Energetics of mice in stable and unstable social conditions: Evidence of an air-borne factor affecting metabolism

The metabolic rates of laboratory mice were compared in three conditions: isolated mice, mice paired together over six days (stable groups), and mice paired with strange partners daily (unstable groups). Stable pairs had 15% lower metabolic rates than either isolated or unstable pairs. In other experiments when two mice were placed in separate metabolic chambers and connected together via an air flow, the metabolic rate of the recipient in the series was 35% lower than the donor. The data suggest that a ‘factor’ produced by the donor mouse was passed via the air supply into the recipient's chamber.     

Calcium and magnesium in Mueller-Hinton agar and their influence on disk diffusion susceptibility results

Concentrations of calcium and magnesium were determined for 39 lots of media, including broth and agar media used for susceptibility tests and plain agar. In addition, the effect that media with and without physiological levels of these divalent cations would have on the disk diffusion susceptibility of 21 strains ofPseudomonas aeruginosa to four antimicrobics was also ascertained. Mueller-Hinton agar showed a wide variation in calcium and magnesium content. Mueller-Hinton broth contained lower concentrations of calcium and magnesium, and there was little lot-to-lot variation. Lots of Mueller-Hinton agar with higher concentrations of calcium and magnesium yielded smaller zone diamaters than those with lower concentrations. Even at equal cation concentration, zones of inhibition varied from lot to lot. Since the addition of calcium and magnesium to Mueller-Hinton agar to obtain a predetermined level did not result in equivalent zone diameters, performance testing of susceptibility media is recommended.     

Candidate protein selection for diagnostic markers of African trypanosomiasis

The ability to accurately diagnose the presence of an infective micro-organism is not only important for individual human and animal health and wellbeing, but is also central to surveillance programmes. Effective and sustainable control of many diseases in the developing world depends on the availability of field applicable diagnostics that are cheap, reliable, simple in design and application, and which provide immediate results. This review examines how the genome sequences can be used in the selection of potential candidate proteins for developing new serodiagnostics for African trypanosomiasis.     

RNAi-mediated depletion of the 15 KH domain protein, vigilin, induces death of dividing and non-dividing human cells but does not initially inhibit protein synthesis

Vigilin/Scp160p/DDP1 is a ubiquitous and highly conserved protein containing 15 related, but non-identical, K-homology (KH) nucleic acid binding domains. While its precise function remains unknown, proposed roles for vigilin include chromosome partitioning at mitosis, facilitating translation and tRNA transport, and control of mRNA metabolism, including estrogen-mediated stabilization of vitellogenin mRNA. To probe sites of vigilin action in vertebrate cells, we performed nucleic acid binding and RNA interference studies. Consistent with a potential role in chromosome partitioning, human vigilin exhibits a higher affinity for Drosophila dodecasatellite single-stranded DNA than for vitellogenin mRNA 3′-UTR. Direct observation and flow cytometry in non-mitotic, serum-starved, HeLa cells showed that RNAi-mediated vigilin knockdown is rapidly lethal, indicating an essential function for vigilin distinct from its proposed role in chromosome partitioning. Pulse labeling experiments revealed that rates of protein synthesis and degradation are unaffected by the several fold reduction in vigilin levels early in siRNA knockdown indicating that vigilin is not a global regulator of translation. These data show that vigilin is an essential protein in human cells, support the view that vigilin’s most essential functions are neither chromosome partitioning nor control of translation, and are consistent with vigilin playing a critical role in cytoplasmic mRNA metabolism.     

Mutation of an essential glutamate residue in folylpolyglutamate synthetase and activation of the enzyme by pteroate binding

Site-directed mutagenesis was performed on Glu143, an essential amino acid in Lactobacillus casei folylpolyglutamate synthetase (FPGS) and the structurally equivalent residue, Glu146, in Escherichia coli FPGS. Glu143 is positioned near the P-loop and interacts with the Mg(2+) of Mg NTP-binding proteins. We have solved the structure of the E143A mutant of L. casei FPGS in the presence of AMPPCP and Mg(2+). The structure showed a water molecule at the place where Mg(2+) bound to the wild type enzyme. Mutant proteins E143A, and even E143D and E143Q with conservative mutations, lacked enzyme activity and failed to complement the methionine auxotrophy of the E. coli folC mutant SF4, showing that Glu143 is an essential residue. Both the L. casei and the E. coli FPGS mutant proteins bound methylene-tetrahydrofolate diglutamate and dihydropteroate normally. The E. coli E146Q mutant FPGS bound ADP with the same affinity as the wild type enzyme but bound ATP with much lower affinity and had higher ATPase activity than the wild type enzyme. The mutant enzyme was defective in forming the acyl-phosphate reaction intermediate from ATP and dihydropteroate. The E. coli FPGS requires activation by dihydropteroate or tetrahydrofolate binding to allow full activity. In the absence of a pteroate substrate, only 30% of the total enzyme binds ATP. We suggest that dihydropteroate causes a conformational change to allow increased ATP binding. The mutant enzyme was similarly activated by dihydropteroate resulting in increased ADP binding.