Effect of arteriovenous flow reversal on blood flow and metabolism in a skin flap

Twelve pig buttock island flaps (10 X 10 cm) were studied for 6 hours after arteriovenous flow reversal at the level of the pedicle. Follow-up was 48 hours. Blood pressure, Po2, pH, and lactate were measured in flap arteries and veins. Oxygen consumption was calculated. Data indicated true flow reversal. Blood pressure and Po2 in flap veins increased to systemic arterial levels. Outflow was provided by the arterial system, demonstrating venous pressure and Po2 values. Lactate increased significantly (1.8 +/- 0.5 to 4.0 +/- 2.3 mmol/liter), while pH dropped from 7.43 +/- 0.03 to 7.11 +/- 0.02. Oxygen consumption remained below baseline. In four flaps thrombosis occurred within 6 hours; no flap survived 48 hours. The results of this study do not encourage clinical application of the concept of flow reversal.     

In vitro toxicity testing for antibacterials against human keratinocytes

The use of cultured human keratinocytes in an in vitro comparison of topical antibacterial toxicity for epithelial cells was examined. The complement of three assessments allows testing of epithelial migration, growth, and survival. The three assessments included (1) flow cytometry for determination of cell survival, (2) a comparison of confluent cell culture growth after antibacterial exposures, and (3) an evaluation of cell migration using a technique of dermal explants to study radial migration. A comparative ranking of the toxicities of the various topical antibacterials was determined with the three assessments. This has confirmed anecdotal reports that many of the topical antibacterials are cell-toxic and may inhibit wound healing. This information can be directly extrapolated to the clinical setting, unlike many of the animal data for wound healing that currently exist.     

Levels of hippocampal calcium and zinc following kindling-induced epilepsy

Hippocampal calcium and zinc content was determined using atomic absorption spectrophotometry in control and commissural-kindled rats. In animals exhibiting 5-10 consecutive motor seizures hippocampal calcium was slightly elevated (356.7 parts per million (ppm), dry weight) but not significantly different from controls (329.8 ppm), whereas the amount of zinc was significantly higher (101.6 ppm) than in nonstimulated animals (88.3 ppm). These results are indicative of certain pathophysiological changes in kindled hippocampi, most likely localized to the granule cells of the dentate gyrus where the bulk of hippocampal zinc is confined.     

Molecular basis for the polymorphic forms of human serum paraoxonase/arylesterase: glutamine or arginine at position 191, for the respective A or B allozymes.

The paraoxonase/arylesterase gene is located close to the cystic fibrosis gene on chromosome 7. Human serum contains two paraoxonase/arylesterase allozymes, A and B, which differ in their substrate specificities and kinetic properties. Purified A, AB, and B esterases were digested with trypsin, and the resultant peptides were compared by high-performance liquid chromatography. The elution profiles were very similar for all three samples, except for (1) one peptide (i.e., peptide A) seen only in the A and AB profiles and (2) another peptide (i.e., peptide B) seen only in the B and AB profiles. Sequencing revealed that peptide A had glutamine at amino acid position 191, whereas peptide B was generated by cleavage on the carboxy side of position 191, presumably because there was a basic (trypsin-specific) amino acid at that position. Working independently, our laboratory and one other laboratory have sequenced the coding region for paraoxonase from human liver cDNA libraries and have identified two polymorphic sites: Arg/Gln at position 191 and Leu/Met at position 54. Using PCR amplification and direct sequencing of nucleotides in both polymorphic regions with genomic DNA, we have estimated the allelic frequencies and have determined their concordance with the serum paraoxonase allozyme phenotypes in 27 unrelated adults and in 16 members of a three-generation pedigree. Among unrelated individuals, the Met/Leu polymorphism at position 54 did not correlate with the serum esterase phenotype. In contrast, the particular amino acid at position 191 correlated perfectly with serum phenotypes: A-type individuals had Gln at position 191, and B-type individuals had Arg at position 191; AB-type serum was found only with the heterozygous (Arg/Gln) combination. Pedigree analysis showed both polymorphisms to be inherited in the expected Mendelian manner and confirmed that only the 191 polymorphism showed concordance with the serum paraoxonase/arylesterase phenotypes.     

Aurora-A-Dependent Control of TACC3 Influences the Rate of Mitotic Spindle Assembly

The essential mammalian gene TACC3 is frequently mutated and amplified in cancers and its fusion products exhibit oncogenic activity in glioblastomas. TACC3 functions in mitotic spindle assembly and chromosome segregation. In particular, phosphorylation on S558 by the mitotic kinase, Aurora-A, promotes spindle recruitment of TACC3 and triggers the formation of a complex with ch-TOG-clathrin that crosslinks and stabilises kinetochore microtubules. Here we map the Aurora-A-binding interface in TACC3 and show that TACC3 potently activates Aurora-A through a domain centered on F525. Vertebrate cells carrying homozygous F525A mutation in the endogenous TACC3 loci exhibit defects in TACC3 function, namely perturbed localization, reduced phosphorylation and weakened interaction with clathrin. The most striking feature of the F525A cells however is a marked shortening of mitosis, at least in part due to rapid spindle assembly. F525A cells do not exhibit chromosome missegregation, indicating that they undergo fast yet apparently faithful mitosis. By contrast, mutating the phosphorylation site S558 to alanine in TACC3 causes aneuploidy without a significant change in mitotic duration. Our work has therefore defined a regulatory role for the Aurora-A-TACC3 interaction beyond the act of phosphorylation at S558. We propose that the regulatory relationship between Aurora-A and TACC3 enables the transition from the microtubule-polymerase activity of TACC3-ch-TOG to the microtubule-crosslinking activity of TACC3-ch-TOG-clathrin complexes as mitosis progresses. Aurora-A-dependent control of TACC3 could determine the balance between these activities, thereby influencing not only spindle length and stability but also the speed of spindle formation with vital consequences for chromosome alignment and segregation.