Formation of spray-dried powder of S-adenosyl-L-methionine

S-Adenosyl-L -methionine (SAM) is an essential metabolite in all living organisms. In clinical research, SAM has also been suggested as a chemotherapeutic agent in various diseases. The main problem of SAM is its instability at high temperatures, at neutral and alkaline pH, and in the presence of humidity. SAM retention in spray-dried powder was determined under various conditions of spray-drying. The highest SAM retention was obtained when maltodextrin (dextrose equivalent, DE, of 25) was used as the carrier solid with the SAM feed liquid at pH 4.0. The water content in the powder had a significant effect on the stability of SAM. SAM powder with lower water content exhibited higher stability.     

Stress responses in Salix gracilistyla cuttings subjected to repetitive alternate flooding and drought

To determine the tolerance of Salix gracilistyla to repetitive alternate flooding and drought, we measured leaf stomatal conductance, pre-dawn water potential, osmotic adjustment, and biomass production under greenhouse conditions. We used a control and nine crossed treatments (F1-D1–F3-D3) in which we combined 1-, 2-, or 3-week floodings (F) and droughts (D). Leaf stomatal conductance was lowest in 3 weeks of flooding or drought when the preceding event (flood or drought) was also of a 3-week duration. Leaf pre-dawn water potential was reduced in 3 weeks of drought when preceded by 2 or 3 weeks of flooding. Cuttings had slight osmotic adjustments in repetitions of long floodings and droughts. During longer durations of drought in crossed experiments, plants had low root and shoot mass, few hypertrophic lenticels, and reduced leaf mass; when flooding duration increased in crossed experiments, root mass was reduced, there were more hypertrophic lenticels, and the leaf area was reduced. Cuttings achieved stress tolerance by inhibition of transpiration, osmotic adjustment, reduction of transpiration area, and development of hypertrophic lenticels. Stress tolerance was weak when repetitive 2- or 3-week floodings were combined with 3-week droughts. The duration of flooding and drought periods under which S. gracilistyla achieves stress tolerance may be critical in determining distributions along riverbanks.     

Valproic Acid-Induced Anxiety and Depression Behaviors are Ameliorated in p39 Cdk5 Activator-Deficient Mice

Neurochemical Research - Valproic acid (VPA) is a drug used for the treatment of epilepsy, seizures, migraines, and bipolar disorders. Cyclin-dependent kinase 5 (Cdk5) is a Ser/Thr kinase activated...     

Stable nuclear transformation of the diatom Chaetoceros sp.

A nuclear transformation system for the centric diatom Chaetoceros sp. has been established using two plasmids pTpfcp/nat and pTpNR/green fluorescent protein (GFP) that had been used for Thalassiosira pseudonana transformation. These contain the nourseothricin resistance gene (nat) with the fucoxanthin chlorophyll a/c binding protein (fcp) promoter/terminator from T. pseudonana and the enhanced green fluorescent protein gene (egfp), with the nitrate reductase (NR) promoter/terminator from T. pseudonana, respectively. Transformants were recovered in the presence of the antibiotic nourseothricin. One to four copies of both nat and egfp genes were integrated into genomic DNA of the transformants. Transformation efficiency was 1.5–6.0 transformants per 10⁸ cells. This work is the first report of stable genetic transformation of Chaetoceros, which is important as not only a constituent member of marine ecosystem but also feed for aquaculture.     

Molecular analysis of X-linked inborn errors of purine metabolism: HPRT1 and PRPS1 mutations

Mutations of two enzyme genes, HPRT1 encoding hypoxanthine guanine phosphoribosyltransferase (HPRT) and PRPS1 encoding a catalytic subunit (PRS-I) of phosphoribosylpyrophosphate synthetase, cause X-linked inborn errors of purine metabolism. Analyzing these two genes, we have identified three HPRT1 mutations in Lesch-Nyhan families following our last report. One of them, a new mutation involving the deletion of 4224 bp from intron 4 to intron 5 and the insertion of an unknown 28 bp, has been identified. This mutation resulted in an enzyme polypeptide with six amino acids deleted due to abnormal mRNA skipping exon 5. The other HPRT1 mutations, a single base deletion (548delT, 183fs189X), and a point mutation causing a splicing error (532+1G>A, 163fs165X) were detected first in Japanese patients but have been reported in European families. On the other hand, in the analysis of PRPS1, no mutation was identified in any patient.     

Enhanced epithelial-mesenchymal transition-like phenotype in N-acetylglucosaminyltransferase V transgenic mouse skin promotes wound healing

N-Acetylglucosaminyltransferase V (GnT-V) catalyzes the β1,6 branching of N-acetylglucosamine on N-glycans. GnT-V expression is elevated during malignant transformation in various types of cancer. However, the mechanism by which GnT-V promotes cancer progression is unclear. To characterize the biological significance of GnT-V, we established GnT-V transgenic (Tg) mice, in which GnT-V is regulated by a β-actin promoter. No spontaneous cancer was detected in any organs of the GnT-V Tg mice. However, GnT-V expression was up-regulated in GnT-V Tg mouse skin, and cultured keratinocytes derived from these mice showed enhanced migration, which was associated with changes in E-cadherin localization and epithelial-mesenchymal transition (EMT). Further, EMT-associated factors snail, twist, and N-cadherin were up-regulated, and cutaneous wound healing was accelerated in vivo. We further investigated the detailed mechanisms of EMT by assessing EGF signaling and found up-regulated EGF receptor signaling in GnT-V Tg mouse keratinocytes. These findings indicate that GnT-V overexpression promotes EMT and keratinocyte migration in part through enhanced EGF receptor signaling.     

A neural-specific F-box protein Fbs1 functions as a chaperone suppressing glycoprotein aggregation

Fbs1 is an F-box protein present abundantly in the nervous system. Similar to the ubiquitously expressed Fbs2, Fbs1 recognizes N-glycans at the innermost position as a signal for unfolded glycoproteins, probably in the endoplasmic reticulum-associated degradation pathway. Here, we show that the in vivo majority of Fbs1 is present as Fbs1-Skp1 heterodimers or Fbs1 monomers but not SCF(Fbs1) complex. The inefficient SCF complex formation of Fbs1 and the restricted presence of SCF(Fbs1) bound on the endoplasmic reticulum membrane were due to the short linker sequence between the F-box domain and the sugar-binding domain. In vitro, Fbs1 prevented the aggregation of the glycoprotein through the N-terminal unique sequence of Fbs1. Our results suggest that Fbs1 assists clearance of aberrant glycoproteins in neuronal cells by suppressing aggregates formation, independent of ubiquitin ligase activity, and thus functions as a unique chaperone for those proteins.     

Reconstitution of the 9 S estrogen receptor with heat shock protein 90

As a first step in the investigation of the reconstitution of steroid hormone receptor systems, we studied the reconstitution of 9 S estrogen receptor (ER) from purified vero ER, which is the estradiol binding subunit, and heat shock protein 90 (hsp 90). By using a phosphate buffer containing molybdate, thiocyanate, dimethylformamide, glycerol, etc., vero ER could be converted to 9 S ER with hsp 90, but not with the control protein, ovalbumin. Inactivation of ER during the reconstitution was suppressed partially by hsp 90, but not by ovalbumin. Like native 8 S ER, the reconstituted ER was sedimented at about 8.9 S and 4.6 S on glycerol gradient centrifugation in low and high salt buffers, respectively.