A new gain‐of‐function mouse line to study the role of Wnt3a in development and disease

Wnt/β‐catenin signals are important regulators of embryonic and adult stem cell self‐renewal and differentiation and play causative roles in tumorigenesis. Purified recombinant Wnt3a protein, or Wnt3a‐conditioned culture medium, has been widely used to study canonical Wnt signaling in vitro or ex vivo. To study the role of Wnt3a in embryogenesis and cancer models, we developed a Cre recombinase activatable Rosa26^Wnt3a allele, in which a Wnt3a cDNA was inserted into the Rosa26 locus to allow for conditional, spatiotemporally defined expression of Wnt3a ligand for gain‐of‐function (GOF) studies in mice. To validate this reagent, we ectopically overexpressed Wnt3a in early embryonic progenitors using the T‐Cre transgene. This resulted in up‐regulated expression of a β‐catenin/Tcf‐Lef reporter and of the universal Wnt/β‐catenin pathway target genes, Axin2 and Sp5. Importantly, T‐Cre; Rosa26^Wnt3a mutants have expanded presomitic mesoderm (PSM) and compromised somitogenesis and closely resemble previously studied T‐Cre; Ctnnb1^ex3 (β‐catenin^GOF) mutants. These data indicate that the exogenously expressed Wnt3a stimulates the Wnt/β‐catenin signaling pathway, as expected. The Rosa26^Wnt3a mouse line should prove to be an invaluable tool to study the function of Wnt3a in vivo.     

Changes of elemental concentrations around and on the surface of fowl sperm membrane during maturation in the male reproductive tract and after in vitro storage

X-ray microprobe analysis was performed to investigate the changes of elemental concentrations around or on the membrane of the head, midpiece, and principal piece regions of individual fowl spermatozoa during maturation in the male reproductive tract and after storage in vitro at 4°C. The pattern of change of elemental concentrations during maturation and postejaculation was, in general, similar in the three different subcellular regions; i.e., concentrations of sodium, potassium, chlorine, and calcium decreased gradually during sperm passage through the male reproductive tract and after storage. Phosphorus concentration remained almost constant in the male tract and decreased gradually after storage. In contrast, magnesium, zinc, and copper concentrations showed an interesting pattern: concentrations increased significantly during maturation to a maximum at ejaculation and decreased again after storage. The ratios of sodium to potassium in the midpiece region showed patterns similar to those of magnesium, zinc, and copper concentrations.     

Insights into the mode of action of 1,2,6,7-tetraoxaspiro [7.11] nonadecane (N-89) against adult Schistosoma mansoni worms

Control of morbidity associated with schistosomiasis via chemotherapy largely relies on the drug praziquantel. Repeated therapy with praziquantel has created concerns about the possible selection of resistant worms and necessitated the search for novel drugs to treat schistosomiasis. Here, a murine model was infected with Schistosoma mansoni and treated with oral 1,2,6,7-tetraoxaspiro [7.11] nonadecane (N-89), which caused a significant reduction in fecundity and egg burden and reduced morbidity when administered at 5-weeks post-infection.The analysis showed that the mode of action occurred through the ingestion of activated N-89 by the worms, and that there was no direct external effect on the S. mansoni worms. Ultrastructural analysis of the treated worms showed disruptions in the gut lumen and the presence of large volumes of material, suggestive of undigested blood meals or red blood cells. In addition, there were reduced vitelline cells in female worms and damage to sub-tegmental musculature in male worms. Eggs recovered from the treated mice showed both damage to the eggs and the production of immature eggs. Expression of mRNA responsible for gut and digestive function and egg production was also significantly affected by N-89 treatment, whereas control genes for musculature showed no significant changes.Thus, N-89 drastically affected the total digestive function and egg production of S. mansoni worms. Physiological processes requiring heme uptake such as egg production and eggshell formation were subsequently affected, suggesting that the compound could be a possible therapeutic drug candidate for schistosomiasis control.     

Three-dimensional structure of gurmarin,a sweet taste-suppressing polypeptide

Summary The solution structure of gurmarin was studied by two-dimensional proton NMR spectroscopy at 600 MHz. Gurmarin, a 35-amino acid residue polypeptide recently discovered in an Indian-originated tree Gymnema sylvestre, selectively suppresses the neural responses of rat to sweet taste stimuli. Sequence-specific protons. The three-dimensional solution structure was determined by simulated-annealing calculations on the basis of 135 interproton distance constraints derived from NOEs, six distance constraints for three hydrogen bonds and 16 dihedral angle constraints derived from coupling constants. A total of 10 structures folded into a well-defined structure with a triple-stranded antiparallel -sheet. The average rmsd values between any two structures were 1.65±0.39 Å for the backbone atoms (N, C, C) and 2.95±0.27 Å for all heavy atoms. The positions of the three disulfide bridges, which could not be deterermined chemically, were estimated to be Cys³–Cys¹⁸, Cys¹⁰–Cys²³ and Cys¹⁷–Cys³³ on the basis of the NMR distance constraints. This disulfide bridge pattern in gurmarin turned out to be analogous to that in -conotoxin and Momordica charantia trypsin inhibitor-II, and the topology of folding was the same as that in -conotoxin.Abbreviations DQF-COSY double-quantum-filtered correlated spectroscopy - HOHAHA homonuclear Hartmann-Hahn spectroscopy - NOESY nuclear Overhauser enhancement spectroscopy - ppm parts per million; rmsd, root-mean-square deviation - TSP 3-(trimethylsilyl)-2,2,3,3-tetradeutero-propionate     

Evaluation of DNA Fingerprinting by PFGE as an Epidemiologic Tool for Salmonella Infections

To evaluate DNA fingerprinting as an epidemiologic tool, pulsed-field gel electrophoresis (PFGE) was performed on isolates of Salmonella, including S. typhimurium, S. thompson, and S. enteritidis. Chromosomal DNA was digested with the restriction endonucleases Bln I and Xba I. The patterns of S. thompson and S. typhimurium isolates from various sources were different from one another. There was no correlation between the phage type and the digestion pattern of S. enteritidis isolates. Some strains belonging to one phage type were distinguished by their PFGE pattern in this study. These results suggest that the Bln I and Xba I digestion patterns of chromosomal DNA are useful for epidemiological analysis of an outbreak of Salmonella infection or food poisoning.     

Optimum medium for large-scale culture of Tetraselmis tetrathele

The prasinophyte Tetraselmis tetrathele is an alga commonly used as livefood for aquatic animal larvae. The alga is usually cultured in Guillard& Ryther medium (Guillard F) or a fertilizer enriched seawater mediumfor the large-scale culture of Nannochloropsis. However, Guillard F is toocomplicated to use for large-scale culture, and some fertilizers are impureand insoluble. A new enriched seawater medium for the large-scale culture ofT. tetrathele (ES-T.T.) was formulated by modifying the Guillard F medium.NaNO₃, NaH₂PO₄, Fe-EDTA andMnCl₂were selected as essential additives for the medium bysystematically removing each additive of Guillard F. Results from axenicculture experiments, using an artificial seawater medium, the requiredamount of these additives was estimated to be, 150 mg NaNO₃,10 mg NaH₂PO₄ · 2H₂0, 15 mgFe-EDTA and 360 µg MnCl₂ · 4H₂O,per liter of seawater. The productive rate of T. tetrathele in ES-T.T. washigher than in a fertilized medium in a 100 liter outdoor cultureexperiment. In 10 liter indoor culture experiments, no significantdifference was detected in production rates between ES-T.T. and Guillard F.Therefore, ES-T.T. is simple and effective to use as a large-scale culturemedium for T. tetrathele.     

Flagellar movement in demembranated preparations of ejaculated fowl spermatozoa

Triton X-100 at a concentration of 0.1% in the extraction medium was optimal for demembranating fowl spermatozoa. The most suitable conditions for reactivation were obtained when a medium composed of 0.5 mM-ATP, 25 mM-potassium glutamate, 10(-7) M-CaCl2, 20 mM-Tris-HCl(pH 7.9), 1 mM-MgSO4, 1 mM-dithiothreitol and 0.2 M-sucrose was used. More than 60% motile spermatozoa were obtained under these conditions. The addition of 1 or 10 microM-cAMP did not appreciably affect motility. Intact and demembranated spermatozoa were immotile at 40 degrees C, whilst at 30 degrees C motility was restored.     

Regio-Selective 10-Hydroxylation of Patchoulol, a Sesquiterpene, by Pithomyces Species

Of some 350 microorganisms screened, four strains of Pithomyces species were found to carry out regio-selective hydroxylation of patchoulol, a sesquiterpene, to 10-hydroxypatchoulol: Pithomyces sp. NRJ201, P. chartarum NRJ210, and, to a lesser extent, P. cynodontis ATCC 26150 and P. atro-olivaceus IFO 6651 were found to catalyze this reaction. A method has been developed by which 10-hydroxypatchoulol was obtained in 25 to 45% yields in 1- to 5-liter fermentation jars at 2 to 4 g of patchoulol per liter and isolated as pure material in 30% yields.     

Aurora B but Not Rho/MLCK Signaling Is Required for Localization of Diphosphorylated Myosin II Regulatory Light Chain to the Midzone in Cytokinesis

Non-muscle myosin II is stimulated by monophosphorylation of its regulatory light chain (MRLC) at Ser19 (1P-MRLC). MRLC diphosphorylation at Thr18/Ser19 (2P-MRLC) further enhances the ATPase activity of myosin II. Phosphorylated MRLCs localize to the contractile ring and regulate cytokinesis as subunits of activated myosin II. Recently, we reported that 2P-MRLC, but not 1P-MRLC, localizes to the midzone independently of myosin II heavy chain during cytokinesis in cultured mammalian cells. However, the mechanism underlying the distinct localization of 1P- and 2P-MRLC during cytokinesis is unknown. Here, we showed that depletion of the Rho signaling proteins MKLP1, MgcRacGAP, or ECT2 inhibited the localization of 1P-MRLC to the contractile ring but not the localization of 2P-MRLC to the midzone. In contrast, depleting or inhibiting a midzone-localizing kinase, Aurora B, perturbed the localization of 2P-MRLC to the midzone but not the localization of 1P-MRLC to the contractile ring. We did not observe any change in the localization of phosphorylated MRLC in myosin light-chain kinase (MLCK)-inhibited cells. Furrow regression was observed in Aurora B- and 2P-MRLC-inhibited cells but not in 1P-MRLC-perturbed dividing cells. Furthermore, Aurora B bound to 2P-MRLC in vitro and in vivo. These results suggest that Aurora B, but not Rho/MLCK signaling, is essential for the localization of 2P-MRLC to the midzone in dividing HeLa cells.