Two alpha subunits and one beta subunit of meprin zinc-endopeptidases are differentially expressed in the zebrafish Danio rerio

Meprins are members of the astacin family of metalloproteases expressed in epithelial tissues, intestinal leukocytes and certain cancer cells. In mammals, there are two homologous subunits, which form complex glycosylated disulfide-bonded homo- and heterooligomers. Both human meprin alpha and meprin beta cleave several basement membrane components, suggesting a role in epithelial differentiation and cell migration. There is also evidence that meprin beta is involved in immune defence owing to its capability of activating interleukin-1beta and the diminished mobility of intestinal leukocytes in meprin beta-knockout mice. Here we show for the first time by reverse transcription PCR, immunoblotting and immunofluorescence analyses that meprins are expressed not only in mammals, but also in the zebrafish Danio rerio. In contrast to the human, mouse and rat enzymes, zebrafish meprins are encoded by three genes, corresponding to two homologous alpha subunits and one beta subunit. Observations at both the mRNA and protein level indicate a broad distribution of meprins in zebrafish. However, there are strikingly different expression patterns of the three subunits, which is consistent with meprin expression in mammals. Hence, D. rerio appears to be a suitable model to gain insight into the basic physiological functions of meprin metalloproteases.

Evaluation of chemically modified SLA implants (modSLA) biofunctionalized with integrin (RGD)- and heparin (KRSR)-binding peptides

Enhancing osseointegration through surface immobilization of multiple short peptide sequences that mimic extracellular matrix (ECM) proteins, such as arginine-glycine-aspartic acid (RGD) and lysine-arginine-serine-arginine (KRSR), has not yet been extensively explored. Additionally, the effect of biofunctionalizing chemically modified sandblasted and acid-etched surfaces (modSLA) is unknown. The present study evaluated modSLA implant surfaces modified with RGD and KRSR for potentially enhanced effects on bone apposition and interfacial shear strength during early stages of bone regeneration. Two sets of experimental implants were placed in the maxillae of eight miniature pigs, known for their rapid wound healing kinetics: bone chamber implants creating two circular bone defects for histomorphometric analysis on one side and standard thread configuration implants for removal torque testing on the other side. Three different biofunctionalized modSLA surfaces using poly-L-lysine-graft-poly(ethylene glycol) (PLL-g-PEG) as a carrier minimizing nonspecific protein adsorption [(i) 20 pmol cm⁻² KRSR alone (KRSR); or in combination with RGD in two different concentrations; (ii) 0.05 pmol cm⁻² RGD (KRSR/RGD-1); (iii) 1.26 pmol cm⁻² RGD (KRSR/RGD-2)] were compared with (iv) control modSLA. Animals were sacrificed at 2 weeks. Removal torque values (701.48-780.28 N mm), bone-to-implant contact (BIC) (35.22%-41.49%), and new bone fill (28.58%-30.62%) demonstrated no significant differences among treatments. It may be concluded that biofunctionalizing modSLA surfaces with KRSR and RGD derivatives of PLL-g-PEG polymer does not increase BIC, bone fill, or interfacial shear strength.

Differential regulation of metzincins in experimental chronic renal allograft rejection: potential markers and novel therapeutic targets

Chronic renal allograft rejection is characterized by alterations in the extracellular matrix compartment and in the proliferation of various cell types. These features are controlled, in part by the metzincin superfamily of metallo-endopeptidases, including matrix metalloproteinases (MMPs), a disintegrin and metalloproteinase (ADAM) and meprin. Therefore, we investigated the regulation of metzincins in the established Fisher to Lewis rat kidney transplant model. Studies were performed using frozen homogenates and paraffin sections of rat kidneys at day 0 (healthy controls) and during periods of chronic rejection at day +60 and day +100 following transplantation. The messenger RNA (mRNA) expression was examined by Affymetrix Rat Expression Array 230A GeneChip and by real-time Taqman polymerase chain reaction analyses. Protein expression was studied by zymography, Western blot analyses, and immunohistology. mRNA levels of MMPs (MMP-2/-11/-12/-14), of their inhibitors (tissue inhibitors of metalloproteinase (TIMP)-1/-2), ADAM-17 and transforming growth factor (TGF)-beta1 significantly increased during chronic renal allograft rejection. MMP-2 activity and immunohistological staining were augmented accordingly. The most important mRNA elevation was observed in the case of MMP-12. As expected, Western blot analyses also demonstrated increased production of MMP-12, MMP-14, and TIMP-2 (in the latter two cases as individual proteins and as complexes). In contrast, mRNA levels of MMP-9/-24 and meprin alpha/beta had decreased. Accordingly, MMP-9 protein levels and meprin alpha/beta synthesis and activity were downregulated significantly. Members of metzincin families (MMP, ADAM, and meprin) and of TIMPs are differentially regulated in chronic renal allograft rejection. Thus, an altered pattern of metzincins may represent novel diagnostic markers and possibly may provide novel targets for future therapeutic interventions.

Influence of different spacers on the biological profile of a DOTA-somatostatin analogue

Radiolabeled somatostatin analogues have been successfully used for targeted radiotherapy and for imaging of somatostatin receptor (sst1-5)-positive tumors. Nevertheless, these analogues are subject to improving their tumor-to-nontarget ratio to enhance their diagnostic or therapeutic properties, preventing nephrotoxicity. In order to understand the influence of lipophilicity and charge on the pharmacokinetic profile of [1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA)]-somatostatin-based radioligands such as [DOTA,1-Nal3]-octreotide (DOTA-NOC), different spacers (X) based on 8-amino-3,6-dioxaoctanoic acid (PEG2), 15-amino-4,7,10,13-tetraoxapentadecanoic acid (PEG4), N-acetyl glucosamine (GlcNAc), triglycine, beta-alanine, aspartic acid, and lysine were introduced between the chelator DOTA and the peptide NOC. All DOTA-X-NOC conjugates were synthesized by Fmoc solid-phase synthesis. The partition coefficient (log D) at pH = 7.4 indicated that higher hydrophilicity than [111In-DOTA]-NOC was achieved with the introduction of the mentioned spacers, except with triglycine and beta-alanine. The high affinity of [InIII-DOTA]-NOC for human sst2 (hsst2) was preserved with the structural modifications, while an overall drop for hsst3 affinity was observed, except in the case of [InIII-DOTA]-beta-Ala-NOC. The new conjugates preserved the good affinity for hsst5, except for [InIII-DOTA]-Asn(GlcNAc)-NOC, which showed decreased affinity. A significant 1.2-fold improvement in the specific internalization rate in AR4-2J rat pancreatic tumor cells (sst2 receptor expression) at 4 h was achieved with the introduction of Asp as a spacer in the parent compound. In sst3-expressing HEK cells, the specific internalization rate at 4 h for [111In-DOTA]-NOC (13.1% +/- 0.3%) was maintained with [111In-DOTA]-beta-Ala-NOC (14.0% +/- 1.8%), but the remaining derivatives showed <2% specific internalization. Biodistribution studies were performed with Lewis rats bearing the AR4-2J rat pancreatic tumor. In comparison to [111In-DOTA]-NOC (2.96% +/- 0.48% IA/g), the specific uptake in the tumor at 4 h p.i. was significantly improved for the 111In-labeled sugar analogue (4.17% +/- 0.46% IA/g), which among all the new derivatives presented the best tumor-to-kidney ratio (1.9).

The effect of matrix bound parathyroid hormone on bone regeneration

INTRODUCTION: Autogenous bone is the most successful bone-grafting material; however, multiple disadvantages continue to drive developments of improved methods for bone regeneration. AIM: The aim of the present study was to test the hypothesis that an arginine-glycine-aspartic acid (RGD) modified polyethylene glycol-based matrix (PEG) containing covalently bound peptides of the parathyroid hormone (PTH(1-34)) enhances bone regeneration to a degree similar to autogenous bone. MATERIAL AND METHODS: Six American foxhounds received a total of 48 cylindrical titanium implants placed in the mandible between the first premolar and the second molar. Five, respectively, 7 months following tooth extraction, implants were placed into the center of surgically created defects. This resulted in a circumferential bone defect simulating an alveolar defect with a circular gap of 1.5 mm. Four treatment modalities were randomly allocated to the four defects per side: (1) PEG-matrix containing 20 microg/ml of PTH(1-34), and 350 microg/ml cys-RGD peptide, (2) PEG alone, (3) autogenous bone and (4) empty defects. Histomorphometric analysis was performed 4 and 12 weeks after implantation. The area fraction of newly formed bone was determined within the former defect and the degree of bone-to-implant contact (BIC) was evaluated both in the defect region and in the apical region of the implant. For statistical analysis ANOVA and subsequent pairwise Student's t-test were applied. RESULTS: Healing was uneventful and all implants were histologically integrated. Histomorphometric analysis after 4 weeks showed an average area fraction of newly formed bone of 41.7+/-1.8% for matrix-PTH, 26.6+/-4.1% for PEG alone, 43.9+/-4.5% for autogenous bone, and 28.9+/-1.5% for empty defects. After 12 weeks, the respective values were 49.4+/-7.0% for matrix-PTH, 39.3+/-5.7% for PEG alone, 50.5+/-3.4% for autogenous bone and 38.7+/-1.9% for empty defects. Statistical analysis after 4 and 12 weeks revealed significantly more newly formed bone in the PTH(1-34) group compared with PEG alone or empty defects, whereas no difference could be detected against autogenous bone. Regarding BIC no significant difference was observed between the four treatment groups neither at 4 nor at 12 weeks. CONCLUSION: It is concluded that an RGD-modified PEG hydrogel containing PTH(1-34) is an effective matrix system to obtain bone regeneration.

Influence of the magnesium aspartate hydrochloride administration to the maternal circuit on the aspartate concentration of the fetal circuit under in vitro perfusion of human placenta

OBJECTIVES: Magnesium aspartate hydrochloride (Magnesiocard, Mg-Asp-HCl) is proposed as a substitute of magnesium sulfate for the treatment of preeclampsia and premature labor. After an i.v. administration of a dose equivalent to that used in the treatment of preeclampsia to nonpregnant volunteers, a 10-fold increase of aspartic acid (Asp) over the physiological level was observed. Animal experiments have demonstrated that highly increased fetal levels of acidic amino acids such as Asp could be associated with neurotoxic damage in the fetal brain. The influence of such an elevation of Asp concentration in the maternal circuit on the fetal level, using the in vitro perfusion model of human placenta, was investigated. STUDY DESIGN: After a control phase (2h), a therapeutic dose of Mg combined with Asp (Magnesiocard, Mg-Asp-HCl) was applied to the maternal circuit approaching 10 times the physiological level of Asp. The administration was performed in two different phases simulating either a peak of maximum concentration (bolus application, 2h) or a steady state level (initially added, 4h). RESULTS: In four experiments, during experimental phases (6h) a slow increase in concentration in the fetal circuit was seen for Mg, AIB (alpha-aminoisobutyric acid, artificial amino acid) and creatinine confirming previous observations. In contrast, no net transfer of Asp across the placenta was seen. A continuous decrease in the concentration of Asp on both maternal and fetal side suggests active uptake and metabolization by the placenta. Viability control parameters remained stable indicating the absence of an effect on placental metabolism, permeability and morphology. CONCLUSION: Elevation of Asp concentration up to 10 times the physiological level by the administration of Mg-Asp-HCl to the maternal circuit under in vitro perfusion conditions of human placenta has no influence on the fetal level of Asp suggesting no transfer of Asp from the maternal to fetal compartment. Therefore, the administration of Mg-Asp-HCl to preeclamptic patients would be beneficial for the patients without any impact on placental or fetal physiology.

The AsaP1 peptidase of Aeromonas salmonicida subsp. achromogenes is a highly conserved deuterolysin metalloprotease (family M35) and a major virulence factor

Infections by the bacterium Aeromonas salmonicida subsp. achromogenes cause significant disease in a number of fish species. In this study, we showed that AsaP1, a toxic 19-kDa metallopeptidase produced by A. salmonicida subsp. achromogenes, belongs to the group of extracellular peptidases (Aeromonas type) (MEROPS ID M35.003) of the deuterolysin family of zinc-dependent aspzincin endopeptidases. The structural gene of AsaP1 was sequenced and found to be highly conserved among gram-negative bacteria. An isogenic Delta asaP1 A. salmonicida subsp. achromogenes strain was constructed, and its ability to infect fish was compared with that of the wild-type (wt) strain. The Delta asaP1 strain was found to infect Arctic charr, Atlantic salmon, and Atlantic cod, but its virulence was decreased relative to that of the wt strain. The 50% lethal dose of the AsaP1 mutant was 10-fold higher in charr and 5-fold higher in salmon than that of the wt strain. The pathology induced by the AsaP1-deficient strain was also different from that of the wt strain. Furthermore, the mutant established significant bacterial colonization in all observed organs without any signs of a host response in the infected tissue. AsaP1 is therefore the first member of the M35 family that has been shown to be a bacterial virulence factor.

Matrix metalloproteinases: multifunctional effectors of inflammation in multiple sclerosis and bacterial meningitis

Matrix metalloproteinases (MMPs) are a family of Zn2+-dependent endopeptidases targeting extracellular matrix (ECM) compounds as well as a number of other proteins. Their proteolytic activity acts as an effector mechanism of tissue remodeling in physiologic and pathologic conditions, and as modulator of inflammation. In the context of neuro-inflammatory diseases, MMPs have been implicated in processes such as (a) blood-brain barrier (BBB) and blood-nerve barrier opening, (b) invasion of neural tissue by blood-derived immune cells, (c) shedding of cytokines and cytokine receptors, and (d) direct cellular damage in diseases of the peripheral and central nervous system. This review focuses on the role of MMPs in multiple sclerosis (MS) and bacterial meningitis (BM), two neuro-inflammatory diseases where current therapeutic approaches are insufficient to prevent severe disability in the majority of patients. Inhibition of enzymatic activity may prevent MMP-mediated neuronal damage due to an overactive or deviated immune response in both diseases. Downregulation of MMP release may be the molecular basis for the beneficial effect of IFN-beta and steroids in MS. Instead, synthetic MMP inhibitors offer the possibility to shut off enzymatic activity of already activated MMPs. In animal models of MS and BM, they efficiently attenuated clinical disease symptoms and prevented brain damage due to excessive metalloproteinase activity. However, the required target profile for the therapeutic use of this novel group of compounds in human disease is not yet sufficiently defined and may be different depending on the type and stage of disease. Currently available MMP inhibitors show little target-specificity within the MMP family and may lead to side-effects due to interference with physiological functions of MMPs. Results from human MS and BM indicate that only a restricted number of MMPs specific for each disease is up-regulated. MMP inhibitors with selective target profiles offer the possibility of a more efficient therapy of MS and BM and may enter clinical trials in the near future.

Crystal structure of the sodium-proton antiporter NhaA dimer and new mechanistic insights

Sodium-proton antiporters rapidly exchange protons and sodium ions across the membrane to regulate intracellular pH, cell volume, and sodium concentration. How ion binding and release is coupled to the conformational changes associated with transport is not clear. Here, we report a crystal form of the prototypical sodium-proton antiporter NhaA from Escherichia coli in which the protein is seen as a dimer. In this new structure, we observe a salt bridge between an essential aspartic acid (Asp163) and a conserved lysine (Lys300). An equivalent salt bridge is present in the homologous transporter NapA, but not in the only other known crystal structure of NhaA, which provides the foundation of most existing structural models of electrogenic sodium-proton antiport. Molecular dynamics simulations show that the stability of the salt bridge is weakened by sodium ions binding to Asp164 and the neighboring Asp163. This suggests that the transport mechanism involves Asp163 switching between forming a salt bridge with Lys300 and interacting with the sodium ion. pKa calculations suggest that Asp163 is highly unlikely to be protonated when involved in the salt bridge. As it has been previously suggested that Asp163 is one of the two residues through which proton transport occurs, these results have clear implications to the current mechanistic models of sodium-proton antiport in NhaA.

Nitrogen metabolism and remobilization during senescence

Senescence is a highly organized and well‐regulated process. As much as 75% of total cellular nitrogen may be located in mesophyll chloroplasts of C3‐plants. Proteolysis of chloroplast proteins begins in an early phase of senescence and the liberated amino acids can be exported to growing parts of the plant (e.g. maturing fruits). Rubisco and other stromal enzymes can be degraded in isolated chloroplasts, implying the involvement of plastidial peptide hydrolases. Whether or not ATP is required and if stromal proteins are modified (e.g. by reactive oxygen species) prior to their degradation are questions still under debate. Several proteins, in particular cysteine proteases, have been demonstrated to be specifically expressed during senescence. Their contribution to the general degradation of chloroplast proteins is unclear. The accumulation in intact cells of peptide fragments and inhibitor studies suggest that multiple degradation pathways may exist for stromal proteins and that vacuolar endopeptidases might also be involved under certain conditions. The breakdown of chlorophyll‐binding proteins associated with the thylakoid membrane is less well investigated. The degradation of these proteins requires the simultaneous catabolism of chlorophylls. The breakdown of chlorophylls has been elucidated during the last decade. Interestingly, nitrogen present in chlorophyll is not exported from senescencing leaves, but remains within the cells in the form of linear tetrapyrrolic catabolites that accumulate in the vacuole. The degradation pathways for chlorophylls and chloroplast proteins are partially interconnected.