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Recombinant N-ras proteins, expressed and produced from synthetic genes cloned into E. coli, have been tested in vitro for GTPase and autophosphorylation activity. The genes corresponding to the assayed proteins were tested for their ability to transform NIH 3T3 cells. Mutations of glutamine to lysine at amino acid position 61 and glycine to valine at position 12 were both found to activate the ability of the N-ras gene to transform NIH 3T3 cells while significantly reducing the GTPase activity of the corresponding protein. N-ras proteins were also found to autophosphorylate in the presence of GTP when a threonine acceptor amino acid is provided at position 59.

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Four lac promoter mutants were constructed. The mutations increased the homology between the lac promoter and the consensus promoter sequences by introducing the consensus -10 and -35 regions and the consensus spacing of 17 residues between these two regions. The promoter mutants were cloned into a pBR322-derivatized vector upstream from the lacZ gene, and levels of beta-galactosidase were an indication of promoter activity. All mutants exhibited higher activity than did the wild-type promoter.

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[3H]Tetracycline (TC) has been shown to photoincorporate into the Escherichia coli ribosome. However, the utility of this process for characterizing the TC binding site on the ribosome is diminished by competing side reactions which also lead to incorporation of radioactivity. In this work we first conducted a detailed study of the labeling processes occurring when ribosomes are irradiated in the presence of [3H]TC with a common, rather low intensity, lamp. On the basis of the results of this study we next explored the usefulness for photoaffinity labeling of the TC site of both irradiation with a high-intensity laser and radioactive, functional TC derivatives having different photochemical properties than TC itself. Labeling patterns determined by polyacrylamide gel electrophoretic analysis of ribosomal proteins extracted from photoaffinity-labeled 30S subunits provided strong evidence that these two approaches offer distinct advantages for characterizing the TC binding site.

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Synthetic oligodeoxyribonucleotides were used to introduce single base changes into the TATAA box of the adenovirus-2 major late promoter. The resultant mutant DNA templates that contained altered TATAA sequences (TATAA leads to TCTAA/TATCA) were tested for their relative transcriptional activity in vitro using HeLa whole cell extracts. The data indicate that the change TATAA leads to TCTAA results in a level of specific transcription reduced to 48.7% of that from the parental template, whereas the change TATAA leads to TATCA reduced transcription to 21%. Neither base change abolished promoter activity.

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By a combination of chemical and enzymatic methods, a 75 base pair DNA duplex containing the sequence of the lambda PR promoter including the OR1 and OR2 cI repressor binding sites was synthesized. The solid support phosphite triester procedure (Caruthers, M. H. et al., Cold Spring Harbor Symposia on Quantitative Biology XLVII, in press) was used for the synthesis of oligonucleotides comprising the sequence. We report here an adaptation of the method of DNA synthesis in test tubes. Assembly of the oligonucleotides involved the use of T4 polynucleotide kinase and T4 DNA ligase. We show that the synthetic DNA is recognized by RNA polymerase and cI repressor in a manner identical to the same control region contained on a restriction fragment isolated from bacteriophage lambda DNA. Our synthetic approach using chemically synthesized promoter variants is thus suitable for studies probing the function of promoters.

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The effects of ischemic arrest and reperfusion on isovolumic end-diastolic pressure, diastolic pressure-volume curves, and indices of ventricular relaxation and contractility were studied in an isolated feline heart preparation. In hearts subjected to 60 min of normothermic (37 degrees C) ischemic arrest, isovolumic developed pressure, and dP/dtmax during reperfusion returned to only approximately 60% of prearrest control levels. Isovolumic end-diastolic pressure (Ped) increased 37.0 +/- 4.3 mmHg and the time constant of ventricular relaxation was prolonged. Hearts maintained at 27 degrees C hypothermia during the 60-min ischemic period demonstrated improved contractile performance (approximately 100% of control), less elevation of Ped (21.4 +/- 4.5 mmHg), and no significant increase in the time constant of relaxation. In both groups of hearts, postarrest end-diastolic pressure-volume curves were shifted up and to the left, whereas indices of ventricular stiffness and muscle stiffness remained unchanged. These data suggest that the rise in isovolumic end-diastolic pressure observed after 1 h of ischemic arrest and reperfusion is the result of an upward and to the left shift of the entire diastolic pressure-volume relationship of the left ventricle. This shift does not appear to be related to diminished contractile performance or incomplete relaxation. Furthermore, the shift is not due to a change in muscle compliance, but to a reduction in the unstressed volume of the ventricle, which most likely results from myocardial contracture and edema.

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To assess the effects of moderate potassium cardioplegia (37 mEq/l KCl) on the severity of myocardial ischemia during arrest and on post arrest ventricular function, 32 isolated, isovolumic feline hearts were studied before, during and 1 hour after ischemic arrest. Normothermia (37 degrees C) was maintained in the remaining 16 hearts, eight without KCl and eight with KCl. Hypothermia (27 degrees C) was maintained in the remaining 16 hearts, eight with KCl and eight without KCl. Myocardial oxygen (PmO2) and carbon dioxide tensions (PmCO2) were measured by mass spectrometry. Maximum developed intraventricular pressure (max DP) and max dP/dt were used as indices of performance. Compared with normothermic or hypothermic arrest alone, the addition of potassium cardioplegia resulted in a significant reduction in the peak PmCO2 measured during the arrest period. Hypothermia alone resulted in morphologic evidence of improved myocardial preservation and a significant reduction in peak PmCO2 compared with normothermia. Post arrest ventricular function was best with the combination of hypothermic arrest and potassium cardioplegia (max DP = 96 +/- 6% of control and max dP/dt = 99 +/- 5% of control). These data suggest that the beneficial effects of postassium cardioplegia and 27 degrees hypothermia are additive, and that reduction in myocardial ischemia as evidenced by a reduction in peak PmCO2 correlated with improvement in ventricular performance in the post arrest period and with preservation of myocardial structure.

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The extent of myocardial protection afforded by a procaine cardioplegic solution during cardiac ischemia has been evaluated and compared with the protection seen using a potassium cardioplegic solution. An isolated cat heart model was employed, and ventricular function parameters, intramyocardial gas tensions, and postischemic myocardial edema were measured and compared following 60 minutes of induced ischemia at 37 degrees C. and 27 degrees C. There was no significant improvement in recovery of postarrest ventricular function when procaine cardioplegia was used during normothermic ischemia. When used at 27 degrees C., however, both cardioplegic solutions were associated with significantly better recovery of postarrest ventricular function, although there was less myocardial edema formation in the potassium-treated hearts. Results of this study indicate that procaine-induced cardioplegia provides myocardial protection during anoxic cardiac arrest which is additive to that afforded by hypothermia alone. In addition, procaine cardioplegia results in postarrest functional recovery which is similar to that seen with potassium cardioplegia.

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