Proteolytic Activities of Kiwifruit Actinidin (Actinidia deliciosa cv. Hayward) on Different Fibrous and Globular Proteins: A Comparative Study of Actinidin with Papain

Actinidin, a member of the papain-like family of cysteine proteases, is abundant in kiwifruit. To date, a few studies have been provided to investigate the proteolytic activity and substrate specificity of actinidin on native proteins. Herein, the proteolytic activity of actinidin was compared to papain on several different fibrous and globular proteins under neutral, acidic and basic conditions. The digested samples were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and densitometry to assess the proteolytic effect. Furthermore, the levels of free amino nitrogen (FAN) of the treated samples were determined using the ninhydrin colorimetric method. The findings showed that actinidin has no or limited proteolytic effect on globular proteins such as immunoglobulins including sheep IgG, rabbit IgG, chicken IgY and fish IgM, bovine serum albumin (BSA), lipid transfer protein (LTP), and whey proteins (α-lactalbumin and β-lactoglobulin) compared to papain. In contrast to globular proteins, actinidin could hydrolyze collagen and fibrinogen perfectly at neutral and mild basic pHs. Moreover, this enzyme could digest pure α-casein and major subunits of micellar casein especially in acidic pHs. Taken together, the data indicated that actinidin has narrow substrate specificity with the highest enzymatic activity for the collagen and fibrinogen substrates. The results describe the actinidin as a mild plant protease useful for many special applications such as cell isolation from different tissues and some food industries as a mixture formula with other relevant proteases.     

Adhesion behaviors of human trophoblast cells by contact with endothelial cells

Although it is still not clear whether migratory trophoblasts reach the spiral arteries by migration within blood vessels against blood flow or by a mechanism of directional cell division/proliferation, this process involves the attachment and adhesion of trophoblasts to endothelial cells lining the blood vessel walls. This raises the possibility that the cell–cell contact with endothelial cells may regulate trophoblast cell adhesion behaviors according to the surrounding flow condition. To test this, the adhesion forces of early gestation human trophoblast cells (TCs) cultured on glass slides coated with type I rat collagen or cultured with human umbilical vein endothelial cells (HUVECs) were measured quantitatively using a micropipette aspiration technique. Then, the resistance of TCs co-cultured with HUVECs to flow-induced shear stress was assessed with a flow chamber technique. The results showed that the adhesion force of TCs to glass slides coated with collagen was positively correlated with the concentration of collagen. By contact with endothelial cells, the adhesion force and the resistance to shear stress for the TCs were significantly enhanced. The interdiction of integrin β1 interaction remarkably reduced the adhesion forces of TCs to endothelial cells, hence their resistance to shear stress. The results therefore suggest that the contacts of TCs with endothelial cells enhance the adhesion forces of human TCs, partially by regulating with the integrin β1 according to the flow condition (i.e., the shear stress) in such a way to prevent the TCs from being carried downstream by flowing blood.     

A tag-less method for direct isolation of human umbilical vein endothelial cells by gravitational field-flow fractionation

The analysis of cellular and molecular profiles represents a powerful tool in many biomedical applications to identify the mechanisms underlying the pathological changes. The improvement of cellular starting material and the maintenance of the physiological status in the sample preparation are very useful. Human umbilical vein endothelial cells (HUVEC) are a model for prediction of endothelial dysfunction. HUVEC are enzymatically removed from the umbilical vein by collagenase. This method provides obtaining a good sample yield. However, the obtained cells are often contaminated with blood cells and fibroblasts. Methods based on negative selection by in vitro passages or on the use of defined marker are currently employed to isolate target cells. However, these approaches cannot reproduce physiological status and they require expensive instrumentation. Here we proposed a new method for an easy, tag-less and direct isolation of HUVEC from raw umbilical cord sample based on the gravitational field-flow fractionation (GrFFF). This is a low-cost, fully biocompatible method with low instrumental and training investments for flow-assisted cell fractionation. The method allows obtaining pure cells without cell culture procedures as starting material for further analysis; for example, a proper amount of RNA can be extracted. The approach can be easily integrated into clinical and biomedical procedures.     

Stimulation of human vascular endothelial cell growth by a platelet-derived growth factor and thrombin

Repair of a vascular wound is mediated by migration and subsequent replication of the endothelial cells that form the inner lining of blood vessels. We have measured the growth response of human umbilical vein endothelial cells (HuE) to two polypeptides that are transiently produced in high concentrations at the site of a wound; the platelet-derived growth factor (PDGF) and the protease thrombin. When 10(4) HuE cells are seeded as a dense island (2-mm diameter) in the center of a 16-mm tissue culture well in medium containing 20% human serum derived from platelet-poor plasma (PDS), no increase in cell number or colony size is observed. With the addition of 0.5 ng/ml partially purified PDGF, colony size increases and the number of cells after 8 days is 4.8 X 10(4). When human thrombin (1 microgram/ml) is added along with the PDGF, the cell number rises to 9.2 X 10(4). Thrombin alone stimulates no increase in cell number. Although partially purified PDGF stimulates endothelial cells maintained in PDS as well as those maintained in whole blood serum (WBS), pure PDGF is active only when assayed in medium that contains WBS and is supplemented with thrombin. These results suggest the existence of a second class of platelet-derived factors that enable HuE cells to respond to the mitogenic activity of the purified platelet mitogen and thrombin.     

Collagenase produced from Aspergillus sp. (UCP 1276) using chicken feather industrial residue

An extracellular collagenolytic serine protease was purified from Aspergillus sp., isolated from the Caatinga biome in northeast Brazil by a two‐step chromatographic procedure, using an anion‐exchanger and gel filtration. The enzyme was produced by submerged fermentation of feather residue as a substrate. The purified collagenase showed a 2.09‐fold increase in specific activity and 22.85% yield. The enzyme was a monomeric protein with a molecular mass of 28.7 kDa, estimated by an SDS–PAGE and AKTA system. The optimum temperature and pH for enzyme activity were around 40°C and pH 8.0, respectively. The enzyme was strongly inhibited by phenyl‐methylsulfonyl fluoride, a serine protease inhibitor, and was thermostable until 65°C for 1 h. We then evaluated the enzyme's potential for degradation of Type I and Type V collagens for producing peptides with antifungal activity. Our results revealed that the cleavage of Type V collagen yielded more effective peptides than Type I, inhibiting growth of Aspergillus terreus , Aspergillus japonicus and Aspergillus parasiticus . Both groups of peptides (Type I and Type V) were identified by SDS–PAGE. To conclude, the thermostable collagenase we purified in this study has various potentially useful applications in the fields of biochemistry, biotechnology and biomedical sciences.     

层层组装构建抗凝血和内皮细胞化协同功能界面

本文采用天然细胞外基质组分、强抗凝血活性的肝素和促细胞黏附生长的胶原蛋白, 在不锈钢支架表面构建肝素/胶原多层膜, 探索利用层层组装多层膜所显示出的协同效应, 实现抗凝血和促进内皮细胞生长的协同功能, 为改善心血管支架抗凝血和抗再狭窄修饰寻求新方法.     

Circular‐shaped microfluidic device to study the effect of shear stress on cellular orientation

Understanding the effects of shear stress on mammalian cells is a crucial factor for understanding a number of biological processes and diseases. Here, we show the development of a circular‐shaped microfluidic device for the facile generation of shear stress gradients. With this microfluidic device, the effect of shear stress on orientation of human umbilical vein endothelial cells was studied. This microfluidic device, which enables to control the alignment of human umbilical vein endothelial cells within a microchannel, can be a valuable tool to mimic blood vessels.     

Angiogenin induces nitric oxide release independently from its RNase activity

Nitric oxide (NO), a biological mediator involved in vascular physiology, was sensed electrochemically using a microelectrode array. Angiogenin was shown to trigger nitric oxide synthase (NOS) activity in human umbilical vein endothelial cells and embryonic stem cell derived endothelial cells independently from its RNase activity.     

RK-805, an endothelial-cell-growth inhibitor produced by Neosartorya sp., and a docking model with methionine aminopeptidase-2

We found that a fungus Neosartorya sp. produced an angiogenesis inhibitor, RK-805. By spectroscopic analyses and semi-synthetic methods from fumagillin, the structure of RK-805 was identified as 6-oxo-6-deoxyfumagillol, which has not been reported as a natural product. RK-805 preferentially inhibited the growth of human umbilical vein endothelial cells (HUVECs) rather than that of human normal fibroblast in cell proliferation assays and blocked endothelial cell migration induced by vascular endothelial growth factor (VEGF). Moreover, RK-805 selectively inhibited methionine aminopeptidase-2 (MetAP2), but not methionine aminopeptidase-1 (MetAP1). The docked structure of RK-805 complexed with human MetAP2 indicated that not only a covalent bond between a nucleophilic imidazole nitrogen atom of His231 and the carbon of the reactive spirocyclic epoxide of RK-805, but also a hydrogen bond between NH (Asn329) and the carbonyl group of RK-805 at C-6 promote close contact in the binding pocket of the enzyme. Taken together, these results suggest that structure activity relationships of RK-805 derivatives at both C-4 and C-6, in comparison with ovalicin and TNP-470, would be useful for development of new angiogenesis inhibitors.     

Divide and conquer: rat liver tissue proteomics based on the analysis of purified constituents

Comparative proteome data of normal and diseased tissue samples are difficult to interpret. Proteins detected in tissues are derived from different cell types and blood constituents. Pathologic or toxicant-induced aberrations may affect the proteome profile of tissues in several ways since different cell types may respond in very different and highly specific manners. The aim of this study was to analyze the proteome profiles of purified rat liver primary cells and of blood plasma in comparison to liver whole tissue. Moreover, we investigated alterations of these profiles induced by the liver toxicant N-nitrosomorpholine (NNM) used as a model compound. Whole liver samples, pure hepatocytes and Kupffer cells as well as blood plasma were obtained from saline- or NNM-treated rats. Proteins were separated by 2-D PAGE and their amounts were estimated by fluorography. Selected proteins were identified by MS analysis of tryptic digests. Among them we identified proteins exclusively expressed in the analysed constituents. Several of these proteins were assigned in the proteome profile of whole-tissue homogenates. Furthermore, we identified several proteins that were modified, up-regulated or down-regulated due to NNM treatment in total liver homogenates. Some of these protein alterations were specifically detected in primary cells isolated from NNM-treated rats. Thus, we demonstrated the successful assignment of NNM-induced proteome alterations in rat liver to the cell type of origin. The currently applied approach may help to better understand pathologic processes at a whole-tissue level.     

Functionalization of multi-walled carbon nanotubes with coumarin derivatives and their biological evaluation

We report the synthesis and the characterization of different multi-walled carbon nanotubes (MWCNTs) linked to natural molecules, 5,7-coumarins and/or oleic acid, obtained from purified pristine MWCNTs by a cascade of chemical functionalization. The activities of these modified MWCNTs were investigated in vitro on human umbilical vein endothelial cells (HUVECs) by evaluating their ability to influence cell viability and to induce cell apoptosis. Our data showed that pristine MWCNTs are markedly cytotoxic; conversely, the carboxylated carbon nanotubes, much more readily dispersed in aqueous solutions and CNT-Link, the key intermediate designed by us for the drug anchorage, are biocompatible at the tested concentrations (1 and 10 μg ml(-1)).     

Mass spectrometric analysis of platelet-activating factor after isolation by solid-phase extraction and direct derivatization with pentafluorobenzoic anhydride

Platelet-activating factor is the term used to denote a class of extremely potent lipid mediators that consist predominantly of 1-O-alkyl- and 1-O-acyl-2-acetyl-sn-glycero-3-phosphocholines. A method has been devised for rapid isolation of these acetylated phospholipids by solid-phase extraction prior to direct derivatization with pentafluorobenzoic anhydride and analysis by gas chromatography (GC)/electron-capture mass spectrometry. Recovery through the entire method (lipid isolation, derivatization, and purification) typically ranged from 70% to 85%. Using the direct derivatization procedure described here, the practical limit of detection for each of the standard alkyl- and acyl-platelet-activating factor homologs was 1 fmol injected into the GC. Results from the application of the method to the analysis of alkyl and acyl homologs of platelet-activating factor isolated from stimulated human umbilical vein endothelial cells are presented, exhibiting excellent accuracy and precision for a wide range of tissue levels of this class of potent autacoids.     

A new fluorescent turn-on chemodosimeter for mercury ions in solution and its application in cells and organisms

Using the Hg²⁺-induced desulfurization reaction of thiosemicarbazide derivative, we designed and synthesized a novel “turn on” coumarin-based fluorescent probe L with a simple structure for detecting mercury ion (II). Spectroscopy revealed that the probe responds selectively to mercury ions over other metal ions with marked fluorescence enhancement. Detection of Hg²⁺ was effective at pH 7.0–9.5, with high selectivity and significant effect in HeLa cells, human umbilical vein endothelial cells and Escherichia coli, but no cytotoxicity. This probe could be an ideal and practical Hg²⁺ probe with important biological significance.     

A palmtop-sized microfluidic cell culture system driven by a miniaturized infusion pump

A palmtop-sized microfluidic cell culture system is presented. The system consists of a microfluidic device and a miniaturized infusion pump that possesses a reservoir of culture medium, an electrical control circuit, and an internal battery. The footprint of the system was downsized to 87 × 57 mm, which is, to the best of our knowledge, the smallest integrated cell culture system. Immortalized human microvascular endothelial cells (HMEC-1) and human umbilical vein endothelial cells (HUVEC) were cultured in the system. HMEC-1 in the system proliferated at the same speed as cells in a microchannel perfused by a syringe pump and cells in a culture flask. HUVEC in the system oriented along the direction of the fluid flow. Claudin-5, a tight junction protein, was localized along the peripheries of the HUVEC. We expect that the present system is applicable to various cell types as a stand-alone and easy-to-use system for microfluidic bioanalysis.     

A microfluidic flow-stretch chip for investigating blood vessel biomechanics

This microfluidic flow-stretch chip integrates fluid shear stress (FSS) and cyclic stretch (CS), two major mechanical stimulations in cardiovascular systems, for cultured cells. The model chip can deliver FSS and CS simultaneously or independently to vascular cells to mimic the haemodynamic microenvironment of blood vessels in vivo. By imposing FSS-only, CS-only, and FSS+CS stimulation on rat mesenchymal stem cells and human umbilical vein endothelial cells, we found the alignment of the cellular stress fibers varied with cell type and the type of stimulation. The flow-stretch chip is a reliable tool for simulating the haemodynamic microenvironment.     

Pyrrole functionalised metalloporphyrins as electrocatalysts for the oxidation of nitric oxide

Pyrrole-functionalised tetracarboxyphenyl porphyrin and trimethoxyphenylcarboxy-phenyl porphyrin containing Ni, Mn and Pd as the central metal ion were used to modify Pt-disk microelectrodes (∅ 50 μm) (by repetitive cyclic voltammetry, dip-dry and pulse-amperometry methods) for the detection of nitric oxide (NO). Electrodes modified with Mn(II) trimethoxyphenylcarboxyphenyl porphyrin using the pulse amperomery approach, were found to be sensitive, stable and fast in response towards the oxidation of NO. Thus, they were used for the detection of NO release from a population of transformed human umbilical vein endothelial cells (T-HUVEC) into a droplet of electrolyte solution following stimulation with vascular endothelial growth factor (VEGF). The electrode surface was covered with an additional layer of Nafion® to prevent interference from anionic molecules such as nitrite.     

RO-heparin Inhibits L-Selectin-mediated Neutrophils Adhesion to Vascular Endothelium Under Flow Conditions

Introduction Migrationandrecruitmentofleukocytesfromblood toinflammatorylesionsitesaresequentiallyregulated byadhesionmoleculesandtheirreceptors[1].These lectinfamilyplaysamajorroleininitiatingattachement ofneutrophilstotheactivatedendothelium.P selectin,…     

Collagen Peptides Derived from Sipunculus nudus Accelerate Wound Healing

Marine collagen peptides have high potential in promoting skin wound healing. This study aimed to investigate wound healing activity of collagen peptides derived from Sipunculus nudus (SNCP). The effects of SNCP on promoting healing were studied through a whole cortex wound model in mice. Results showed that SNCP consisted of peptides with a molecular weight less than 5 kDa accounted for 81.95%, rich in Gly and Arg. SNCP possessed outstanding capacity to induce human umbilical vein endothelial cells (HUVEC), human immortalized keratinocytes (HaCaT) and human skin fibroblasts (HSF) cells proliferation and migration in vitro. In vivo, SNCP could markedly improve the healing rate and shorten the scab removal time, possessing a scar-free healing effect. Compared with the negative control group, the expression level of tumor necrosis factor-α, interleukin-1β and transforming growth factor-β1 (TGF-β1) in the SNCP group was significantly down-regulated at 7 days post-wounding (p < 0.01). Moreover, the mRNA level of mothers against decapentaplegic homolog 7 (Smad7) in SNCP group was up-regulated (p < 0.01); in contrast, type II TGF-β receptors, collagen I and α-smooth muscle actin were significantly down-regulated at 28 days (p < 0.01). These results indicate that SNCP possessed excellent activity of accelerating wound healing and inhibiting scar formation, and its mechanism was closely related to reducing inflammation, improving collagen deposition and recombination and blockade of the TGF-β/Smads signal pathway. Therefore, SNCP may have promising clinical applications in skin wound repair and scar inhibition.     

Fabrication of Stiffness Gradients of GelMA Hydrogels Using a 3D Printed Micromixer

Many properties in both healthy and pathological tissues are highly influenced by the mechanical properties of the extracellular matrix. Stiffness gradient hydrogels are frequently used for exploring these complex relationships in mechanobiology. In this study, the fabrication of a simple, cost‐efficient, and versatile system is reported for creation of stiffness gradients from photoactive hydrogels like gelatin‐methacryloyl (GelMA). The setup includes syringe pumps for gradient generation and a 3D printed microfluidic device for homogenous mixing of GelMA precursors with different crosslinker concentration. The stiffness gradient is investigated by using rheology. A co‐culture consisting of human adipose tissue‐derived mesenchymal stem cells (hAD‐MSCs) and human umbilical cord vein endothelial cells (HUVECs) is encapsulated in the gradient construct. It is possible to locate the stiffness ranges at which the studied cells displayed specific spreading morphology and migration rates. With the help of the described system, variable mechanical gradient constructs can be created and optimal 3D cell culture conditions can be experientially identified.     

Total syntheses of the bromotyrosine-derived natural products ianthelline, 5-bromoverongamine and JBIR-44

The total syntheses of the bromotyrosine-derived natural products ianthelline, 5-bromoverongamine and JBIR-44 are described and their cytotoxic activity in a cervical cancer (HeLa) cell line and human umbilical vein endothelial cells (HUVECs) are reported.