Expression of a fibrinogen fusion peptide in Escherichia coli: a model thrombin substrate for structure/function analysis

The initial event in fibrin clot formation is the thrombin-catalyzed cleavage of the A alpha chain of human fibrinogen. Most of the information required for thrombin recognition and cleavage of the A alpha chain lies in the amino terminal 51 residue CNBr fragment. By selective modification of residues in this region, we probed the features that participate in thrombin interactions. We constructed a vector which expressed a tripartite protein (tribrid) consisting of amino acids 1 to 50 of the A alpha chain followed by 60 amino acids of chicken collagen and the beta-galactosidase protein from Escherichia coli. Cell lysates run on NaDodSO4-polyacrylamide gels contained the predicted band of molecular weight (mol wt) 125,000. The tribrid reacted with a monoclonal antibody, Mab-Y18, which recognizes the amino terminus of the A alpha chain. When cell lysates were incubated with thrombin, FPA was released. By including one heterogeneous oligonucleotide in the construction, we generated plasmids that encoded three specific amino acid substitutions. Surprisingly, changing Gly14 to Val did not alter thrombin cleavage, although recognition by Mab-Y18 was lost. Substitution of lie for Arg23 did not alter either thrombin cleavage or monoclonal recognition. Substitution of Leu for Arg 16 altered thrombin cleavage; unexpectedly, recognition by Mab-Y18 was not changed.     

Left mesial temporal sclerosis and verbal memory: a magnetoencephalography study.

The purpose of this study is to examine the pattern of mesial temporal lobe activity in patients with left mesial temporal sclerosis (LMTS) and normal control subjects during a verbal memory task using magnetoencephalography. Six LMTS patients and seven normal control subjects performed a word recognition task while event-related neuromagnetic responses were recorded for 248 trials in each of two runs. An equivalent current dipole model was used to localize activity sources and determine if the right or left mesial temporal lobe was active. All six (100%) LMTS patients but only two (28%) normal control subjects had right mesial temporal activity (P < 0.02). Two (33%) LMTS patients and 6 (86%) normal control subjects had left mesial temporal activity (P = 0.27). No significant differences in latency or duration of activity were seen between left and right sides or between groups. Our data suggest that patients with left hippocampal dysfunction are more likely to recruit the right mesial temporal lobe during verbal memory tasks than normal control subjects. A trend toward less left mesial temporal activity in LMTS patients is seen as well. Further study is needed to determine the predictive value of this technique regarding postresection memory outcome.     

The effects of obesity and exercise on the pharmacokinetics of caffeine in lean and obese volunteers

Summary The effects of obesity, exercise, and the interaction of obesity and exercise were examined in 6 caffeine naive, untrained, nonsmoking, college males (3 lean (LV), 3 obese (OV)). Each subject received caffeine (oral, 5.83 mg·kg^–1 lean body weight) or placebo (50 mg citrate) prior to 3 h of seated rest and prior to 90 min of treadmill walking (40% of their maximal aerobic power) followed by 90 min of seated recovery. Serum samples were collected at various times and analyzed for caffeine by HPLC. Pharmacokinetic analysis indicated that at rest, OV had a significantly higher absorption rate constant (Ka 0.0757 vs. 0.0397 min^–1), lower elimination rate constant (Ke 0.0027 vs. 0.0045 min^–1), and longer serum half-life (t_1/2 4.37 vs. 2.59 h) in comparison to LV. In exercise, as well as at rest LV and OV had a large difference in the volume of distribution (43.2 vs. 101. 1) (rest, 54.1 vs. 103.1). Exercise consistently resulted in a decrease in the maximal serum concentration of caffeine and the area under the curve in OV while having no consistent effect on LV. The interactive effects of obesity and exercise could not be dissociated. However, these results demonstrate that both obesity and exercise have modified the pharmacokinetics of caffeine.This work was presented at the annual meeting of the American Society for Pharmacology and Experimental Therapeutics, Boston, August 22, 1985     

Venous and fingertip blood to calculate plasma volume shift following exercise

This study determined whether fingertip blood samples used to calculate percentage change in calculated plasma volume following exercise were in agreement with values obtained from venous blood samples. Twenty-five subjects engaged in two cycle ergometer exercises at 100 and 200 W, with percentage plasma volume shift (% PVS) determined after each from venous (VB) and fingertip (FT) blood. Values for % PVS were FT -6.25% compared with VB -8.04% (P less than 0.05), with the correlation between the two methods at r = 0.88. The following equation was established: corrected FT % PVS = (0.8662 * FT) - 2.625; SEE = 2.60%. In order to cross-validate this equation, fifteen additional subjects submitted to VB and FT. Corrected FT % PVS using the established equation resulted in a mean value of 9.53 compared with 10.53% for actual VB % PVS. Although these means were not significantly different, there was approximately a 25% chance that the corrected FT % PVS would be more than one standard error of estimate from the regression line. It was concluded that FT underestimates VB % PVS. However, when limited to group data, FT can be corrected to favorably represent VB % PVS following moderate to heavy cycle ergometer exercise.