The identification and purification of a mammalian-like protein kinase C in the yeast Saccharomyces cerevisiae.

We have purified a yeast protein kinase that is phospholipid-dependent and activated by Diacylglycerol (DAG) in the presence of Ca2+ or by the tumour-promoting agent tetradecanoyl-phorbol acetate (TPA). The properties of this enzyme are similar to those of the mammalian protein kinase C (PKC). The enzyme was purified using chromatography on DEAE-cellulose followed by hydroxylapatite. The latter chromatography separated the activity to three distinguishable sub-species, analogous to the mammalian PKC isoenzymes. The fractions enriched in PKC activity contain proteins that specifically bind TPA, are specifically phosphorylated in the presence of DAG and recognized by anti-mammalian PKC antibodies.     

Defensive Medicine among Obstetricians and Gynecologists in Tertiary Hospitals

Objective

To describe the daily work practice under the threat of defensive medicine among obstetricians and gynecologists.

Study Design

A prospective cross-sectional survey of obstetricians and gynecologists working at tertiary medical centers in Israel.

Results

Among the 117 obstetricians and gynecologists who participated in the survey, representing 10% of the obstetricians and gynecologists registered by the Israel Medical Association, 113 (97%) felt that their daily work practice is influenced by concern about being sued for medical negligence and not only by genuine medical considerations. As a result, 102 (87%) physicians are more likely to offer the cesarean section option, even in the absence of a clear medical indication, 70 (60%) follow court rulings concerning medical practices, and 85 (73%) physicians mentioned that discussions about medical negligence court rulings are included in their departments'' meetings.

Conclusions

Defensive medicine is a well-embedded phenomenon affecting the medical decision process of obstetricians and gynecologists.     

The geometry of the Pareto front in biological phenotype space

When organisms perform a single task, selection leads to phenotypes that maximize performance at that task. When organisms need to perform multiple tasks, a trade‐off arises because no phenotype can optimize all tasks. Recent work addressed this question, and assumed that the performance at each task decays with distance in trait space from the best phenotype at that task. Under this assumption, the best‐fitness solutions (termed the Pareto front) lie on simple low‐dimensional shapes in trait space: line segments, triangles and other polygons. The vertices of these polygons are specialists at a single task. Here, we generalize this finding, by considering performance functions of general form, not necessarily functions that decay monotonically with distance from their peak. We find that, except for performance functions with highly eccentric contours, simple shapes in phenotype space are still found, but with mildly curving edges instead of straight ones. In a wide range of systems, complex data on multiple quantitative traits, which might be expected to fill a high‐dimensional phenotype space, is predicted instead to collapse onto low‐dimensional shapes; phenotypes near the vertices of these shapes are predicted to be specialists, and can thus suggest which tasks may be at play.     

MiR-449a Affects Epithelial Proliferation during the Pseudoglandular and Canalicular Phases of Avian and Mammal Lung Development

Congenital diaphragmatic hernia is associated with pulmonary hypoplasia and respiratory distress, which result in high mortality and morbidity. Although several transgenic mouse models of lung hypoplasia exist, the role of miRNAs in this phenotype is incompletely characterized. In this study, we assessed microRNA expression levels during the pseudoglandular to canalicular phase transition of normal human fetal lung development. At this critical time, when the distal respiratory portion of the airways begins to form, microarray analysis showed that the most significantly differentially expressed miRNA was miR-449a. Prediction algorithms determined that N-myc is a target of miR-449a and identified the likely miR-449a:N-myc binding sites, confirmed by luciferase assays and targeted mutagenesis. Functional ex vivo knock-down in organ cultures of murine embryonic lungs, as well as in ovo overexpression in avian embryonic lungs, suggested a role for miR-449a in distal epithelial proliferation. Finally, miR-449a expression was found to be abnormal in rare pulmonary specimens of human fetuses with Congenital Diaphragmatic Hernia in the pseudoglandular or canalicular phase. This study confirms the conserved role of miR-449a for proper pulmonary organogenesis, supporting the delicate balance between expansion of progenitor cells and their terminal differentiation, and proposes the potential involvement of this miRNA in human pulmonary hypoplasia.     

Deterministic Somatic Cell Reprogramming Involves Continuous Transcriptional Changes Governed by Myc and Epigenetic-Driven Modules

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Nuclear localization of non-receptor protein tyrosine phosphatase epsilon is regulated by its unique N-terminal domain

Precise subcellular localization is an important factor in regulation of the functions of protein tyrosine phosphatases. The non-receptor form of protein tyrosine phosphatase epsilon (cyt-PTP(epsilon)) can be found in cell nuclei, among other cellular locations, while p67 PTP(epsilon), a naturally occurring isoform which lacks the 27 N terminal residues of cyt-PTP(epsilon), is exclusively cytosolic. Using deletion and scanning mutagenesis we report that the first 10 amino acid residues of cyt-PTP(epsilon), in particular residues R4, K5, and R9, are critical components for its nuclear localization. We also establish that increased oxidative stress enhances accumulation of cyt-PTP(epsilon) in cell nuclei. Of the four known protein forms of PTP(epsilon), cyt-PTP(epsilon) is the only one which includes the extreme N-terminal sequence containing R4, K5, and R9. The role of the unique N terminus of cyt-PTP(epsilon) is therefore to regulate its subcellular localization. The existence of naturally occurring forms of PTP(epsilon) which lack this sequence and which are generated by translational and posttranslational mechanisms, suggests that nuclear localization of cyt-PTP(epsilon) can be actively regulated by cells.     

X-ray structures of Torpedo californica acetylcholinesterase complexed with (+)-huperzine A and (-)-huperzine B: structural evidence for an active site rearrangement

Kinetic and structural data are presented on the interaction with Torpedo californica acetylcholinesterase (TcAChE) of (+)-huperzine A, a synthetic enantiomer of the anti-Alzheimer drug, (-)-huperzine A, and of its natural homologue (-)-huperzine B. (+)-Huperzine A and (-)-huperzine B bind to the enzyme with dissociation constants of 4.30 and 0.33 microM, respectively, compared to 0.18 microM for (-)-huperzine A. The X-ray structures of the complexes of (+)-huperzine A and (-)-huperzine B with TcAChE were determined to 2.1 and 2.35 A resolution, respectively, and compared to the previously determined structure of the (-)-huperzine A complex. All three interact with the "anionic" subsite of the active site, primarily through pi-pi stacking and through van der Waals or C-H.pi interactions with Trp84 and Phe330. Since their alpha-pyridone moieties are responsible for their key interactions with the active site via hydrogen bonding, and possibly via C-H.pi interactions, all three maintain similar positions and orientations with respect to it. The carbonyl oxygens of all three appear to repel the carbonyl oxygen of Gly117, thus causing the peptide bond between Gly117 and Gly118 to undergo a peptide flip. As a consequence, the position of the main chain nitrogen of Gly118 in the "oxyanion" hole in the native enzyme becomes occupied by the carbonyl of Gly117. Furthermore, the flipped conformation is stabilized by hydrogen bonding of Gly117O to Gly119N and Ala201N, the other two functional elements of the three-pronged "oxyanion hole" characteristic of cholinesterases. All three inhibitors thus would be expected to abolish hydrolysis of all ester substrates, whether charged or neutral.