Created pools and food availability for fishes in a restored salt marsh

Trophic support functions for fishes are a key goal of salt marsh restoration. Food availability in restored sites may be enhanced by creation of shallow pools, which are important sources of prey items in tidal wetlands. Young restored salt marshes are typically sparsely vegetated and are subject to rapidly changing geomorphology. Scouring and sedimentation create and fill shallow depressions, producing a shifting mosaic of tidal pools. In a large (8-ha) southern California experimental restoration site, we created shallow pools and assessed their development of foods for fishes. Created pools quickly developed abundant invertebrate prey, with densities exceeding those found in older, naturally formed pools (P < 0.0001). Opportunistic mobile and disturbance-associated taxa (calanoid copepods, nematodes, Polydora complex, and Trichocorixa reticulata) accounted for higher invertebrate densities in created pools. We repeated experiments in spring, summer, and fall and found seasonal variability in trophic development. We also applied bottom-up (nitrogen addition) and top-down (fish exclusion) treatments to pools. Some measures of algal biomass were increased by nitrogen fertilization (P = 0.001–0.06), but there were no upward-cascading effects on invertebrate composition or abundance. Fish abundance in the site varied seasonally, but there were no compelling effects of fish exclusion treatments on algal or invertebrate abundance. Incorporating shallow depressions into salt marsh restoration projects is a potential tool to jumpstart fish-support functions.     

Regulation of Heparan Sulfate and Chondroitin Sulfate Glycosaminoglycan Biosynthesis by 4-Fluoro-glucosamine in Murine Airway Smooth Muscle Cells

The importance of the pathological changes in proteoglycans has driven the need to study and design novel chemical tools to control proteoglycan synthesis. Accordingly, we tested the fluorinated analogue of glucosamine (4-fluoro-N-acetyl-glucosamine (4-F-GlcNAc)) on the synthesis of heparan sulfate (HS) and chondroitin sulfate (CS) by murine airway smooth muscle (ASM) cells in the presence of radiolabeled metabolic precursors. Secreted and cell-associated CS and HS were assessed for changes in size by Superose 6 chromatography. Treatment of ASM cells with 4-F-GlcNAc (100 μm) reduced the quantity (by 64.1–76.6%) and decreased the size of HS/CS glycosaminoglycans associated with the cell layer (K_av shifted from 0.30 to 0.45). The quantity of CS secreted into the medium decreased by 65.7–73.0%, and the size showed a K_av shift from 0.30 to 0.50. Treatment of ASM cells with 45 μm and 179 μm 4-F-GlcNAc in the presence of a stimulator of CS synthesis, 4-methylumbelliferyl-β-d-xyloside, reduced the amount of the xyloside-CS chains by 65.4 and 87.0%, respectively. The size of xyloside-CS chains synthesized in the presence of 4-F-GlcNAc were only slightly larger than those with xyloside treatment alone (K_av of 0.55 compared with that of 0.6). The effects of 4-F-GlcNAc to inhibit CS synthesis were not observed with equimolar concentrations of glucosamine. We propose that 4-F-GlcNAc inhibits CS synthesis by inhibiting 4-epimerization of UDP-GlcNAc to UDP-GalNAc, thereby depleting one of the substrates required, whereas HS elongation is inhibited by truncation when the nonreducing terminus of the growing chain is capped with 4-F-GlcNAc.The synthesis and physical properties (size and charge) of proteoglycans are altered under some pathological conditions such as cancer (1), spinal cord injury (2), atherosclerosis (3), and asthma (4). The importance of these pathological changes in proteoglycans has driven the need to study and design novel chemical tools which can control proteoglycan biosynthesis. Thus, we have studied the effect of a fluorinated analogue of glucosamine on proteoglycan synthesis in murine airway smooth muscle cells.Mono-, di-, and oligosaccharides that contain fluorine have been developed to study the enzymes involved in carbohydrate metabolism, and some of these have been shown to be inhibitors. The atomic size of fluorine is only slightly smaller (van der Waals'' radius (r′) of 135 pm) than that of oxygen (140 pm), and the C-F bond has a higher energy (485 kJ/mol) compared with that of C-O (370 kJ/mol) (5). The substitution of fluorine for oxygen at the 4-position of N-acetylglucosamine (4-F-GlcNAc)² confers a greater electronegativity on the bond and makes it less likely to be removed from the GlcN carbon ring. It is the properties of fluorine that contribute to the unique characteristics of 4-F-GlcNAc.4-F-GlcNAc used for cell culture experiments has O-acetyl groups at several of its ring positions, which in effect increases its cell permeability compared with that of unmodified forms (6). After hydrolysis to remove the O-acetyl residues, 4-F-GlcNAc, like GlcNAc, must be converted to UDP-4-F-GlcNAc, which in turn can be a substrate (or inhibitor) of enzyme reactions that use UDP-GlcNAc. GlcN is typically used as a control compound for 4-F-GlcNAc in vitro because of its superior cell permeability characteristics when compared with acetylated GlcN derivatives. Although acetylated GlcN derivatives enter the cell via passive diffusion, GlcN can enter by both passive diffusion and through the glucose transporter 4 (7).4-F-GlcNAc and 4-F-N-acetylgalactosamine (4-F-GalNAc) have been specifically studied as potential inhibitors of cell growth for the treatment of leukemia. The IC₅₀ values for 4-F-GlcNAc and 4F-GalNAc inhibition of leukemic cell proliferation are 34 and 35 μm, respectively (8). Moreover, by blocking polylactosamine synthesis necessary for elaboration of selectin ligands, 4-F-GlcNAc exhibits anti-inflammatory effects by reducing leukocyte homing to areas of contact allergic hypersensitivity in mice in vivo (9). Beyond effects on cell membrane glycoproteins, it has been proposed that the 4-fluorinated analogue of glucosamine truncates the GlcNAc-hexuronic acid chains on heparan sulfate (HS) by preventing the formation of the normal 1,4-glycosidic linkage between glucuronate (GlcUA) and on the nonreducing end of the growing chain (10). Thus, 4-F-GlcNAc has been suggested as a therapy for reducing amyloid deposition, which can feature HS accumulation (10, 11). Treatment of cultured hepatocytes in vitro with 4-F-GlcNAc and 4F-GalNAc (10–1000 μm) for 24 h reduced [³H]glucosamine and [³⁵S]sulfate incorporation into cellular glycosaminoglycans (11). However, total protein synthesis was also reduced at 1000 μm (11).Although the effects of 4-F-GlcNAc on HS production have been described (10), its effects on other extracellular matrix glycosaminoglycans, chondroitin/dermatan sulfate (CS/DS) and hyaluronan (HA), have not been reported.Airway smooth muscle (ASM) cells produce HS- and CS/DS-containing proteoglycans, including perlecan, versican, and decorin (12). Using these cells, we observed that 4F-GlcNAc inhibits CS/DS synthesis nearly as effectively as it inhibits HS synthesis. Although the 4-F on a nonreducing terminal F-GlcNAc-HS chain would block further HS synthesis by preventing the formation of the GlcUAβ1,4 bond required for elongation, the glycosidic bond in CS/DS is β1,3 between hexuronic acid and GalNAc. Thus, UDP-4-F-GlcNAc could not interfere with CS/DS synthesis via the same mechanism because it cannot be 4-epimerized to UDP-4F-GalNAc. Thus, we hypothesized that UDP-4-F-GlcNAc is a potent inhibitor of the 4-epimerase required to convert UDP-GlcNAc to UDP-GalNAc, thereby depleting the cell of UDP-GalNAc, a necessary substrate for CS/DS synthesis. To explore this putative mechanism, we analyzed the inhibitory effects of 4-F-GlcNAc on intrinsic and xyloside-stimulated CS synthesis in ASM cells (13).     

Identification of Specific Hemopexin-like Domain Residues That Facilitate Matrix Metalloproteinase Collagenolytic Activity

Collagen serves as a structural scaffold and a barrier between tissues, and thus collagen catabolism (collagenolysis) is required to be a tightly regulated process in normal physiology. In turn, the destruction or damage of collagen during pathological states plays a role in tumor growth and invasion, cartilage degradation, or atherosclerotic plaque formation and rupture. Several members of the matrix metalloproteinase (MMP) family catalyze the hydrolysis of collagen triple helical structure. This study has utilized triple helical peptide (THP) substrates and inhibitors to dissect MMP-1 collagenolytic behavior. Analysis of MMP-1/THP interactions by hydrogen/deuterium exchange mass spectrometry followed by evaluation of wild type and mutant MMP-1 kinetics led to the identification of three noncatalytic regions in MMP-1 (residues 285–295, 302–316, and 437–457) and two specific residues (Ile-290 and Arg-291) that participate in collagenolysis. Ile-290 and Arg-291 contribute to recognition of triple helical structure and facilitate both the binding and catalysis of the triple helix. Evidence from this study and prior studies indicates that the MMP-1 catalytic and hemopexin-like domains collaborate in collagen catabolism by properly aligning the triple helix and coupling conformational states to facilitate hydrolysis. This study is the first to document the roles of specific residues within the MMP-1 hemopexin-like domain in substrate binding and turnover. Noncatalytic sites, such as those identified here, can ultimately be utilized to create THP inhibitors that target MMPs implicated in disease progression while sparing proteases with host-beneficial functions.The mechanism of collagenolysis, by which proteases catalyze the hydrolysis of amide bonds within triple helical structures, has been investigated for over 30 years. Despite this lengthy period, few inroads have been made in the identification of specific enzyme residues that facilitate collagenolysis. The primary mammalian collagenases have been identified as cathepsin K and several members of the matrix metalloproteinase (MMP)³ family. Most of the early work on MMP collagenolysis focused on analysis of the sites of hydrolysis, and how unique features within these sites may direct collagen catabolism (1). More recent work has evaluated the active sites and domains of MMPs to better understand the dynamic role that the enzyme plays in collagen hydrolysis (2–4).Collagenolytic members of the MMP family possess similar domain organizations, including propeptide, catalytic (CAT), linker, and hemopexin-like (HPX) domains (5). Several of these domains and/or regions within them have been implicated in collagenolysis. For example, MMP-1 residues 183–191, which are on the V-B loop between the fifth β-strand and the second α-helix in the CAT domain, as well as the active site cleft itself, have substantial roles in collagenolysis (6, 7). MMP-1 residue Gly-233 has been implicated as necessary for conformational flexibility of the active site (8). Within the MMP-1 linker domain, residues 262–276 were proposed to form a polyproline type II helix and interact with and destabilize the MMP cleavage site in collagen (9), whereas Gly-272 may allow bending of the linker domain to aid in interaction between the CAT and HPX domains (10).The HPX domain has a critical role in collagenolysis, as removal of the MMP-1, MMP-8, MMP-13, or MMP-14 (MT1-MMP) HPX domain results in a loss of collagenolytic activity (11–16). However, no information has been obtained as to the identity of specific residues within the HPX domain that participate in collagenolysis. Secondary binding sites (exosites) may promote interaction of proteases with large, macromolecular substrates, such as collagen. The identification of exosites involved in collagenolysis may aid in the design of selective MMP inhibitors (17–20). Ultimately, as exosites are identified, the manner in which the CAT, linker, and HPX domains work together to facilitate collagenolysis can be revealed.One approach for the rapid analysis of protein structure and identification of binding sites within proteins involves hydrogen/deuterium exchange (HDX) of protein backbone amide hydrogens with detection by mass spectrometry (MS) (21–23). A protein or protein/ligand pair is incubated for defined intervals in a deuterated environment. After rapid quenching of the HDX reaction, the partially deuterated protein is digested, and the resulting peptide fragments are analyzed by LC-MS. The deuterium buildup curve measured for each fragment yields an average amide exchange rate that reflects the environment of the peptide in the intact protein. HDX MS has been used previously to monitor the interaction between doxycycline and MMP-7 (24). The interaction sites identified were consistent with other biophysical studies mapping doxycycline binding outside of the catalytic Zn²⁺ (24). This present study has utilized HDX MS with a triple helical peptide (THP) substrate to identify nonactive site MMP-1 regions involved in collagenolysis. Subsequently, site-specific mutagenesis of MMP-1 in combination with THP inhibitors and substrates was utilized to identify, for the first time, specific HPX domain residues that participate in collagenolysis and to provide insight as to how these residues function mechanistically.     

Ion transport and osmotic adjustment in Escherichia coli in response to ionic and non-ionic osmotica

Bacteria respond to osmotic stress by a substantial increase in the intracellular osmolality, adjusting their cell turgor for altered growth conditions. Using Escherichia coli as a model organism we demonstrate here that bacterial responses to hyperosmotic stress specifically depend on the nature of osmoticum used. We show that increasing acute hyperosmotic NaCl stress above ∼1.0 Os kg⁻¹ causes a dose-dependent K⁺ leak from the cell, resulting in a substantial decrease in cytosolic K⁺ content and a concurrent accumulation of Na⁺ in the cell. At the same time, isotonic sucrose or mannitol treatment (non-ionic osmotica) results in a gradual increase of the net K⁺ uptake. Ion flux data are consistent with growth experiments showing that bacterial growth is impaired by NaCl at the concentration resulting in a switch from net K⁺ uptake to efflux. Microarray experiments reveal that about 40% of upregulated genes shared no similarity in their responses to NaCl and sucrose treatment, further suggesting specificity of osmotic adjustment in E. coli to ionic and non-ionic osmotica. The observed differences are explained by the specificity of the stress-induced changes in the membrane potential of bacterial cells highlighting the importance of voltage-gated K⁺ transporters for bacterial adaptation to hyperosmotic stress.     

Genomic dissection of microbial pathogenesis in cultured Drosophila cells

Recent RNA interference screens that were performed at a genome-wide level have identified host factors that are important for the growth of Listeria monocytogenes in cultured cells from the fruit fly Drosophila melanogaster. The screens identified genes that are involved in phagocytosis but did not detect genes known to be involved in immune signaling pathways. These studies provide a foundation for the identification of host factors and virulence mechanisms.     

Induction of the unfolded protein response in familial amyotrophic lateral sclerosis and association of protein-disulfide isomerase with superoxide dismutase 1

Mutations in Cu/Zn superoxide dismutase (SOD1) are linked to motor neuron death in familial amyotrophic lateral sclerosis (ALS) by an unclear mechanism, although misfolded SOD1 aggregates are commonly associated with disease. Proteomic analysis of the transgenic SOD1(G93A) ALS rat model revealed significant up-regulation of endoplasmic reticulum (ER)-resident protein-disulfide isomerase (PDI) family members in lumbar spinal cords. Expression of SOD1 mutants (mSOD1) led to an up-regulation of PDI in motor neuron-like NSC-34 cells but not other cell lines. Inhibition of PDI using bacitracin increased aggregate production, even in wild type SOD1 transfectants that do not readily form inclusions, suggesting PDI may protect SOD1 from aggregation. Moreover, PDI co-localized with intracellular aggregates of mSOD1 and bound to both wild type and mSOD1. SOD1 was also found in the microsomal fraction of cells despite being a predominantly cytosolic enzyme, confirming ER-Golgi-dependent secretion. In SOD1(G93A) mice, a significant up-regulation of unfolded protein response entities was also observed during disease, including caspase-12, -9, and -3 cleavage. Our findings therefore implicate unfolded protein response and ER stress-induced apoptosis in the patho-physiology of familial ALS. The possibility that PDI may be a therapeutic target to prevent SOD1 aggregation is also raised by this study.     

Midazolam as an adjunctive therapy for capture myopathy in Bar-tailed Godwits (Limosa lapponica baueri) with prognostic indicators

Capture myopathy is a complication of capture and handling in many species of birds and mammals. Muscular necrosis leads to ataxia, paralysis, and pain, whereas metabolic disturbances can result in death. We conducted an opportunistic clinical trial on Bar-tailed Godwits (Limosa lapponica baueri) that developed capture myopathy after a cannon-net capture in New Zealand in October 2008. We assessed the beneficial effects of midazolam, a benzodiazepine with the effects of anxiolysis, muscle relaxation, and sedation, in the adjunctive treatment of capture myopathy. Physical and biochemical parameters were analyzed retrospectively for their potential as indicators for survival until release. Birds (n=16) were treated with subcutaneous fluid therapy, a nonsteroidal anti-inflammatory (meloxicam), gavage feeding, and sling therapy twice daily. The treatment group (n=8) was treated twice daily with intramuscular midazolam injections, 1.5 mg/kg. Surviving godwits were released over 1-9 days, with 6 of 8 treated birds (75%) surviving to release, compared with 3 of 8 controls (38%). Inability to counteract weight loss in captivity was the most significant problem for both groups. Lack of waterproofing and predation were contributing causes of death for at least two godwits after release. Birds treated with midazolam showed subjective benefits including improved tolerance of handling and sling therapy. Clinical parameters (change in body mass, packed cell volume [PCV], plasma creatine kinase [CK], aspartate aminotransferase [AST], total protein, and uric acid [UA] over time) were not statistically different between groups, although peak average values for CK, AST, and UA were lower in the treatment group. Decline in body mass (%), PCV, final plasma UA, and peak plasma CK were the most useful prognostic indicators. Midazolam shows potential as an ancillary treatment for capture myopathy in birds and is worthy of continued study and use.