Primase activity of human DNA polymerase alpha-primase. Divalent cations stabilize the enzyme activity of the p48 subunit

DNA polymerase alpha-primase consists of four subunits, p180, p68, p58, and p48, and comprises two essential enzymatic functions. To study the primase activity of the complex, we expressed cDNAs encoding for the human p58 and p48 subunits either as single proteins or together using Escherichia coli expression vectors. Co-expression of both primase subunits allowed the purification of a heterodimer in high yields that revealed stable primase activity. Purified recombinant p48 subunit showed enzyme activity, whereas purified p58 did not. In contrast to the heterodimer, the primase activity of p48 was unstable. The activity of p48 could be stabilized by the addition of the divalent cations Mg2+ and Mn2+ but not Zn2+. On a poly(dC) template the primase activity was hardly influenced by the monovalent cation potassium. However, by using poly(dT) as a template the recombinant p48 activity was sensitive to salt, whereas recombinant p58-p48 and the bovine DNA polymerase alpha-primase purified from thymus were less sensitive to the addition of monovalent cations. A complex of bacterially expressed primase and baculovirus-expressed p180 and p68 was assembled in vitro and shown to support replication of simian virus 40 DNA in a cell-free system.     

Kinetics of Hydrogen Absorption and Release in Zirconium Alloys During Steam Oxidation

The kinetics of hydrogen absorption during steam oxidation in Zr–Sn and Zr–Nb alloys in the temperature range of 1,000–1,400 °C was investigated by neutron radiography. Hydrogen uptake can be subdivided into two steps: an initial phase and a state of equilibrium. The initial phase is controlled by the kinetics of hydrogen diffusion through the growing oxide layer. In the state of equilibrium, transport kinetics does not determine the hydrogen content of the material. An equilibrium is established between the hydrogen content of the gas environment and the metal phases. The temperature dependence of hydrogen absorption is Arrhenius-like at temperatures between 1,100 and 1,300 °C.     

Laser-Welded Steel Foils with Sapphire Substrates

A nanosecond pulsed laser was used to weld stainless steel foils of 10 μm thickness to the sapphire substrates.The microstructure of the bonded interface was studied.Both materials were partially ablated under the influence of the laser beam.An inhomogeneous distribution of the steel and sapphire along the bonded interface is observed.The electrical resistance of the steel foil was measured before and after laser welding,showing that the weld slightly increases the electrical resistance but still keeps the values acceptable for electrical contacts.     

Maverick picking: the impact of modifications in work schedules on manual order picking processes

Order picking, the process of retrieving items from their storage locations to fulfil customer orders, ranks among the most labour- and time-intensive processes in warehousing. Prior research in this area had a strong focus on the development of operating policies that increase the efficiency of manual order picking, for example by calculating optimal routes for the order pickers or by assigning products to storage locations. One aspect that poses a major challenge to many warehouse managers in practice has, curiously enough, remained largely unexplored by academic research: modifications in workflows (i.e. workplace deviance in a positive or negative sense) in order picking, which we define as ‘maverick picking’. The purpose of this paper is to characterise maverick picking and to study its causes, its forms of appearance and its potential impact on order picking performance. To gain insights into maverick picking, we first survey the literature to illustrate the state-of-knowledge of maverick picking. Subsequently, we report the results of a multi-case study on maverick picking and deduct a related content framework. The results of our case study support the proposition that maverick picking is highly relevant in practice and that it is a major determinant of order picking performance.     

Influence of the depth position on the neutron embrittlement of the VVER reactor pressure vessel steel 15Cr2MoV(A) consequences for the assessment of reactor safety

The dependence of the mechanical properties on the depth position in the unirradiated state and after irradiation up to neutron fluences of approximately 5 × 10¹⁸ and 70 × 10¹⁸ cm⁻² (E > 0.5 MeV) is tested on a forging made out of VVER 440 reactor pressure vessel (RPV) steel 15CrMoV. The near-surface position shows a higher strength and a lower transition temperature than the positions greater than 1/4 wall thickness. Irradiation does not change these differences in a significant manner. The testing of specimens from the 1/4 depth position within the surveillance programme, as normally laid down in the legal rules relating to nuclear power plants, results in a conservative safety assessment against brittle failure up to the EOL fluence. On taking into account fluence attenuation, the flux effect, etc., the toughness gradually increases from the inside to the outside of the wall after longer RPV operating times.     

C-erbB-2 overexpression in primary breast cancer: independent prognostic factor in patients at high risk

The prognostic value of c-erbB-2 protein overexpression has been evaluated in 463 patients with operable breast cancer after a median follow-up of 66 months. Overexpression was observed in 99/463 (21%) of the breast tumors. It showed significant positive correlation to histological grade (p < 0.0001) and tumor size (p < 0.02). A relationship of borderline significance was observed between c-erbB-2 protein overexpression and negative or low estrogen receptor (ER) content. No significant correlation was found to lymph node involvement or proliferating tumor cell fraction as determined by the proliferating cell nuclear antigen (PCNA). After a median follow-up of 66 months (range 6 to 109 months), the overall survival of all patients amounted to 63%. Multivariate analysis revealed lymph node involvement, tumor size, histological grade, histological type, c-erbB-2 protein overexpression, progesterone receptor (PR) content, and oral contraceptive use as independent prognostic factors. In an univariate analysis, the overall survival amounted to 72% and 38% of tumor patients with negative and positive c-erbB-2 protein overexpression, respectively. The most significant finding is that c-erbB-2 overexpression has been recognized as an independent predictive factor in subsets of tumor patients who would be expected to have a generally poor prognosis, such as those indicating axillary lymph node involvement, large tumor size (> 2 cm), and PR negativity.     

Strategies and perspectives in ion-channel engineering

Membranes form natural barriers that need to be permeable to diverse matter like ions and substrates. This permeability is controlled by ion-channel proteins, which have attracted great interest for pharmaceutical applications. Ion-channel engineering (ICE) modifies biological ion channels by chemical/biological synthetis means. The goal is to obtain ion channels with modified or novel functionality. Three functional strategies exist. The first is the manipulation of the wider pores with robust β-barrel structures, such as those of α-hemolysin and porins. The second engineering approach focuses on the modification of narrow (mostly α-helical) pores to understand selectivity and modes of action. A third functional approach addresses channel gating by (photo)triggering the biological receptor that controls the channel. Several synthetis strategies have been developed and successfully utilized for the synthetic modification of biological ion-channels: the S-alkylation of specifically introduced Cys, protein semisynthesis by native chemical ligation, protein semisynthesis by protein trans-splicing, as well as nonsense-suppression methods. Structural studies (X-ray crystallography, NMR spectroscopy) are necessary to support the functional studies and to afford predictable engineering. The reprogramming and re-engineering of channels can be used for sensing applications, treatment of channelopathies, chemical neurobiology, and providing novel lead compounds for targeting ion channels.     

Steroide. LXIX [1]. Ringschlußreaktionen vicinaler Halogen-urethane der Steroidreihe

Steroids. LXIX. Cyclisation Reactions of Vicinal Substituted Halogen Carbamoyloxy Steroids Vicinal halogen urethanes of the cholestane, androstane and estratriene series react by heating under solvolytic conditions to cyclic carbonates of the corresponding cis-diols. By heating under basic conditions the same compounds react preferably to 2-oxazolidinones and if the basic conditions are strong enough by hydrolysis of the oxazolidinones to cis-amine alcohols too. Connections between the nature of the substituent at the urethane group or the steric arrangement of the vicinal groups on one hand and the reactivity to ring closure on the other hand are presented.