ECM Protein Nanofibers and Nanostructures Engineered Using Surface-initiated Assembly

The extracellular matrix (ECM) in tissues is synthesized and assembled by cells to form a 3D fibrillar, protein network with tightly regulated fiber diameter, composition and organization. In addition to providing structural support, the physical and chemical properties of the ECM play an important role in multiple cellular processes including adhesion, differentiation, and apoptosis. In vivo, the ECM is assembled by exposing cryptic self-assembly (fibrillogenesis) sites within proteins. This process varies for different proteins, but fibronectin (FN) fibrillogenesis is well-characterized and serves as a model system for cell-mediated ECM assembly. Specifically, cells use integrin receptors on the cell membrane to bind FN dimers and actomyosin-generated contractile forces to unfold and expose binding sites for assembly into insoluble fibers. This receptor-mediated process enables cells to assemble and organize the ECM from the cellular to tissue scales. Here, we present a method termed surface-initiated assembly (SIA), which recapitulates cell-mediated matrix assembly using protein-surface interactions to unfold ECM proteins and assemble them into insoluble fibers. First, ECM proteins are adsorbed onto a hydrophobic polydimethylsiloxane (PDMS) surface where they partially denature (unfold) and expose cryptic binding domains. The unfolded proteins are then transferred in well-defined micro- and nanopatterns through microcontact printing onto a thermally responsive poly(N-isopropylacrylamide) (PIPAAm) surface. Thermally-triggered dissolution of the PIPAAm leads to final assembly and release of insoluble ECM protein nanofibers and nanostructures with well-defined geometries. Complex architectures are possible by engineering defined patterns on the PDMS stamps used for microcontact printing. In addition to FN, the SIA process can be used with laminin, fibrinogen and collagens type I and IV to create multi-component ECM nanostructures. Thus, SIA can be used to engineer ECM protein-based materials with precise control over the protein composition, fiber geometry and scaffold architecture in order to recapitulate the structure and composition of the ECM in vivo.     

A Cysteine Endopeptidase (“Dionain”) Is Involved in the Digestive Fluid of Dionaea muscipula (Venus’s Fly-trap)

The carnivorous plant Dionaea muscipula (Venus’s flytrap) secretes proteinases into the digestive fluid to digest prey proteins. In this study, we obtained evidence that the digestive fluid contains a cysteine endopeptidase, presumably belonging to the papain family, through inhibitor studies and partial amino acid sequencing of the major SDS–PAGE band protein. The name “dionain” is proposed for the enzyme.     

Multiple mechanisms contribute to myenteric plexus ablation induced by benzalkonium chloride in the guinea-pig ileum

Ablation of rat myenteric plexus with benzalkonium chloride has provided a model of intestinal aganglionosis, but the degenerative responses are not well understood. We examined the effects of this detergent on neurons and glia, including expression of c-Myc, c-Jun, JunB, and c-Fos, and on immunocytes in the guinea-pig ileum. Benzalkonium chloride (0.1%) or saline was applied to the serosal surface of distal ileum. Tissues were analyzed 2, 3, or 7 days later and compared with cyclosporine-treated and untreated animals. More than 90% of myenteric neurons were destroyed in ileal segments 3–7 days after benzalkonium-chloride treatment. Glia withdrew processes from around neurons after 2 days and were mostly gone after 3 days. Neuronal c-Myc began to disappear while c-Fos, c-Jun, and JunB were evident in some neuronal nuclei after 2 or 3 days. After 3 days, widespread apoptosis was evident in the myenteric plexus. Populations of T cells, B cells, and macrophage-like cells in untreated and saline-treated myenteric plexuses were substantially increased 3 and 7 days after benzalkonium-chloride treatment. Cyclosporine delayed significant neuronal loss. We conclude that a variety of degenerative mechanisms may be active in this model, including an immune response which may actively contribute to tissue destruction. Received: 13 September 1996 / Accepted: 20 January 1997     

A multiple chemostat system for consortia studies on microbially influenced corrosion

A multiple chemostat system has been developed in which metal specimens can be exposed to a consortium of bacteria. The system comprises a single test chemostat containing the test specimen operated at a high dilution rate to facilitate the wash out of planktonic bacteria, selecting for attached or biofilm growth. This chemostat is fed at a steady low rate by a number of separate chemostats each of which contains a pure axenic culture of one member of the consortium being tested. This system has the advantage of providing a continual inoculum of the test species to the test specimen allowing both aerobic and anaerobic bacteria to be grown in the same system. Constant levels of three bacterial types were maintained in the system: Pseudomonas aeruginosa, Thiobacillus ferrooxidans and Desulfovibrio vulgaris. Exposure of 316L stainless steel electrodes to this system resulted in increased corrosion of coupons exposed biotically, as compared to those exposed abiotically. A current monitoring technique and electrochemical impedance spectroscopy were used to evaluate effects of bacteria on metallic corrosion.     

Genetic diversity assessment and estimation of phylogenetic relationships among 26 Fagaceae species using ISSRs

The family Fagaceae includes several species and presents huge genetic variability. In the last two decades, several genetic studies about phylogenetics and genetic diversity of Fagaceae have emerged. ISSR markers were used to evaluate the genetic diversity of 26 species of Fagaceae belonging to the genera Castanea, Fagus and Quercus. Among several primers tested, 17 were selected for the evaluation of diversity and estimation of genetic relationships. A total of 371 ISSR markers were produced and each primer revealed high polymorphism. Specific ISSR markers for the Quercus infrageneric groups were amplified. ISSRs proved to be a reliable tool for the discrimination of the analyzed species per genus, infrageneric group and/or ecological origin.     

Introduction: Metals in Biology: α-Ketoglutarate/Iron-Dependent Dioxygenases

Four minireviews deal with aspects of the α-ketoglutarate/iron-dependent dioxygenases in this eighth Thematic Series on Metals in Biology. The minireviews cover a general introduction and synopsis of the current understanding of mechanisms of catalysis, the roles of these dioxygenases in post-translational protein modification and de-modification, the roles of the ten-eleven translocation (Tet) dioxygenases in the modification of methylated bases (5mC, T) in DNA relevant to epigenetic mechanisms, and the roles of the AlkB-related dioxygenases in the repair of damaged DNA and RNA. The use of α-ketoglutarate (alternatively termed 2-oxoglutarate) as a co-substrate in so many oxidation reactions throughout much of nature is notable and has surprisingly emerged from biochemical and genomic analysis. About 60 of these enzymes are now recognized in humans, and a number have been identified as having critical functions.     

In vivo antiplasmodial activity of 11(13)-dehydroivaxillin from Carpesium ceruum

The whole plants of Carpesium genus are used in traditional medicine as anti-pyretic, analgesic and vermifugic, including a topical application for sores and inflammation. A previous study on Carpesium genus suggested that the antiplasmodial activity against Plasmodium falciparum was due to the existence of 11(13)- dehydroivaxillin (DDV) from EtOAc extracts of C. ceruum (Compositae). Here, the antimalarial activity of DDV was evaluated against Plasmodium berghei in mice. The LD₅₀ of the compound was determined as 51.2 mg/kg, while doses of 124 mg/kg and above were found to be lethal to mice. DDV (2, 5, 10 mg/kg/day) exhibited a significant blood schizontocidal activity in 4-day early infection, repository evaluation and in an established infection with a significant mean survival time comparable to that of the standard drug, chloroquine, 5 mg/kg/day. DDV possesses a promising antiplasmodial activity, which can be exploited in malaria therapy.     

Recruitment of Nox4 to a Plasma Membrane Scaffold Is Required for Localized Reactive Oxygen Species Generation and Sustained Src Activation in Response to Insulin-like Growth Factor-I

Nox4-derived ROS is increased in response to hyperglycemia and is required for IGF-I-stimulated Src activation. This study was undertaken to determine the mechanism by which Nox4 mediates sustained Src activation. IGF-I stimulated sustained Src activation, which occurred primarily on the SHPS-1 scaffold protein. In vitro oxidation experiments indicated that Nox4-derived ROS was able to oxidize Src when they are in close proximity, and Src oxidation leads to its activation. Therefore we hypothesized that Nox4 recruitment to the plasma membrane scaffold SHPS-1 allowed localized ROS generation to mediate sustained Src oxidation and activation. To determine the mechanism of Nox4 recruitment, we analyzed the role of Grb2, a component of the SHPS-1 signaling complex. We determined that Nox4 Tyr-491 was phosphorylated after IGF-I stimulation and was responsible for Nox4 binding to the SH2 domain of Grb2. Overexpression of a Nox4 mutant, Y491F, prevented Nox4/Grb2 association. Importantly, it also prevented Nox4 recruitment to SHPS-1. The role of Grb2 was confirmed using a Pyk2 Y881F mutant, which blocked Grb2 recruitment to SHPS-1. Cells expressing this mutant had impaired Nox4 recruitment to SHPS-1. IGF-I-stimulated downstream signaling and biological actions were also significantly impaired in Nox4 Y491F-overexpressing cells. Disruption of Nox4 recruitment to SHPS-1 in aorta from diabetic mice inhibited IGF-I-stimulated Src oxidation and activation as well as cell proliferation. These findings provide insight into the mechanism by which localized Nox4-derived ROS regulates the sustained activity of a tyrosine kinase that is critical for mediating signal transduction and biological actions.     

Structural and Functional Impact of SRP54 Mutations Causing Severe Congenital Neutropenia

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