Metabolic enzyme response in the pressure-overloaded heart of weanling and adult rats

Weanling and adult rats were subjected to left ventricular pressure overload induced by abdominal aortic constriction. At 5 days or 5 weeks postsurgery, the left ventricle (LV) was dissected, weighed, and metabolic marker enzyme activities (mumole/g/min) of tissue homogenates were measured. Enzymes representing glycolytic (phosphofructokinase (PFK] and mitochondrial (citrate synthase (CS) and malate dehydrogenase (MDH] metabolisms were evaluated. Five days of pressure overload had detectable, but statistically nonsignificant effects on left ventricles of both weanling and adult rats. Sustained pressure overload (5 weeks) increased LV weight by 52 and 39% in weanling and adult rats, respectively. PFK activity was 24 +/- 1 (mean +/- SE) in control weanlings and was unaltered in any of the other groups. LDH isoenzyme composition was estimated by substrate inhibition (ratio 0.33/10 mM pyruvate). With normal heart development, the LDH ratio increased from 1.89 +/- 0.06 to 2.03 +/- 0.08. Pressure overload had no influence on the adult LDH ratio. Developmental LDH responses were not observed in weanling LV after 5 weeks of aortic constriction (1.74 +/- 0.06). The product of CS activity and LV weight was used to estimate mitochondrial mass in the ventricle. Mitochondria accumulated at a rate of about 5% increase per day over the intervening 5-week period of normal heart growth. Pressure overload for 5 weeks in weanling rats elicited net accumulation of mitochondria at a rate of about 9% increase per day. Mitochondrial accumulation in the adapting adult rat heart amounted to less than 1% increase per day. The results indicate that qualitative and quantitative differences exist between young and adult animals in their heart enzyme adaptive responses to pressure overloading. Divergent metabolic adaptations may contribute to heart functional differences in the enlarged heart of weanlings and adults.     

Functional evaluation of the rat heart in situ.

We have developed methods for evaluating muscle function in the intact rat heart in situ using a contractility index (dP/dt)P-1, calculated from left ventricular pressure derivative-left ventricular pressure loop plots. Aortic flow measurements were also taken to further characterize in situ rat heart function. The preparation remained functionally stable and was within physiological blood gas and pH limits for at least 30 min following surgical procedures. The contractility index was not influenced by increased afterload, decreased preload or increased heart rate; however, appropriate changes were observed following isoproterenol and propranolol administration. Appropriate changes in aortic flow measurements were observed also with the above interventions. These studies demonstrate that the in situ rat heart is a stable physiological experimental preparation. It should be useful for evaluating heart function since a contractility index derived from pressure-velocity relationships and measurements necessary for pump function analysis can be obtained simultaneously.     

Purification and Some Properties of Bacteriophage ST-1

Bacteriophage ST-1 is shown to be a small, isometric, single-stranded deoxyribonucleic acid (SS-DNA) virus with a diameter of about 260 nm. Standard methods for growth, assay, preparation of high-titer lysates, and purification of the phage are suggested. ST-1 infects K-12 and not C strains of Escherichia coli and requires a divalent cation to adsorb to susceptible bacteria. Adsorption also requires an activation of the particle brought on by incubation at 37 C. The latent and eclipse periods are essentially identical (9 to 11 min) in ST-1 infections, with an average burst size about 250 phages per cell. Multiple densities of ST-1 infectivity are observed during purification in CsCl gradients. The virus recovered from different densities has the same sedimentation coefficient and, therefore, all phage containing fractions are pooled during purification. The purified ST-1 particle has a sedimentation coefficient of 121S relative to phiX-174 (114S) in a sucrose gradient and a molecular weight of 6.8 x 10(6) (as estimated from its relative sedimentation). The nucleic acid is assumed to be SS-DNA on the basis of (i) the specific incorporation of (3)H-thymine, (ii) the dependence of its UV absorption on temperature, and (iii) its reaction with formaldehyde. ST-1 SS-DNA sediments at 24.4S relative to phiX-174 SS-DNA (23.8S).     

Cold-Sensitive Mutants of Bacteriophage φX174. II. Comparison of Two Cold-Sensitive Mutants

Cold-sensitive bacteriophage phiX174 mutants, another class of conditional lethals, were examined with regard to growth parameters, DNA synthesis, and particle properties. Two mutants, cs70 and cs82, were examined. Mutant cs70 was eclipse defective, showing altered eclipse kinetics at permissive temperature (40 C) and failing entirely to eclipse at restrictive temperature (25 C). Mutant cs70 replicated well at 25 C if allowed prior eclipse at 40 C. Mutant cs82 had wild-type eclipse at both temperatures but was defective in single-strand synthesis at 25 C, which led to delayed progeny phage appearance, decreased progeny phage synthesis rate, and greatly reduced burst size. The cs82 block could not be bypassed by temperature shift. Since complementation analysis of cs70 and cs82 was not feasible due to the unique properties of these mutants, those phiX174 properties affected by the virus coat were examined as an index of a mutation in a coat protein gene. Mutant cs70 had aberrant attachment kinetics at both 25 C and 40 C, evidence of a coat protein alteration. Mutant cs70 also exhibited significantly decreased thermal stability, further evidence of an altered virus structure. Mutant cs82 had increased thermal stability, but the difference was not sufficient to allow unequivocal assignment of this mutant to a coat protein gene. Both mutants had wild-type antiserum inactivation and host range, although cs70 was subject to less of (low-level) plating restriction by endogenous F(+) factors.     

Mapping of a yeast G protein betagamma signaling interaction.

The mating pathway of Saccharomyces cerevisiae is widely used as a model system for G protein-coupled receptor-mediated signal transduction. Following receptor activation by the binding of mating pheromones, G protein betagamma subunits transmit the signal to a MAP kinase cascade, which involves interaction of Gbeta (Ste4p) with the MAP kinase scaffold protein Ste5p. Here, we identify residues in Ste4p required for the interaction with Ste5p. These residues define a new signaling interface close to the Ste20p binding site within the Gbetagamma coiled-coil. Ste4p mutants defective in the Ste5p interaction interact efficiently with Gpa1p (Galpha) and Ste18p (Ggamma) but cannot function in signal transduction because cells expressing these mutants are sterile. Ste4 L65S is temperature-sensitive for its interaction with Ste5p, and also for signaling. We have identified a Ste5p mutant (L196A) that displays a synthetic interaction defect with Ste4 L65S, providing strong evidence that Ste4p and Ste5p interact directly in vivo through an interface that involves hydrophobic residues. The correlation between disruption of the Ste4p-Ste5p interaction and sterility confirms the importance of this interaction in signal transduction. Identification of the Gbetagamma coiled-coil in Ste5p binding may set a precedent for Gbetagamma-effector interactions in more complex organisms.     

Contrasting dynamics of water and mineral nutrients in stems shown by stable isotope tracers and cryo‐SIMS

Lateral exchange of water and nutrients between xylem and surrounding tissues helps to de‐couple uptake from utilization in all parts of a plant. We studied the dynamics of these exchanges, using stable isotope tracers for water (H₂¹⁸O), magnesium (²⁶Mg), potassium (⁴¹K) and calcium (⁴⁴Ca) delivered via a cut stem for various periods to the transpiration stream of bean shoots (Phaseolus vulgaris cv. Fardenlosa Shiny). Tracers were subsequently mapped in stem cross‐sections with cryo‐secondary ion mass spectrometry. The water tracer equilibrated within minutes across the entire cross‐section. In contrast, the nutrient tracers showed a very heterogeneous exchange between xylem vessels and the different stem tissues, even after 4 h. Dynamics of nutrients in the tissues revealed a fast and extensive exchange of nutrients in the xylem parenchyma, with, for example, calcium being completely replaced by tracer in less than 5 min. Dilution of potassium tracer during its 30 s transit in xylem sap through the stem showed that potassium concentration was up‐regulated over many hours, to the extent that some of it was probably supplied by phloem recirculation from the shoot.