Monodisperse 130 kDa and 260 kDa Recombinant Human Hemoglobin Polymers as Scaffolds for Protein Engineering of Hemoglobin-Based Oxygen Carriers.

A recombinant 130 kDa dihemoglobin which is made up of a single-chain tetra-α globin and four ß globins has been expressed as a soluble protein in E. coli. The sequence of the single chain tetra-α is: αI-Gly-αII-(SerGlyGly)5Ser-αIII-Gly-αIV. This dihemoglobin has been purified and characterized in vitro by size exclusion chromatography, electrospray mass spectroscopy, equilibrium oxygen binding, and analytical ultracentrifugation. The observed values of P50 and nmax for the dihemoglobin are slightly lower than those observed for the recombinant hemoglobin rHb1.1 (a "monohemoglobin" comprised of two ß globins and an αI-Gly-αII diα-globin chain). Titration of the deoxy form of dihemoglobin with CO shows that all eight heme centers bind ligand. In vivo, dihemoglobin showed increased circulating halflife and a reduced pressor response in conscious rats when compared to rHb1.1. These observations suggest that dihemoglobin is an oxygen carrying molecule with desirable in vivo properties and provides a platform for an isooncotic hemoglobin solution derived solely from a recombinant source. A 260 kDa tetrahemoglobin has also been produced by chemical crosslinking of a dihemoglobin that contains a Lys16Cys mutation in the C-terminal α-globin subunit. Tetrahemoglobin also shows reduced vasoactivity in conscious rats that is comparable to that observed for dihemoglobin.

In vitro elution studies of amikacin and cefazolin from polymethylmethacrylate.

OBJECTIVE: To compare the in vitro elution characteristics of amikacin and cefazolin from polymethylmethacrylate (PMMA) alone and in combination. STUDY DESIGN: A prospective, controlled, experimental study. METHODS: Three aliquots of 6 g sterile PMMA were measured and to them added (1) 750 mg amikacin; (2) 1050 mg cefazolin; and (3) 750 mg amikacin and 1050 mg cefazolin. Ten beads of each antimicrobial/PMMA combination were placed in 5 mL phosphate-buffered saline (PBS) at pH 7.4 and room temperature with constant agitation. PBS was sampled at 15 time points between 1 hour and 30 days. Amikacin concentrations were determined by fluorescence polarization immunoassay and cefazolin concentrations by high-performance liquid chromatography. RESULTS: Amikacin and cefazolin eluted at concentrations greater than 8 and 4 times, respectively, above the minimum inhibitory concentration (MIC) for susceptible bacteria over 30 days. Co-elution of the antibiotics resulted in a greater rate and proportion of antibiotic eluted. Concentrations of amikacin and cefazolin in the co-eluted fluid were not maintained sufficiently above the MIC for selected bacteria over 30 days. CONCLUSIONS: PMMA beads of only amikacin or cefazolin-eluted concentrations greater than the MIC for selected bacteria for 30 days. Co-elution of the antibiotics at the selected doses resulted in a significantly shorter duration of elution and may not be effective for treatment of wound infection. CLINICAL RELEVANCE: Co-elution of amikacin and cefazolin from PMMA at the selected doses cannot be recommended for sustained treatment of infection.

Urodynamic effects of a percutaneously controlled static hydraulic urethral sphincter in canine cadavers.

OBJECTIVE: To describe a percutaneously controlled static hydraulic urethral sphincter (SHUS) and evaluate urodynamic effects of the SHUS in canine cadavers. SAMPLE POPULATION: Cadavers of 6 adult female dogs. PROCEDURE: Cadavers were obtained immediately after dogs were euthanatized. Baseline maximal urethral closure pressure (MUCP) and cystourethral leak point pressure (CLPP) were measured by use of a urethral pressure profilometer. An SHUS system was constructed by use of a silicone vascular occluder and subcutaneous infusion port. The SHUS system was then placed around the pelvic urethra in each cadaver. Measurements of MUCP and CLPP were repeated after varying occlusion of the SHUS (0%, 25%, and 50% occlusion). Baseline MUCP and CLPP values were compared with values obtained at 0%, 25%, and 50% occlusion of the SHUS by use of repeated-measures ANOVA. RESULTS: Mean +/- SD MUCP for canine cadavers was 7 +/- 1.3 cm H2O at baseline, which increased to 127 +/- 53 cm H2O after 50% occlusion of the SHUS. Mean CLPP was 11 +/- 8.6 cm H2O at baseline, which increased to 73 +/- 38 cm H2O after 50% occlusion of the SHUS. Mean MUCP and CLPP were significantly associated with the amount of occlusion. CONCLUSIONS AND CLINICAL RELEVANCE: The SHUS had positive effects on MUCP and CLPP in canine cadavers. Therefore, additional evaluation of the SHUS in live dogs is warranted.