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AbstractPhenological patterns of the components within forest ecosystems, such as understory vegetation, are important indicators of climate variability, productivity, and additional ecosystem services such as food and habitat availability for wildlife. Proximal sensing systems can provide detailed phenological records at a relatively low cost. As interest in these datasets increases, we need additional information regarding the effect of different approaches on the scale of observations and camera field of view. In this research, we examine the impact of field of view on the capacity of cameras to detect changes in phenology of individual species in an image time series. We examine two co-located series of oblique images acquired using a fine and broad field of view and compare a number of phenological indicators, including the start and end of season derived for individual plant species. Our results indicate both fine and broad field of view camera systems are highly effective at detecting key markers of plant phenology with no significant differences between the two. This result supports environmental monitoring using cost-effective broad field of view cameras, or even—subject to some constraints—readily available camera stations installed for tourism or traffic monitoring.

As part of a program aimed at generating compounds with affinity for the α2-δ subunit of voltage-gated calcium channels, several novel β-amino acids were prepared using an efficient nitroalkane-mediated cyclopropanation as a key step. Depending on the ester that was chosen, the target amino acids could be prepared in as few as three steps. The cyclopropyl amino acids derived from ketones proved to be potent binders of the α2-δ subunit of voltage-gated calcium channels, but did not interact with the large neutral amino acid system L (leucine) transporter. Anticonvulsant effects were observed in vivo with compound 34 but only after intracerebroventricular (icv) administration, presumably due to inadequate brain concentrations of the drug being achieved following oral dosing. However, pregabalin 1 was active in the DBA/2 model after oral (and icv) dosing, supporting a hypothesis that active transport is a prerequisite for such zwitterionic species to cross the blood−brain barrier.

Pregabalin exhibits robust activity in preclinical assays indicative of potential antiepileptic, anxiolytic, and antihyperalgesic clinical efficacy. It binds with high affinity to the α2-δ subunit of voltage-gated calcium channels and is a substrate of the system L neutral amino acid transporter. A series of pregabalin analogues were prepared and evaluated for their α2-δ binding affinity as demonstrated by their ability to inhibit binding of [³H]gabapentin to pig brain membranes and for their potency to inhibit the uptake of [³H]leucine into CHO cells, a measure of their ability to compete with the endogenous substrate at the system L transporter. Compounds were also assessed in vivo for their ability to promote anxiolytic, analgesic, and anticonvulsant actions. These studies suggest that distinct structure activity relationships exist for α2-δ binding and system L transport inhibition. However, both interactions appear to play an important role in the in vivo profile of these compounds.

We have successfully used the major subunit ClpG of Escherichia coli CS31A fimbriae as an antigenic and immunogenic exposure-delivery vector for various heterologous peptides varying in nature and length. However, the ability of ClpG as a carrier to maintain in vitro and in vivo the native biological properties of passenger peptide has not yet been reported. To address this possibility, we genetically fused peptides containing all or part of the E. coli human heat-stable enterotoxin (STh) sequence to the amino or carboxyl ends of ClpG. Using antibodies to the ClpG and STh portions for detecting the hybrids; AMS (4-acetamido-4'-maleimidylstilbene-2, 2'-disulfonate), a potent free thiol-trapping reagent, for determining the redox state of STh in the fusion; and the suckling mouse assay for enterotoxicity, we demonstrated that all ClpG-STh proteins were secreted in vitro and in vivo outside the E. coli cells in a heat-stable active oxidized (disulfide-bonded) form. Indeed, in contrast to many earlier studies, blocking the natural NH(2) or COOH extremities of STh had, in all cases, no drastic effect on cell release and toxin activity. Only antigenicity of STh C-terminally extended with ClpG was strongly affected in a conformation-dependent manner. These results suggest that the STh activity was not altered by the chimeric structure, and therefore that, like the natural toxin, STh in the fusion had a spatial structure flexible enough to be compatible with secretion and enterotoxicity (folding and STh receptor recognition). Our study also indicates that disulfide bonds were essential for enterotoxicity but not for release, that spontaneous oxidation by molecular oxygen occurred in vitro in the medium, and that the E. coli cell-bound toxin activity in vivo resulted from an effective export processing of hybrids and not cell lysis. None of the ClpG-STh subunits formed hybrid CS31A-STh fimbriae at the cell surface of E. coli, and a strong decrease in the toxin activity was observed in the absence of CS31A helper proteins. In fact, chimeras translocated across the outer membrane as a free folded monomer once they were guided into the periplasm by the ClpG leader peptide through the CS31A-dependent secretory pathway. In summary, ClpG appears highly attractive as a carrier reporter protein for basic and applied research through the engineering of E. coli for culture supernatant delivery of an active cysteine-containing protein, such as the heat-stable enterotoxin.

To study the folding of human Escherichia coli heat-stable enterotoxin STh, we used the major protein subunit of CS31A fimbriae (ClpG) as a marker of STh secretion and a provider of a signal peptide. We established that STh genetically fused to the N or C terminus of ClpG was able to mobilize ClpG to the culture supernatant while still retaining full enterotoxicity. These features indicate that the STh activity was not altered by the chimeric structure and suggest that spatial conformation of STh in the fusion is close to that of the native toxin, thus permitting recognition and activation of the intestinal STh receptor in vivo. In contrast to other studies, we showed that disulfide bond formation did not occur in the periplasm through the DsbA pathway and that there was no correlation between DsbA and secretion, folding, or activity. This discrepancy was not attributable to the chimeric nature of STh since there was no effect of dsbA or dsbB mutations on secretion and activity of recombinant STh from which ClpG had been deleted. Periplasmic and lysate fractions of dsbA(+) and dsbA(-) cells did not have any STh activity. In addition, the STh chimera was exclusively found in an inactive reduced form intracellularly and in an active oxidized form extracellularly, irrespective of the dsbA background. Subsequently, a time course experiment in regard to the secretion of STh from both dsbA(+) and dsbA(-) cells indicated that the enterotoxin activity (proper folding) in the extracellular milieu increased with time. Overall, these findings provide evidence that STa toxins can be cell-released in an unfolded state before being completely disulfide-bonded outside the cell.

PNQX (1,4,7,8,9,10-hexahydro-9-methyl-6-nitropyrido[3,4-f]quinoxaline-2,3-dione) is a potent AMPA (IC50 = 0.063 μM) and GlyN (IC50 = 0.37 μM) receptor antagonist that was developed in our laboratories. While possessing a desirable in vitro and in vivo activity profile, this compound suffers from low aqueous solubility. In an effort to improve its potency and physical properties, we have designed and synthesized novel ring-opened analogues 4, 6, 9, and 11. Modeling analyses demonstrated that, while the 5-substituent in these analogues was forced to adopt an out-of-plane conformation due to steric contacts with neighboring substituents, the overall structure retained a good fit to a previously described AMPA pharmacophore model. This nonplanar orientation may lessen efficient packing in the solid state, compared to PNQX, leading to increased water solubility. Indeed, several nonplanar analogues containing appropriate functionalities, for example, the sarcosine analogue 9, were found to retain AMPA (IC50 = 0.14 μM) and GlyN (IC50 = 0.47 μM) receptor affinity and possess improved aqueous solubility compared to PNQX. The synthesis and the SAR of these compounds are discussed.

Wrinkles, porosity, delaminations and other defects introduced during the manufacturing processing can compromise mechanical performance of advanced composites. This paper describes a method of using fiber optic sensors for monitoring the formation of graphite fiber bending in real time during manufacturing process. Theoretical formulation of the sensor behavior and experimental results are presented. The response of the sensor to composite fiber bending is characterized. The application to analyzing the formation of wrinkles in compression molding of graphite/epoxy composites is demonstrated.

Two classes of structurally different tachykinin neurokinin3 (NK3) antagonists were used to evaluate species difference in antagonist binding between human and rat NK3 receptors. In competition binding experiments with [125I-MePhe7]NKB as radioligand, PD 154740, PD 157672, SR 48968, and SR 142801 displayed lower Ki values for the human NK3 receptor (40 +/- 4, 12 +/- 1,350 +/- 50, and 0.40 +/- 0.05 nM, respectively) than for the rat NK3 receptor (2450 +/- 130, 288 +/- 25, > 10,000, and 11.0 +/- 0.5 nM, respectively). Data from in vitro functional assay showed similar species preference as observed with the competition binding assay. It was shown previously that substitution of only two amino acid residues in the rat receptor to their human counterparts could change the species selectivity of SR 48968, a weak NK3 antagonist. In the double-substituted rat mutant, all three antagonists (PD 154740, PD 157672, and SR 142801) displayed Ki values (76 +/- 8, 16 +/- 2, and 0.50 +/- 0.05 nM, respectively) very similar to the Ki values for the wild-type human NK3 receptor. Thus, in addition to their previously reported effects on SR 48968, these two amino acid residues are responsible for the species selectivity of these three additional NK3 antagonists. Because PD 154740 and PD 157672 are very different structurally from SR 48968 and SR 142801, our results indicate that the two identified residues may be involved in adopting a receptor conformation that favors the binding of NK3 antagonists that display species preference for the human NK3 receptor.

Secretion of aerobactin is thought to play an important part in the virulence of invasive Escherichia coli also capable of synthesizing enterochelin. Why, despite its markedly lower affinity for iron than that of enterochelin, aerobactin proves to be the predominant active siderophore for bacterial growth in transferrin was investigated. We studied the action of two iron chelators, 2,2'-dipyridyl and transferrin, in expression of the aerobactin and enterochelin genes. Specifically, we describe the sequential localization of the two siderophores in the cell compartments during bacterial growth under different iron limitation conditions. Our results demonstrated that, whatever the exogenous iron-chelating agent used, aerobactin was rapidly excreted, whereas enterochelin accumulated early in periplasm before its very belated release into the external medium. This work also showed that the advantage of aerobactin over enterochelin in competition with transferrin was not due to (i) lack of enterochelin activity, (ii) a cell-bound aerobactin-dependent mechanism, (iii) antagonism between the two siderophores, and probably (iv) genetic preferential induction of aerobactin. We propose that the superiority of aerobactin in competing with transferrin for iron(III) was a consequence of its more rapid excretion with respect to enterochelin. In contrast to transferrin, 2,2'-dipyridyl induced a greater efficiency of enterochelin, possibly by a more permanent function as iron-binding compounds in the bacterial envelope. In summary, unlike aerobactin, enterochelin appears to be a weakly secreted high-affinity iron ligand.

Wrinkles, porosity, delaminations and other defects introduced during the manufacturing processing can compromise mechanical performance of advanced composites. This paper describes a method of using fiber optic sensors for monitoring the formation of graphite fiber bending in real time during manufacturing process. Theoretical formulation of the sensor behavior and experimental results are presented. The response of the sensor to composite fiber bending is characterized. The application to analyzing the formation of wrinkles in compression molding of graphite/epoxy composites is demonstrated.

The nature of the common surface antigen of six hemagglutinating and adhesive piliated Escherichia coli strains isolated from diarrheic or septicemic calves was studied. By electron microscopy studies, the E. coli surface antigen designated CS31A was found on bacterial cells and in purified form to consist of thin (2-nm) "fibrillar" fimbriae. E. coli 31A, which was cured of a 105-megadalton plasmid, failed to express CS31A fimbriae, but retained the ability to hemagglutinate and to adhere in vitro on intestinal cells. Conversely, E. coli K-12, harboring the 105-megadalton plasmid originating from strain 31A, produced CS31A fimbriae but was not able to hemagglutinate or adhere on intestinal cells. A single fimbrial subunit of 29 kilodaltons was observed when purified fimbriae from the 105-megadalton plasmid-containing E. coli K-12 strain was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis or eluted by gel filtration after dissociation by 8.5 M guanidium hydrochloride from an S300 Sephacryl column. Western immunoblot analysis and the N-terminal sequence and amino acid composition of CS31A indicate structural and immunological relatedness between CS31A and K88 protein subunits.

CS31A is a K88-related capsule-like surface protein that mediates Escherichia coli and Klebsiella pneumoniae adhesion to the human Caco-2 and Intestine-407 cell lines. In this study, we demonstrate that ClpG, the major subunit of CS31A, contains the adhesive domain of the polymerized structure. We mapped this domain within the ClpG protein by performing adhesion inhibition experiments with Intestine-407 cells with nine synthetic peptides (CLP1 to CLP9) covering the dominant antigenic regions of ClpG and with the corresponding rabbit antipeptide antibodies. The peptides CLP1 (amino acid positions in parentheses) (5-18), CLP2 (44-56), CLP3 (82-96), CLP7 (174-190), CLP8 (185-199), and CLP9 (235-249) and corresponding antipeptide antibodies targeting parts of the N- and C-terminal regions of ClpG had no effect on the adhesion of the TCFF15 recombinant strain expressing CS31A. Only the CLP5 (132-146) peptide, corresponding to the central part of the protein, and relevant antibodies inhibited bacterial adhesion to intestinal epithelial cells. Anti-CLP4 (97-109) and anti-CLP6 (148-162) antibodies targeting regions surrounding the CLP5 sequence also inhibited bacterial adhesion. Site-directed mutagenesis experiments inducing changes in the amino acid sequence of the ClpG protein corresponding to the CLP5 peptide resulted in the expression of nonadhesive CS31A antigen. These findings indicate that the ClpG receptor-binding domain is located in the central variable V2 region.

To assess the role of the aerobactin-related system in the virulence of bovine opportunistic Escherichia coli, and to determine the stage(s) of the overall infectious process at which it is acting, germfree lambs were mixedly infected orally with two derivative strains of this bacterium differing in their ability (Iut+) or inability (Iut-) to express a functional aerobactin-mediated iron transport system. The Iut- strain was compared with the Iut+ strain for colonization of the gut, translocation to the mesenteric lymph nodes (MLN), and spread to other organs and to the body fluids of diassociated lambs. The Iut- mutant was found in smaller numbers in the duodenum, suggesting that aerobactin conferred a significant selective advantage for colonization of this intestinal segment. Although the two challenge strains translocated to MLN, the population level in the MLN was always higher for the Iut+ strain. Moreover, experimental infections resulted in recovery of only the Iut+ strain in the organs other than the MLN and in the body fluids. These results indicate a role for aerobactin in promoting systemic spread of the bacteria from the intestine. Direct evidence was obtained that aerobactin secretion occurred in vivo at both intestinal and extraintestinal sites of infection. In contrast to enterobactin, aerobactin was detected in the duodenum, jejunum, ileum, cecum, liver, spleen, kidney, urine, cerebrospinal fluid, and bile. The highest concentration of aerobactin was found in the urine, even when the samples were devoid of infecting bacteria. All of these findings suggest that aerobactin is released in vivo in a diffusible form and that it may be an important step in the production of disease by intestinal opportunistic E. coli.

The clpG gene coding for the CS31A subunit was localized on a 0.9-kb SphI fragment from the recombinant plasmid pAG315. This was established by testing the ability of subclones to hybridize with a 17-meric oligonucleotide probe obtained from N-terminal analysis of the CS31A subunit. The nucleotide sequence of the region coding for CS31A was determined. From primer extension analysis, two initiation translation start sites were detected. Two possible promoterlike sequences were identified; the ribosome binding site and the translation terminator are proposed. Inverted repeat sequences leading to the formation of possible hairpin structures of the transcripts were found on the 5' untranslated region of clpG. The deduced amino acid composition was in close agreement with the chemical amino acid composition and sequence match with the first 25 N-terminal amino acids from the published N-terminal sequence of the purified CS31A subunit. The clpG gene codes for a mature protein of 257 amino acids with a molecular size of 26,777 Da. An obvious homology was observed when the amino acid sequence of CS31A was compared with those of K88 and F41. This homology includes five different conserved sequences of up to 19 identical amino acids, which is associated with conserved proline. An extensive change in the CS31A region homologous to that identified to contain the K88 receptor binding site might be responsible for the functional divergence between CS31A and K88.

CS31A is a K88-related capsule-like surface protein that mediates Escherichia coli and Klebsiella pneumoniae adhesion to the human Caco-2 and Intestine-407 cell lines. In this study, we demonstrate that ClpG, the major subunit of CS31A, contains the adhesive domain of the polymerized structure. We mapped this domain within the ClpG protein by performing adhesion inhibition experiments with Intestine-407 cells with nine synthetic peptides (CLP1 to CLP9) covering the dominant antigenic regions of ClpG and with the corresponding rabbit antipeptide antibodies. The peptides CLP1 (amino acid positions in parentheses) (5-18), CLP2 (44-56), CLP3 (82-96), CLP7 (174-190), CLP8 (185-199), and CLP9 (235-249) and corresponding antipeptide antibodies targeting parts of the N- and C-terminal regions of ClpG had no effect on the adhesion of the TCFF15 recombinant strain expressing CS31A. Only the CLP5 (132-146) peptide, corresponding to the central part of the protein, and relevant antibodies inhibited bacterial adhesion to intestinal epithelial cells. Anti-CLP4 (97-109) and anti-CLP6 (148-162) antibodies targeting regions surrounding the CLP5 sequence also inhibited bacterial adhesion. Site-directed mutagenesis experiments inducing changes in the amino acid sequence of the ClpG protein corresponding to the CLP5 peptide resulted in the expression of nonadhesive CS31A antigen. These findings indicate that the ClpG receptor-binding domain is located in the central variable V2 region.

CS31A is a multimeric surface protein surrounding certain enterotoxigenic and septicemic bovine Escherichia coli strains. The possibility of using CS31A as a carrier of foreign antigenic determinants was investigated. Introduction of heterologous viral epitopes into the CS31A major subunit, ClpG, was successfully performed in the V3 region of the molecule which encodes for an immunodominant linear epitope. E. coli K-12 strains producing the hybrid CS31A molecules or the purified chimeric proteins were used for mice immunization. By using the C epitope derived from the S protein of the porcine transmissible gastroenteritis virus, significant antiviral antibody titers were elicited and seroneutralization of the virus was demonstrated, confirming that the molecular environment in V3 is favorable for antigen presentation. These results indicate that synthesis of CS31A hybrid proteins by the wild-type strain 31A could become a powerful tool for the development of oral vaccines directed against mucosal pathogens.

Two mutant strains (denoted as DCMUr-I and DCMUr-II) of the blue-green alga Aphanocapsa 6714, capable of growing in the presence of 10−5 M DCMU (an inhibitor of electron transport on the acceptor side of photosystem II), were isolated. The DCMU sensitivity of growth rates and of two photosystem II activities, O2 emission and fluorescence, was investigated in the mutant cells and compared with that of the wild type. The DCMU sensitivity of isolated thylakoids was also studied. The sensitivity of the mutant cells to other DCMU-type inhibitors (o-phenanthroline and atrazine) was tested. The results suggest that strain DCMUr-I resistance could be due to the acquisition of a cellular permeability barrier to DCMU, expressed only after an adaptation phase in the presence of the inhibitor. DCMUr-II resistance seems to be due primarily to an alteration of the thylakoid membranes of the photosynthetic apparatus itself.