Detection of cross-links between FtsH, YidC, HflK/C suggests a linked role for these proteins in quality control upon insertion of bacterial inner membrane proteins

Little is known about the quality control of proteins upon integration in the inner membrane of Escherichia coli. Here, we demonstrate that YidC and FtsH are adjacent to a nascent, truncated membrane protein using in vitro photo cross-linking. YidC plays a critical but poorly understood role in the biogenesis of E. coli inner membrane proteins (IMPs). FtsH functions as a membrane chaperone and protease. Furthermore, we show that FtsH and its modulator proteins HflK and HflC copurify with tagged YidC and, vice versa, that YidC copurifies with tagged FtsH. These results suggest that FtsH and YidC have a linked role in the quality control of IMPs.     

Effect of lecithin and MgCO3 as additives on the enzymatic activity of carbonic anhydrase encapsulated in poly(lactide-co-glycolide) (PLGA) microspheres

A model enzyme, carbonic anhydrase, was encapsulated and released from poly(lactide-co-glycolide) (PLGA) microspheres (1-3 microm) made by a novel phase inversion technique. Lecithin was used as a surfactant in the encapsulation process and was incorporated in either the organic phase, aqueous phase, both phases, or not at all. Additional microspheres were also made with lecithin incorporated in the aqueous phase and a basic salt, MgCO3, in the polymeric phase. Released carbonic anhydrase, protein extracted from microspheres, or enzyme incubated with lecithin and PLGA were analyzed via HPLC and activity assay to determine the effect of these additives on protein integrity and activity. Lecithin in the aqueous phase appeared to increase the fraction of enzyme in monomeric form as well as its activity for both extracted protein and released protein as compared to the other formulations without MgCO3. Incubation of enzyme with PLGA degradation products indicated that the acidic environment within the microspheres aids in the irreversible inactivation of the encapsulated protein. Addition of MgCO3 further increased the amount of monomer in both the extracted and released protein by decreasing the amount of acid-induced cleavage and noncovalent aggregation, but still greatly decreased the activity of the enzyme.     

Disturbance and Seasonal Dynamics of Mycorrhizae in a Tropical Deciduous Forest in Mexico1

Mycorrhizal fungi were sampled in a deciduous tropical forest on the Pacific coast of Mexico during different seasons and in natural treefall gaps and pastures. All 12 plant species sampled in the forest were arbuscular mycorrhizal. The percent root infection and spore production were closely related to the phenology of the plants. Most tree species and all herbaceous species had the highest infection in the summer rainy season, but two species, Opuntia excelsa and Jacquinia pungens, had highest infection in the dry season. Unusually high rainfall during the dry season was associated with increased infection but not increased spore production. Spore density was low for all species at all sample times, except at the beginning of the July 1993 rainy season in, when we observed up to 28 spores/g soil. The percent cover of shrubs or herbs did not increase in gaps after two years, and we observed no colonizing seedlings. No plant species with cover higher than 2.7 percent occurred exclusively in gaps or forest. The percent mycorrhizal infection did not differ significantly between gaps and forest. Spore counts were as high in the gaps as in the forest in two of the three gaps but lower in the third gap. The lack of significant response of plants in these gaps after two years differed from the rapid response in tropical rainforests. It is likely related to the small size of the gaps and to light infiltration to the forest floor. Pastures were dominated by two species of exotic grasses and one species of mycorrhizal fungus, whereas forests had 15 fungal species. The slow regrowth of vegetation in gaps was not limited by mycorrhizal fungi, since they were still abundant after the treefalls, but recovery in pastures could be affected by low fungal diversity and dominance of grasses.     

Multiplex Real-Time PCR Assay Using TaqMan Probes for the Identification of Trypanosoma cruzi DTUs in Biological and Clinical Samples

Background

Trypanosoma cruzi has been classified into six Discrete Typing Units (DTUs), designated as TcI–TcVI. In order to effectively use this standardized nomenclature, a reproducible genotyping strategy is imperative. Several typing schemes have been developed with variable levels of complexity, selectivity and analytical sensitivity. Most of them can be only applied to cultured stocks. In this context, we aimed to develop a multiplex Real-Time PCR method to identify the six T. cruzi DTUs using TaqMan probes (MTq-PCR).

Methods/Principal Findings

The MTq-PCR has been evaluated in 39 cultured stocks and 307 biological samples from vectors, reservoirs and patients from different geographical regions and transmission cycles in comparison with a multi-locus conventional PCR algorithm. The MTq-PCR was inclusive for laboratory stocks and natural isolates and sensitive for direct typing of different biological samples from vectors, reservoirs and patients with acute, congenital infection or Chagas reactivation. The first round SL-IR MTq-PCR detected 1 fg DNA/reaction tube of TcI, TcII and TcIII and 1 pg DNA/reaction tube of TcIV, TcV and TcVI reference strains. The MTq-PCR was able to characterize DTUs in 83% of triatomine and 96% of reservoir samples that had been typed by conventional PCR methods. Regarding clinical samples, 100% of those derived from acute infected patients, 62.5% from congenitally infected children and 50% from patients with clinical reactivation could be genotyped. Sensitivity for direct typing of blood samples from chronic Chagas disease patients (32.8% from asymptomatic and 22.2% from symptomatic patients) and mixed infections was lower than that of the conventional PCR algorithm.

Conclusions/Significance

Typing is resolved after a single or a second round of Real-Time PCR, depending on the DTU. This format reduces carryover contamination and is amenable to quantification, automation and kit production.     

Environmental scanning electron microscopy (ESEM)—a versatile tool in studying plants

Environmental scanning electron microscopy (ESEM) enables the investigation of hydrated and uncoated plant samples and the in situ observation of dynamic processes. Water vapor in the microscope chamber takes part in secondary electron detection and charge prevention. Two ESEM modes are available and offer a broad spectrum of applications. The environmental or wet mode prevents sample dehydration by the combination of sample cooling (5°C) and a vapor pressure of 4–6 Torr. In the low vacuum mode, the maximum chamber pressure is limited to 1 Torr (corresponding to about 5% relative humidity in the chamber) and allows the simultaneous use of a backscattered electron detector for imaging material contrast. A selection of characteristic plant samples and various applications are presented as a guide to ESEM for plant scientists. Leaf surfaces, trichomes, epicuticular waxes, and inorganic surface layers represent samples being comparatively resistant to dehydration, whereas callus cells and stigmatic tissue are examples for dehydration- and beam-sensitive samples. The potential of investigating dynamic processes in situ is demonstrated by studying anther opening, by tensile testing of leaves, and by performing hydration/dehydration experiments by changing the vapor pressure. Additionally, automated block-face imaging and serial sectioning using in situ ultramicrotomy is presented. The strengths and weaknesses of ESEM are discussed and it is shown that ESEM is a versatile tool in plant science.     

The structure of arylamine N-acetyltransferase from Mycobacterium smegmatis--an enzyme which inactivates the anti-tubercular drug,isoniazid

Arylamine N-acetyltransferases which acetylate and inactivate isoniazid, an anti-tubercular drug, are found in mycobacteria including Mycobacterium smegmatis and Mycobacterium tuberculosis. We have solved the structure of arylamine N-acetyltransferase from M. smegmatis at a resolution of 1.7 A as a model for the highly homologous NAT from M. tuberculosis. The fold closely resembles that of NAT from Salmonella typhimurium, with a common catalytic triad and domain structure that is similar to certain cysteine proteases. The detailed geometry of the catalytic triad is typical of enzymes which use primary alcohols or thiols as activated nucleophiles. Thermal mobility and structural variations identify parts of NAT which might undergo conformational changes during catalysis. Sequence conservation among eubacterial NATs is restricted to structural residues of the protein core, as well as the active site and a hinge that connects the first two domains of the NAT structure. The structure of M. smegmatis NAT provides a template for modelling the structure of the M. tuberculosis enzyme and for structure-based ligand design as an approach to designing anti-TB drugs.