An engineered bifunctional high affinity iron uptake protein in the yeast plasma membrane.

High affinity iron uptake in fungi is supported by a plasma membrane protein complex that includes a multicopper ferroxidase enzyme and a ferric iron permease. In Saccharomyces cerevisiae, this complex is composed of the ferroxidase Fet3p and the permease Ftr1p. Fe(II) serves as substrate for Fe-uptake by being substrate for Fet3p; the resulting Fet3p-produced Fe(III) is then transported across the membrane via Ftr1p. A model of metabolite channeling of this Fe(III) is tested here by first constructing and kinetically characterizing in Fe-uptake two Fet3p-Ftr1p chimeras in which the multicopper oxidase/ferroxidase domain of Fet3p has been fused to the Ftr1p iron permease. Although the bifunctional chimeras are as kinetically efficient in Fe-uptake as is the wild type two-component system, they lack the adaptability and fidelity in Fe-uptake of the wild type. Specifically, Fe-uptake through the Fet3p, Ftr1p complex is insensitive to a potential Fe(III) trapping agent - citrate - whereas Fe-uptake via the chimeric proteins is competitively inhibited by this Fe(III) chelator. This inhibition does not appear to be due to scavenging Fet3p-produced Fe(III) that is in equilibrium with bulk solvent but could be due to leakiness to citrate found in the bifunctional but not the two-component system. The data are consistent with a channeling model of Fe-trafficking in the Fet3p, Ftr1p complex and suggest that in this system, Fet3p serves as a redox sieve that presents Fe(III) specifically for permeation through Ftr1p.

Spectroscopy and reactivity of the type 1 copper site in Fet3p from Saccharomyces cerevisiae: correlation of structure with reactivity in the multicopper oxidases.

Fet3p is a multicopper oxidase recently isolated from the yeast, Saccharomyces cerevisiae. Fet3p is functionally homologous to ceruloplasmin (Cp) in that both are ferroxidases. However, by sequence homology Fet3p is more similar to fungal laccase, and both contain a type 1 Cu site that lacks the axial methionine ligand present in the functional type 1 sites of Cp. To determine the contribution of the electronic structure of the type 1 Cu site of Fet3p to the ferroxidase mechanism, we have examined the absorption, circular dichroism, magnetic circular dichroism, electron paramagnetic resonance, and resonance Raman spectra of wild-type Fet3p and type 1 and type 2 Cu-depleted mutants. The spectroscopic features of the type 1 Cu site of Fet3p are nearly identical to those of fungal laccase, indicating a very similar three-coordinate geometry. We have also examined the reactivity of the type 1 Cu site by means of redox titrations and stopped-flow kinetics. From poised potential redox titrations, the E degrees of the type 1 Cu site is 427 mV, which is low for a three-coordinate type 1 Cu site. The kinetics of reduction of the type 1 Cu sites of four different multicopper oxidases with two different substrates were compared. The type 1 site of a plant laccase (Rhus vernicifera) is reduced moderately slowly by both Fe(II) and a bulky organic substrate, 1,4-hydroquinone (with 6 equiv of substrate, k(obs) = 0.029 and 0.013 s(-)(1), respectively). On the other hand, the type 1 site of a fungal laccase (Coprinus cinereus) is reduced very rapidly by both substrates (k(obs) > 23 s(-)(1)). In contrast, both Fet3p and Cp are rapidly reduced by Fe(II) (k(obs) > 23 s(-)(1)), but only very slowly by 1,4-hydroquinone (10- and 100-fold more slowly than plant laccase, respectively). Semiclassical theory is used to analyze the origin of these differences in reactivity in terms of type 1 Cu site accessibility to specific substrates.

Transmission failure rate for computed tomography examinations in a filmless imaging department.

The purpose of the study was to determine the frequency and causes of unsuccessful computed tomography (CT) transmissions in a filmless imaging department and to determine the added efficiency gains provided by the sequential addition of modality worklist software and a major network upgrade. Prospective data on CT transmission error rates were recorded over an 18-month period. During the study interval, modality worklist functionality was added, followed by a network upgrade. Failed transmissions were categorized as to the source of the error (human v technical), and the specific problem encountered. Prior to the introduction of modality worklist software, the initial CT transmission failure rate was 7.6%, which was primarily the result of human error (69%), in the form of data entry error. Upon the introduction of modality worklist software, the transmission failure rate decreased to 3.5%, with human error accounting for only 16% of all failed transmissions. The subsequent addition of a network upgrade from shared Ethernet to switched Ethernet further reduced the transmission failure rate to 2.0%, which was believed to be the result of a reduction in the number of network collisions. Other sources of failed transmission occur at the levels of the CT scanner (network interface card), picture archiving and communication system (PACS)/hospital information system (HIS) interface, and modality gateway. When planning the transmission from film-based to filmless operation, one should consider various hardware, software, and infrastructural requirements to ensure successful PACS implementation. Software upgrades, in the form of modality worklist software, serve to improve technologist productivity by minimizing data entry error. Infrastructural changes, in the form of network upgrading, ensure proper dissemination of electronic data with decreased frequency of network collisions. Collectively, these improvements lead to enhanced transmission of digital images, resulting in productivity gains within the filmless CT department.

Challenges associated with interfacing computed tomography to a picture archiving and communication system at the Baltimore Veterans Affairs Medical Center--a historical perspective.

The interfacing of digital image acquisition modalities to the picture archiving and communication system (PACS) plays a major part in the conversion from a traditional film-based radiology practice to one that relies almost entirely on soft-copy reading. The Baltimore Veterans Affairs Medical Center (VAMC) is one of the first filmless hospitals in the world. Since 1993, it has used computed tomography (CT) scanners connected to a commercial PACS to provide digitized patient images for filmless reading. Over the years, the evolution of Digital Imaging and Communications in Medicine (DICOM) standards, advances in networking technologies, and enhancements in PACS and hospital information system (HIS) software have greatly improved this system's robustness and patient/study identification accuracy. The result has been a major increase in productivity.

Analysis of a Zebrafish semaphorin reveals potential functions in vivo.

The semaphorin/collapsin gene family is a large and diverse family encoding both secreted and transmembrane proteins, some of which are thought to act as repulsive axon guidance molecules. However, the function of most semaphorins is still unknown. We have cloned and characterized several semaphorins in the zebrafish in order to assess their in vivo function. Zebrafish semaZ2 is expressed in a dynamic and restricted pattern during the period of axon outgrowth that indicates potential roles in the guidance of several axon pathways. Analysis of mutant zebrafish with reduced semaZ2 expression reveals axon pathfinding errors that implicate SemaZ2 in normal guidance.

Molecular cloning and expression of two novel zebrafish semaphorins.

The large, conserved semaphorin/collapsin gene family encodes putative axon guidance molecules. We describe the cloning and expression of two n ovel zebrafish semaphorins that represent an increase in the size and diversity of the family. These semaphorins are expressed in unique and dynamic patterns during development.