Is neutrophilia the key to diagnosing appendicitis in pregnancy?

BACKGROUND: The diagnosis of acute appendicitis (AA) in pregnancy remains challenging. We investigated which preoperative clinical factors are most predictive of AA in pregnant women. METHODS: 164 pregnant patients undergoing magnetic resonance imaging for suspected AA were retrospectively reviewed. Logistic regression was used to compare those with pathologically confirmed AA and those without. RESULTS: 28 patients (17.1%) had pathologically confirmed AA. 42.9% (n = 12) were perforated at the time of operation. Factors associated with AA included history of emesis (p = 0.005), migratory abdominal pain (p = 0.006), rebound tenderness (p = 0.01), elevated white blood cell count (p = 0.003), elevated Alvarado Score (p < 0.001), elevated neutrophil count (p = 0.021), and left shift (p = 0.001). As a screening test, a left shift with neutrophils >70% provided a sensitivity and negative predictive value of 100.0%. DISCUSSION: Every patient in our series with AA had a left shift. Neutrophil count and percentage should be considered in the diagnostic evaluation of these patients to better guide resource utilization and treatment.

Harvesting of novel polyhydroxyalkanaote (PHA) synthase encoding genes from a soil metagenome library using phenotypic screening.

We previously reported the construction of metagenomic libraries in the IncP cosmid vector pRK7813, enabling heterologous expression of these broad-host-range libraries in multiple bacterial hosts. Expressing these libraries in Sinorhizobium meliloti, we have successfully complemented associated phenotypes of polyhydroxyalkanoate synthesis mutants. DNA sequence analysis of three clones indicates that the complementing genes are homologous to, but substantially different from, known polyhydroxyalkanaote synthase-encoding genes. Thus we have demonstrated the ability to isolate diverse genes for polyhydroxyalkanaote synthesis by functional complementation of defined mutants. Such genes might be of use in the engineering of more efficient systems for the industrial production of bioplastics. The use of functional complementation will also provide a vehicle to probe the genetics of polyhydroxyalkanaote metabolism and its relation to carbon availability in complex microbial assemblages.

Phospholipid association is essential for dynamin-related protein Mgm1 to function in mitochondrial membrane fusion.

Mgm1, the yeast ortholog of mammalian OPA1, is a key component in mitochondrial membrane fusion and is required for maintaining mitochondrial dynamics and morphology. We showed recently that the purified short isoform of Mgm1 (s-Mgm1) possesses GTPase activity, self-assembles into low order oligomers, and interacts specifically with negatively charged phospholipids (Meglei, G., and McQuibban, G. A. (2009) Biochemistry 48, 1774-1784). Here, we demonstrate that s-Mgm1 binds to a mixture of phospholipids characteristic of the mitochondrial inner membrane. Binding to physiologically representative lipids results in approximately 50-fold stimulation of s-Mgm1 GTPase activity. s-Mgm1 point mutants that are defective in oligomerization and lipid binding do not exhibit such stimulation and do not function in vivo. Electron microscopy and lipid turbidity assays demonstrate that s-Mgm1 promotes liposome interaction. Furthermore, s-Mgm1 assembles onto liposomes as oligomeric rings with 3-fold symmetry. The projection map of negatively stained s-Mgm1 shows six monomers, consistent with two stacked trimers. Taken together, our data identify a lipid-binding domain in Mgm1, and the structural analysis suggests a model of how Mgm1 promotes the fusion of opposing mitochondrial inner membranes.

Folding and association of thermophilic dimeric and trimeric DsrEFH proteins: Tm0979 and Mth1491.

Although the majority of natural proteins exist as protein-protein complexes, the molecular basis for the formation and regulation of such interactions and the evolution of protein interfaces remain poorly understood. We have investigated these phenomena by characterizing the thermal and chemical denaturation of thermophilic DsrEFH proteins that have a common subunit fold but distinct quaternary structures: homodimeric Tm0979 and homotrimeric Mth1491. Tm0979 forms a moderate affinity dimer, and a monomeric intermediate is readily populated at equilibrium and during folding kinetics. In contrast, the Mth1491 trimer has extremely high stability, so that a monomeric form is not measurably populated at equilibrium, although it may be during folding kinetics. A common mechanism for evolution of quaternary structures may be facile formation of a relatively stable monomeric species, with stabilizing intermolecular interactions centering on alternative environments for a beta-strand at the edge of the monomer, augmented by malleable hydrophobic interactions. The exceptional trimer stability arises from a remarkably slow unfolding rate constant, 6.5 x 10(-13) s(-1), which is a common characteristic of highly stable thermophilic and/or oligomeric proteins. The folding characteristics of Tm0979 and Mth1491 have interesting implications for assembly and regulation of homo- and heterooligomeric proteins in vivo.

The dynamin-related protein Mgm1p assembles into oligomers and hydrolyzes GTP to function in mitochondrial membrane fusion.

Mitochondrial dynamics resulting from competing membrane fusion and fission reactions are required for normal cellular function in eukaryotes. Mgm1p, a dynamin-related protein, is a key component in yeast mitochondrial fusion and is evolutionarily conserved. Previous studies suggest that Mgm1p mediates mitochondrial inner membrane fusion in a manner similar to that of other dynamin proteins that use GTP hydrolysis and oligomerization to induce structural changes in lipid bilayers; however, a direct demonstration of these activities has yet to be presented. Here we show that purified Mgm1p forms low-order oligomers that are dependent on protein concentration, suggesting a dynamic and reversible interaction. We further demonstrate that Mgm1p has GTPase activity and kinetic properties consistent with a mechanoenzyme and with a role in inner membrane mitochondrial fusion. Mutations of key residues in conserved motifs of the GTPase domain show markedly reduced or diminished GTPase activity. A mutation in the GTPase effector domain, involved in assembly and assembly-stimulated GTP hydrolysis, has basal GTPase activity similar to that of wild-type Mgm1p but has a weaker propensity to form oligomers. Finally, our data indicate that Mgm1p interacts specifically with negatively charged phospholipids found in mitochondrial membranes, and point mutations in the predicted lipid-binding domain abrogate these interactions. These findings suggest the presence of a putative lipid-binding domain, providing insight into how this protein mediates inner membrane fusion. Together, these data indicate that Mgm1p mediates fusion through oligomerization, GTP hydrolysis, and lipid binding in a manner similar to those of other dynamin mechanoenzymes.

A novel member of the YchN-like fold: solution structure of the hypothetical protein Tm0979 from Thermotoga maritima.

We report herein the NMR structure of Tm0979, a structural proteomics target from Thermotoga maritima. The Tm0979 fold consists of four beta/alpha units, which form a central parallel beta-sheet with strand order 1234. The first three helices pack toward one face of the sheet and the fourth helix packs against the other face. The protein forms a dimer by adjacent parallel packing of the fourth helices sandwiched between the two beta-sheets. This fold is very interesting from several points of view. First, it represents the first structure determination for the DsrH family of conserved hypothetical proteins, which are involved in oxidation of intracellular sulfur but have no defined molecular function. Based on structure and sequence analysis, possible functions are discussed. Second, the fold of Tm0979 most closely resembles YchN-like folds; however the proteins that adopt these folds differ in secondary structural elements and quaternary structure. Comparison of these proteins provides insight into possible mechanisms of evolution of quaternary structure through a simple mechanism of hydrophobicity-changing mutations of one or two residues. Third, the Tm0979 fold is found to be similar to flavodoxin-like folds and beta/alpha barrel proteins, and may provide a link between these very abundant folds and putative ancestral half-barrel proteins.