Expression of Axwnt-8 and Axszl in the urodele, axolotl: comparison with Xenopus

In both the urodele axolotl and the anuran Xenopus, Wnt-8 is expressed in posterior lateral plate mesoderm (LPM) in neurula and tailbud stages. In contrast to Xenopus, expression in axolotl is more prominent in gastrula endoderm, is not initiated in mesoderm until late gastrulation, and is present in the tailbud and in the brain at tailbud stages. Sizzled is expressed in axolotl in the ventral region, similar to its pattern in Xenopus. In axolotl, the Wnt-8-expressing LPM remains relatively dorsal through tailbud stages, while ventral blood island (VBI) markers appear in a wide ventral arc.     

Natamycin blocks fungal growth by binding specifically to ergosterol without permeabilizing the membrane

Natamycin is a polyene antibiotic that is commonly used as an antifungal agent because of its broad spectrum of activity and the lack of development of resistance. Other polyene antibiotics, like nystatin and filipin are known to interact with sterols, with some specificity for ergosterol thereby causing leakage of essential components and cell death. The mode of action of natamycin is unknown and is investigated in this study using different in vitro and in vivo approaches. Isothermal titration calorimetry and direct binding studies revealed that natamycin binds specifically to ergosterol present in model membranes. Yeast sterol biosynthetic mutants revealed the importance of the double bonds in the B-ring of ergosterol for the natamycin-ergosterol interaction and the consecutive block of fungal growth. Surprisingly, in strong contrast to nystatin and filipin, natamycin did not change the permeability of the yeast plasma membrane under conditions that growth was blocked. Also, in ergosterol containing model membranes, natamycin did not cause a change in bilayer permeability. This demonstrates that natamycin acts via a novel mode of action and blocks fungal growth by binding specifically to ergosterol.     

CO metabolism, sensing, and signaling

CO is a colorless and odorless gas produced by the incomplete combustion of hydrocarbons, both of natural and anthropogenic origin. Several microorganisms, including aerobic and anaerobic bacteria and anaerobic archaea, use exogenous CO as a source of carbon and energy for growth. On the other hand, eukaryotic organisms use endogenous CO, produced during heme degradation, as a neurotransmitter and as a signal molecule. CO sensors act as signal transducers by coupling a "regulatory" heme-binding domain to a "functional" signal transmitter. Although high CO concentrations inhibit generally heme-protein actions, low CO levels can influence several signaling pathways, including those regulated by soluble guanylate cyclase and/or mitogen-activated protein kinases. This review summarizes recent insights into CO metabolism, sensing, and signaling.     

Functional and structural interactions between osteoblastic and preosteoclastic cells in vitro

Osteoblasts are involved in the bone resorption process by regulating osteoclast maturation and activity. In order to elucidate the mechanisms underlying osteoblast/preosteoclast cell interactions, we developed an in vitro model of co-cultured human clonal cell lines of osteoclast precursors (FLG 29.1) and osteoblastic cells (Saos-2), and evaluated the migratory, adhesive, cytochemical, morphological, and biochemical properties of the co-cultured cells. In Boyden chemotactic chambers, FLG 29.1 cells exhibited a marked migratory response toward the Saos-2 cells. Moreover, they preferentially adhered to the osteoblastic monolayer. Direct co-culture of the two cell types induced: (1) positive staining for tartrate-resistant acid phosphatase in FLG 29.1 cells; (2) a decrease of the alkaline phosphatase activity expressed by Saos-2 cells; (3) the appearance of typical ultrastructural features of mature osteoclasts in FLG 29.1 cells; (4) the release into the culture medium of granulocyte-macrophage colony stimulating factor. The addition of parathyroid hormone to the co-culture further potentiated the differentiation of the preosteoclasts, the cells tending to fuse into large multinucleated elements. These in vitro interactions between osteoblasts and osteoclast precursors offer a new model for studying the mechanisms that control osteoclastogenesis in bone tissue.     

Role of the reticulum in the stability and shape of the isolated human erythrocyte membrane

In order to examine the widely held hypothesis that the reticulum of proteins which covers the cytoplamsic surface of the human erythrocyte membrane controls cell stability and shape, we have assessed some of its properties. The reticulum, freed of the bilayer by extraction with Triton X-100, was found to be mechanically stable at physiological ionic strength but physically unstable at low ionic strength. The reticulum broke down after a characteristic lag period which decreased 500-fold between 0 degrees and 37 degrees C. The release of polypeptide band 4.1 from the reticulum preceded that of spectrin and actin, suggesting that band 4.1 might stabilize the ensemble but is not essential to its integrity. The time-course of breakdown was similar for ghosts, the reticulum inside of ghosts, and the isolated reticulum. However, at very low ionic strength, the reticulum was less stable within the ghost than when free; at higher ionic strength, the reverse was true. Over a wide range of conditions the membrane broke down to vesicles just as the reticulum disintegrated, presumably because the bilayer was mechanically stabilized by this network. The volume of both ghosts and naked reticula varied inversely and reversibly with ionic strength. The volume of the naked reticulum varied far more widely than the ghost, suggesting that its deformation was normally limited by the less extensible bilayer. The contour of the isolated reticulum was discoid and often dimpled or indented, as visualized in the fluorescence microscope after labeling of the ghosts with fluoroscein isothiocyanate. Reticula derived from ghosts which had lost the ability to crenate in isotonic saline were shriveled, even though the bilayer was smooth and expanded. Conversly, ghosts crenated by dinitrophenol yielded smooth, expanded reticula. We conclude that the reticulum is a durable, flexible, and elastic network which assumes and stabilizes the contour of the membrane but is not responsible for its crenation.     

High-performance liquid chromatography and radioimmunoassay of rat plasma corticosterone

Problems inherent in corticosterone radioimmunoassay (RIA) led to consideration of alternative methods. A high-performance liquid chromatography (HPLC) procedure was evaluated that separated and quantitated dichloromethane-extracted corticosterone by reverse-phase chromatography. The results were correlated (r = 0.92) with an RIA procedure. The HPLC recovered nearly 100% of corticosterone added to rat plasma and had excellent reproducibility. In addition, chromatogram profiles of dichloromethane-soluble components obtained from rat plasma, derived from drug effect studies, could have value for characterizing response patterns. Without automated sample injection equipment, HPLC is more appropriately applied in monitoring RIA results than in processing large numbers of samples.     

Inhibitors of efflux pumps in Gram-negative bacteria

In Gram-negative bacteria, efflux complexes, consisting of an inner-membrane pump, a periplasmic adaptor protein and outer-membrane channel, provide an efficient means for the export of structurally unrelated drugs, causing the multidrug-resistance phenotype. Resistance due to this antibiotic efflux is an increasing problem worldwide. A new molecular challenge is to combat this transport by searching for new molecules to block efflux and thus restore drug susceptibility to resistant clinical strains. Recent data shed new light on the structure and activity of the archetypal efflux pumps AcrAB-TolC and MexAB-OprM. Here, we describe recent insights into the molecular mechanisms of bacterial efflux pumps and their inhibitors. Current progress for the clinical use of efflux-pump inhibitors and new strategies to combat the drug-efflux mechanisms will be discussed.