Fibulin-5 binds urokinase-type plasminogen activator and mediates urokinase-stimulated β1-integrin-dependent cell migration

uPA (urokinase-type plasminogen activator) stimulates cell migration through multiple pathways, including formation of plasmin and extracellular metalloproteinases, and binding to the uPAR (uPA receptor; also known as CD87), integrins and LRP1 (low-density lipoprotein receptor-related protein 1) which activate intracellular signalling pathways. In the present paper we report that uPA-mediated cell migration requires an interaction with fibulin-5. uPA stimulates migration of wild-type MEFs (mouse embryonic fibroblasts) (Fbln5+/+ MEFs), but has no effect on fibulin-5-deficient (Fbln5-/-) MEFs. Migration of MEFs in response to uPA requires an interaction of fibulin-5 with integrins, as MEFs expressing a mutant fibulin-5 incapable of binding integrins (Fbln(RGE/RGE) MEFs) do not migrate in response to uPA. Moreover, a blocking anti-(human β1-integrin) antibody inhibited the migration of PASMCs (pulmonary arterial smooth muscle cells) in response to uPA. Binding of uPA to fibulin-5 generates plasmin, which excises the integrin-binding N-terminal cbEGF (Ca2+-binding epidermal growth factor)-like domain, leading to loss of β1-integrin binding. We suggest that uPA promotes cell migration by binding to fibulin-5, initiating its cleavage by plasmin, which leads to its dissociation from β1-integrin and thereby unblocks the capacity of integrin to facilitate cell motility.     

Characterization of a thermotolerant laccase produced by Streptomyces sp. SB086

Laccases have become desirable enzymes for application in many industrial processes. Nowadays, most of these enzymes are obtained from fungi. Among prospective studies for bacterial laccase genes, some have included actinomycetes, but only a few studies have characterized the enzyme produced. Thus, we have isolated a laccase-producing actinomycete from forest soil under restoration process and further aimed to characterize its produced enzyme. The isolate SB086 was assigned to the Streptomyces genus by a combination of phenotypical, chemical and phylogenetic properties. Our data indicate that the bacterium produces a thermotolerant laccase. The maximum activity was obtained in the pH range 4.0–5.0 and at 50 °C in reaction mixture containing 5 mM CuSO₄; thermal stability was noted at 60 °C and 70 °C—a well-desired characteristic for industry. The active enzyme presented a high molecular mass (over 100 kDa) and was less sensitive to inhibition by metal ions than generally described for bacterial laccases. Our findings support in silico data of bacterial laccase secretion, and reinforce the view that actinomycetes may be a rich source of laccase for industrial application.     

Brain Barrier Breakdown as a Cause and Consequence of Neuroinflammation in Sepsis

The blood-brain barrier (BBB) and the blood-cerebrospinal fluid barrier (BCSFB) are important for the maintenance of brain homeostasis. During sepsis, peripheral production of proinflammatory cytokines and reactive oxygen species are responsible for structural alterations in those brain barriers. Thus, an increasing permeability of these barriers can lead to the activation of glial cells such as microglia and the production of cytotoxic mediators which in turn act on the brain barriers, damaging them further. Thereby, in this review, we try to highlight how the brain barrier’s permeability is not only a cause but a consequence of brain injury in sepsis.     

The brain structure of selected trypanorhynch tapeworms

The brain architecture in four species of tapeworms from the order Trypanorhyncha has been studied. In all species, the brain consists of paired anterior and lateral lobes, and an unpaired central lobe. The anterior lobes connect by dorsal and ventral semicircular commissures; the central and lateral lobes connect by a median and an X-shaped crisscross commissure. In the center of the brain, five well-developed compact neuropils are present. The brain occupies a medial position in the scolex pars bothrialis. The ventral excretory vessels are situated outside the lateral lobes of the brain; the dorsal excretory vessels are located inside the brain and dorsal to the median commissure. The brain gives rize four anterior proboscis nerves and four posterior bulbar nerves with myelinated giant axons (GAs). The cell bodies of the GAs are located within the X-commissure and in the bulbar nerves. Highly developed serotonergic neuropils are present in the anterior and lateral lobes; numerous 5-HT neurons are found in the brain lobes including the central unpaired lobe. The X-cross commissure consists of the α-tub-immunoreactive and 5-HT-IR neurites. Eight ultrastructural types of neurons were found in the brain of the three species investigated. In addition, different types of synapses were present in the neuropils. Glial cells ensheath the brain lobes, the neuropils, the GAs, and the bulbar nerves. Glia cell processes form complex branching patterns of thin cytoplasmic sheets sandwiched between adjacent neural processes and filling the space between neurons. Multilayer myelin-like envelopes and a mesaxon-like structure have been found in Trypanorhyncha nervous system. We compared the brain architecture of Trypanorhyncha with that of an early basal cestode taxon, that is, Diphyllobothriidea, and present a hypothesis about the homology of the anterior brain lobes in order Trypanorhyncha; and the lateral lobes and median commissure are homologous brain structures within Eucestoda.     

Differences in PGE2 Production between Primary Human Monocytes and Differentiated Macrophages: Role of IL-1β and TRIF/IRF3

Prostaglandin E2 (PGE₂) is induced in vivo by bacterial products including TLR agonists. To determine whether PGE₂ is induced directly or via IL-1β, human monocytes and macrophages were cultured with LPS or with Pam3CSK4 in presence of caspase-1 inhibitor, ZVAD, or IL-1R antagonist, Kineret. TLR agonists induced PGE₂ in macrophages exclusively via IL-1β-independent mechanisms. In contrast, ZVAD and Kineret reduced PGE₂ production in LPS-treated (but not in Pam3CSK4-treated) monocytes, by 30–60%. Recombinant human IL-1β augmented COX-2 and mPGES-1 mRNA and PGE₂ production in LPS-pretreated monocytes but not in un-primed or Pam3CSK4-primed monocytes. This difference was explained by the finding that LPS but not Pam3CSK4 induced phosphorylation of IRF3 in monocytes suggesting activation of the TRIF signaling pathway. Knocking down TRIF, TRAM, or IRF3 genes by siRNA inhibited IL-1β-induced COX-2 and mPGES-1 mRNA. Blocking of TLR4 endocytosis during LPS priming prevented the increase in PGE₂ production by exogenous IL-1β. Our data showed that TLR2 agonists induce PGE₂ in monocytes independently from IL-1β. In the case of TLR4, IL-1β augments PGE₂ production in LPS-primed monocytes (but not in macrophages) through a mechanism that requires TLR4 internalization and activation of the TRIF/IRF3 pathway. These findings suggest a key role for blood monocytes in the rapid onset of fever in animals and humans exposed to bacterial products and some novel adjuvants.     

Stress-dependent Proteolytic Processing of the Actin Assembly Protein Lsb1 Modulates a Yeast Prion

Yeast prions are self-propagating amyloid-like aggregates of Q/N-rich protein that confer heritable traits and provide a model of mammalian amyloidoses. [PSI⁺] is a prion isoform of the translation termination factor Sup35. Propagation of [PSI⁺] during cell division under normal conditions and during the recovery from damaging environmental stress depends on cellular chaperones and is influenced by ubiquitin proteolysis and the actin cytoskeleton. The paralogous yeast proteins Lsb1 and Lsb2 bind the actin assembly protein Las17 (a yeast homolog of human Wiskott-Aldrich syndrome protein) and participate in the endocytic pathway. Lsb2 was shown to modulate maintenance of [PSI⁺] during and after heat shock. Here, we demonstrate that Lsb1 also regulates maintenance of the Sup35 prion during and after heat shock. These data point to the involvement of Lsb proteins in the partitioning of protein aggregates in stressed cells. Lsb1 abundance and cycling between actin patches, endoplasmic reticulum, and cytosol is regulated by the Guided Entry of Tail-anchored proteins pathway and Rsp5-dependent ubiquitination. Heat shock-induced proteolytic processing of Lsb1 is crucial for prion maintenance during stress. Our findings identify Lsb1 as another component of a tightly regulated pathway controlling protein aggregation in changing environments.     

Cytotoxic Effects of the Selective Ligands of Membrane Progesterone Receptors in Human Pancreatic Adenocarcinoma Cells BxPC3

Biochemistry (Moscow) - Progesterone and its synthetic analogues act on cells through different types of receptors, affecting proliferation and apoptosis. These compounds exert their effect through...