Improving d-mannitol productivity of Escherichia coli: Impact of NAD,CO2 and expression of a putative sugar permease from Leuconostoc pseudomesenteroides

The highly productive whole-cell biotransformation of d-fructose to d-mannitol with recombinant, resting cells of Escherichia coli BL21(DE3) requires the combined expression of mdh, fdh and glf which encode mannitol and formate dehydrogenases and a sugar facilitator, respectively. However, long-term stability of the system was restricted, possibly due to loss of the cofactor NAD, high concentrations of formate, formation of CO₂ affecting the internal pH of the cells, accumulation of high intracellular concentrations of d-mannitol, and export of d-mannitol. Downstream of the mdh gene of Leuconostoc pseudomesenteroides, we identified an open reading frame encoding for a putative mannitol permease. The gene was cloned and expressed in E. coli. Biochemical analyses revealed an activity as secondary carrier for d-fructose. Therefore, the carrier was named FupL and participation in d-mannitol transport was excluded. In biotransformation experiments, the productivity of d-mannitol formation obtained with the strain expressing the additional fupL gene was enhanced by 20%.     

Does prescribed burning mean a threat to the rare satyrine butterfly Hipparchia fagi? Larval-habitat preferences give the answer

The ecological effects of fire management, especially regarding arthropods are poorly investigated. Burning in winter was assumed to pose a threat to butterfly species hibernating as larvae. To assess the impact of prescribed burning on population viability, we analysed larval-habitat preferences of the highly endangered, xero-thermophilous butterfly Hipparchia fagi in vineyards of the Kaiserstuhl region (southern Germany). Microhabitat preference analyses for mature larvae and egg-laying females revealed a preference of H. fagi for Bromus erectus-dominated communities with sparse vegetation coverage and a distinct tuft growth of the host plant B. erectus on microclimatically benefited slopes. We explain the preference of B. erectus by a preference of vegetation structure. The grass tufts offer a suitable climatically buffered living space for larvae. Egg deposition took place on dry substrate at positions of high solar radiation, thus adapted to hot and dry microclimate. As the larval habitat was sparsely vegetated as well as generally legally protected, fire management was not applicable and therefore not affecting the populations. We think it is conceivable that H. fagi, occurring here at its northern range limit, might expand its larval habitat into denser, combustible B. erectus stands in the course of global warming. A change in habitat preferences would necessitate a re-evaluation of management options.     

Reduced number of CFTR molecules in erythrocyte plasma membrane of cystic fibrosis patients

Cystic fibrosis (CF), the most common genetic disease among Caucasians, is caused by mutations in the gene encoding CFTR (cystic fibrosis transmembrane conductance regulator). The most frequent mutation, DeltaF508, results in protein misfolding and, as a consequence, prevents CFTR from reaching its final location at the cell surface. CFTR is expressed in various cell types including red blood cells. The functional role of CFTR in erythrocytes is still unclear. Since the number of CFTR copies in a single erythrocyte of healthy donors and CF patients with a homozygous DeltaF508 mutation is unknown, we counted CFTR, localized in erythrocyte plasma membrane, at the single molecule level. A novel experimental approach combining atomic force microscopy with quantum-dot-labeled anti-CFTR antibodies, used as topographic surface markers, was employed to detect individual CFTR molecules. Analysis of erythrocyte plasma membranes taken from healthy donors and CF patients with a homozygous DeltaF508 mutation reveals mean (SEM) values of 698 (12.8) (n=542) and 172 (3.8) (n=538) CFTR molecules per red blood cell, respectively. We conclude that erythrocytes reflect the CFTR status of the organism and that quantification of CFTR in a blood sample could be useful in the diagnosis of CFTR related diseases.     

Unfolding barriers in bacteriorhodopsin probed from the cytoplasmic and the extracellular side by AFM

Selecting an individual membrane protein and probing its mechanical properties has become possible by AFM-based single-molecule force spectroscopy. In contrast to earlier studies, we extracted and unfolded bacteriorhodopsin monomers from the purple membrane not only from the cytoplasmic side, but also from the extracellular side, and recorded the force extension profiles. This way different pathways through the potential landscape are explored. A map of the 21 most dominant barriers with their positions relative to the amino acid sequences is given at an accuracy of +/-3 aa. Most barriers were found to provide resistance to forced unfolding only when extracted toward one of the sides. However, certain barriers have identical positions to within a few amino acids when probed from either of the sides, which typifies them as structural traps.     

Genomic organization of the human fibroblast growth factor receptor 2 (FGFR2) gene and comparative analysis of the human FGFR gene family

The human fibroblast growth factor receptor (FGFR) genes play important roles in normal vertebrate development. Mutations in the human FGFR2 gene have been associated with many craniosynostotic syndromes and malformations, including Crouzon, Pfeiffer, Apert, Jackson-Weiss, Beare-Stevenson cutis gyrata, and Antley-Bixler syndromes, and Kleeblaatschadel (cloverleaf skull) deformity. The mutations identified to date are concentrated in the previously characterized region of FGFR2 that codes for the extracellular IgIII domain of the receptor protein. The search for mutations in other regions of the gene, however, has been hindered by lack of knowledge of the genomic structure. Using a combination of genomic library screening, long-range PCR, and genomic walking, we have characterized the genomic structure of nearly the entire human FGFR2 gene, including a delineation of the organization and size of all introns and exons and determination of the DNA sequences at the intron/exon boundaries. Comparative analysis of the human FGFR gene family reveals that the genomic organization of the FGFRs is relatively conserved. Moreover, alignment of the amino acid sequences shows that the four corresponding proteins share 46% identity overall, with up to 70% identity between individual pairs of FGFR proteins. However, the FGFR2 gene contains an additional exon not found in other members of the family, and it also has much larger intronic sequences throughout the gene. Remarkable similarities in genomic organization, intron/exon boundaries, and intron sizes are found between the human and mouse FGFR2 genes. Knowledge gained from this study of the human FGFR2 gene structure may prove useful in future screening studies designed to find additional mutations associated with craniosynostotic syndromes, and in understanding the molecular and cell biology of this receptor family.     

Twisting biomaterials around your little finger: environmental impacts of bio-based wrappings

Background, aim, and scope  

Packaging uses nearly 40% of all polymers, a substantial share of which is used for sensitive merchandise such as moisture-sensitive food. To find out if bio-based materials are environmentally advantageous for this demanding application, we compared laminated, printed film across the whole life cycle.     

Target-approaching behavior of barn owls (Tyto alba): influence of sound frequency

We studied the influence of frequency on sound localization in free-flying barn owls by quantifying aspects of their target-approaching behavior to a distant sound source during ongoing auditory stimulation. In the baseline condition with a stimulus covering most of the owls hearing range (1–10 kHz), all owls landed within a radius of 20 cm from the loudspeaker in more than 80% of the cases and localization along the azimuth was more accurate than localization in elevation. When the stimulus contained only high frequencies (>5 kHz) no changes in striking behavior were observed. But when only frequencies from 1 to 5 kHz were presented, localization accuracy and precision decreased. In a second step we tested whether a further border exists at 2.5 kHz as suggested by optimality models. When we compared striking behavior for a stimulus having energy from 2.5 to 5 kHz with a stimulus having energy between 1 and 2.5 kHz, no consistent differences in striking behavior were observed. It was further found that pre-takeoff latency was longer for the latter stimulus than for baseline and that center frequency was a better predictor for landing precision than stimulus bandwidth. These data fit well with what is known from head-turning studies and from neurophysiology.     

Assembly and oligomerization of human ATP synthase lacking mitochondrial subunits a and A6L

Here we study ATP synthase from human ρ⁰ (rho zero) cells by clear native electrophoresis (CNE or CN-PAGE) and show that ATP synthase is almost fully assembled in spite of the absence of subunits a and A6L. This identifies subunits a and A6L as two of the last subunits to complete the ATP synthase assembly. Minor amounts of dimeric and even tetrameric forms of the large assembly intermediate were preserved under the conditions of CNE, suggesting that it associated further into higher order structures in the mitochondrial membrane. This result was reminiscent to the reduced amounts of dimeric and tetrameric ATP synthase from yeast null mutants of subunits e and g detected by CNE. The dimer/oligomer-stabilizing effects of subunits e/g and a/A6L seem additive in human and yeast cells. The mature IF₁ inhibitor was specifically bound to the dimeric/oligomeric forms of ATP synthase and not to the monomer. Conversely, nonprocessed pre-IF₁ still containing the mitochondrial targeting sequence was selectively bound to the monomeric assembly intermediate in ρ⁰ cells and not to the dimeric form. This supports previous suggestions that IF₁ plays an important role in the dimerization/oligomerization of mammalian ATP synthase and in the regulation of mitochondrial structure and function.