Constitutive activation of sonic hedgehog signaling in the chicken mutant talpid(2): Shh-independent outgrowth and polarizing activity.

We have examined the developmental properties of the polydactylous chicken mutant, talpid(2). Ptc, Gli1, Bmp2, Hoxd13, and Fgf4 are expressed throughout the anteroposterior axis of the mutant limb bud, despite normal Shh expression. The expression of Gli3, Ihh, and Dhh appears to be normal, suggesting that the Shh signaling pathway is constitutively active in talpid(2) mutants. We show that preaxial talpid(2) limb bud mesoderm has polarizing activity in the absence of detectable Shh mRNA. When the postaxial talpid(2) limb bud (including all Shh-expressing cells) is removed, the preaxial cells reform a normal-shaped talpid(2) limb bud (regulate). However, a Shh-expressing region (zone of polarizing activity) does not reform; nevertheless Fgf4 expression in the apical ectodermal ridge is maintained. Such reformed talpid(2) limb buds develop complete talpid(2) limbs. After similar treatment, normal limb buds downregulate Fgf4, the preaxial cells do not regulate, and a truncated anteroposterior deficient limb forms. In talpid(2) limbs, distal outgrowth is independent of Shh and correlates with Fgf4, but not Fgf8, expression by the apical ectodermal ridge. We propose a model for talpid(2) in which leaky activation of the Shh signaling pathway occurs in the absence of Shh ligand.     

Engineering Pichia pastoris for stereoselective nitrile hydrolysis by co-producing three heterologous proteins

A Pichia pastoris strain with stereoselective nitrile hydratase activity has been constructed by engineering the co-expression of three genes derived from Pseudomonas putida. Using a technique that could be widely applicable, the genes encoding nitrile hydratase α and β structural subunits and P14K accessory protein were first assembled as individual expression cassettes and then incorporated onto one plasmid, which was integrated into the P. pastoris chromosome. The resulting strain can be used as a catalyst for bioconversions requiring stereospecific nitrile hydrolysis. Received: 3 November 1998 / Received revision: 25 February1999 / Accepted: 14 March 1999     

A Gram-negative bacterium producing a heat-stable nitrilase highly active on aliphatic dinitriles

A Gram-negative bacterial strain, identified as Acidovorax facilis strain 72W, has been isolated from soil by enrichment using 2-ethylsuccinonitrile as the sole nitrogen source. This strain grows on a variety of aliphatic mono- and dinitriles. Experiments using various heating regimes indicate that nitrile hydratase, amidase and nitrilase activities are present. The nitrilase is efficient at hydrolyzing aliphatic dinitriles to cyanoacid intermediates. It has a strong bias for C₃–C₆ dinitriles over mononitriles of the same chain length. Whole, resting cell hydrolysis of 2-methylglutaronitrile results in 4-cyanopentanoic acid and 2-methylglutaric acid as the major products. Heating, at least 20 min at 50 °C, eliminates nitrile hydratase and amidase activities, resulting in greater than 97% selectivity to 4-cyanopentanoic acid. The nitrilase activity has good heat stability, showing a half-life of 22.7 h at 50 °C and a temperature optimum of at least 65 °C for activity. The strain has been deposited as ATCC 55746. Received: 26 January 1999 / Received revision: 10 June 1999 / Accepted: 27 June 1999     

Elite athletes and the gene for angiotensin-converting enzyme.

The deletion (D) allele of the gene for angiotensin-converting enzyme (ACE) is associated with higher plasma and tissue levels of the enzyme and has also been related to a variety of cardiovascular complications, particularly myocardial infarction. On the basis of indirect evidence, we hypothesized that inheritance of the D allele would contribute to elite athletic ability. Over a period of 4 yr, 120 Caucasian athletes who were national (Australian) representatives in sports demanding a high level of aerobic fitness were recruited. Their ACE genotypes were compared with those of a community control group recruited randomly from the electoral roll. There was no difference in ACE genotype frequencies between the two groups. The DD genotype frequency was 30% in athletes and 29% in the control group, and the II genotype frequency was 22.5 and 22%, respectively. The results do not exclude the possibility that ACE genotype could be related to some attribute relating to a specific type of elite athletic ability or that there may be a difference between genders. Larger studies are desirable.     

Increased ribonucleotide reductase activity in hydroxyurea-resistant mosquito cells

Hydroxyurea-resistant Aedes albopictus mosquito cells were selected by incremental exposure of unmutagenized cells to hydroxyurea concentrations ranging from 0.1 to 8 mM. Clonal populations that had become 40-fold more resistant to hydroxyurea than wild-type cells varied in morphology, and their growth rate decreased to a ∼45 h doubling time, relative to an 18 h doubling time in unselected cells. At this level of resistance, the cells remained diploid, with a modal chromosome number of 6. When labelled with ³⁵S[methionine/cysteine], clone HU1062, which grew in the presence of 8 mM hydroxyurea, overproduced a labeled protein with the approximate size of the 45,000 dalton M2 subunit of ribonucleotide reductase. Consistent with this observation, ribonucleotide reductase activity in HU-1062 cells was approximately 10-fold higher than in wild-type control cells. This is the first example of an hydroxyurea-resistant insect cell line. © 1997 Wiley-Liss, Inc.     

Thiophene Rings Improve the Device Performance of Conjugated Polymers in Polymer Solar Cells with Thick Active Layers

Developing novel materials that tolerate thickness variations of the active layer is critical to further enhance the efficiency of polymer solar cells and enable large‐scale manufacturing. Presently, only a few polymers afford high efficiencies at active layer thickness exceeding 200 nm and molecular design guidelines for developing successful materials are lacking. It is thus highly desirable to identify structural factors that determine the performance of semiconducting conjugated polymers in thick‐film polymer solar cells. Here, it is demonstrated that thiophene rings, introduced in the backbone of alternating donor–acceptor type conjugated polymers, enhance the fill factor and overall efficiency for thick (>200 nm) solar cells. For a series of fluorinated semiconducting polymers derived from electron‐rich benzo[1,2‐b:4,5‐b′]dithiophene units and electron‐deficient 5,6‐difluorobenzo[2,1,3]thiazole units a steady increase of the fill factor and power conversion efficiency is found when introducing thiophene rings between the donor and acceptor units. The increased performance is a synergistic result of an enhanced hole mobility and a suppressed bimolecular charge recombination, which is attributed to more favorable polymer chain packing and finer phase separation.     

Cytological properties of an Aedes albopictus mosquito cell line infected with Wolbachia strain wAlbB

In vitro production of the obligate intracellular bacterium, Wolbachia pipientis, is essential to its manipulation as a genetic tool to spread transgenes within vector populations. We have adapted the Wolbachia-infected Aa23 Aedes albopictus mosquito cell line to Eagle's minimal medium, supplemented with nonessential amino acids, glutamine, and 20% fetal bovine serum. When plated at low densities, Aa23E cells grew as patchy monolayers, comprised of non-contiguous clusters of cells that gave rise to solid clumps of tightly adherent cells. Multicellular clumps eventually detached from the substrate and floated freely in the medium. Removal of Wolbachia by treatment with tetracycline did not alter the cytological properties of the host cells, which had a population doubling time of 4-5 d. The presence of Wolbachia was monitored by Giemsa staining of cytological preparations, polymerase chain reaction (PCR) amplification of Wolbachia 16S ribosomal DNA, and by simultaneous PCR amplification of ribosomal protein genes from Wolbachia and mosquito host cell genomes. Wolbachia morphology was pleomorphic, and Wolbachia DNA persisted in the culture medium for several weeks after degradation of PCR-amplifiable host cell DNA.