In vitro antitumor and anti-angiogenic activities of a shrimp chondroitin sulfate.

Thrombin triggers cellular responses that are crucial for development and progression of cancer, such as proliferation, migration, oncogene expression and angiogenesis. Thus, biomolecules capable of inhibiting this protease have become targets in cancer research. The present work describes the in vitro antitumor properties of a chondroitin sulfate with anti-thrombin activity, isolated from the Litopenaeus vannamei shrimp (sCS). Although the compound was unable to induce cytotoxicity or cell death and/or cell cycle changes after 24 h incubation, it showed a long-term antiproliferative effect, reducing the tumor colony formation of melanoma cells by 75% at 100 µg/mL concentration and inhibiting the anchorage-independent colony formation. sCS reduced 66% of melanoma cell migration in the wound healing assay and 70% in the transwell assay. The compound also decreased melanin and TNF-α content of melanoma cells by 52% and 75% respectively. Anti-angiogenic experiments showed that sCS promoted 100% reduction of tubular structure formation at 100 µg/mL. These results are in accordance with the sCS-mediated in vitro expression of genes related to melanoma development (Cx-43, MAPK, RhoA, PAFR, NFKB1 and VEGFA). These findings bring a new insight to CS molecules in cancer biology that can contribute to ongoing studies for new approaches in designing anti-tumor therapy.

New Imaging Parameters to Predict Sudden Cardiac Death in Chagas Disease.

Chronic Chagas' cardiomyopathy is the most severe and frequent manifestation of Chagas disease, and has a high social and economic burden. New imaging modalities, such as strain echocardiography, nuclear medicine, computed tomography and cardiac magnetic resonance imaging, may detect the presence of myocardial fibrosis, inflammation or sympathetic denervation, three conditions associated with risk of sudden death, providing additional diagnostic and/or prognostic information. Unfortunately, despite its high mortality, there is no clear recommendation for early cardioverter-defibrillator implantation in patients with Chagas heart disease in the current guidelines. Ideally, the risk of sudden cardiac death may be evaluated in earlier stages of the disease using new image methods to allow the implementation of primary preventive strategies.

A further unique chondroitin sulfate from the shrimp Litopenaeus vannamei with antithrombin activity that modulates acute inflammation.

The detailed structure of a further Chondroitin Sulfate from Litopenaeus vannamei shrimp (sCS) is described. The backbone structure was established by 1H/13C NMR, which identified 3-O-sulfated GlcA, 4-O-sulfated GalNAc, 6-O-sulfated GalNAc, and 4,6-di-O-sulfated GalNAc residues. GlcA is linked to GalNAc 4,6 di S and GlcA 3S is linked to GalNAc 4S, GalNAc 4,6 di-S and GalNAc6S residues. The anticoagulant properties of this sCS were evaluated by activated partial thromboplastin time, anti-IIa, anti-Xa and anti-heparin cofactor II-mediated activities, and sCS failed to stabilise antithrombin in a fluoresence shift assay. The anti-inflammatory effect of sCS was explored using a model of acute peritonitis, followed by leukocyte count and measurement of the cytokines, IL-1ß, IL-6 and TNF-α. The compound showed low clotting effects, but high anti-IIa activity and HCII-mediated thrombin inhibition. Its anti-inflammatory effect was shown by leukocyte recruitment inhibition and a decrease in pro-inflammatory cytokine levels. Although the biological role of sCS remains unknown, its properties indicate that it is suitable for studies of multi-potent molecules obtained from natural sources.

Anti-IIa activity and antitumor properties of a hybrid heparin/heparan sulfate-like compound from Litopenaeus vannamei shrimp.

In this present study, the anti-IIa activity and the antitumor properties of a hybrid heparin/heparan sulfate-like compound (sH/HS) from Litopenaeus vannamei shrimp heads are related. In addition to inhibiting 90.7% of thrombin activity at the lowest tested concentration (0.5 µg/mL), sH/HS compound stimulated the synthesis of antithrombotic heparan sulfate by endothelial cells in a dose-dependent manner. In vitro experiments demonstrated that the molecule from shrimp displayed a potent anti-angiogenic effect, reducing over 80% of the tubular structures formation at 50 and 100 µg/mL. In addition, sH/HS compound was able to inhibit the migration of B16F10 cells at all tested concentrations without affecting the cell viability. Although the studied compound had no effect on the proliferation of such cells during a period of 24 h, it had a significant long-term anti-proliferative effect, reducing about 80% of colony formation and anchorage-independent growth at 50 and 100 µg/mL concentrations. When its effectiveness was tested in vivo, it was demonstrated that sH/HS promoted a reduction of more than 90% of tumor growth. In the context of thromboembolic disorders associated with cancer, such findings make the sH/HS compound an excellent target for studies on inhibiting of development and tumor progression, and the prevention of coagulopathies.

2,3-Di-O-sulfo glucuronic acid: An unmodified and unusual residue in a highly sulfated chondroitin sulfate from Litopenaeus vannamei.

The occurrence of a natural and unmodified highly sulfated chondroitin sulfate from Litopenaeus vannamei heads (sCS) is herein reported. Its partial digestion by Chondroitinases AC and ABC together with its electrophoretic migration profile revealed it as a highly sulfated chondroitin sulfate despite its average molecular weight being similar to CSA. Using orthogonal 1D/2D NMR experiments, the anomeric signals (δ 4.62/106.0) corresponding to unusual 2,3-di-O-Sulfo-GlcA (∼36%), U33S (δ 4.42/84.1, ∼63%) and U22S (4.12/80.1, ∼50%) substitutions were confirmed. In addition, non-sulfated GlcA (δ 4.5/106.3) linked to 4-O- (A14S, 36%) or 6-O-Sulfo (A16S, 28%) GalNAc (δ 4.64/103.5) was observed. Although the biological role of sCS in shrimp is unknown, its influence on hemostasis was also demonstrated. The sCS identification brings to light new questions about the hierarchical model of GAGs biosynthesis and contributes to the better understanding of the subtle relationship between GAGs structure and function.

Cardiac 123I-MIBG uptake in de novo Brazilian patients with Parkinson's disease without clinically defined dysautonomia.

Myocardial scintigraphy with meta-iodo-benzyl-guanidine (123I cMIBG) has been studied in Parkinson's disease (PD), especially in Asian countries, but not in Latin America. Most of these studies include individuals with PD associated to a defined dysautonomia. Our goal is to report the cardiac sympathetic neurotransmission in de novo Brazilian patients with sporadic PD, without clinically defined dysautonomia. We evaluated retrospectively a series of 21 consecutive cases with PD without symptoms or signs of dysautonomia assessed by the standard bedside tests. This number was reduced to 14 with the application of exclusion criteria. 123I cMIBG SPECT up-take was low or absent in all of them and the heart/mediastinum ratio was low in 12 of 14. We concluded that 123I cMIBG has been able to identify cardiac sympathetic neurotransmission disorder in Brazilian de novo PD patients without clinically defined dysautonomia.

Cardiac 123I-MIBG uptake in de novo Brazilian patients with Parkinson's disease without clinically defined dysautonomia / Cintilografia Miocárdica com 123I-MIBG em pacientes brasileiros com doença de Parkinson recentemente diagnosticada sem disautonomia clinicamente manifesta

Myocardial scintigraphy with meta-iodo-benzyl-guanidine (123I cMIBG) has been studied in Parkinson's disease (PD), especially in Asian countries, but not in Latin America. Most of these studies include individuals with PD associated to a defined dysautonomia. Our goal is to report the cardiac sympathetic neurotransmission in de novo Brazilian patients with sporadic PD, without clinically defined dysautonomia. We evaluated retrospectively a series of 21 consecutive cases with PD without symptoms or signs of dysautonomia assessed by the standard bedside tests. This number was reduced to 14 with the application of exclusion criteria. 123I cMIBG SPECT up-take was low or absent in all of them and the heart/mediastinum ratio was low in 12 of 14. We concluded that 123I cMIBG has been able to identify cardiac sympathetic neurotransmission disorder in Brazilian de novo PD patients without clinically defined dysautonomia.

A cintilografia miocárdica com meta-iodo-benzil-guanidina (123I cMIBG) foi estudada na doença de Parkinson (DP), especialmente nos países asiáticos, mas não na América Latina. A quase totalidade desses estudos inclui indivíduos com DP com disautonomia definida. Nosso objetivo é relatar a neurotransmissão simpática cardíaca em doentes brasileiros com DP de novo esporádica, sem disautonomia clinicamente definida. Foi avaliada retrospectivamente uma série de 21 casos consecutivos com DP sem sintomas ou sinais de disautonomia observáveis pelos testes de beira-de-leito. Com a aplicação dos critérios de exclusão, este número foi reduzido para 14. A captação do 123I MIBG pelo SPECT foi baixa ou ausente em todos os pacientese; a relação coração / mediastino foi baixa em 12 dos 14. Concluímos que a 123c MIBG é capaz de identificar alteração da neurotransmissão simpática cardíaca em doentes com DP de novo sem disautonomia clinicamente definida.

A heparin-like glycosaminoglycan from shrimp containing high levels of 3-O-sulfated D-glucosamine groups in an unusual trisaccharide sequence.

The detailed characterization of a novel heparin-like glycosaminoglycan purified from the viscera (heads) of the shrimp Litopenaeus vannamei is reported. Structural analysis performed by mono- and two-dimensional nuclear magnetic resonance (NMR) spectroscopy revealed it to be rich in both glucuronic acid and N,6-sulfated glucosamine residues. The key peculiarities were its high 3-O-sulfated glucosamine content compared to mammalian heparins; a residue which is usually associated with the antithrombin (AT) binding site, and the location of these residues within 2-O-sulfated iduronate and glucuronate-containing sequences (I2S-A(∗)-G), a situation not found in mammalian heparin. It also exhibited higher molecular weight (∼36kDa) than conventional heparin (∼16kDa) but, negligible anticoagulant activity (∼5IU/mg compared to heparin ∼190IU/mg) and stabilization of AT, which has been linked directly to anticoagulation activity. A high affinity fraction, eluting at a similar salt concentration (0.75-1.5M NaCl) from an antithrombin affinity column, to the high affinity fraction of heparin, also showed only weak thermal stabilization of AT (+∼2°C). These structural peculiarities may help elucidate more clearly the relationship between structure and function of sulfated polysaccharides, and provide useful model compounds with which to better understand interactions of biological significance.

A non-hemorrhagic hybrid heparin/heparan sulfate with anticoagulant potential.

The structural characterization and the anticoagulant potential of a novel heparin/heparan sulfate-like compound from the heads of Litopenaeus vannamei shrimp are described. While it is distinct from either heparin or heparan sulfate, enzymatic depolymerization and nuclear magnetic resonance spectroscopy analyses revealed that this molecule does share some structural features with heparin, such as the high degree of N- and 6-O-sulfation and minor N-acetylation, and with heparan sulfate, in the glucuronic acid content. Its ability to stabilize human antithrombin explains its significant anticoagulant activity in aPTT and Factor-Xa inhibition assays. Interestingly, in contrast to mammalian heparin, the shrimp compound displayed negligible hemorrhagic effect. Together, these findings have particular interest since they reveal a novel molecule with significant anti-Xa activity coupled with low bleeding effects which make the shrimp heparin/HS-like compound a potential alternative for mammalian heparin.

Anti-inflammatory properties of a heparin-like glycosaminoglycan with reduced anti-coagulant activity isolated from a marine shrimp.

The anti-inflammatory properties of a heparin-like compound from the shrimp Litopenaeus vannamei are related. Besides reducing significantly (p<0.001) the influx of inflammatory cells to injury site in a model of acute inflammation, shrimp heparin-like compound was able to reduce the matrix metalloproteinase (MMPs) activity in the peritoneal lavage of inflamed animals. Moreover, this compound also reduced almost 90% the activity of MMP-9 secreted by human activated leukocytes. Negligible anti-coagulant activities in aPPT assay and a poor bleeding potential make this compound a better alternative than mammalian heparin as a possible anti-inflammatory drug.