An Emerging Approach for Parallel Quantification of Intracellular Protozoan Parasites and Host Cell Characterization Using TissueFAXS Cytometry

Characterization of host-pathogen interactions is a fundamental approach in microbiological and immunological oriented disciplines. It is commonly accepted that host cells start to change their phenotype after engulfing pathogens. Techniques such as real time PCR or ELISA were used to characterize the genes encoding proteins that are associated either with pathogen elimination or immune escape mechanisms. Most of such studies were performed in vitro using primary host cells or cell lines. Consequently, the data generated with such approaches reflect the global RNA expression or protein amount recovered from all cells in culture. This is justified when all host cells harbor an equal amount of pathogens under experimental conditions. However, the uptake of pathogens by phagocytic cells is not synchronized. Consequently, there are host cells incorporating different amounts of pathogens that might result in distinct pathogen-induced protein biosynthesis. Therefore, we established a technique able to detect and quantify the number of pathogens in the corresponding host cells using immunofluorescence-based high throughput analysis. Paired with multicolor staining of molecules of interest it is now possible to analyze the infection profile of host cell populations and the corresponding phenotype of the host cells as a result of parasite load.     

A rRNA-mRNA base pairing model for UGA-dependent termination.

A series of site-directed mutations has been constructed in E coli 16S rRNA and shown to suppress UGA-dependent translational termination. With the exception of the C726 to G base change, all were constructed in helix 34. Characterization of these mutations is reviewed here and from these data and mRNA-rRNA base pairing model for the termination event is presented. The interaction functions via antiparallel base pairing between either 1 of the 2 UCA motifs in helix 34 and the complementary UGA stop codon on the message, thus forming a quasicontinuous A-type helical structure that is further stabilized by stacking enthalpy. Finally, rRNA motifs potentially required for UAA and UAG-dependent translational termination are discussed.     

TMTC1 and TMTC2 Are Novel Endoplasmic Reticulum Tetratricopeptide Repeat-containing Adapter Proteins Involved in Calcium Homeostasis

The endoplasmic reticulum (ER) is organized in part by adapter proteins that nucleate the formation of large protein complexes. Tetratricopeptide repeats (TPR) are well studied protein structural motifs that support intermolecular protein-protein interactions. TMTC1 and TMTC2 were identified by an in silico search as TPR-containing proteins possessing N-terminal ER targeting signal sequences and multiple hydrophobic segments, suggestive of polytopic membrane proteins that are targeted to the secretory pathway. A variety of cell biological and biochemical assays was employed to demonstrate that TMTC1 and TMTC2 are both ER resident integral membrane proteins with multiple clusters of TPR domains oriented within the ER lumen. Proteomic analysis followed by co-immunoprecipitation verification found that both proteins associated with the ER calcium uptake pump SERCA2B, and TMTC2 also bound to the carbohydrate-binding chaperone calnexin. Live cell calcium measurements revealed that overexpression of either TMTC1 or TMTC2 caused a reduction of calcium released from the ER following stimulation, whereas the knockdown of TMTC1 or TMTC2 increased the stimulated calcium released. Together, these results implicate TMTC1 and TMTC2 as ER proteins involved in ER calcium homeostasis.     

Asymmetrical Effect of Levodopa on the Neural Activity of Motor Regions in PD

Parkinson''s disease (PD) is a neurodegenerative illness often characterized by asymmetrical symptoms. However, the reason for this asymmetry and the cerebral correlates underlying symptom asymmetry are still not well understood. Furthermore, the effects of levodopa on the cerebral correlates of disease asymmetry have not been investigated. In this study, right-handed PD patients performed self-initiated, externally triggered and repetitive control finger movements with both their right and left hands during functional magnetic resonance imaging (fMRI) to investigate asymmetrical effects of levodopa on the hemodynamic correlates of finger movements. Patients completed two experimental sessions OFF and ON medication after a minimum of 12 hours medication withdrawal. We compared the effect of levodopa on the neural activation patterns underlying the execution of both the more affected and less affected hand for self-initiated and externally triggered movements. Our results show that levodopa led to larger differences in cerebral activity for movements of the more affected, left side: there were significant differences in activity after levodopa administration in regions of the motor cortico-striatal network when patients performed self-initiated and externally triggered movements with their left hand. By contrast, when patients used their right hand, levodopa led to differences in cerebellar activity only. As our patients were affected more severely on their left side, we propose that levodopa may help provide additional dopaminergic input, improving movements for the more severely affected side. These results suggest that the impact of reduced dopamine in the cortico-striatal system and the action of levodopa is not symmetrical.     

Negative ion ammonia chemical ionization and electron impact ionization mass spectrometric analysis of steryl fatty acyl esters

Synthesis of steryl palmitates, varied in the nature of the steryl moiety, provided model compounds for investigation of the mass spectrometric behavior of steryl long-chain fatty acyl esters. The structure of the steryl moiety was varied according to: (i) position and degree of unsaturation in the steroid nucleus and C-17 side-chain, (ii) position and degree of methylation, (iii) presence or absence of a 9 beta, 19-cyclopropane ring. Compounds were chosen so as to be representative of biochemically important steryl esters. Electron impact (EI) behavior of steryl palmitate esters closely resembles that of their short-chain (e.g. acetate) counterparts. M+.ions were generally weak or absent and the major high mass ions arose from characteristic fragmentations of the steroid nucleus following loss of the acyl moiety ([M-RCO2H]+.). Fragment ions characteristic of the acyl moiety were lacking. Negative ion chemical ionization (NICI) using ammonia as reagent gas, on the other hand, afforded spectra containing characteristic fragment ions [RCO2]-, [RCO2-18]-, and [RCO2-19]- from which the nature of the fatty acyl moiety can be readily deduced. Hence, NICI and EI provide complementary means of ionization for the mass spectrometric determination of structures of steryl esters.     

Pulsed-field gel electrophoresis for genotypic comparison of Rhizobium bacteria that nodulate leguminous trees

Abstract Pulsed-field gel electrophoresis (PFGE) was applied to characterize Rhizobium bacteria isolated from the root nodules of Acacia senegal and Prosopis chilensis trees growing in Sudan and Keya. For the electrophoresis, the total DNA of 42 isolates, embedded in agarose, was digested by a rare-cutting restriction endonuclease, Xba I. The PFGE run resulted in good resolution of the DNA fragments and gave the strains distinctive fingerprint patterns. The patterns were analysed visually and using automated clustering analysis, which divided the strains into groups resembling the results generated by numerical taxonomy. However, several strains had unique banding patterns, which indicates that these strains are genetically very diverse.