In cellulo crystallization of Trypanosoma brucei IMP dehydrogenase enables the identification of genuine co-factors.

Sleeping sickness is a fatal disease caused by the protozoan parasite Trypanosoma brucei (Tb). Inosine-5'-monophosphate dehydrogenase (IMPDH) has been proposed as a potential drug target, since it maintains the balance between guanylate deoxynucleotide and ribonucleotide levels that is pivotal for the parasite. Here we report the structure of TbIMPDH at room temperature utilizing free-electron laser radiation on crystals grown in living insect cells. The 2.80 Å resolution structure reveals the presence of ATP and GMP at the canonical sites of the Bateman domains, the latter in a so far unknown coordination mode. Consistent with previously reported IMPDH complexes harboring guanosine nucleotides at the second canonical site, TbIMPDH forms a compact oligomer structure, supporting a nucleotide-controlled conformational switch that allosterically modulates the catalytic activity. The oligomeric TbIMPDH structure we present here reveals the potential of in cellulo crystallization to identify genuine allosteric co-factors from a natural reservoir of specific compounds.

Real-time investigation of dynamic protein crystallization in living cells.

X-ray crystallography requires sufficiently large crystals to obtain structural insights at atomic resolution, routinely obtained in vitro by time-consuming screening. Recently, successful data collection was reported from protein microcrystals grown within living cells using highly brilliant free-electron laser and third-generation synchrotron radiation. Here, we analyzed in vivo crystal growth of firefly luciferase and Green Fluorescent Protein-tagged reovirus µNS by live-cell imaging, showing that dimensions of living cells did not limit crystal size. The crystallization process is highly dynamic and occurs in different cellular compartments. In vivo protein crystallization offers exciting new possibilities for proteins that do not form crystals in vitro.

Effect of media composition on yield values of bacteria growing on binary and ternary substrate mixtures in continuous culture.

Pseudomonas aeruginosa 142 and a presumed variant were grown axenically in chemostats on salicylate/benzoate or salicylate/glucose binary feeds. Each substrate was supplied at 2, 10, 50, 90, 98, or 100% of the total energy flux. Two experiments were also run with ternary mixtures using the same substrates. Aliquots were transferred to fed-batch reactors receiving the same substrates at the same specific rates as the chemostat, but with one substrate radiolabeled with 14C. Radiolabel incorporated into biomass, 14CO2, and soluble microbial products over a period of 8 minutes was used to establish the biomass yield, CO2 yield, and product yield, respectively, associated with a given substrate. The effect of the percent substrate in the feed on the yields depended on the pair of substrates supplied. When benzoate comprised 50% or more of the applied substrate in salicylate/benzoate feeds, the fraction of benzoate in the feed had a small effect on the yield values associated with benzoate. However, when benzoate constituted 2% or 10% of the feed, CO2 yields were lower, biomass yields were slightly lower, and product yields were higher. In contrast, the percent of salicylate in the feed had little effect on any of the salicylate yields for cells growing on the salicylate/benzoate feeds. When salicylate was mixed with glucose, the yields associated with salicylate behaved quite differently. Biomass and CO2 yields were lower and product yields higher when salicylate was 2% or 10% of the feed than when it was higher. In the same substrate mixtures, glucose-based biomass yields were higher and CO2 yields were lower when glucose constituted 2% or 10% of the feed but were constant for higher percentages. The results suggest that the fate of a substrate is relatively independent of the feed composition as long as the substrate in question constitutes a significant percentage of the mixture. Thus, in those situations the assumption of a constant biomass yield in multicomponent substrate modeling is justified. However, when a given substrate constitutes a small percentage of the feed, significant changes in yield may occur.

The reliability of radioactive phosphorus (32P) in the diagnosis of intraocular tumors; experience with 912 patients.

The 32P uptake test used in the differential diagnosis of 912 cases yielded an accuracy rate of 94.82% for intraocular lesions behind the equator, and 85.15% for lesions of the anterior uvea. The highest error rate occurred in patients with suspected tumors of the iris and ciliary body. It seems advisable to take a 40% difference on the uptake as the limit between a positive or negative test result.

Difficult issues in health planning, development, and review.

To give an overview of the kinds of issues expected to be encountered by the new Health Systems Agencies being established in conformity with Public Law 93-641, the authors draw from the experiences of the Comprehensive Health Planning agency and Regional Medical Program of an Upstate New York area. Problems faced together in the years 1972 to 1975 in health planning, development, review, and public policy are described. The unusual geographic and working relationship of these agencies makes them, together, a useful prototype of the HSA to examine.