Biomedical subjects
Publications and source records attributed to M A Fabian.
Synthetic genes for avian pancreatic polypeptide (aPP) and for the miniature DNA binding protein PPBR4 were cloned and expressed on the surface of M13 bacteriophage. We anticipate that these constructs will have utility optimizing the properties of miniature proteins based on aPP that result from our previously described protein grafting procedure.
Biology is replete with examples of protein-induced DNA bending, yet the forces responsible for bending have been neither established nor quantified. Mirzabekov and Rich proposed in 1979 that asymmetric neutralization of the anionic phosphodiester backbone by basic histone proteins could provide a thermodynamic driving force for DNA bending in the nucleosome core particle [Mirzabekov, A. D., & Rich, A. (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 1118-1121]. Strauss and Maher lent support to this proposal in 1994 by demonstrating that replacement of six proximal phosphate residues with neutral methylphosphonates resulted in DNA bent spontaneously toward the neutralized face [Strauss, J. K., & Maher, L. J., III (1994) Science 266, 1829-1834; Strauss, J. K., Prakash, T. P., Roberts, C., Switzer, C., & Maher, L. J., III (1996) Chem. Biol. 3, 671-678; Strauss, J. K., Roberts, C.; Nelson, M. G.; Switzer, C., & Maher, J. L., III (1996) Proc. Natl. Acad. Sci. U.S.A. 93, 9515-9520]. Here it is shown that bZIP proteins bend DNA via a mechanism involving direct contacts between one or two basic side chains and a symmetry-related pair of unique, nonbridging phosphate oxygens. The locations of these phosphates provide direct experimental support for a protein-induced bending mechanism based on asymmetric charge neutralization. This straightforward mechanism is compatible with many DNA-recognition motifs and may represent a general strategy for the assembly of protein-DNA complexes of defined stereochemistries.
An NMR titration method has been developed to simultaneously measure the difference in acid dissociation constants (delta pKa) of two or more compounds with high precision and accuracy. The delta pKa between the conjugate acids of the two stereoisomers of 4-tert-butylcyclohexylamine 1 was determined in a single 1H NMR titration experiment. A mixture of the two stereoisomers was titrated with DCI in a 3:1 (v/v) mixture of CD3OD/D2O. From the variations of the H1 chemical shifts the ratio of the acidity constants was determined. The trans stereoisomer 1t was found to be the more basic by 0.121 +/- 0.002 pK unit. A repeat titration in DMSO-d6 also found 1t to be the more basic, by 0.217 +/- 0.003 pK unit. The delta pKa between the two stereoisomers of 4-tert-butylcyclohexanecarboxylic acid 2 was determined in both of these solvents using 1H and 13C NMR. Thermodynamic parameters delta delta H degree and delta delta S degree were evaluated from the temperature dependence of delta pKa. To further demonstrate the utility of this method, the delta pKas of the conjugate acids of the four stereoisomers of 2-decalylamine 3 were determined in a single 1H NMR titration experiment. The cis,cis stereoisomer (3cc) was found to be the most basic, with the cis,trans (3ct), trans,cis (3tc), and trans,trans (3tt) less basic by 0.012 +/- 0.003, 0.037 +/- 0.004, and 0.141 +/- 0.005 pK unit, respectively. A second four-component titration was also performed with the two 4-tert-butylcyclohexylamines (1) and the two trans-2-decalylamines (3tc, 3tt).
Warm ischemia of the intestine is a medical emergency which results from mesenteric vascular occlusion. In addition, intestinal transplantation techniques will also inevitably result in intestinal ischemia. The recovery of organ function following ischemia depends on the extent of irreversible damage produced by the ischemia and the extent of reflow upon reperfusion. In some organs energy homeostasis has been found to correlate with organ recovery and graft survival following ischemia-reperfusion. Investigating the usefulness of the determination of adenine and pyridine nucleotides as indicators of the extent of ischemic injury in intestinal segments, we found that after an initial 40% decrease in ATP following 30 min of ischemia there was no further decrease despite increasing the ischemia period to 120 min. Similarly, the decrease in NAD+ and NADP which occurred after 30 min of ischemia did not decrease further after 60, 90, or 120 min of ischemia. Xanthine was the only biochemical where an increase appeared to correlate with ischemia duration while energy charge was of no value in indicating injury extent. Additionally, after reperfusion there was at best a poor correlation between recovery of ATP content and the duration of ischemia. Microcirculation reflow after reperfusion indicated ischemia time-related endothelial cell injury. Thus, the measurement of high-energy phosphates in intestinal segments is not of value as an indicator of the extent of intestinal ischemic injury.
Explore the source record for details and available documents.
Explore the source record for details and available documents.
Explore the source record for details and available documents.
Explore the source record for details and available documents.
Explore the source record for details and available documents.
Explore the source record for details and available documents.
A number of organ preservation solutions have been formulated to slow the inevitable progression of ischemic injury, thus prolonging the storage time between removal and implantation. As adenine nucleotide content has been shown to correlate with the functional recovery of transplanted livers and hearts, this study investigated the effects of 24 hr of storage in three preservation solutions, saline (SA), Euro-Collins (CO), and University of Wisconsin (UW) on adenine and nicotinamide adenine nucleotides and inosine content of the rat small intestine. Significant biochemical differences were found between segments as early as after the initial perfusion when the inosine content was higher in UW-perfused than CO- or SA-perfused segments. After 2 hr of storage in CO solution and after 6 and 24 hr in both CO and UW solutions, the ATP content was higher than in SA-stored segments. In addition to inosine, which was significantly higher at all time points for UW-stored segments, the AMP and total adenine nucleotide content of UW-stored segments at 24 hr was significantly higher than SA- or CO-stored segments. After 24 hr of storage, those segments stored in UW were able to utilize significantly more oxygen than SA-stored. These data provide biochemical evidence supporting the advantages of CO and UW storage solutions over SA for preservation of small intestine segments.
Explore the source record for details and available documents.
The aforementioned technique offers an expeditious and safe means of extending the cadaveric right renal vein for 3 to 5 centimeters, while greatly minimizing the ischemic time of the kidney during preparatory dissection. The cuff of the cadaveric right renal artery can also be preserved, and the danger of kinking of the arterial graft is minimized. Our experience has shown that separation of the left renal vein 3 to 4 millimeters away from the vena cava allows a left renal vein of adequate length for transplantation of the left kidney. Between June 1985 and August 1989, we have used this technique successfully in more than 120 cadaveric right renal transplants and have seen no vascular complications attributable to the stapled conduit of the vena cava.
Both surgical fundusectomy and pharmacologic acid inhibition have been shown to produce antral G cell hyperplasia in rats. Fundusectomy has also been shown to be a reliable, reproducible means to produce hypergastrinemia. In contrast, treatment of animals with famotidine, a histamine H2 receptor antagonist, or omeprazole, a H+-K+-ATPase enzyme inhibitor, failed to produce hypergastrinemia. This suggests that elimination of acid inhibition may not be the sole mechanism of hypergastrinemia after fundusectomy. We hypothesized that removal of the gastric fundus may remove a factor which controls G cell activity.
Hypergastrinemia is often attributed to loss of acid inhibition in the gastric antrum. Prior studies suggest, however, that hypergastrinemia after fundusectomy occurs earlier and to a greater degree than hypergastrinemia after total acid inhibition with high dose (8 mg/kg/day) famotidine. To explain the very early (0 to 21-day) gastrin response, 45 male Sprague-Dawley rats (250 to 350 gm) were equally allocated to fundusectomy alone, famotidine alone, and fundusectomy plus famotidine groups. Vena cava blood samples and antral tissue were harvested at 0, 4, 7, 9, 14, and 21 days. Fundusectomy produced dramatic, sustained hypergastrinemia as early as 4 days after the procedure. Minimal, transient hypergastrinemia was seen with famotidine treatment, but this returned to baseline in spite of total acid inhibition. Antral gastrin was comparable in all three groups at the intervals assessed. Furthermore, fundusectomy plus famotidine did not demonstrate a significant difference from fundusectomy alone. Thus it appears that fundusectomy produces early and sustained hypergastrinemia by a mechanism other than acid inhibition.