A Screening Approach to Assess the Impact of Various Commercial Sources of Crude Marine λ-Carrageenan on the Production of Oligosaccharides with Anti-heparanase and Anti-migratory Activities.

Oligosaccharides derived from λ-carrageenan (λ-COs) are gaining interest in the cancer field. They have been recently reported to regulate heparanase (HPSE) activity, a protumor enzyme involved in cancer cell migration and invasion, making them very promising molecules for new therapeutic applications. However, one of the specific features of commercial λ-carrageenan (λ-CAR) is that they are heterogeneous mixtures of different CAR families, and are named according to the thickening-purpose final-product viscosity which does not reflect the real composition. Consequently, this can limit their use in a clinical applications. To address this issue, six commercial λ-CARs were compared and differences in their physiochemical properties were analyzed and shown. Then, a H2O2-assisted depolymerization was applied to each commercial source, and number- and weight-averaged molar masses (Mn and Mw) and sulfation degree (DS) of the λ-COs produced over time were determined. By adjusting the depolymerization time for each product, almost comparable λ-CO formulations could be obtained in terms of molar masses and DS, which ranged within previously reported values suitable for antitumor properties. However, when the anti-HPSE activity of these new λ-COs was screened, small changes that could not be attributed only to their small length or DS changes between them were found, suggesting a role of other features, such as differences in the initial mixture composition. Further structural MS and NMR analysis revealed qualitative and semi-quantitative differences between the molecular species, especially in the proportion of the anti-HPSE λ-type, other CARs types and adjuvants, and it also showed that H2O2-based hydrolysis induced sugar degradation. Finally, when the effects of λ-COs were assessed in an in vitro migration cell-based model, they seemed more related to the proportion of other CAR types in the formulation than to their λ-type-dependent anti-HPSE activity.

A Marine λ-Oligocarrageenan Inhibits Migratory and Invasive Ability of MDA-MB-231 Human Breast Cancer Cells through Actions on Heparanase Metabolism and MMP-14/MMP-2 Axis.

Sugar-based molecules such as heparins or natural heparan sulfate polysaccharides have been developed and widely studied for controlling heparanase (HPSE) enzymatic activity, a key player in extracellular matrix remodelling during cancer pathogenesis. However, non-enzymatic functions of HPSE have also been described in tumour mechanisms. Given their versatile properties, we hypothesized that sugar-based inhibitors may interfere with enzymatic but also non-enzymatic HPSE activities. In this work, we assessed the effects of an original marine λ-carrageenan derived oligosaccharide (λ-CO) we previously described, along with those of its native counterpart and heparins, on cell viability, proliferation, migration, and invasion of MDA-MB-231 breast cancer cells but also of sh-MDA-MB-231 cells, in which the expression of HPSE was selectively downregulated. We observed no cytotoxic and no anti-proliferative effects of our compounds but surprisingly λ-CO was the most efficient to reduce cell migration and invasion compared with heparins, and in a HPSE-dependent manner. We provided evidence that λ-CO tightly controlled a HPSE/MMP-14/MMP-2 axis, leading to reduced MMP-2 activity. Altogether, this study highlights λ-CO as a potent HPSE "modulator" capable of reducing not only the enzymatic activity of HPSE but also the functions controlled by the HPSE levels.

New insights in bacterial and eukaryotic diversity of microbial mats inhabiting exploited and abandoned salterns at the Ré Island (France).

In order to understand the effect of human practices on microbial mats organisation, the study aimed to investigate the biodiversity within microbial mats from exploited and abandoned salterns. Despite several attempts, archaeal 16S rRNA gene fragment sequences were not obtained, indicating that microbial mats were probably dominated by Bacteria with very low abundance of Archaea (< 1%). Thus, the study compared the bacterial and meiofaunal diversity of microbial mats from abandoned and exploited salterns. The higher salinity (101 ± 3.7 psu vs. 51.1 ± 0.7 psu; Welch t-test p < 0.05) of the exploited site maintained lower bacterial diversity in comparison to the abandoned site where the salinity gradient was no longer maintained. However, the microbial mats exhibited similar bacterial class composition while the eukaryotic diversity was significantly higher in the exploited saltern. The abandoned saltern was dominated by sulfate-reducing bacteria and Nematoda, while the exploited saltern was characterized by the presence of halophilic bacteria belonging to Marinobacter, Salinivibrio and Rhodohalobacter genera, and the larger abundance of Hypotrichia (ciliates). Such bacterial and eukaryotic diversity difference might be explained by human actions for salt recovery in exploited salterns such as scraping the surface of microbial mat and increasing salinity renewing the microbial mat each year. Such action decreases the bacterial diversity changing the food web structure that favour the presence of a larger diversity of eukaryotic organisms. Our study provides new insights on microbial mat communities inhabiting salterns, especially the consequences of abandoning saltern exploitation.

Anticancer activity of limonene: A systematic review of target signaling pathways.

Limonene (LIM) is a monoterpene, which is abundant in essential oils of Citrus fruits peels (Rutaceae). More recently, LIM, as a potential natural anticancer compound, has attracted major attention and exerted a chemopreventive activity, stimulating the detoxification of carcinogenic compounds and limiting tumor growth and angiogenesis in various cancer models. Twenty-six (26) articles were selected based on previously established criteria. Anticancer activity of LIM was related to the inhibition of tumor initiation, growth, and angiogenesis and the induction of cancer cells apoptosis. LIM was able to increase Bax expression, release cytochrome c, and activate the caspase pathway. In addition, LIM increased the expression of p53 and decreased the activity of Ras/Raf/MEK/ERK and PI3K/Akt pathways. LIM also decreased the expression of VEGF and increased the activities of the Man-6-P / IGF2R and TGF-ßIIR receptors. These results highlight LIM as an abundant natural molecule with low toxicity and pleiotropic pharmacological activity in cancer cells, targeting various cell-signaling pathways critically involved in the initiation, growth, and chemoresistance of cancer cells.

Heparin length in the coating of extremely small iron oxide nanoparticles regulates in vivo theranostic applications.

The positive contrast of extremely small iron oxide nanoparticles (ESIONP) in magnetic resonance imaging (MRI) rejuvenates this class of metal nanoparticles (NP).Yet, the current synthesis often lacks the possibility of adjusting the core size (while it is a key element for ESIONP-based MRI contrast behaviour), and also involved multiple complex steps before obtaining a ready-to-use probe for medical applications. In this study, we faced these challenges by applying heparin oligosaccharides (HO) of different lengths as coatings for the preparation of HEP-ESIONP with a one-pot microwave method. We demonstrated that the HO length could control the core size during the synthesis to achieve optimal positive MRI contrast, and that HEP-ESIONP were endowed directly with anticoagulant properties and/or a specific antitumor activity, according to the HO used. Relevantly, positron emission tomography (PET)-based in vivo biodistribution study conducted with 68Ga core-doped HEP-ESIONP analogues revealed significant changes in the probe behaviours, the shortening of HO promoting a shift from hepatic to renal clearance. The different conformations of HO coatings and a thorough in vitro characterisation of the probes' protein coronas provided insight into this crucial impact of HO length on opsonization-mediated immune response and elimination. Overall, we were able to identify a precise HO length to get an ESIONP probe showing prolonged vascular lifetime and moderate accumulation in a tumor xenograft, balanced with a low uptake by non-specific organs and favourable urinary clearance. This probe met all prerequisites for advanced theranostic medical applications with a dual MRI/PET hot spot capability and potential antitumor activity.

λ-Carrageenan Oligosaccharides of Distinct Anti-Heparanase and Anticoagulant Activities Inhibit MDA-MB-231 Breast Cancer Cell Migration.

In tumor development, the degradation of heparan sulfate (HS) by heparanase (HPSE) is associated with cell-surface and extracellular matrix remodeling as well as the release of HS-bound signaling molecules, allowing cancer cell migration, invasion and angiogenesis. Because of their structural similarity with HS, sulfated polysaccharides are considered a promising source of molecules to control these activities. In this study, we used a depolymerisation method for producing λ-carrageenan oligosaccharides (λ-CO), with progressive desulfation over time. These were then used to investigate the influence of polymeric chain length and degree of sulfation (DS) on their anti-HPSE activity. The effects of these two features on λ-CO anticoagulant properties were also investigated to eliminate a potential limitation on the use of a candidate λ-CO as a chemotherapeutic agent. HPSE inhibition was mainly related to the DS of λ-CO, however this correlation was not complete. On the other hand, both chain length and DS modulated λ-CO activity for factor Xa and thrombin IIa inhibition, two enzymes that are involved in the coagulation cascade, and different mechanisms of inhibition were observed. A λ-carrageenan oligosaccharide of 5.9 KDa was identified as a suitable anticancer candidate because it displayed one of the lowest anticoagulant properties among the λ-CO produced, while showing a remarkable inhibitory effect on MDA-MB-231 breast cancer cell migration.

Immunotherapy of triple-negative breast cancer with cathepsin D-targeting antibodies.

BACKGROUND: Triple-negative breast cancer (TNBC) treatment is currently restricted to chemotherapy. Hence, tumor-specific molecular targets and/or alternative therapeutic strategies for TNBC are urgently needed. Immunotherapy is emerging as an exciting treatment option for TNBC patients. The aspartic protease cathepsin D (cath-D), a marker of poor prognosis in breast cancer (BC), is overproduced and hypersecreted by human BC cells. This study explores whether cath-D is a tumor cell-associated extracellular biomarker and a potent target for antibody-based therapy in TNBC. METHODS: Cath-D prognostic value and localization was evaluated by transcriptomics, proteomics and immunohistochemistry in TNBC. First-in-class anti-cath-D human scFv fragments binding to both human and mouse cath-D were generated using phage display and cloned in the human IgG1 λ format (F1 and E2). Anti-cath-D antibody biodistribution, antitumor efficacy and in vivo underlying mechanisms were investigated in TNBC MDA-MB-231 tumor xenografts in nude mice. Antitumor effect was further assessed in TNBC patient-derived xenografts (PDXs). RESULTS: High CTSD mRNA levels correlated with shorter recurrence-free survival in TNBC, and extracellular cath-D was detected in the tumor microenvironment, but not in matched normal breast stroma. Anti-cath-D F1 and E2 antibodies accumulated in TNBC MDA-MB-231 tumor xenografts, inhibited tumor growth and improved mice survival without apparent toxicity. The Fc function of F1, the best antibody candidate, was essential for maximal tumor inhibition in the MDA-MB-231 model. Mechanistically, F1 antitumor response was triggered through natural killer cell activation via IL-15 upregulation, associated with granzyme B and perforin production, and the release of antitumor IFNγ cytokine. The F1 antibody also prevented the tumor recruitment of immunosuppressive tumor-associated macrophages M2 and myeloid-derived suppressor cells, a specific effect associated with a less immunosuppressive tumor microenvironment highlighted by TGFß decrease. Finally, the antibody F1 inhibited tumor growth of two TNBC PDXs, isolated from patients resistant or not to neo-adjuvant chemotherapy. CONCLUSION: Cath-D is a tumor-specific extracellular target in TNBC suitable for antibody-based therapy. Immunomodulatory antibody-based strategy against cath-D is a promising immunotherapy to treat patients with TNBC.

Family of Bioactive Heparin-Coated Iron Oxide Nanoparticles with Positive Contrast in Magnetic Resonance Imaging for Specific Biomedical Applications.

Unfractionated heparin (UFH) and low-molecular-weight heparins (LMWH) are well-known for their anticoagulant properties. There is also currently a growing interest in using LMWH in targeted cancer therapy. In particular, several types inhibit heparanase, a key enzyme overexpressed in the tumor microenvironment that promotes angiogenesis progression and metastasis spreading. Here, we propose iron oxide nanoparticles (HEP-IONP) coated with different heparins of distinct anticoagulant/anti-heparanase activity ratios and suitable for positive contrast in magnetic resonance imaging. As a proof of concept, magnetic resonance angiography (MRA) was conducted in mice up to 3 h after intravenous administration. This new IONP-based positive contrast appropriate for clinic together with the long vascular circulating times can enable innovative theranostic applications if combined with the various bioactivities of the heparins. Indeed, we showed, using advanced in vitro tests, how HEP-IONP anticoagulant or anti-heparanase activities were maintained depending on the heparin species used for the coating. Overall, the study allowed presenting an IONP coated with a commercial LMWH (Lovenox) suggested as a theranostic translational probe for MRA diagnostic and treatment of thrombosis, and an antitumor IONP coated with a specific depolymerized heparin to be used in targeted therapy and diagnostic modalities.

Assessment of Heparanase-Mediated Angiogenesis Using Microvascular Endothelial Cells: Identification of λ-Carrageenan Derivative as a Potent Anti Angiogenic Agent.

Heparanase is overexpressed by tumor cells and degrades the extracellular matrix proteoglycans through cleavage of heparan sulfates (HS), allowing pro-angiogenic factor release and thus playing a key role in tumor angiogenesis and metastasis. Here we propose new HS analogs as potent heparanase inhibitors: Heparin as a positive control, Dextran Sulfate, λ-Carrageenan, and modified forms of them obtained by depolymerization associated to glycol splitting (RD-GS). After heparanase activity assessment, 11 kDa RD-GS-λ-Carrageenan emerged as the most effective heparanase inhibitor with an IC50 of 7.32 ng/mL compared to 10.7 ng/mL for the 16 kDa unfractionated heparin. The fractionated polysaccharides were then tested in a heparanase-rich medium-based in vitro model, mimicking tumor microenvironment, to determine their effect on microvascular endothelial cells (HSkMEC) angiogenesis. As a preliminary study, we identified that under hypoxic and nutrient poor conditions, MCF-7 cancer cells released much more mature heparanase in their supernatant than in normal conditions. Then a MatrigelTM assay using HSkMEC cultured under hypoxic conditions in the presence (or not) of this heparanase-rich supernatant was realized. Adding heparanase-rich media strongly enhanced angiogenic network formation with a production of twice more pseudo-vessels than with the control. When sulfated polysaccharides were tested in this angiogenesis assay, RD-GS-λ-Carrageenan was identified as a promising anti-angiogenic agent.

Production of heparin and λ-carrageenan anti-heparanase derivatives using a combination of physicochemical depolymerization and glycol splitting.

Strongly associated with tumor angiogenesis and metastasis, the enzyme heparanase is an endo-ß-d-glucuronidase which is overexpressed in the tumor microenvironment. Its inhibition could be one of the most promising anti-angiogenic approaches to date. Although heparin is known as a good heparanase inhibitor, it also possesses major anticoagulant properties that may be incompatible with its use as an anti-angiogenic agent, hence the considerable interest for other sources of sulfated polysaccharides. Recent investigations point to λ-carrageenans, highly sulfated galactans with a tremendous potential that are found in red algae. This study describes the production of low-molecular-weight (LMW) heparins and λ-carrageenans, using a combination of glycol splitting and ultrasonically-assisted radical hydrolysis using hydrogen-peroxide. The structural characteristics, as well as the anticoagulant and antiheparanase activities of the resulting products were assessed. The best candidate was a LMW glycol-split λ-carrageenan that displayed major anti-heparanase properties, with an IC50 of 7.32ng/mL and a close-to-zero anticoagulant activity.

Short-Term and Long-Term Biological Effects of Chronic Chemical Contamination on Natural Populations of a Marine Bivalve.

Understanding the effects of chronic chemical contamination on natural populations of marine organisms is complex due to the combined effects of different types of pollutants and environmental parameters that can modulate the physiological responses to stress. Here, we present the effects of a chronic contamination in a marine bivalve by combining multiple approaches that provide information on individual and population health. We sampled variegated scallops (Mimachlamys varia) at sites characterized by different contaminants and contamination levels to study the short and long-term (intergenerational) responses of this species to physiological stress. We used biomarkers (SOD, MDA, GST, laccase, citrate synthase and phosphatases) as indicators of oxidative stress, immune system alteration, mitochondrial respiration and general metabolism, and measured population genetic diversity at each site. In parallel, concentration of 14 trace metals and 45 organic contaminants (PAHs, PCBs, pesticides) in tissues were measured. Scallops were collected outside and during their reproductive season to investigate temporal variability in contaminant and biomarker levels. Our analyses revealed that the levels of two biomarkers (Laccase-type phenoloxidase and malondialdehyde) were significantly correlated with Cd concentration. Additionally, we observed significant seasonal differences for four of the five biomarkers, which is likely due to the scallop reproductive status at time of sampling. As a source of concern, a location that was identified as a reference site on the basis of inorganic contaminant levels presented the same level of some persistent organic pollutants (DDT and its metabolites) than more impacted sites. Finally, potential long-term effects of heavy metal contamination were observed for variegated scallops as genetic diversity was depressed in the most polluted sites.

Alteration of cathepsin D trafficking induced by hypoxia and extracellular acidification in MCF-7 breast cancer cells.

The microenvironment that surrounds tumor cells is characterized by hypoxic conditions and extracellular acidity. These hostile conditions induce crucial changes in cell behavior and can promote the secretion of many soluble factors such as growth factors, cytokines and enzymes. The lysosomal aspartyl-endopeptidase cathepsin D (CD) is a marker of poor prognosis in breast cancer and is associated with a metastatic risk. In this study, the transport of CD was investigated in a model of breast cancer cells line (MCF-7) cultivated under hypoxia and acidification of media. CD secretion was assessed using Western blot analysis and protease activity was measured in conditioned culture media. We demonstrate that cultured MCF-7 cells secrete an active 52 kDa pCD precursor and report that under hypoxia there was an increased amount of pCD secreted. More surprisingly, extracellular acidification (pH 6 and 5.6) induced the secretion of the fully-mature and active (34 kDa + 14 kDa) double chain CD. Our findings reflect the fact that chemical anomalies influence the secretion path of CD in a breast cancer cell model, resulting in altered trafficking of the mature form. This important result may provide new arguments in favor of the role of extracellular CD in the degradation of the matrix proteins that constitute the breast tumor microenvironment.

Anti-heparanase activity of ultra-low-molecular-weight heparin produced by physicochemical depolymerization.

Heparanase is an endo-ß-D-glucuronidase that plays an important role in cancer progression, in particular during tumor angiogenesis and metastasis. Inhibiting this enzyme is considered as one of the most promising approaches in cancer therapy. Heparin is a complex glycoaminoglycan known as a strong inhibitor of heparanase. It is primarily used in clinical practice for its anticoagulant activities, which may not be compatible with its use as anti-angiogenic agent. In this study, we described the production of ultra-low-molecular-weight heparins (ULMWH) by a physicochemical method that consists in a hydrogen peroxide-catalyzed radical hydrolysis assisted by ultrasonic waves. We assessed the structural characteristics, anticoagulant and anti-heparanase activities of the obtained heparin derivatives and compared them with three commercial low-molecular-weight heparins (LMWH), glycol-split non-anticoagulant heparins and heparins produced by enzymatic methods. ULMWH generated by the physicochemical method were characterized by high anti-heparanase and moderate anticoagulant activities. These heparin derivatives might be potential candidates for cancer therapy when a compromise is needed between anti-heparanase and anticoagulant activities.

Factors other than metalloprotease are required for full virulence of French Vibrio tubiashii isolates in oyster larvae.

Vibrio tubiashii is a marine pathogen isolated from larval and juvenile bivalve molluscs that causes bacillary necrosis. Recent studies demonstrated the isolation of this species in a French experimental hatchery/nursery affecting Crassostrea gigas spat in 2007. Here, using larvae of C. gigas as an interaction model, we showed that the French V. tubiashii is virulent to larvae and can cause bacillary necrosis symptoms with an LD50 of about 2.3 × 10(3) c.f.u. ml(-1) after 24 h. Moreover, complete or gel permeation HPLC fractionated extracellular products (ECPs) of this strain appeared toxic to larvae. MS-MS analysis of the different ECP fractions revealed the existence of an extracellular metalloprotease and other suspected virulence factors. This observation is also supported by the expression level of some potential virulence factors. The overall results suggest that the pathology caused by the French V. tubiashii in C. gigas oysters is caused by a group of toxic factors and not only the metalloprotease.

First description of French V. tubiashii strains pathogenic to mollusk: II. Characterization of properties of the proteolytic fraction of extracellular products.

Extracellular products (ECPs) of the French Vibrio tubiashii strain 07/118 T2 were previously reported to be toxic for the Pacific oyster Crassostrea gigas. In this study we now assessed host cellular immune responses and bacterial potential effectors by which these ECPs can be associated with host damages. The adhesion capacity (28% inhibition) and phagocytosis ability (56% inhibition) of oyster hemocytes were the main functions affected following in vitro contact between hemocytes and V. tubiashii ECPs. This may be linked to the demonstration of the capability of ECPs to cleave various cellular substrates as oyster collagen. Moreover, a strong metalloproteolytic activity was recorded with general (azocasein) and specific (ADAM) substrates and characterized by the use of standard inhibitors and metal ions. The addition of 1,10-phenanthroline and Zn2+ decreased proteolytic activity by about 80% and 50% respectively, confirming the presence of zinc metalloproteolytic activity in the ECPs. Mass spectrometry analyses of crude ECPs identified an extracellular zinc metalloprotease encoded by a gene with an open reading frame of 1821 bp (606 aa). Consensus zinc-binding motifs specific to thermolysin family and some glycosylation and phosphorylation sites were located on the deduced protein sequence. Taken together, our results suggest that this (these) zinc metalloprotease(s) might contribute to the impairment of hemocyte immunological functions; however, their direct involvement in ECPs toxicity remains to be demonstrated.

First description of French V. tubiashii strains pathogenic to mollusk: I. Characterization of isolates and detection during mortality events.

Nine dominant bacterial isolates were obtained from different batches of Crassostrea gigas spat experiencing high mortality rates in a French experimental hatchery/nursery in 2007. Using phenotypic analysis combined with multilocus sequence analysis, the isolates were shown to be genetically close to the Vibrio tubiashii type strain. Based on (1) analyses of the recA gene sequences; (2) the results of DNA-DNA hybridization assays between 07/118 T2 (LMG 27884=CECT 8426), which is a representative strain, and the V. tubiashii type strain (69%); and (3) phenotypic traits, the bacteria were classified in a group close to American V. tubiashii strain. Its virulence (70% of mortalities) and the toxicity of the extracellular products of 07/118 T2 was demonstrated (41% of mortalities). Moreover, a QPCR diagnostic tool targeting the gyrB gene was developed to investigate the epidemiological significance of V. tubiashii in French oyster mortality outbreaks recorded by the national surveillance network. Of the 21 batches originating from hatcheries, only two were positive, whereas V. tubiashii DNA could not be detected in any of the batches of moribund animals collected in field/outdoor facilities. These results demonstrate the existence of a group of virulent V. tubiashii in France that episodically infect C. gigas.

Ultrasonic-assisted preparation of a low molecular weight heparin (LMWH) with anticoagulant activity.

Low molecular weight heparin (LMWH) is currently used as an anticoagulant agent and constitutes an alternative to unfractionated heparin, which is the cause of serious adverse drug reaction such as heparin-induced thrombocytopenia (HIT). Commercially available LMWH is produced by enzymatic depolymerization that is costly or by chemical methods that are generally carried out under conditions that could imply side reactions that reduce final product efficiency and yields. In this work, we present the use of a physicochemical method for the production of LMWH. This method consists in the use of hydrogen peroxide-catalyzed radical hydrolysis assisted by ultrasonic waves. LMWH that are produced using this physicochemical method have an average molecular weight and anticoagulant properties (Anti-Xa and Anti-IIa) that are comparable to some of commercial LMWH that are currently used. Ultrasonic-assisted radical depolymerization of heparin leads to products with a remarkably low polydispersity index. Moreover, in comparison to other LMWH such as those produced by enzymatic ß-elimination, this physicochemical depolymerization of heparin induces fewer oligosaccharides with less than five monosaccharide units. This contributes to the better preservation of the ATIII pentasaccharide binding sequence, which results in a high Anti-Xa/Anti-IIa ratio (1.86). However, LMWH obtained using this physicochemical method have a lower degree of sulfation than other LMWH, which seems to be the cause of a lower Anti-Xa and Anti-IIa activity (143.62±5.42 and 77.07±4.4, respectively).

Serum hemorphin-7 levels are decreased in obesity.

OBJECTIVE: Hemorphin peptides exhibit biological activities that interfere with the endorphin system, the inflammatory response, and blood-pressure control. VV-hemorphin-7 and LVV-hemorphin-7 peptides exert a hypotensive effect, in particular, by inhibiting the renin-angiotensin system. Furthermore, levels of circulating hemorphin-7 peptides have been found to be decreased in diseases such as type 1 and type 2 diabetes. DESIGN AND METHODS: Because type 2 diabetes and obesity share common features, such as insulin resistance, microinflammation, high glomerular-filtration rate (GFR), and cardiovascular risk, we evaluated serum VV-hemorphin-7 like immunoreactivity (VVH7-i.r.) levels, using an enzyme-linked immunosorbent assay method, on a group of 54 obese subjects without diabetes or hypertension, compared with a group of 33 healthy normal-weight subjects. RESULTS: Circulating VVH7-i.r. levels were significantly decreased in the obese group compared with the control group (1.98 ± 0.19 vs. 4.86 ± 0.54 µmol/l, respectively, P < 0.01), and a significant negative correlation between VVH7-i.r. and diastolic blood pressure (DBP) was found in obese patients (r = -0.35, P = 0.011). There was no significant correlation between VVH7-i.r. level and insulin resistance, metabolic syndrome, or GFR. CONCLUSIONS: The decreased serum hemorphin-7 found in obese subjects, as in diabetes, may contribute to the development of hypertension and to the cardiovascular risk associated with these metabolic diseases.

Differential tissue distribution and specificity of phenoloxidases from the Pacific oyster Crassostrea gigas.

Phenoloxidases (POs) play a key role in melanin production, are involved in invertebrate immune mechanisms, and have been detected in different bivalves. Recently, we identified catecholase- and laccase-like PO activities in plasma and haemocyte lysate supernatant (HLS) of the Pacific oyster Crassostrea gigas. To go further in our investigations, the aims of this study were (i) to determine the tissue distribution of PO activities in C. gigas, and (ii) to identify and characterise the different sub-classes of POs (i.e. tyrosinase, catecholase and/or laccase) involved in these oxido-reductase activities. With dopamine and p-phenylenediamine (PPD) but not with l-tyrosine used as substrates, PO-activities were detected by spectrophotometry in the gills, digestive gland, mantle, and muscle. These results suggest the presence of catecholase and laccase but not of tyrosinase activities in oyster tissues. The highest activity was recovered in the digestive gland. PO-like activities were all inhibited by 1-phenyl-2-thiourea (PTU) and by the specific laccase inhibitor, cethyltrimethylammonium bromide (CTAB). With dopamine as substrate, the catecholase inhibitor 4-hexylresorcinol (4-HR) only inhibited PO in the muscle. SDS-PAGE zymographic assays with dopamine and PPD elicited a unique ~40kDa protein band in the muscle. In the other tissues, laccase-like activities could be related to ~10kDa and/or ~200kDa protein bands. The ~10kDa protein band was also detected in plasma and HLS, confirming the presence of a laccase in the later compartments, and probably in most of the tissues of C. gigas. This is the first time to our knowledge that a ~10kDa protein band is associated to a laccase-like activity in a mollusc species, contributing to the characterisation of phenoloxidase activities in marine bivalves.

Proteolytic degradation by cathepsin D of glycated hemoglobin from diabetes patients gives rise to hemorphin-7 peptides.

Previous studies showed a significantly reduced level of hemorphins in the serum of diabetes patients. In order to elucidate the biochemical mechanisms responsible for this anomaly, the influence of hemoglobin glycation on hemorphin generation was studied. The glycation of hemoglobin occurs in the blood of diabetes patients and this could modify its enzymatic digestion and the resulting proteolytic products. Several samples of hemoglobin were obtained from the blood of type 1 diabetes patients (n=8) and normal healthy control subjects (n=2). The glycated hemoglobin samples were classified on the basis of their HbA1c values expressed as a percentage of total hemoglobin. Four solutions of glycated hemoglobin characterized by HbA1c values of 6%, 9.1%, 10.7% and 12.1% were treated with cathepsin D and the hemorphins obtained following the proteolysis were compared to controls. It was found that hemorphins were produced whatever the level of glycation of hemoglobin and also that the degree of glycation had no effect on the quantity of hemorphins released. Thus the alteration of hemoglobin does not seem to be the essential reason for the decrease in hemorphin concentrations in the sera of diabetic patients.