Using secretion to solve a solubility problem: high-yield expression in Escherichia coli and purification of the bacterial glycoamidase PNGase F.

PNGase F is a widely used deglycosidase, secreted in small amounts by the gram-negative bacterium Flavobacterium meningosepticum. We have designed a T7 promoter-based Escherichia coli expression system to provide a high-yield source of recombinant enzyme. When expressed intracellularly, the enzyme was produced in a largely insoluble state. However, when expressed as a fusion with the leader sequence from the ompA gene, hexahistidine-tagged PNGase F was efficiently processed and exported to the E. coli periplasm. Single-step purification using immobilized metal affinity chromatography yielded 8 mg of pure enzyme per liter of culture, which is fully active on a range of protein and peptide substrates.     

Mitochondrial DNA variation in human evolution and disease

Analysis of mitochondrial DNA (mtDNA) variation has permitted the reconstruction of the ancient migrations of women. This has provided evidence that our species arose in Africa about 150000 years before present (YBP), migrated out of Africa into Asia about 60000 to 70000 YBP and into Europe about 40000 to 50000 YBP, and migrated from Asia and possibly Europe to the Americas about 20000 to 30000 YBP. Although much of the mtDNA variation that exists in modern populations may be selectively neutral, studies of the mildly deleterious mtDNA mutations causing Leber's hereditary optic neuropathy (LHON) have demonstrated that some continent-specific mtDNA lineages are more prone to manifest the clinical symptoms of LHON than others. Hence, all mtDNA lineages are not equal, which may provide insights into the extreme environments that were encountered by our ancient ancestor, and which may be of great importance in understanding the pathophysiology of mitochondrial disease.     

Correction to: Metabolic growth hypothesis for the evolution of the nuchal hump in swordtail fishes

Environmental Biology of Fishes -     

Structure of the bacterial type II NADH dehydrogenase: a monotopic membrane protein with an essential role in energy generation

Non‐proton pumping type II NADH dehydrogenase (NDH‐2) plays a central role in the respiratory metabolism of bacteria, and in the mitochondria of fungi, plants and protists. The lack of NDH‐2 in mammalian mitochondria and its essentiality in important bacterial pathogens suggests these enzymes may represent a potential new drug target to combat microbial pathogens. Here, we report the first crystal structure of a bacterial NDH‐2 enzyme at 2.5 Å resolution from Caldalkalibacillus thermarum. The NDH‐2 structure reveals a homodimeric organization that has a unique dimer interface. NDH‐2 is localized to the cytoplasmic membrane by two separated C‐terminal membrane‐anchoring regions that are essential for membrane localization and FAD binding, but not NDH‐2 dimerization. Comparison of bacterial NDH‐2 with the yeast NADH dehydrogenase (Ndi1) structure revealed non‐overlapping binding sites for quinone and NADH in the bacterial enzyme. The bacterial NDH‐2 structure establishes a framework for the structure‐based design of small‐molecule inhibitors.     

Quantification of tartrate resistant acid phosphatase distribution in mouse tibiae using image analysis.

Tartrate resistant acid phosphatase (TRAP) activity of bone is a suitable biochemical marker for osteoclastic bone resorption. Qualitatively, the histochemical distribution of TRAP has been used to identify osteoclasts responsible for bone resorption; however, there have been few attempts to quantify TRAP localization. We describe a method for evaluating bone resorption by quantifying area percentages of positive TRAP localization using image analysis. Mouse tibiae were paraffin embedded following demineralization in disodium ethylenediamine tetraacetic acid. Longitudinal sections of tibia were cut from 15 levels in the left and the right limbs of six mice (180 sections total) and stained for TRAP distribution. Positive TRAP localization was quantified by pixel area count and reported as a percentage of the total tissue area specified. The 1.85 mm2 region of interest was placed at the midpoint of the epiphyseal growth plate containing the provisional calcification layer and the primary spongiosa, while excluding cortical bone of each mouse tibia. The percentage of TRAP localization ranged from 0.95 to 1.31% and was not significantly different from level to level or limb to limb in each mouse (p > 0.100). Within the same region of interest, an osteoclast count along the bone perimeter also was performed. We demonstrated a strong correlation (r2 = 0.903) between the conventional histomorphometric osteoclast index and positive TRAP localization, validating the latter as an alternative method to assess bone resorption. Quantitative analysis of TRAP is significant because it allows statistical comparisons between treatment groups, promotes precise pathological diagnoses and facilitates a reference data base that may aid the study of bone related diseases involving increased bone resorption.     

Myoclonic epilepsy and ragged-red fiber disease (MERRF) is associated with a mitochondrial DNA tRNA(Lys) mutation

An A to G transition mutation at nucleotide pair 8344 in human mitochondrial DNA (mtDNA) has been identified as the cause of MERRF. The mutation alters the T psi C loop of the tRNA(Lys) gene and creates a CviJI restriction site, providing a simple molecular diagnostic test for the disease. This mutation was present in three independent MERRF pedigrees and absent in 75 controls, altered a conserved nucleotide, and was heteroplasmic. All MERRF patients and their less-affected maternal relatives had between 2% and 27% wild-type mtDNAs and showed an age-related association between genotype and phenotype. This suggests that a small percentage of normal mtDNAs has a large protective effect on phenotype. This mutation provides molecular confirmation that some forms of epilepsy are the result of deficiencies in mitochondrial energy production.     

Copper toxicity: evidence for the conversion of cupric to cuprous copper in vivo under anaerobic conditions.

We have determined the toxicity to cells of Escherichia coli B of cupric copper applied under aerobic and anaerobic conditions in two ways. The growth of cells in liquid medium incorporating cupric copper shows differential inhibition, comparing aerobic and anaerobic conditions--toxicity being greater under anoxia. The growth of colonies upon agar plates incorporating cupric copper does not show such a differential effect. We conclude that colonies on plates are largely anoxic even when incubated aerobically. EPR spectra of cells obtained at various times after application of cupric copper under anoxic conditions indicate the conversion of a considerable proportion of the Cu(II) to a non-paramagnetic species, probably Cu(I). We demonstrate that Cu(I) is more toxic than Cu(II) to cells when applied under anoxic conditions and conclude that the difference in toxicity of Cu(II) applied to cells under aerobic and anaerobic conditions results from the greater extent of reduction of Cu(II) to Cu(I) under anaerobic conditions.     

A new simple-leaved species of Recchia (Simaroubaceae) from southeastern Mexico

Recchia simplicifolia, a new species from southern Veracruz, southern Tabasco and northern Chiapas, represents the first non-microphyllous simple-leaved New World species of Simaroubaceae. It differs from the other two species of Recchia in its simple leaves, cauliflory, and rain forest habitat. Its discovery may necessitate a reevaluation of the relationships of Surianaceae and Simaroubaceae. Although its distribution corresponds to a high-precipitation area which demonstrates strong affinities to the wet Central American flora and may have served as a refuge area for rain forest species during parts of the Pleistocene, its relationships are strictly with Mexican species of the dry Pacific slope, supporting the hypothesis of exchange between the wet and dry Mexican floras during the climatic cycles of the Pleistocene.