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Extracorporeal life support (ECLS) is fast becoming more common place for use in adult patients failing mechanical ventilation. Management of coagulation and thrombosis has long been a major complication in the use of ECLS therapies. Scanning electron microscopy (SEM) of membrane oxygenators (MOs) after use in ECLS circuits can offer novel insight into any thrombotic material deposition on the MO. In this pilot study, we analyzed five explanted MOs immediately after use in a sheep model of different acute respiratory distress syndrome (ARDS). We describe our methods of MO dissection, sample preparation, image capture, and results. Of the five MOs analyzed, those that received continuous heparin infusion showed very little thrombosis formation or other clot material, whereas those that were used with only initial heparin bolus showed readily apparent thrombotic material.

Azotobacter vinelandii has recently been used for a variety of genetic experiments which take advantage of its facile transformation system and its high-frequency homologous recombination. One gene that has been cloned and sequenced is the fdxA gene that encodes a small Fe-S protein called A. vinelandii ferredoxin I (AvFdI). Because this protein has been extensively characterized by X-ray crystallography and spectroscopic methods, we are using it as a model to address some general questions in Fe-S biochemistry. AvFdI is not a very abundant protein in wild-type cells, so to facilitate our biochemical studies we have developed the overexpression system described herein. The results show that AvFdI can be easily overproduced ca. 50-fold in its native background, by introducing multiple copies of the fdxA gene into A. vinelandii, on the broad-host-range multicopy plasmid, pKT230. The protein can be expressed from its own constitutive promoter or from the controlled nifH promoter. The overproduced protein has no deleterious effects on the organism and is identical to the protein produced by wild-type cells. This A. vinelandii-based system should be generally useful for the overproduction of other A vinelandii proteins or for the expression of genes from thermophilic or other organisms with similarly high G-C contents, or for the expression of O2-sensitive metalloproteins that are unstable in other systems.

The isolation and regeneration of tobacco mesophyll protoplasts from fully developed leaves, an important methodological step in plant genetic engineering as well as in plant cell biology and physiology, has been proven unreliable to the extent that it has become a significant setback to basic research. This unfortunate situation is primarily due to the suboptimal physiological state of greenhouse-grown protoplast donor plants. A technically simple and inexpensive method, based on the utilization of commercial Phototron units, is described for the production of suitable tobacco donor plants. Furthermore, a modified version of such a culture unit can be used to regenerate plants from protoplast-derived calli.