Glutamate dehydrogenase from the hyperthermophile Pyrococcus furiosus. Thermal denaturation and activation.

Pyrococcus furiosus is a marine hyperthermophile that grows optimally at 100 degrees C. Glutamate dehydrogenase (GDH) from P. furiosus is a hexamer of identical subunits and has an M(r) = 270,000 +/- 5500 at 25 degrees C. Electron micrographs showed that the subunit arrangement is similar to that of GDH from bovine liver (i.e. 3/2 symmetry in the form of a triangular antiprism). However, GDH from P. furiosus is inactive at temperatures below 40 degrees C and undergoes heat activation above 40 degrees C. Both NAD+ and NADP+ are utilized as cofactors. Apparently the inactive enzyme also binds cofactors, since the enzyme maintains the ability to bind to an affinity column (Cibacron blue F3GA) and is specifically eluted with NADP+. Conformational changes that accompany activation and thermal denaturation were detected by precision differential scanning microcalorimetry. Thermal denaturation starts at 110 degrees C and is completed at 118 degrees C. delta(cal) = 414 Kcal [mol GDH]-1. Tm = 113 degrees C. This increase in heat capacity indicates an extensive irreversible unfolding of the secondary structure as evidenced also by a sharp increase in absorbance at 280 nm and inactivation of the enzyme. The process of heat activation of GDH from 40 to 80 degrees C is accompanied by a much smaller increase in absorbance at 280 nm and a reversible increase in heat capacity with delta(cal) = 187 Kcal [mol GDH]-1 and Tm = 57 degrees C. This absorbance change as well as the moderate increase in heat capacity suggest that thermal activation leads to some exposure of hydrophobic groups to solvent water as the GDH structure is opened slightly. The increase in absorbance at 280 nm during activation is only 12% of that for denaturation. Overall, GDH appears to be well adapted to correspond with the growth response of P. furiosus to temperature.

Microbial manganese reduction mediated by bacterial strains isolated from aquifer sediments.

One hundred and five strains isolated from aquifer sediments andEscherichia coli ML30S were tested for their ability to reduce manganese oxides. Eighty-two strains, includingE. coli, reduced manganese. In most cases the bacterial activity decreased the pH and Eh below 6.75 and 350 mV, respectively, enhancing a spontaneous and nonspecific reduction of manganese. However, for 12 strains the reduction was specifically catalyzed by bacteria; the high pH and Eh values would not permit a spontaneous reduction of manganese. Some of the most active strains were identified as genera common in soils and waters, i.e.,Pseudomonas, Bacillus, Corynebacterium, andAcinetobacter. Two strains were studied in detail. One of the strains, identified asPseudomonas fluorescens, required contact between the cells and the manganese oxides for reduction to occur. The reduction was inhibited by 15 mM of sodium azide. The other strain, identified asAcinetobacter johnsonii, catalyzed manganese reduction by an inductive and dialyzable substance which was excreted by the bacteria. The mechanism involved has not been previously demonstrated.

Cancer of the piriform sinus: treatment by radiation therapy alone and with surgery.

To ascertain the optimal treatment for carcinoma of the piriform sinus, the authors determined survival rates and local and regional tumor growth for two groups of patients: those treated by radiation therapy alone (n = 209) and those treated by radiation therapy combined with surgery (n = 154). The two groups were similar with respect to the characteristics of primary tumor stage and degree of nodal involvement. The overall 3-year and 5-year actuarial survival rates were 19.2% and 15.5%, respectively. For 5-year actuarial survival, there was no significant difference between patients with T1 and T2 tumors, but there was a significant difference between patients with T1 + T2 tumors versus those with T3 tumor. There was no significant difference in 3- and 5-year survival between patients with N0 and N1 nodal involvement and those with N1 and N2 involvement, but there was a significant difference between patients with N0 versus those with N3 involvement. The 5-year actuarial survival rate is significantly better for patients who underwent surgery followed by radiation therapy than for those who received only radiation therapy. However, for patients with early-stage (T1 and T2) tumors, radiation therapy alone controls local tumor growth as well as the combination of surgery and radiation therapy does. For each treatment group, the causes of death and patterns of failure were studied and compared with investigations to date.