotoxicity, defined by the Cardiac Review and Evaluation Committee criteria, was assessed every 3 months for up to 15 months. Hazard ratios (HRs) of cardiotoxicity risk were assessed for each biomarker at baseline, at visit 2 (3 months), and as a function of the difference between visit 2 and baseline. Joint models were assessed for the most promising biomarkers.

RESULTS

TnI, CRP, GDF-15, MPO, PlGF, and sFlt-1 levels increased from baseline to visit 2 (p < 0.05). A greater risk of cardiotoxicity was associated with interval changes in TnI (HR: 1.38 per SD; 95% confidence interval: 1.05 to 1.81; p = 0.02) and MPO (HR: 1.34 per SD; 95% confidence interval: 1.00 to 1.80; p = 0.048) and in models combining both markers (p = 0.007 and p = 0.03, respectively). The risk of cardiotoxicity was 46.5% in patients with the largest changes in both markers (ΔTnI >121.8 μg/l; ΔMPO >422.6 pmol/l).

CONCLUSIONS

Early increases in TnI and MPO levels offer additive information about the risk of cardiotoxicity in patients undergoing doxorubicin and trastuzumab therapy. Independent validation of these findings is necessary before application to clinical practice.

The first full-length hexon protein DNA and deduced amino acid sequences of a subgenus D adenovirus (AV) were determined from candidate AV48 (85-0844). Comprehensive comparison of this sequence with hexon protein sequences from human subgenera A, B, C, D, F, bovine AV3, and mouse AV1 revealed seven discrete hypervariable regions (HVRs) among the 250 variable residues in loops 1 and 2. These regions differed in length between serotypes, from 2 to 38 residues, and contained>> 00% of hexon serotype-specific residues among human serotypes. Alignment with the published crystal structure of AV2 established the location and structure of the type-specific regions. Five HVRs were shown to be part of linear loops on the exposed surfaces of the protein, analogous to the serotype-specific loops or "puffs" in picornavirus capsid proteins. The HVRs were supported by a common framework of conserved residues, of which 68 to 75% were hydrophobic. Unique sequences were limited to the seven HVRs, so that one or more of these regions contain the type-specific neutralization epitopes. A neutralizing AV48 hexon-specific antiserum recognized linear peptides that corresponded to six HVRs by enzyme immunoassay. Affinity-purification removal of all peptide-reactive antibodies did not significantly decrease the neutralization titer. Eluted peptide-reactive antibodies did not neutralize. Human antisera that neutralized AV48 did not recognize linear peptides. Purified trimeric native hexon inhibited neutralization, but monomeric heat-denatured hexon did not. We conclude that the AV48 neutralization epitope(s) is complex and conformational.

The ubiquitylation of cell-cycle regulatory proteins by the large multimeric anaphase-promoting complex (APC/C) controls sister chromatid segregation and the exit from mitosis. Selection of APC/C targets is achieved through recognition of destruction motifs, predominantly the destruction (D)-box and KEN (Lys-Glu-Asn)-box. Although this process is known to involve a co-activator protein (either Cdc20 or Cdh1) together with core APC/C subunits, the structural basis for substrate recognition and ubiquitylation is not understood. Here we investigate budding yeast APC/C using single-particle electron microscopy and determine a cryo-electron microscopy map of APC/C in complex with the Cdh1 co-activator protein (APC/C(Cdh1)) bound to a D-box peptide at ∼10 Å resolution. We find that a combined catalytic and substrate-recognition module is located within the central cavity of the APC/C assembled from Cdh1, Apc10--a core APC/C subunit previously implicated in substrate recognition--and the cullin domain of Apc2. Cdh1 and Apc10, identified from difference maps, create a co-receptor for the D-box following repositioning of Cdh1 towards Apc10. Using NMR spectroscopy we demonstrate specific D-box-Apc10 interactions, consistent with a role for Apc10 in directly contributing towards D-box recognition by the APC/C(Cdh1) complex. Our results rationalize the contribution of both co-activator and core APC/C subunits to D-box recognition and provide a structural framework for understanding mechanisms of substrate recognition and catalysis by the APC/C.

The importance of NADPH oxidase (Nox) in hypoxic responses in hypoxia-sensing cells, including pulmonary artery smooth muscle cells (PASMCs), remains uncertain. In this study, using Western blot analysis we found that the major Nox subunits Nox1, Nox4, p22(phox), p47(phox), and p67(phox) were equivalently expressed in mouse pulmonary and systemic (mesenteric) arteries. However, acute hypoxia significantly increased Nox activity and translocation of p47(phox) protein to the plasma membrane in pulmonary, but not mesenteric, arteries. The Nox inhibitor apocynin and p47(phox) gene deletion attenuated the hypoxic increase in intracellular concentrations of reactive oxygen species and Ca(2+) ([ROS](i) and [Ca(2+)](i)), as well as contractions in mouse PASMCs, and abolished the hypoxic activation of Nox in pulmonary arteries. The conventional/novel protein kinase C (PKC) inhibitor chelerythrine, specific PKCepsilon translocation peptide inhibitor, and PKCepsilon gene deletion, but not the conventional PKC inhibitor GO6976, prevented the hypoxic increase in Nox activity in pulmonary arteries and [ROS](i) in PASMCs. The PKC activator phorbol 12-myristate 13-acetate could increase Nox activity in pulmonary and mesenteric arteries. Inhibition of mitochondrial ROS generation with rotenone or myxothiazol prevented hypoxic activation of Nox. Glutathione peroxidase-1 (Gpx1) gene overexpression to enhance H(2)O(2) removal significantly inhibited the hypoxic activation of Nox, whereas Gpx1 gene deletion had the opposite effect. Exogenous H(2)O(2) increased Nox activity in pulmonary and mesenteric arteries. These findings suggest that acute hypoxia may distinctively activate Nox to increase [ROS](i) through the mitochondrial ROS-PKCepsilon signaling axis, providing a positive feedback mechanism to contribute to the hypoxic increase in [ROS](i) and [Ca(2+)](i) as well as contraction in PASMCs.