Effects of a large-scale, natural sediment deposition event on plant cover in a Massachusetts salt marsh.

In mid-winter 2018, an unprecedented sediment deposition event occurred throughout portions of the Great Marsh in Massachusetts. Evaluation of this event in distinct marsh areas spanning three towns (Essex, Ipswich, and Newbury) revealed deposition covering 29.2 hectares with an average thickness of 30.1±2.1 mm measured shortly after deposition. While sediment deposition helps marshes survive sea level rise by building elevation, effects of such a large-scale deposition on New England marshes are unknown. This natural event provided an opportunity to study effects of large-scale sediment addition on plant cover and soil chemistry, with implications for marsh resilience. Sediment thickness did not differ significantly between winter and summer, indicating sediment is not eroding or compacting. The deposited sediment at each site had similar characteristics to that of the adjacent mudflat (e.g., texture, bivalve shells), suggesting that deposited materials resulted from ice rafting from adjacent flats, a natural phenomenon noted by other authors. Vegetative cover was significantly lower in plots with rafted sediment (75.6±2.3%) than sediment-free controls (93.1±1.6%) after one growing season. When sorted by sediment thickness categories, the low thickness level (1-19 mm) had significantly greater percent cover than medium (20-39 mm) and high (40-90 mm) categories. Given that sediment accretion in the Great Marsh was found to average 2.7 mm per year, the sediment thickness documented herein represents ~11 years of sediment accretion with only a 25% reduction in plant cover, suggesting this natural sediment event will likely increase long-term marsh resilience to sea level rise.

Generation of an LFA-1 antagonist by the transfer of the ICAM-1 immunoregulatory epitope to a small molecule.

The protein-protein interaction between leukocyte functional antigen-1 (LFA-1) and intercellular adhesion molecule-1 (ICAM-1) is critical to lymphocyte and immune system function. Here, we report on the transfer of the contiguous, nonlinear epitope of ICAM-1, responsible for its association with LFA-1, to a small-molecule framework. These LFA-1 antagonists bound LFA-1, blocked binding of ICAM-1, and inhibited a mixed lymphocyte reaction (MLR) with potency significantly greater than that of cyclosporine A. Furthermore, in comparison to an antibody to LFA-1, they exhibited significant anti-inflammatory effects in vivo. These results demonstrate the utility of small-molecule mimics of nonlinear protein epitopes and the protein epitopes themselves as leads in the identification of novel pharmaceutical agents.

Preferential adsorption, internalization and resistance to degradation of the major isoform of the Alzheimer's amyloid peptide, A beta 1-42, in differentiated PC12 cells.

A central question in Alzheimer's disease (AD) is the role of amyloid in pathogenesis. Recent discoveries implicating the longer A beta 1-42 form of amyloid in pathogenesis led us to characterize the interaction of A beta with cells to elucidate differences that might account for these observations. We characterized the adsorption, internalization and degradation of radiolabeled A beta in NGF-differentiated PC12 cells under conditions that are not acutely toxic. All A beta peptides examined absorb to the surface of PC12 cells and are internalized; however the adsorption and internalization of A beta 1-42 is significantly greater than that of A beta 1-40 and A beta 1-28. The adsorption of A beta 1-42 is decreased by treatment of the cells with neuraminidase, but not heparitinase. The fate of the internalized A beta 1-42 is also very different than shorter A beta peptides; a fraction of the internalized A beta 1-42 accumulates intracellularly and is resistant to degradation for at least 3 days while A beta 1-40 and shorter peptides are eliminated with a half life of about 1 h. A beta 1-42 does not appear to inhibit lysosomal hydrolases, since A beta 1-28 is degraded at the same rate in the presence or absence of A beta 1-42. The intracellular A beta 1-42 is located in a dense organellar compartment and colocalizes with the lysosomal markers Lucifer Yellow and horseradish peroxidase. These data indicate that there are significant differences in the cell surface adsorption, internalization and catabolism of A beta 1-42 compared to A beta 1-40 and A beta 1-28. These differences may be important for the preferential accumulation of the longer A beta 1-42 isoform and its association with AD pathogenesis.

Analysis of peptide synthesis products by electrospray ionization mass spectrometry.

Electrospray ionization mass spectrometry is an easy, rapid method for the verification of proper peptide synthesis and for the identification of most synthetic by-products. A synthesis-purification scheme has been described that uses mass analysis to (1) confirm the presence of the proper product in the crude peptide mixture, (2) guide the purification process, and (3) confirm the mass and purity of the final product. Even though many of these steps could be performed just as well with other ionization techniques, the liquid-flow characteristics of electrospray source are clearly an advantage when LC-MS is required. In addition, the ease with which fragment ions can be generated to provide structural information, even with the least sophisticated instruments, is a further advantage of ESI-MS. Although much of the operation described here was done manually, many of the steps could be automated with little additional effort (e.g., use of an autosampler). Quadrupole and ion trap instruments are widely available at present and provide the chemist with a variety of instruments from which to choose. Electrospray time-of-flight instruments will be commercially have just become available and should also provide similar results. As electrospray instruments continue to evolve, the instruments display greater performance and enhanced user-friendly interfaces, yet are lower in price and smaller in size. These features should lead to even more widespread use for the characterization of synthetic peptides.

Vesicular transport: implications for cell polarity.

In polarized cells intracellular sorting of plasma membrane proteins occurs to a large extent at the trans-Golgi network, giving rise to vesicles destined for distinct plasma membrane domains. This review discusses the several pathways, both direct and indirect, which lead to protein incorporation into the correct cell surface, as well as the mechanisms involved. Proteins contain signals which direct their incorporation into the distinct vesicles destined for plasma membrane microdomains. Specific coat proteins are involved in vesicle assembly and are likely to play a role in the generation of discrete vesicle populations. Molecules involved in vesicle docking and fusion may also add specificity to the targeting process.

Growth hormone secretagogues: characterization, efficacy, and minimal bioactive conformation.

Another class of growth hormone (GH) secretagogues has been discovered by altering the backbone structure of a flexible linear GH-releasing peptide (GHRP). In vitro and in vivo characterization confirms these GH secretagogues as the most potent and smallest (M(r) < 500) reported. Anabolic efficacy is demonstrated in rodents with intermittent delivery. A convergent model of the bioactive conformation of GHRPs is developed and is supported by the NMR structure of a highly potent cyclic analog of GHRP-2. The model and functional data provide a logical framework for the further design of low-molecular weight secretagogues and illustrate the utility of an interdisciplinary approach to elucidating potential bound-state conformations of flexible peptide ligands.

Cultured rat microglia express C1q and receptor for C1q: implications for amyloid effects on microglia.

Senile plaques, the pathological hallmark of Alzheimer's disease (AD), are associated with complement components, including C1q. Reactive microglia appear to be involved in the later stages of plaque development. Since tissue macrophages are known to synthesize C1q, cultured rat microglia were examined for C1q immunoreactivity. Anti-C1q staining was detected, particularly in process-bearing microglia, indicating constitutive expression of C1q. Thus, microglia could provide a source of C1q for plaques even before becoming reactive. Since it has been previously shown that C1q binds beta 1-42, the major constituent of senile plaques, and since beta 1-42 is toxic to microglia in vitro, we asked if preincubation of beta 1-42 with C1q alters either metabolic indices of amyloid-induced degeneration in microglial cultures or the formation of amyloid deposits on these cells. While electron microscopic analysis of negatively stained amyloid fibrils confirmed that pre-incubation with C1q induced the association of C1q with the fibrils, no effect of the binding of C1q to beta 1-42 on beta 1-42 toxicity in microglia was observed. Interestingly, immunoreactivity for the C1q receptor that is known to modulate phagocytosis was found and was up-regulated in non-process-bearing microglia by interferon-gamma. While these data exclude a role for the C1q receptor in beta 1-42 toxicity in microglia, the observed expression and up-regulation of C1q receptor on microglia by interferon-gamma would be consistent with a role for C1q in complement-mediated inflammatory responses in AD and as a potential activator of microglial function in plaques.

Structural determinants of calcium signaling by RGD peptides in rat osteoclasts: integrin-dependent and -independent actions.

Extensive characterization of the vitronectin receptor (VNR), a member of the integrin group of cell adhesion molecules, which is abundantly expressed in osteoclasts, has revealed a role for this receptor in osteoclast adhesion as well as bone resorption. Earlier evidence from our laboratory suggests that VNR is also capable of transducing intracellular signals following receptor ligand interaction, although this function is poorly understood. Thus, addition of peptides containing the minimal tripeptide Arg-Gly-Asp (RGD) integrin recognition sequence elicits transient increases in intracellular free calcium ions, with maximal responses seen with a bone sialoprotein peptide, BSP-IIA. In the present study we have attempted to determine some of the structural requirements for calcium signaling in osteoclasts using derivatives of the peptide PRGDN/T sequence found in bone sialoprotein. While some peptides, such as the parent sequence PRGDN, can induce both signaling and retractile events, it was found that minor structural modifications yielded peptides such as PRADN which elicited a transient increase in intracellular free calcium ions without promoting a reduction in osteoclast spread area (retraction). Conversely, certain other modifications resulted in peptides, such as PrGDN and benzoyl-RGDN, which effect osteoclast retraction, while having minimal Ca2+ signaling capabilities. Osteoclast adhesion, and hence retraction, are known to be RGD-dependent and integrin-dependent events. However, intracellular Ca2+ signaling is RGD-independent and, based on lack of inhibition by an anti-beta 3 integrin antibody F11 and echistatin, very likely integrin-independent. These data suggest that signaling is not always via VNR and as yet unknown receptors on the osteoclast membrane play a role in osteoclast signaling and hence function.

Intracellular A beta 1-42 aggregates stimulate the accumulation of stable, insoluble amyloidogenic fragments of the amyloid precursor protein in transfected cells.

We have analyzed the effect of internalized amyloid beta-protein (A beta) 1-42 aggregates on the metabolism of the amyloid precursor protein (APP) in stably transfected 293 cells. The amount of potentially amyloidogenic fragments of APP immunoprecipitated by anti-carboxyl-terminal APP and anti-A beta antibodies is dramatically enhanced by the treatment of the cells with A beta 1-42, which is resistant to degradation, but not A beta 1-28, which does not accumulate in cells. This accumulation of amyloidogenic carboxyl-terminal fragments is specific, since there is relatively little effect of A beta 1-42 on the amount of the nonamyloidogenic alpha-secretase carboxyl-terminal fragment. The amyloidogenic fragments accumulate in the same nonionic detergent-insoluble fraction of the cell that contains the internalized A beta 1-42. Western analysis indicates that a subset of the amyloidogenic fragments react with antibodies that recognize a conformation of A beta that is specifically associated with aggregated forms of A beta, suggesting that the adoption of this aggregation-related conformation may be an early event which precedes the final processing that produces A beta. Pulse-chase analysis of the [35S]Met-labeled 16-kDa amyloidogenic fragment indicates that it is relatively stable in A beta 1-42-treated cells, with a half-life of approximately 50 h. This fragment is degraded with a half-life of 30 min in control cells treated with A beta 1-28. In contrast, the turnover of the nonamyloidogenic alpha-secretase product is not significantly altered by the presence of A beta 1-42. The continuous uptake of A beta 1-42 from the medium is not required for the stimulation of amyloidogenic fragment accumulation, suggesting that the presence of intracellular A beta 1-42 aggregates establishes a new pathway for APP catabolism in cells which leads to the long term stability of the fragments. If these amyloidogenic fragments of APP ultimately give rise to A beta, then the production of A beta may be an autocatalytic, "runaway" process in cells containing A beta 1-42 nuclei. It is conceivable that the accumulation of insoluble APP and amyloidogenic fragments of APP in response to A beta 1-42 aggregates may mimic the pathophysiology of dystrophic neurites, where the accumulation of intracellular APP and APP fragments has been documented by immunohistochemistry.

Benzodiazepine peptidomimetic inhibitors of farnesyltransferase.

A structural survey of protein Zn2+ binding geometries was instigated based upon the functional requirement of Ras farnesyltransferase for Zn2+. The Cys-X-X-Cys motif found in Zn(2+)-binding proteins such as aspartate transcarbamylase was used as a template to devise a bidentate-coordination model for Cys-A1-A2-X peptide inhibitors. Accordingly, replacement of the central dipeptide with the hydrophobic scaffold 3-amino-1-carboxymethyl-2,3-dihydro-5- phenyl-1H-1,4-benzodiazepin-2-one (BZA) yielded a peptidomimetic inhibitor, Cys(BZA)Met, of moderate potency (IC50 = 400 nM). N-Methylation of the cysteine amide improved potency almost 100-fold (IC50 = 0.3-1 nM). The increased affinity presumably correlates with a preferred conformation of the inhibitor which maximizes a hydrophobic interaction between the scaffold and the enzyme, and the proper presentation of cysteine and methionine to allow bidentate coordination at Zn2+. These non-peptide inhibitors have been shown to block farnesylation of the Ras protein in intact cells and provide lead compounds for the development of new cancer therapeutic agents.

beta-Amyloid activates complement by binding to a specific region of the collagen-like domain of the C1q A chain.

beta-amyloid peptides that accumulate within the brain of individuals with Alzheimer's disease bind to C1q and activate the classical C pathway via a specific interaction with a site within the collagen-like domain of C1q (C1q-CLF). Synthetic analogues of beta-amyloid peptides, beta 1-42 and beta 1-40, bound to C1q and were strong activators of C as assessed by both total C consumption and C4 consumption. beta 1-42 was significantly more effective than beta 1-40 in binding to C1q and triggering C activation, whereas beta 1-28 demonstrated little or no binding or C activation. This C-activating capacity seems to be largely correlated with the assembly of the beta 1-42 into low speed sedimentable aggregates and/or macromolecular fibrils. Radiolabeled C1q and C1q-CLF bind specifically to these aggregates or amyloid fibrils. In addition, using synthetic C1q peptides in a solid phase binding assay, the major binding site of beta 1-42 to C1q was localized to the C1q A chain collagen-like residues 14-26, a region previously described as a novel interaction site for Ab-independent activators of C1. C1q A chain peptide 14-26 blocked the ability of beta-amyloid peptides to activate the classical C pathway, providing evidence that this relatively unrecognized mechanism of C activation (via binding to the C1q-CLF) may have crucial physiologic consequences. Finally, these observations provide further support for the hypothesis that C activation and inflammation may be a component in the pathogenesis of AD and suggest possibilities for modulating the progression of AD.

Neurodegeneration induced by beta-amyloid peptides in vitro: the role of peptide assembly state.

The progressive neurodegeneration of Alzheimer's disease has been hypothesized to be mediated, at least in part, by beta-amyloid protein. A relationship between the aggregation state of beta-amyloid protein and its ability to promote degeneration in vitro has been previously suggested. To evaluate this hypothesis and to define a structure-activity relationship for beta-amyloid, aggregation properties of an overlapping series of synthetic beta-amyloid peptides (beta APs) were investigated and compared with beta AP neurotoxic properties in vitro. Using light microscopy, electrophoresis, and ultracentrifugation assays, we found that few beta APs assembled into aggregates immediately after solubilization, but that over time peptides containing the highly hydrophobic beta 29-35 region formed stable aggregations. In short-term neuronal cultures, toxicity was associated specifically with those beta APs that also exhibited significant aggregation. Further, upon the partial reversal of beta 1-42 aggregation, a concomitant loss of toxicity was observed. A synthetic peptide derived from a different amyloidogenic protein, islet amyloid polypeptide, exhibited aggregation but not toxicity, suggesting that beta AP-induced neurotoxicity in vitro is not a nonspecific reaction to aggregated protein. The correlation between beta AP aggregation and neurotoxicity was also observed in long-term neuronal cultures but not in astrocyte cultures. These data are consistent with the hypothesis that beta-amyloid protein contributes to neurodegeneration in Alzheimer's disease.

Estimating the 95% effective defibrillation dose.

Minimum squared error (MinSE) testing protocols and a MinSE estimator are presented which accurately estimate the voltage that defibrillates 95% of the time (the ED95). The MinSE experimental procedures, presented in the form of lookup tables, detail the response to successful and unsuccessful trials. The lookup tables also show the ED95 estimates calculated from the observed results using the MinSE estimator. Two assumptions are required to develop the look-up tables: 1) the dose-response curve, chosen using a statistical analysis of a retrospective sample, and 2) the distribution of the ED95's in the population. The MinSE estimator and experimental procedure are examined in a prospective study of five dogs (19-25 kg, heart weights 139.3-236.9 gm) using nonthoracotomy implantable defibrillator electrodes and a biphasic defibrillation waveform (3.5 ms first phase, 2.0 ms second phase). Employing an ED95 population distribution assumption applicable to most implantable defibrillator electrodes and waveforms, e.g., the ED95 is between 0.0 and 800.0 V, the measured rms error was 15% of the mean measured ED95 for the MinSE, four test shock, ED95 estimates. If the protocols are designed with an ED95 population distribution assumption for animals of the same species and size, and defibrillation is constrained to one electrode configuration and waveform, the estimates improve by 3.8%. Using techniques from the Bayesian statistics literature, the MinSE approach can be extended to a variety of defibrillation parameter estimation problems.

Intracellular accumulation and resistance to degradation of the Alzheimer amyloid A4/beta protein.

The A4 or beta protein is a peptide that constitutes the major protein component of senile plaques in Alzheimer disease. The A4/beta protein is derived from a larger, transmembrane amyloid precursor protein (APP). The putative abnormal processing events leading to amyloid accumulation are largely unknown. Here we report that a 42-residue synthetic peptide, beta 1-42, corresponding to one of the longer forms of the A4/beta protein, accumulates in cultured human skin fibroblasts and is stable for at least 3 days. The peptide appears to accumulate intracellularly, since it does not accumulate under conditions that prevent endocytosis and accumulation is correlated with the acquisition of resistance to removal by trypsin digestion. This intracellular accumulation is also correlated with the ability of the peptide to aggregate as determined by SDS/polyacrylamide gel electrophoresis. At low concentrations of the beta 1-42 peptide, which favor the nonaggregated state, no accumulation is observed. Shorter peptide analogs (28 or 39 residues) that are truncated at the C terminus, which lack the ability to aggregate in SDS gels, fail to accumulate. The accumulated intracellular beta 1-42 peptide is in an aggregated state and is contained in a dense organellar compartment that overlaps the distribution of late endosomes or secondary lysosomes. Immunofluorescence of the internalized peptide in permeabilized cells reveals that it is contained in granular deposits, consistent with localization in late endosomes or secondary lysosomes. Sequence analysis indicates that some of the internalized peptide is subject to N-terminal trimming. These results suggest that the aggregated A4/beta protein may be resistant to degradation and suggest that the A4/beta protein may arise, at least in part, by endosomal or lysosomal processing of APP. Our results also suggest that relatively nonspecific proteolysis may be sufficient to generate the A4/beta protein if this part of APP is selectively resistant to proteolysis.

Cyclic RGD peptide analogues as antiplatelet antithrombotics.

Stimulation of platelets activates GPIIbIIIa, the heterodimeric integrin receptor, to bind fibrinogen (Fg), which results in platelet aggregation. GPIIbIIIa/Fg binding inhibitors are potentially suitable for acute use during and after thrombolytic therapy as antithrombotic agents. Incorporation of the tripeptide sequence Arg-Gly-Asp (RGD), a common structural element of many integrin ligands, into cyclic peptides produced a series of peptides of the general structure BrAc-(AA1)-RGD-Cys-OH, which were prepared by solid-phase peptide synthesis. Cyclization was accomplished by reaction of the N-terminal bromoacetyl group with the cysteine sulfhydryl at pH 8 at high dilution, resulting in thioether-bridged cyclic peptides [cyclo-S-Ac-(AA1)-RGD-Cys-OH]. Use of alpha-substituted bromoacetyl groups gave rise to an analogous series of acetyl-substituted thioether-bridged cyclic peptides. Oxidation of the thioethers produced separable diastereomeric sulfoxide-bridged cyclic peptides. After thorough evaluation in a GPIIbIIIa ELISA assay and a platelet aggregation assay, G-4120 (70A; AA1 = D-Tyr; sulfoxide bridge) was selected for further investigation as an antithrombotic agent. G-4120 was equipotent in the platelet aggregation assay to kistrin, a highly potent inhibitor of fibrinogen-mediated platelet aggregation isolated from snake venom (IC50 = 0.15 microM).

Assembly and aggregation properties of synthetic Alzheimer's A4/beta amyloid peptide analogs.

The amyloid A4 or beta peptide is a major component of extracellular amyloid deposits that are a characteristic feature of Alzheimer's disease. We synthesized a series of peptide analogs of the A4/beta peptide which are progressively longer at their carboxyl termini, including 42- and 39-residue peptides which represent the major forms of the A4/beta peptide in senile plaque and the hereditary cerebral hemorrhage with amyloidosis form, respectively. All peptides tested, beta 1-28 through beta 1-42, formed amyloid-like fibrils and previously unreported thin sheet-like structures which stained with thioflavin T and Congo Red. The solubility of beta 1-42 and shorter peptides was pH and concentration dependent, with a broad insolubility profile in the pH range of 3.5-6.5 and at concentrations above 0.75 mg/ml. Only peptides of 42 residues or longer were significantly insoluble at pH 7.4. beta 1-47 and beta 1-52 peptides are highly insoluble in aqueous media but are soluble at 40 mg/ml in the alpha helix-promoting solvent, 1,1,1,3,3,3-hexafluoro-2-propanol. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the beta 1-42 peptide migrates as a series of higher molecular mass aggregates whereas shorter peptides migrate as monomers. Aggregation is also dependent on pH, peptide concentration, and time of incubation in aqueous medium. These results indicate that the length of the hydrophobic carboxyl terminus of the A4/beta peptide is important in determining the solubility and aggregation properties of the A4/beta peptide and that acid pH environment, high peptide concentration, and long incubation time would be predicted to be important factors in promoting amyloid deposition.

Nerve growth factor stimulation of GAP-43 phosphorylation in intact isolated growth cones.

Phosphorylation of the nervous system-specific growth cone protein GAP-43 by kinase C in vivo occurs exclusively in growth cones and distal axons, and the onset of this phosphorylation is delayed relative to the onset of axonogenesis, with the delay predicted on the time needed for axons to reach the vicinity of their targets (Meiri et al., 1991). We have used a subcellular fraction of intact growth cones (IGCs) to investigate whether this induction of GAP-43 phosphorylation can be influenced by target-derived substances, and show here that increased phosphorylation of GAP-43 can be both stimulated and maintained by NGF at concentrations of 2 x 10(-10) M. This low concentration of NGF and the subsequent phosphorylation of GAP-43 are both consistent with the interpretation that phosphorylation is due to the binding of NGF to a biologically active high-affinity receptor. Second, we used the monoclonal antibody 2G12 to show that the NGF-stimulated phosphorylation of GAP-43 occurs on serine, the kinase C phosphorylation site, consistent with the results seen in vivo. Levels of phosphorylated GAP-43 in the intact IGCs are also modulated by calcium-stimulated dephosphorylation that could be inhibited by EGTA but not okadaic acid and that therefore resembled the calcineurin-stimulated dephosphorylation reported in vitro. The results suggest that the spatial and temporal regulation of GAP-43 phosphorylation that occurs during axonogenesis in vivo can be regulated by target-derived neurotropic molecules, specifically NGF.

A new host and locality record for Mesocestoides sp. tetrathyridia (Cestoidea: Cyclophyllidea), with a summary of the genus from snakes of the world.

A new host and distribution record is reported for tetrathyridia of Mesocestoides sp. One of 5 (20%) Namib tiger snakes, Telescopus beetzi, from South Africa was infected. Numerous tetrathyridia were found encapsulated in mesentery attached to the small intestine. Morphological examination of tetrathyridia revealed absence of buds, multiple scoleces, or any other evidence of asexual proliferation. A summary of the snakes of the world reported as hosts of tetrathyridia of Mesocestoides sp. is presented.