Inhibition and transcriptional silencing of a subtilisin-like proprotein convertase, PACE4/SPC4, reduces the branching morphogenesis of and AQP5 expression in rat embryonic submandibular gland

The submandibular gland (SMG) develops through the epithelial-mesenchymal interaction mediated by many growth/differentiation factors including activin and BMPs, which are synthesized as inactive precursors and activated by subtilisin-like proprotein convertases (SPC) following cleavage at their R-X-K/R-R site. Here, we found that Dec-RVKR-CMK, a potent inhibitor of SPC, inhibited the branching morphogenesis of the rat embryonic SMG, and caused low expression of a water channel AQP5, in an organ culture system. Dec-RVKR-CMK also decreased the expression of PACE4, a SPC member, but not furin, another SPC member, suggesting the involvement of PACE4 in the SMG development. Heparin, which is known to translocate PACE4 in the extracellular matrix into the medium, and an antibody specific for the catalytic domain of PACE4, both reduced the branching morphogenesis and AQP5 expression in the SMG. The inhibitory effects of Dec-RVKR-CMK were partially rescued by the addition of recombinant BMP2, whose precursor is one of the candidate substrates for PACE4 in vivo. Further, the suppression of PACE4 expression by siRNAs resulted in decreased expression of AQP5 and inhibition of the branching morphogenesis in the present organ culture system. These observations suggest that PACE4 regulates the SMG development via the activation of some growth/differentiation factors.     

Synthesis in vitro of glycoprotein in regenerating rat liver

The metabolism of glucosamine in regenerating rat liver was studied in liver slices. [1-¹⁴C]Glucosamine was incorporated into acid-soluble fraction, rapidly converted to UDP-N-acetylhexosamine and transferred to acid-insoluble fraction. Electrophoretic analysis revealed that most of the radioactive macromolecules released from the slices to the incubation medium were plasma glycoproteins.The incorporation of [1-¹⁴c]glucosamine into UDP-N-acetylhexosamine significantly increased from 6 h to 48 h after partial hepatectomy. On the contrary, the incorporation into acid-insoluble fractions of slice and medium decreased to about 50% of the control values. The rate of transfer of N-acetylhexosamine from UDP-N-acetylhexosamine to acid-insoluble fractions also decreased at 12 h and 48 h respectively. This indicates that the transfer of N-acetylhexosamine to glycoproteins decreases during 48 h of liver regeneration.The enhancement of [1-¹⁴C]glucosamine incorporation into UDP-N-acetylhexosamine is due to an accumulation of the label in the larger pool of this compound. Evidently, some control mechanism may operate on the transfer of N-acetylhexosamine from UDP-N-acetylhexosamine to glycoproteins in regenerating rat liver.     

New sesquiterpene lactone glucosides from the roots of Ferula varia

Five new eudesmane- (1–5), two new guaiane- (6 and 7) and one new germacrane-type (8) sesquiterpene lactone glucosides were isolated from the H₂O-soluble fraction of the roots of Ferula varia. Their structures were elucidated by extensive spectroscopic analyses. The absolute configuration of 1 was determined by modified Mosher's method.     

A noninvasive method for extracting bivalve DNA from the water-filled mantle cavity

Genetic studies play a great role for determining the biology of bivalves, particularly those covering population genetics, phylogeny, breeding, stock management, and conservation. However, DNA sampling methods that require removal of bivalves from the water and/or opening of their shells often cause stress and damage to bivalves, which can be lethal. The invasiveness of DNA sampling has made it difficult to conduct genetic studies in threatened species, rare species, and/or breeding lineages. In the present study, we developed a non-invasive method for bivalve DNA sampling using the water-filled mantle cavity (WMC). Our method can extract DNA from a small WMC sample (about 100 µl), collected using a fine needle and syringe without opening the shell. We demonstrated that the WMC sample contains intact mitochondrial and nuclear DNA. DNA contamination from other organisms, such as adjacent bivalve individuals, did not affect the resulting PCR and DNA sequencing analyses. Finally, the individuals from whom WMC was collected remained alive for more than 2 months after the experiments. This non-invasive method will be of great assistance in investigating the genetics of bivalves.

     

Synergistic effects of TAL1 over-expression and PHO13 deletion on the weak acid inhibition of xylose fermentation by industrial Saccharomyces cerevisiae strain

In the industrial production of bioethanol from lignocellulosic biomass, a strain of Saccharomyces cerevisiae that can ferment xylose in the presence of inhibitors is of utmost importance. The recombinant, industrial-flocculating S. cerevisiae strain NAPX37, which can ferment xylose, was used as the parent to delete the gene encoding p-nitrophenylphosphatase (PHO13) and overexpress the gene encoding transaldolase (TAL1) to evaluate the synergistic effects of these two genes on xylose fermentation in the presence of weak acid inhibitors, including formic, acetic, or levulinic acids. TAL1 over-expression or PHO13 deletion improved xylose fermentation as well as the tolerance of NAPX37 to all three weak acids. The simultaneous deletion of PHO13 and the over-expression of TAL1 had synergistic effects and improved ethanol production and reduction of xylitol accumulation in the absence and presence of weak acid inhibitors.     

An essential role of phosphatidylglycerol in the formation of the osmotically stable liposomes of Escherichia coli phospholipids

A temperature sensitive auxotroph of Escherichia coli K-12 requiring unsaturated fatty acids can grow normally at 28 degrees C, but requires an osmotic stabilizer such as a high amount of salt or sugar in the medium for the growth at 42 degrees C. Namely, the apparent osmotic stability of the cells at 28 degrees C and 42 degrees C is quite different. The osmotic properties of liposomes of the phospholipids extracted from these cells were investigated. The osmotically induced volume change of the multilamellar liposomes was examined by the turbidimetric method. The liposomes prepared from cells grown at 28 degrees C can swell and shrink under a wide range of hypo-and hypertonic conditions. However, those from cells grown at 42 degrees C could not swell under hypotonic conditions. These results exhibit a good correlation between the apparent osmotic stability of E. coli cells and the osmotic properties of the liposomes prepared from the extracted total phospholipids. To clarify the role of each phospholipid component, the osmotic properties of the liposomes reconstituted from the purified phospholipid species were further investigated. The results clearly showed that phosphatidylglycerol is the key factor that stabilizes the membranes of E. coli phospholipids against osmotic pressure.     

Isotopic Determination and Optimum Reaction Conditions of Pantothenic Acid Synthetase

When yeast protoplasts that were producing repressible acid phosphatase (r-APase) were treated with tunicamycin (TM), three specific proteins of 59k, 57k, and 55k daltons were accumulated in the membrane fraction in addition to the usual membrane proteins and these proteins were not detected in the secreted fraction. These proteins were immunoprecipitated with anti r-APase antiserum. Their molecular sizes were almost the same as those endo-H treated r-APase. Therefore these proteins were considered to be nonglycosylated forms of r-APase proteins. These results proved that nonglycosylated forms of r-APase produced by TM-treatment were not secreted by yeast protoplasts.