Mutant and chimeric recobinant plasminogen activatorsproduction in eukaryotic cellsand preliminary characterization

Mutant urokinase-type plasminogen activator (u-PA) genes and hybrid genes between tissue-type plasminogen activator (t-PA) and u-PA have been designed to direct the synthesis of new plasminogen activators and to investigate the structure-function relationship in these molecules. The following classes of constructs were made starting from cDNA encoding human t-PA or u-PA: 1) u-PA mutants in which the Arg156 and Lys158 were substituted with threonine, thus preventing cleavage by thrombin and plasmin; 2) hybrid molecules in which the NH2-terminal regions of t-PA (amino acid residues 1-67, 1-262, or 1-313) were fused with the COOH-terminal region of u-PA (amino acids 136-411, 139-411, or 195-411, respectively); and 3) a hybrid molecule in which the second kringle of t-PA (amino acids 173-262) was inserted between amino acids 130 and 139 of u-PA. In all cases but one, the recombinant proteins, produced by transfected eukaryotic cells, were efficiently secreted in the culture medium. The translation products have been tested for their ability to activate plasminogen after in situ binding to an insolubilized monoclonal antibody directed against urokinase. All recombinant enzymes were shown to be active, except those in which Lys158 of u-PA was substituted with threonine. Recombination of structural regions derived from t-PA, such as the finger, the kringle 2, or most of the A-chain sequences, with the protease part or the complete u-PA molecule did not impair the catalytic activity of the hybrid polypeptides. This observation supports the hypothesis that structural domains in t-PA and u-PA fold independently from one to another

Minimal symmetric Darlington synthesis

We consider the symmetric Darlington synthesis of a p x p rational symmetric Schur function S with the constraint that the extension is of size 2p x 2p. Under the assumption that S is strictly contractive in at least one point of the imaginary axis, we determine the minimal McMillan degree of the extension. In particular, we show that it is generically given by the number of zeros of odd multiplicity of I-SS*. A constructive characterization of all such extensions is provided in terms of a symmetric realization of S and of the outer spectral factor of I-SS*. The authors's motivation for the problem stems from Surface Acoustic Wave filters where physical constraints on the electro-acoustic scattering matrix naturally raise this mathematical issue

A Seamless Convergence of the Digital and Physical Factory Aiming in Personalized Product Emergence Process (PPEP) for Smart Products within ESB Logistics Learning Factory at Reutlingen University

AbstractA seamless convergence of the digital and physical factory aiming in personalized Product Emergence Process (PPEP) for smart products within ESB Logistics Learning Factory at Reutlingen University.A completely new business model with reference to Industrie4.0 and facilitated by 3D Experience Software in today's networked society in which customers expect immediate responses, delightful experience and simple solutions is one of the mission scenarios in the ESB Logistics Learning Factory at ESB Business School (Reutlingen University).The business experience platform provides software solutions for every organization in the company respectively in the factory. An interface with dashboards, project management apps, 3D - design and construction apps with high end visualization, manufacturing and simulation apps as well as intelligence and social network apps in a collaborative interactive environment help the user to learn the creation of a value end to end process for a personalized virtual and later real produced product.Instead of traditional ways of working and a conventional operating factory real workers and robots work semi-intuitive together. Centerpiece in the self-planned interim factory is the smart personalized product, uniquely identifiable and locatable at all times during the production process – a scooter with an individual colored mobile phone – holder for any smart phone produced with a 3D printer in lot size one. Smart products have in the future solutions incorporated internet based services – designed and manufactured - at the costs of mass products. Additionally the scooter is equipped with a retrievable declarative product memory. Monitoring and control is handled by sensor tags and a raspberry positioned on the product. The engineering design and implementation of a changeable production system is guided by a self-execution system that independently find amongst others esplanade workplaces.The imparted competences to students and professionals are project management method SCRUM, customization of workflows by Industrie4.0 principles, the enhancements of products with new personalized intelligent parts, electrical and electronic self-programmed components and the control of access of the product memory information, to plan in a digital engineering environment and set up of the physical factory to produce customer orders. The gained action-orientated experience refers to the chances and requirements for holistic digital and physical systems

High resolution crystal structures of the <i>Cerebratulus lacteus</i> mini-Hb in the unligated and carbomonoxy states

The nerve tissue mini-hemoglobin from Cerebratulus lacteus (CerHb) displays an essential globin fold hosting a protein matrix tunnel held to allow traffic of small ligands to and from the heme. CerHb heme pocket hosts the distal TyrB10/GlnE7 pair, normally linked to low rates of O2 dissociation and ultra-high O2 affinity. However, CerHb affinity for O2 is similar to that of mammalian myoglobins, due to a dynamic equilibrium between high and low affinity states driven by the ability of ThrE11 to orient the TyrB10 OH group relative to the heme ligand. We present here the high resolution crystal structures of CerHb in the unligated and carbomonoxy states. Although CO binds to the heme with an orientation different from the O2 ligand, the overall binding schemes for CO and O2 are essentially the same, both ligands being stabilized through a network of hydrogen bonds based on TyrB10, GlnE7, and ThrE11. No dramatic protein structural changes are needed to support binding of the ligands, which can freely reach the heme distal site through the apolar tunnel. A lack of main conformational changes between the heme-unligated and -ligated states grants stability to the folded mini-Hb and is a prerequisite for fast ligand diffusion to/from the heme

Novel Human/Non-Human Primate Cross-Reactive Anti-Transferrin Receptor Nanobodies for Brain Delivery of Biologics

The blood-brain barrier (BBB), while being the gatekeeper of the central nervous system (CNS), is a bottleneck for the treatment of neurological diseases. Unfortunately, most of the biologicals do not reach their brain targets in sufficient quantities. The antibody targeting of receptor-mediated transcytosis (RMT) receptors is an exploited mechanism that increases brain permeability. We previously discovered an anti-human transferrin receptor (TfR) nanobody that could efficiently deliver a therapeutic moiety across the BBB. Despite the high homology between human and cynomolgus TfR, the nanobody was unable to bind the non-human primate receptor. Here we report the discovery of two nanobodies that were able to bind human and cynomolgus TfR, making these nanobodies more clinically relevant. Whereas nanobody BBB00515 bound cynomolgus TfR with 18 times more affinity than it did human TfR, nanobody BBB00533 bound human and cynomolgus TfR with similar affinities. When fused with an anti-beta-site amyloid precursor protein cleaving enzyme (BACE1) antibody (1A11AM), each of the nanobodies was able to increase its brain permeability after peripheral injection. A 40% reduction of brain Aβ1–40 levels could be observed in mice injected with anti-TfR/BACE1 bispecific antibodies when compared to vehicle-injected mice. In summary, we found two nanobodies that could bind both human and cynomolgus TfR with the potential to be used clinically to increase the brain permeability of therapeutic biologicals

On the class SI of J-contractive functions intertwining solutions of linear differential equations

In the PhD thesis of the second author under the supervision of the third author was defined the class SI of J-contractive functions, depending on a parameter and arising as transfer functions of overdetermined conservative 2D systems invariant in one direction. In this paper we extend and solve in the class SI, a number of problems originally set for the class SC of functions contractive in the open right-half plane, and unitary on the imaginary line with respect to some preassigned signature matrix J. The problems we consider include the Schur algorithm, the partial realization problem and the Nevanlinna-Pick interpolation problem. The arguments rely on a correspondence between elements in a given subclass of SI and elements in SC. Another important tool in the arguments is a new result pertaining to the classical tangential Schur algorithm.Comment: 46 page

Scanning Tunneling Spectroscopy in MgB2

We present scanning tunneling microscopy measurements of the surface of superconducting MgB2 with a critical temperature of 39K. In zero magnetic field the conductance spectra can be analyzed in terms of the standard BCS theory with a smearing parameter Gamma. The value of the superconducting gap is 5.2 meV at 4.2 K, with no experimentally significant variation across the surface of the sample. The temperature dependence of the gap follows the BCS form, fully consistent with phonon-mediated superconductivity in this novel superconductor. The application of a magnetic field induces strong pair-breaking as seen in the conductance spectra in fields up to 6 T.Comment: 4 pages, 4 figure

Effect of bilayer coupling on tunneling conductance of double-layer high T_c cuprates

Physical effects of bilayer coupling on the tunneling spectroscopy of high T$_{c}$ cuprates are investigated. The bilayer coupling separates the bonding and antibonding bands and leads to a splitting of the coherence peaks in the tunneling differential conductance. However, the coherence peak of the bonding band is strongly suppressed and broadened by the particle-hole asymmetry in the density of states and finite quasiparticle life-time, and is difficult to resolve by experiments. This gives a qualitative account why the bilayer splitting of the coherence peaks was not clearly observed in tunneling measurements of double-layer high-T$_c$ oxides.Comment: 4 pages, 3 figures, to be published in PR