Structural variation of the 3-acetamido-4,5,6-trihydroxyazepane iminosugar through epimerization and C-alkylation leads to low micromolar HexAB and NagZ inhibitors.

We report the synthesis of seven-membered iminosugars derived from a 3S-acetamido-4R,5R,6S-trihydroxyazepane scaffold and their evaluation as inhibitors of functionally related exo-N-acetylhexosaminidases including human O-GlcNAcase (OGA), human lysosomal ß-hexosaminidase (HexAB), and Escherichia coli NagZ. Capitalizing on the flexibility of azepanes and the active site tolerances of hexosaminidases, we explore the effects of epimerization of stereocenters at C-3, C-5 and C-6 and C-alkylation at the C-2 or C-7 positions. Accordingly, epimerization at C-6 (L-ido) and at C-5 (D-galacto) led to selective HexAB inhibitors whereas introduction of a propyl group at C-7 on the C-3 epimer furnished a potent NagZ inhibitor.

Emerging roles of protein O-GlcNAcylation in cardiovascular diseases: Insights and novel therapeutic targets.

Cardiovascular diseases (CVDs) are the leading cause of death globally. While the major focus of pharmacological and non-pharmacological interventions has been on targeting disease pathophysiology and limiting predisposing factors, our understanding of the cellular and molecular mechanisms underlying the pathogenesis of CVDs remains incomplete. One mechanism that has recently emerged is protein O-GlcNAcylation. This is a dynamic, site-specific reversible post-translational modification of serine and threonine residues on target proteins and is controlled by two enzymes: O-linked ß-N-acetylglucosamine transferase (OGT) and O-linked ß-N-acetylglucosaminidase (OGA). Protein O-GlcNAcylation alters the cellular functions of these target proteins which play vital roles in pathways that modulate vascular homeostasis and cardiac function. Through this review, we aim to give insights on the role of protein O-GlcNAcylation in cardiovascular diseases and identify potential therapeutic targets in this pathway for development of more effective medicines to improve patient outcomes.

Kinetic and thermodynamic insights into the inhibitory mechanism of TMG-chitotriomycin on Vibrio campbellii GH20 exo-ß-N-acetylglucosaminidase.

We investigated the inhibition kinetics of VhGlcNAcase, a GH20 exo-ß-N-acetylglucosaminidase (GlcNAcase) from the marine bacterium Vibrio campbellii (formerly V. harveyi) ATCC BAA-1116, using TMG-chitotriomycin, a natural enzyme inhibitor specific for GH20 GlcNAcases from chitin-processing organisms, with p-nitrophenyl N-acetyl-ß-d-glucosaminide (pNP-GlcNAc) as the substrate. TMG-chitotriomycin inhibited VhGlcNAcase with an IC50 of 3.0 ± 0.7 µM. Using Dixon plots, the inhibition kinetics indicated that TMG-chitotriomycin is a competitive inhibitor, with an inhibition constant Ki of 2.2 ± 0.3 µM. Isothermal titration calorimetry experiments provided the thermodynamic parameters for the binding of TMG-chitotriomycin to VhGlcNAcase and revealed that binding was driven by both favorable enthalpy and entropy changes (ΔH° = -2.5 ± 0.1 kcal/mol and -TΔS° = -5.8 ± 0.3 kcal/mol), resulting in a free energy change, ΔG°, of -8.2 ± 0.2 kcal/mol. Dissection of the entropic term showed that a favorable solvation entropy change (-TΔSsolv° = -16 ± 2 kcal/mol) is the main contributor to the entropic term.

Synthesis of novel homoazanucleosides and their peptidyl analogs.

Synthesis of novel homoazanucleosides and their peptidyl analogs as hybrid molecules comprised of amino acids, an iminosugar and natural nucleobases is reported for the first time. A pluripotent amino-substituted chiral polyhydroxypyrrolidine, possessing orthogonally different functional groups on either arm of the pyrrolidine ring, served as an ideal substrate for the synthesis of the proposed peptidyl homoazanucleosides. The acid sensitive primary benzyloxy group, on one arm of the pyrrolidine ring, after selective deprotection, was utilized for the introduction of nucleobases to obtain the homoazanucleosides. The amino group on the other side offered the opportunity to be coupled with amino acids to deliver the desired peptidyl homoazanucleosides. Glycosidase inhibition studies revealed that the acetamido derivatives of homoazanucleosides were found to be sub-millimolar inhibitors of ß-N-acetyl-glucosaminidase.

NAG-thiazoline is a potent inhibitor of the Vibrio campbellii GH20 ß-N-Acetylglucosaminidase.

Vibrio spp. play a vital role in the recycling of chitin in oceans, but several Vibrio strains are highly infectious to aquatic animals and humans. These bacteria require chitin for growth; thus, potent inhibitors of chitin-degrading enzymes could serve as candidate drugs against Vibrio infections. This study examined NAG-thiazoline (NGT)-mediated inhibition of a recombinantly expressed GH20 ß-N-acetylglucosaminidase, namely VhGlcNAcase from Vibrio campbellii (formerly V. harveyi) ATCC BAA-1116. NGT strongly inhibited VhGlcNAcase with an IC50 of 11.9 ± 1.0 µm and Ki 62 ± 3 µm, respectively. NGT was also found to completely inhibit the growth of V. campbellii strain 650 with an minimal inhibitory concentration value of 0.5 µm. ITC data analysis showed direct binding of NGT to VhGlcNAcase with a Kd of 32 ± 1.2 µm. The observed ΔG°binding of -7.56 kcal·mol-1 is the result of a large negative enthalpy change and a small positive entropic compensation, suggesting that NGT binding is enthalpy-driven. The structural complex shows that NGT fully occupies the substrate-binding pocket of VhGlcNAcase and makes an exclusive hydrogen bond network, as well as hydrophobic interactions with the conserved residues around the -1 subsite. Our results strongly suggest that NGT could serve as an excellent scaffold for further development of antimicrobial agents against Vibrio infections. DATABASE: Structural data are available in PDB database under the accession number 6K35.

Novel GH-20 ß-N-acetylglucosaminidase inhibitors: Virtual screening, molecular docking, binding affinity, and anti-tumor activity.

ß-N-acetylglucosaminidases (GlcNAcases) play a crucial role in the metabolism of glycan-conjugated proteins/lipids in humans. Elevated levels of serum GlcNAcases have been associated with certain types of cancer, and GlcNAcases therefore serve as drug targets. Here, we employed virtual screening to identify two novel GlcNAcase inhibitors from the National Cancer Institute (NCI) Drug Library using a bacterial GH-20 GlcNAcase (VhGlcNAcase) as a search model. NSC73735 was shown to be most potent with IC50 of 12.7 ±â€¯1.2 µM, agreeing with Kd of 0.94 ±â€¯0.2 µM obtained by ITC. Molecular docking refinement indicated that Trp582 the key residue that interacted with all the inhibitor molecules. Docking NSC7373 into the active site of human O-GlcNAcase (hOGA) yielded reasonably good fit with the estimated Kd of 44.7 µM, indicating its possibility to be a true binding partner. NSC73735 was shown to significantly suppress both cell growth and GlcNAcase activity of five cancer cell lines (U937, THP-1, MCF-7, HepG2 and PC-3) that express endogenous GlcNAcases. The cell cytotoxicity assay indicated the inherent effects of the lead compound on GlcNAcase expression with cancer cell proliferation, and therefore this novel GlcNAcase inhibitor may serve as a virtuous candidate for further development of highly potent anti-tumor agents.

High-level expression of ß-N-Acetylglucosaminidase BsNagZ in Pichia pastoris to obtain GlcNAc.

ß-N-Acetylglucosaminidases (NAGase) can remove N-acetylglucosamine (GlcNAc) from the non-reducing end of chitin or chitosan. GlcNAc has many important physiological functions in organism, which can be used for the treatment of rheumatoid arthritis clinically and be used as food antioxidant, infant food additive and diabetic sweetener. Thus, it is very important to develop genetic-engineering strains with high-yield NAGase to hydrolyze chitin into GlcNAc. Here, the NAGase gene of Bacillus subtilis 168 (BsnagZ) was synthesized according to the codon bias of Pichia pastoris and expressed in P. pastoris. The expression level of BsNagZ in P. pastoris increased over the induced time and the highest activity reached 0.76 U/mL at the 7th day. The recombinant BsNagZ was purified for characterization. The optimal temperature and pH are 60 °C and 6.0, respectively. It can both keep over 80% activities after pre-incubation at 55 °C for one hour and at 4 °C for 12 h from pH 4.5 to 10.0. To further improve the expression level of BsNagZ, a recombinant strain with four copy BsnagZs was screened using a high concentration of zeocin. The highest BsNagZ activity reached 3.2 U/mL at the 12th day, which was fourfold higher than that of single-copy strain. Combined with commercial chitinase CtnSg, GlcNAc can be produced by recombinant BsNagZ when used colloidal chitin as the substrate. Our study highlights that the NAGase was first successfully expressed in P. pastoris and GlcNAc can be produced via NAGase hydrolyzing the colloidal chitin.

A mechanism-based GlcNAc-inspired cyclophellitol inactivator of the peptidoglycan recycling enzyme NagZ reverses resistance to ß-lactams in Pseudomonas aeruginosa.

The development of a potent mechanism-based inactivator of NagZ, an enzyme critical to the production of inducible AmpC ß-lactamase in Gram-negative bacteria, is presented. This inactivator significantly reduces MIC values for important ß-lactams against a clinically relevant strain of Pseudomonas aeruginosa.

Application of fragment based virtual screening towards inhibition of bacterial N-acetyglucosaminidase$.

A structure-based approach is applied for the development of inhibitors of bacterial N-acetyglucosaminidase (autolysin). Autolysins are enzymes involved in the degradation of peptidoglycan and therefore participate in bacterial cell growth and different lysis phenomena. Several studies indicate that by the inhibition of autolysins, and consequently of bacterial cell division, antibacterial activity can be obtained, thus paving the road to a novel group of therapeutics against human pathogens. As crystal structures of the autolysin E (AtlE)-ligand complexes were obtained in our laboratories, fragment-based virtual screening was the method of choice for the initial studies. Fragment libraries from various databases were merged to increase the number of compounds for the virtual screening. Twenty-four commercially available virtual hits were selected and subjected to quantitative analysis of binding interactions using the surface plasmon resonance technique. Twelve fragments showed fragment-AtlE interactions. For F1, the top hit of the virtual screening, a KD of 228 µM was determined, while other fragments displayed non-stoichiometric binding. Blind docking of potential binders uncovers three possible allosteric sites. Ligands of N-acetyglucosaminidase identified in our study represent valuable information for the further development of AtlE inhibitors, which could in future represent antibacterial agents acting by a novel mode of action.

The influence of inhibition of acid phosphatase, ß-N-acetylglucosaminidase and lactate dehydrogenase present in the sperm of ide (Leuciscus idus) on the percentage of fertilised eggs.

This study investigated how the inhibition of certain enzymes present in ide sperm influences sperm motility and the percentage of fertilised eggs. The enzymes studied were acid phosphatase (AcP), ß-N-acetylglucosaminidase (ß-NAGase) and lactate dehydrogenase (LDH). None of the inhibitors affected ide sperm motility parameters. The addition of gossypol (a LDH inhibitor) caused a considerable increase in the percentage of fertilised eggs (92-95% compared to 63% in the control). The inhibition of AcP caused a considerable decrease in fertility rate - at the highest inhibitor dose, the percentage of fertilised eggs decreased to 26%. A similar effect was seen after the addition of acetamide (a ß-NAGase inhibitor), but in this case the highest dose caused complete inhibition of fertilisation. The results presented here indicate the importance of AcP and ß-NAGase in the process of ide fertilisation.

Potent inhibition of a GH20 exo-ß-N-acetylglucosaminidase from marine Vibrio bacteria by reaction intermediate analogues.

Exo-ß-N-acetylglucosaminidases (GlcNAcases) are hydrolytic enzymes involved in the metabolism of chitin in bacteria and in eukaryotic glycosphingolipid metabolism, with genetic defects in human GlcNAcases (HexA and HexB) resulting in Tay-Sachs and Sandhoff diseases, respectively. Here, we determined the effects of three known inhibitors of exo-ß-N-acetylglucosaminidases (PUGNAc, NHAcCAS and NHAcDNJ) on a GH20 exo-ß-N-GlcNAcase (VhGlcNAcase) from the pathogenic bacterium Vibrio harveyi, in dose-response experiments. The inhibitors were shown to modify the kinetic parameters (both Km and kcat), yielding significant decreases in the overall efficiency of the enzyme in hydrolyzing the natural substrate diNAG. Molecular interactions between the inhibitors and the enzyme were investigated by isothermal calorimetry (ITC), and were confirmed using molecular docking. VhGlcNAcase was strongly inhibited by these compounds, with PUGNAc having the lowest IC50 value, of 1.2 µM. Molecular docking suggested that the inhibitors mimicked reaction intermediates, with enzyme-inhibitor interactions being similar to those of the enzyme with diNAG. The equilibrium dissociation constants (Kd) obtained from ITC were 0.19 µM for PUGNAc, 12.9 µM for NHAcCAS and 25.6 µM for NHAcDNJ, confirming that PUGNAc was the most potent inhibitor. The ITC data indicated that the binding of the enzyme to the inhibitors was driven by enthalpy. The negative heat capacity change (ΔCp) of -0.34 ±â€¯0.05 kcal·mol-1·K-1 indicates that hydrophobic interactions make a substantial contribution to the molecular interactions between PUGNAc and the enzyme. Our results suggest that PUGNAc is a highly potent inhibitor, and suggest its usefulness as a scaffold for potential drugs targeting GlcNAcase-related metabolic diseases.

Diamide Inhibitors of the Bacillus subtilis N-Acetylglucosaminidase LytG That Exhibit Antibacterial Activity.

N-Acetylglucosaminidases (GlcNAcases) play an important role in the remodeling and recycling of bacterial peptidoglycan by degrading the polysaccharide backbone. Genetic deletions of autolysins can impair cell division and growth, suggesting an opportunity for using small molecule autolysin inhibitors both as tools for studying the chemical biology of autolysins and also as antibacterial agents. We report here the synthesis and evaluation of a panel of diamides that inhibit the growth of Bacillus subtilis. Two compounds, fgkc (21) and fgka (5), were found to be potent inhibitors (MIC 3.8 ± 1.0 and 21.3 ± 0.1 µM, respectively). These compounds inhibit the B. subtilis family 73 glycosyl hydrolase LytG, an exo GlcNAcase. Phenotypic analysis of fgkc (21)-treated cells demonstrates a propensity for cells to form linked chains, suggesting impaired cell growth and division.

Thiamet G mediates neuroprotection in experimental stroke by modulating microglia/macrophage polarization and inhibiting NF-κB p65 signaling.

Inflammatory responses are accountable for secondary injury induced by acute ischemic stroke (AIS). Previous studies indicated that O-GlcNAc modification (O-GlcNAcylation) is involved in the pathology of AIS, and increase of O-GlcNAcylation by glucosamine attenuated the brain damage after ischemia/reperfusion. Inhibition of ß-N-acetylglucosaminidase (OGA) with thiamet G (TMG) is an alternative option for accumulating O-GlcNAcylated proteins. In this study, we investigate the neuroprotective effect of TMG in a mouse model of experimental stroke. Our results indicate that TMG administration either before or after middle cerebral artery occlusion (MCAO) surgery dramatically reduced infarct volume compared with that in untreated controls. TMG treatment ameliorated the neurological deficits and improved clinical outcomes in neurobehavioral tests by modulating the expression of pro-inflammatory and anti-inflammatory cytokines. Additionally, TMG administration reduced the number of Iba1+ cells in MCAO mice, decreased expression of the M1 markers, and increased expression of the M2 markers in vivo. In vitro, M1 polarization of BV2 cells was inhibited by TMG treatment. Moreover, TMG decreased the expression of iNOS and COX2 mainly by suppressing NF-κB p65 signaling. These results suggest that TMG exerts a neuroprotective effect and could be useful as an anti-inflammatory agent for ischemic stroke therapy.

Azide anions inhibit GH-18 endochitinase and GH-20 Exo ß-N-acetylglucosaminidase from the marine bacterium Vibrio harveyi.

Vibrio harveyi is a bioluminescent marine bacterium that utilizes chitin as its sole source of energy. In the course of chitin degradation, the bacterium primarily secretes an endochitinase A (VhChiA) to hydrolyze chitin, generating chitooligosaccharide fragments that are readily transported into the cell and broken down to GlcNAc monomers by an exo ß-N-acetylglucosaminidase (VhGlcNAcase). Here we report that sodium salts, especially sodium azide, inhibit two classes of these chitin-degrading enzymes (VhChiA and VhGlcNAcase) with distinct modes of action. Kinetic analysis of the enzymatic hydrolysis of pNP-glycoside substrates reveals that sodium azide inhibition of VhChiA has a mixed-type mode, but that it inhibits VhGlcNAcase competitively. We propose that azide anions inhibit chitinase activity by acting as strong nucleophiles that attack Cγ of the catalytic Glu or Cß of the neighbouring Asp residues. Azide anions may bind not only to the catalytic centre, but also to the other subsites in the substrate-binding cleft of VhChiA. In contrast, azide anions may merely occupy the small-binding pocket of VhGlcNAcase, thereby blocking the accessibility of its active site by short-chain substrates.

O-GlcNAcylation of the human epidermal growth factor receptor.

The reversible O-linked attachment of single ß-D-N-acetylglucosamine (GlcNAc) moieties to serine/threonine residues in target proteins is a frequently occurring post-translational modification affecting the functionality of many cellular systems. In this report we present experimental evidence suggesting that the epidermal growth factor receptor (EGFR) is subjected to O-GlcNAcylation in human carcinoma epidermoid A431 cells and human lung carcinoma A549 cells. However, no signal was detected in human cervix adenocarcinoma HeLa cells or in mouse EGFR-T17 fibroblasts ectopically expressing the human EGFR. We detected a positive O-GlcNAcylation signal in the immunoprecipitated EGFR by Western blotting using two distinct specific anti-O-GlcNAc antibodies even after N-deglycosylation of the receptor using peptide-N-glycosidase F (PNGase F). Conversely, the presence of EGFR was detected by Western blotting using an anti-EGFR antibody in the immunocomplex of O-GlcNAcylated proteins immunoprecipitated with an anti-O-GlcNAc antibody. These signals were enhanced when the O-linked ß-N-acetylglucosaminidase (OGA) inhibitor Thiamet G was added to prevent the deglycosylation of the GlcNAc moiety(ies). Moreover, we also detected a positive signal in the immunoprecipitated and N-deglycosylated EGFR using PNGase F, and tunicamycin when the cells were metabolically labeled with azido-GlcNAc (GlcNAz), biotinylated and probed with a streptavidin-labeled peroxidase. Finally, EGFR and O-linked ß-N-acetylglucosamine transferase (OGT) co-immunoprecipitate, and incubation of the immunoprecipitated EGFR with the immunoprecipitated OGT in the presence of uridine 5'-diphospho-N-acetylglucosamine (UDP-GlcNAc) resulted in a significant enhancement of the EGFR O-GlcNAcylation signal as detected by Western blotting using an anti-O-GlcNAc antibody. We conclude that the human EGFR is subjected to O-GlcNAcylation in the A431 and A549 tumor cell lines.

Improvement of glycosylation structure by suppression of ß-N-acetylglucosaminidases in silkworm.

The baculovirus-silkworm recombinant protein expression system is an excellent method for achieving high-level expression and post-translational modifications, especially glycosylation. However, the presence of paucimannosidic-type N-glycan in glycoproteins restricts their clinical use. Paucimannosidic-type N-glycan is produced by insect-specific membrane-binding-type ß-N-acetylglucosaminidase (GlcNAcase). In the silkworm, BmGlcNAcase1, BmGlcNAcase2, and BmFDL are membrane-binding-type GlcNAcases. We investigated the localization of these GlcNAcases and found that BmFDL and BmGlcNAcase2 were mainly located in the fat body and hemolymph, respectively. The fat body is the main tissue of recombinant protein expression by baculovirus, and many glycoproteins are secreted into the hemolymph. These results suggest that inhibition of BmFDL and BmGlcNAcase2 could increase GlcNAc-type N-glycan levels. We therefore injected a GlcNAcase inhibitor into silkworms to investigate changes in the N-glycan structure of the glycoprotein expressed by baculovirus; modest levels of GlcNAc-type N-glycan were observed (0.8% of total N-glycan). Next, we generated a transgenic silkworm in which RNA interference (RNAi) reduced the BmFDL transcript level and enzyme activity to 25% and 50%, respectively, of that of the control silkworm. The proportion of GlcNAc-type N-glycan increased to 4.3% in the RNAi-transgenic silkworm. We conclude that the structure of N-glycan can be changed by inhibiting the GlcNAcases in silkworm.

Exploring NAG-thiazoline and its derivatives as inhibitors of chitinolytic ß-acetylglucosaminidases.

NAG-thiazoline (NGT) and its derivatives are well-known inhibitors against most ß-acetylglucosaminidases (ß-GlcNAcases) except for insect and bacterial chitinolytic ß-GlcNAcases, including the molting-indispensable OfHex1 from the insect Ostrinia furnacalis. Here, we report the co-crystal structure of OfHex1 in complex with NGT. This structure reveals a large active pocket in OfHex1 that may account for the poor inhibitory activity of NGT. To test this hypothesis, a bulky substituent was designed and synthesized on the thiazoline ring of NGT. The resulting compound (NMAGT) was determined to be a submicromolar inhibitor of OfHex1 with a Ki value of 0.13 µM, which is 600-fold lower than Ki value of NGT. Molecular dynamics simulation analysis supported the good fit of NMAGT to the active pocket.

A crystal structure-guided rational design switching non-carbohydrate inhibitors' specificity between two ß-GlcNAcase homologs.

Selective inhibition of function-specific ß-GlcNAcase has great potential in terms of drug design and biological research. The symmetrical bis-naphthalimide M-31850 was previously obtained by screening for specificity against human glycoconjugate-lytic ß-GlcNAcase. Using protein-ligand co-crystallization and molecular docking, we designed an unsymmetrical dyad of naphthalimide and thiadiazole, Q2, that changes naphthalimide specificity from against a human glycoconjugate-lytic ß-GlcNAcase to against insect and bacterial chitinolytic ß-GlcNAcases. The crystallographic and in silico studies reveal that the naphthalimide ring can be utilized to bind different parts of these enzyme homologs, providing a new starting point to design specific inhibitors. Moreover, Q2-induced closure of the substrate binding pocket is the structural basis for its 13-fold increment in inhibitory potency. Q2 is the first non-carbohydrate inhibitor against chitinolytic ß-GlcNAcases. This study provides a useful example of structure-based rationally designed inhibitors as potential pharmaceuticals or pesticides.

Irreversible inhibitory kinetics of mercuric ion on N-acetyl-ß-D-glucosaminidase from Nile tilapia (Oreochromis niloticus).

N-acetyl-ß-D-glucosaminidase (EC 3.2.1.52, NAGase), hydrolyzes dimers or trimers of N-acetyl-ß-D-glucosamine (NAG) into monomers and is shown to be important for the reproduction of male animals. NAGase is purified from the spermary of Nile tilapia, and its enzyme activity can be strongly inhibited by mercuric chloride (HgCl2). In this paper, we determined the kinetics of HgCl2-mediated inhibition of NAGase, and our results showed that it was irreversible inhibition with an IC50 value at 2.70±0.02 µM. Moreover, Hg(2+) reduced the thermal and pH stability of the enzyme. We determined the inhibition kinetics of Hg(2+) by using the kinetic method of substrate reaction. With this inhibition model, the microscopic rate constants for the reaction of Hg(2+) with free enzyme (k1) and the enzyme-substrate complex ( [Formula: see text] ) were determined to be 4.42×10(-4) mM(-1) s(-1) and 7.06×10(-5) mM(-1) s(-1), respectively, indicating that the presence of substrate can protect NAGase from Hg(2+) inhibition.

Effects of two PBDE congeners on the moulting enzymes of the freshwater amphipod Gammarus pulex.

Polybrominated diphenyl ethers (PBDEs) are abundant in aquatic environment. However, only few studies have investigated their impacts on freshwater invertebrates. This work aimed to study the effects of BDE-47 and BDE-99 congeners on the chitobiase and chitinolytic enzymes activities of the freshwater amphipod Gammarus pulex, according to gender, PBDE concentration and time of exposure. In addition, the bioaccumulation of BDE-47 and BDE-99 were measured. Results revealed that females have bioaccumulated more PBDE than males, and BDE-99 was more accumulated than BDE-47. PBDE exposures for 96 h have caused chitobiase and chitinolytic enzymes inhibition. This study not only indicate the importance of taking into account various confounding factors (gender, congeners, concentration) to understand PBDE effects, but underline also disruptions of molting enzymes activities. These disturbances suggest effects on the gammarid development and reproduction, and consequently effects on the gammarid population, and on a larger scale, a dysfunction of the ecosystem.