Phylogeny to function: PE/PPE protein evolution and impact on Mycobacterium tuberculosis pathogenicity

The pe/ppe genes represent one of the most intriguing aspects of the Mycobacterium tuberculosis genome. These genes are especially abundant in pathogenic mycobacteria, with more than 160 members in M. tuberculosis. Despite being discovered over 15 years ago, their function remains unclear, although various lines of evidence implicate selected family members in mycobacterial virulence. In this review, we use PE/PPE phylogeny as a framework within which we examine the diversity and putative functions of these proteins. We report on the evolution and diversity of the respective gene families, as well as the implications thereof for function and host immune recognition. We summarize recent findings on pe/ppe gene regulation, also placing this in the context of PE/PPE phylogeny. We collate data from several large proteomics datasets, providing an overview of PE/PPE localization, and discuss the implications this may have for host responses. Assessment of the current knowledge of PE/PPE diversity suggests that these proteins are not variable antigens as has been so widely speculated; however, they do clearly play important roles in virulence. Viewing the growing body of pe/ppe literature through the lens of phylogeny reveals trends in features and function that may be associated with the evolution of mycobacterial pathogenicity.     

Clinical assay of the human erythrocyte lactate transporter. II. Analysis and display of normal human data

We have applied a lactate efflux assay to human red cells at two temperatures and with initial lactic acid loads up to 8 mM, metabolically generated. Efflux was about 1.5 times faster at external pH of 8.5 than at 7.5; the latter was the standard pH used thereafter. Multiple lactate loads in a single blood specimen demonstrated clear evidence of saturation kinetics at both pH levels, since the efflux rate did not increase proportionally with the lactate load. Best-fitting rectangular hyperboles were determined for 129-131 assays from 43 volunteers at 20 degrees and 30 degrees. In most cases high and low lactate loads permitted a two-point evaluation of saturation kinetics, and a positive indication was obtained in 88 of 89 tests. The apparent efflux Km and Vm values may be influenced by pH as well as by lactate levels and cannot be taken as rigorous, although they agree reasonably well with literature data on influx and exchange velocities. The data displayed a Hill constant of 1, a 30 degrees/20 degrees velocity ratio of 2.7, and no significant clustering by sex or age. A single assay with initial lactate level above 5 mM at 30 degrees should be sufficient to identify cases with a defective transporter, using the 95% tolerance limits developed in this report.     

Expression of the alpha 1-proteinase inhibitor gene family during evolution of the genus Mus

alpha 1-Proteinase inhibitors (alpha 1-PIs) are members of the serpin superfamily of proteinase inhibitors, and are important in the maintenance of homeostasis in a wide variety of animal taxa. Previous studies have shown that in mice (genus Mus), evolution of alpha 1-PIs is characterized by gene amplification, region-specific concerted evolution, and rapid accumulation of amino acid substitutions. The latter occurs primarily in the reactive center, which is the region of the alpha 1-PI molecule that determines the inhibitor's specificity for target proteinases. The P1 residue within the reactive center, which is methionine in so-called orthodox alpha 1-PIs and an amino acid other than methionine in unorthodox alpha 1-PIs, is a primary determinant of inhibitor specificity. In the present study, we find that the expression of mRNAs encoding unorthodox alpha 1-PIs is polymorphic within Mus species, i.e., among individuals or inbred strains. This is in striking contrast to mRNAs that encode orthodox alpha 1-PIs, whose concentrations are relatively invariant. The intraspecies variations in mRNA expression represent polymorphisms in the structure of the alpha 1- PI gene family. The results, taken together with previously described aspects of alpha 1-PI evolution, indicate that the dissimilar levels of polymorphism exhibited by orthodox and unorthodox alpha 1-PIs, which likely have distinct physiological functions, may reflect different levels of selective constraint. The significance of this finding to the evolution of gene families is discussed.      

Electrical connections between left superior pulmonary vein, left atrium, and ligament of Marshall: implications for mechanisms of atrial fibrillation

The importance of the ligament of Marshall (LOM) to rapid activations within the left superior pulmonary vein (LSPV) during atrial fibrillation (AF) remains poorly understood. We aimed to characterize the importance of electrical coupling between the LSPV with the left atrium (LA) and the LOM in the generation of high-frequency activations within this PV. We performed high-density mapping of the LSPV-LA-LOM junction in eight dogs, using 1,344 electrodes with a 1-mm resolution before and after posterior ostial ablation to diminish PV-LA electrical connections. A LOM potential was recordable up to 6.5 mm (SD 2.2) into the LSPV in all dogs during sinus rhythm (SR) and LA pacing. Functional LOM-LSPV electrical connections bypassing the PV-LA junction were present in five of eight dogs. Direct LOM-LSPV connections contributed to 46.5% (SD 16.0) of LSPV activations during AF, resulting in a greater propensity to develop focal activations (P < 0.05) and a higher activation rate during AF of LSPVs with direct LOM connections compared with those without (P < 0.03). Posterior LSPV ostial ablation without damaging the anterior wall or LOM slowed residual LA-PV conduction (P < 0.001). This diminished PV-LA coupling prevented the reinduction of LSPV focal activations in all dogs. However, persistent LOM focal activations in two dogs continued to activate the LSPV rapidly [cycle length 151.8 ms (SD 4.8)] via direct LOM-LSPV connections. LOM-LSPV connection forms an accessory pathway that contributes to the electrical coupling between LSPV and LA during SR and AF. This pathway may contribute to rapid activations within the LSPV during AF.