Nearest-neighbour transition-state analysis for nucleic acid kinetics

We used stopped-flow to monitor hypochromicity for 43 oligonucleotide duplexes to study nucleic acid kinetics and extract transition-state parameters for association and dissociation. Reactions were performed in 1.0 M NaCl (for literature comparisons) and 2.2 mM MgCl₂ (PCR conditions). Dissociation kinetics depended on sequence, increased exponentially with temperature, and transition-state parameters inversely correlated to thermodynamic parameters (r = −0.99). Association had no consistent enthalpic component, varied little with temperature or sequence, and poorly correlated to thermodynamic parameters (r = 0.28). Average association rates decreased 78% in MgCl₂ compared to NaCl while dissociation was relatively insensitive to ionic conditions. A nearest-neighbour kinetic model for dissociation predicted rate constants within 3-fold of literature values (n = 11). However, a nearest-neighbour model for association appeared overparameterized and inadequate for predictions. Kinetic predictions were used to simulate published high-speed (<1 min) melting analysis and extreme (<2 min) PCR experiments. Melting simulations predicted apparent melting temperatures increase on average 2.4°C when temperature ramp rates increased from 0.1 to 32°C/s, compared to 2.8°C reported in the literature. PCR simulations revealed that denaturation kinetics are dependent on the thermocycling profile. Simulations overestimated annealing efficiencies at shorter annealing times and suggested that polymerase interactions contribute to primer-template complex stability at extension temperatures.     

Oligosaccharides at each glycosylation site make structure-dependent contributions to biological properties of human tissue plasminogen activator.

The effect of altering oligosaccharide structures at sites 184 and 448 of tissue plasminogen activator (tPA) has been examined. Alteration to high-mannose forms at sites 184 and 448 was accomplished by the growth of cells in the presence of deoxymannojirimycin (dMM). Modification to neutral, unsialylated forms at these sites was achieved by neuraminidase treatment of control preparations of tPA. Oligosaccharides at site 117 were not markedly affected by either treatment because structures at this site are high-mannose and not sialylated in untreated preparations. The effect on enzymatic activity and on a related property, lysine affinity, was determined. dMM treatment was found to increase both the lysine affinity and catalytic activity of tPA. Neuraminidase treatment increased enzyme activity, but was without effect on affinity for lysine. To evaluate the effects of alterations at site 184 and site 448, the catalytic activity and lysine affinity of type I and type II tPA were monitored individually. In the dMM-treated sample, type I tPA (with sugars at sites 117, 184 and 448) was found to have 2- to 3-fold increased catalytic activity and an affinity for lysine which was greater than that of type I from untreated preparations, but less than that of control type II tPA (containing sugar only at sites 117 and 448). In neuraminidase-treated type I, catalytic activity was also enhanced but lysine affinity remained unchanged. Type II from dMM- and neuraminidase-treated preparations had catalytic activity that was increased approximately 1.5-fold compared to untreated controls, whereas affinity for lysine was unchanged.(ABSTRACT TRUNCATED AT 250 WORDS)     

SOME EFFECTS OF KINETIN ON THE GROWTH AND FLOWERING OF INTACT GREEN PLANTS

Wittwer , S. H., and R. R. Dedolph . (Michigan State U., E. Lansing.) Some effects of kinotin on the growth and flowering of intact green plants. Amer. Jour. Bot. 50(4): 330–336. Illus. 1963.—Dry matter accumulation of aerial parts, and heights of tomato, cucumber, and pea plants were markedly reduced when kinetin was incorporated into the culture solution root medium in concentrations ranging from 10^–5 to 10^–7 M. Concentrations which suppressed top growth (height, dry weight) generally had lesser effects on root growth and, in some instances, enhanced it. Thus top/root ratios were greatly reduced and approached unity in kinetin-treated peas and tomatoes. Flowering was inhibited in tomatoes and accelerated in peas. There were marked changes in root morphology, including the formation of pseudonodules. Kinetin had an effect which was opposite to that of gibberellin on internode elongation, root extension, top/root ratios and flowering of dwarf peas. N⁶-benzyladenine was more active than kinetin in suppressing the growth of intact green plants. The data show that kinetin can markedly alter the behavior of intact plants when absorbed by the roots from culture solutions in which the concentrations are comparable to those which are biologically active on explants.     

Nutrient element accumulation and distribution in an intensively cultured American sycamore plantation

Summary American sycamore (Platanus occidentalis L.) plantations were intensively cultured on floodplain and terrace sites near the Ohio River in western Kentucky. Biomass and nutrient uptake under various spacing and fertilizer treatments were investigated by total tree analysis techniques. Nutrient element concentrations of sample trees were affected by site and fertilizer treatment but not by spacing. The order of nutrient concentrations were: N> Ca>K>Mg>P>Mn. Branches and foliage of saplings harvested 3 growing seasons after planting accounted for 45 to 70 per cent of the nutrient elements found in the above-ground tree parts. Comparison of the estimated annual nutrient removals with estimated annual inputs from precipitation, mineral weathering and mineralization of organic matter indicates that removal of the total above-ground tree components can have an important effect on soil nutrient reserves.The investigation reported in this paper (76-8-129) is in connection with a project of the Kentucky Agricultural Experiment Station and is published with approval of the Director.The authors are Graduate Assistant, Michigan State University; Assistant Professor and Associate Professor, Department of Forestry University of Kentucky.The authors are Graduate Assistant, Michigan State University; Assistant Professor and Associate Professor, Department of Forestry University of Kentucky.     

Human DNA topoisomerase II: evaluation of enzyme activity in normal and neoplastic tissues

We have used both a quantitative filter binding assay and a decatenation assay to measure DNA topoisomerase II activity. The filter binding assay, which measures catenating activity, is able to detect topoisomerase II activity at 50-100-fold lower protein concentrations than the decatenation assay. Because of this remarkable sensitivity, we have been able to quantitate topoisomerase II activity in a variety of normal and neoplastic human tissues. The highest level of enzyme activity in normal tissues was found in the spleen and thymus. The highest level of enzyme activity in neoplasms was found in those that clinically behave in an aggressive manner and had a high proliferative status by flow cytometry. Surprisingly, these high topoisomerase II values in the neoplastic specimens are in the same range of values found in normal nonproliferating tissue. Since much previous data indicate that the enzyme is apparently a property of only proliferating cells, this finding might suggest that human tissues contain more than one form of the enzyme. The finding that 35-65% of the topoisomerase II activity in human tissues is resistant to teniposide suggests that more than one enzyme form exists.