Isolation and characterization of a beta-primeverosidase-like enzyme from Penicillium multicolor

p-Nitrophenyl and eugenyl beta-primeveroside (6-O-beta-D-xylopyranosyl-beta-D-glucopyranoside) hydrolytic activity was found in culture filtrate from Penicillium multicolor IAM7153, and the enzyme was isolated. The enzyme was purified as a beta-primeverosidase-like enzyme by precipitation with ammonium sulfate followed by successive chromatographies on Phenyl Sepharose, Mono Q, and beta-galactosylamidine affinity columns. The molecular mass was estimated to be 50 kDa by SDS-PAGE and gel filtration. The purified enzyme was highly specific toward the substrate p-nitrophenyl beta-primeveroside, which was cleaved in an endo-manner into primeverose and p-nitrophenol, but a series of beta-primeveroside as aroma precursors were hydrolyzed only slightly as substrates for the enzyme. In analyses of its hydrolytic action and kinetics, the enzyme showed narrow substrate specificity with respect to the aglycon and glycon moieties of the diglycoside. We conclude that the present enzyme is a kind of beta-diglycosidase rather than beta-primeverosidase.     

CYP707A3, a major ABA 8'-hydroxylase involved in dehydration and rehydration response in Arabidopsis thaliana

Abscisic acid (ABA) catabolism is one of the determinants of endogenous ABA levels affecting numerous aspects of plant growth and abiotic stress responses. The major ABA catabolic pathway is triggered by ABA 8'-hydroxylation catalysed by the cytochrome P450 CYP707A family. Among four members of Arabidopsis CYP707As, the expression of CYP707A3 was most highly induced in response to both dehydration and subsequent rehydration. A T-DNA insertional cyp707a3-1 mutant contained higher ABA levels in turgid plants, which showed a reduced transpiration rate and hypersensitivity to exogenous ABA during early seedling growth. On dehydration, the cyp707a3-1 mutant accumulated a higher amount of stress-induced ABA than the wild type, an event that occurred relatively later and was coincident with slow drought induction of CYP707A3. The cyp707a3 mutant plants exhibited both exaggerated ABA-inducible gene expression and enhanced drought tolerance. Conversely, constitutive expression of CYP707A3 relieved growth retardation by ABA, increased transpiration, and a reduction of endogenous ABA in both turgid and dehydrated plants. Taken together, our results indicate that CYP707A3 plays an important role in determining threshold levels of ABA during dehydration and after rehydration.     

Preference for dietary fat induced by release of beta-endorphin in rats

AimsTo determine whether beta-endorphin contributes to the ingestion of and preference for dietary oil, we examined the relationship between the dynamics of beta-endorphin, before and after the ingestion of corn oil, and the intake volume of corn oil.Main methodsRats were offered 5% corn oil for 20 min for 5 consecutive days so they could acquire a preference for corn oil. On day 6, seven groups of rats were presented with the oil for defined time periods, and we measured the beta-endorphin levels in the serum and cerebrospinal fluid (CSF) before and after the presentation of corn oil as well as the consumed volume of corn oil at defined time points.Key findingsBeta-endorphin levels in serum and CSF were significantly increased 15 min after the ingestion of corn oil, followed by a rapid decrease and maintenance at the basal level throughout the rest of the experimental period. The intake of corn oil was the lowest in the time period of 15–30 min, when the beta-endorphin level reached a peak value. The intake volume of corn oil might be inversely correlated with beta-endorphin levels in serum and CSF. The pretreatment of naloxone, an antagonist of the opioid receptor, decreased the initial licking rate for corn oil and increased the latency for corn oil in the licking test.SignificanceThe beta-endorphin was rapidly released after oil ingestion, which contributed to the hedonic preference and ingestive behavior for fat.     

Boron Nutrition of Tobacco BY-2 Cells. V. Oxidative Damage is the Major Cause of Cell Death Induced by Boron Deprivation

Boron (B) is an essential micronutrient for vascular plants.However, it remains unclear how B deficiency leads to variousmetabolic disorders and cell death. To understand this mechanism,we analyzed the physiological changes in suspension-culturedtobacco (Nicotiana tabacum) BY-2 cells upon B deprivation. When3-day-old cells were transferred to B-free medium, cell deathwas detectable as early as 12 h after treatment. The B-deprivedcells accumulated more reactive oxygen species and lipid peroxidesthan control cells, and showed a slight but significant decreasein the cellular ascorbate pool. Supplementing the media withlipophilic antioxidants effectively suppressed the death ofB-deprived cells, suggesting that the oxidative damage is theimmediate and major cause of cell death under B deficiency.Dead cells in B-free culture exhibited a characteristic morphologywith a shrunken cytoplasm, which is often seen in cells undergoingprogrammed cell death (PCD). However, they did not display otherhallmarks of PCD such as internucleosomal DNA fragmentation,decreased ascorbate peroxidase expression and protection fromdeath by cycloheximide. These results suggest that the deathof tobacco cells induced by B deprivation is not likely to bea typical PCD.     

Mesenchymal to embryonic incomplete transition of human cells by chimeric OCT4/3 (POU5F1) with physiological co-activator EWS

POU5F1 (more commonly known as OCT4/3) is one of the stem cell markers, and affects direction of differentiation in embryonic stem cells. To investigate whether cells of mesenchymal origin acquire embryonic phenotypes, we generated human cells of mesodermal origin with overexpression of the chimeric OCT4/3 gene with physiological co-activator EWS (product of the EWSR1 gene), which is driven by the potent EWS promoter by translocation. The cells expressed embryonic stem cell genes such as NANOG, lost mesenchymal phenotypes, and exhibited embryonal stem cell-like alveolar structures when implanted into the subcutaneous tissue of immunodeficient mice. Hierarchical analysis by microchip analysis and cell surface analysis revealed that the cells are subcategorized into the group of human embryonic stem cells and embryonal carcinoma cells. These results imply that cells of mesenchymal origin can be traced back to cells of embryonic phenotype by the OCT4/3 gene in collaboration with the potent cis-regulatory element and the fused co-activator. The cells generated in this study with overexpression of chimeric OCT4/3 provide us with insight into cell plasticity involving OCT4/3 that is essential for embryonic cell maintenance, and the complexity required for changing cellular identity.     

Stability of folding structure of Zic zinc finger proteins

Zic family proteins have five C₂H₂-type zinc finger (ZF) motifs. We physicochemically characterized the folding properties of Zic ZFs. Alteration of chelation with zinc ions and of hydrophobic interactions changed circular dichroism spectra, suggesting that they caused structural changes. The motifs were heat stable, but electrostatic interactions had little effect on structural stability. These results highlight the importance of chelating interactions and hydrophobic interactions for the stability of the folding structure of Zic ZF proteins.     

Suppression of Side Reactions During Final Deprotection Employing a Strong Acid in Boc Chemistry: Regeneration of Methionyl Residues from Their Sulfonium Salts

During global deprotection using a strong acid in Boc chemistry, the electrophilic alkylating species, i.e. carbocations, and formaldehyde generated from the side-chain protecting groups and the benzyloxymethyl group on the His residue, respectively, can cause alkylation of susceptible residues. To reduce these side reactions, a deprotection procedure using a strong acid such as HF or trifuloromethanesulfonic acid must always be carried out in the presence of scavengers. We found that addition of hydroxylamine derivatives could efficiently suppress the side reactions associated with formaldehyde and that addition of 2-mercaptopyridine could not only specifically circumvent alkylation of the Met residue but also convert its sulfonium salt to the Met residue regardless of the substituent species.     

Relationships Between Slc1a5 and Osteoclastogenesis

Slc1a5 (ASCT2) encodes a small neutral amino-acid exchanger and is the most well-studied glutamine transporter in cancer cells. To investigate the role of Slc1a5 in osteoclastogenesis, we developed Slc1a5-deficient mice by using a conventional gene-targeting approach. The Slc1a5^−/− mice showed no obvious abnormalities in growth. Glutamine uptake was assessed in Slc1a5^+/+ and Slc1a5^−/− bone marrow cells stimulated with RANKL. The rate of glutamine uptake in Slc1a5^−/− bone marrow cells was reduced to 70% of that of cells from Slc1a5^+/+ bone marrow. To confirm the involvement of Slc1a5 in osteoclast formation, bone marrow cells derived from Slc1a5^+/+ or Slc1a5^−/− mice were stimulated with RANKL and macrophage colony-stimulating factor and stained with tartrate-resistant acid phosphatase. The bone resorption activity and actin ring formation of stimulated cells were measured. The formation of multinucleated osteoclasts in bone marrow cells isolated from Slc1a5^−/− mice was severely impaired compared with those from Slc1a5^+/+ mice. RANKL-induced expression of ERK, NFκB, p70S6K, and NFATc1 was suppressed in Slc1a5^−/− osteoclasts. These results show that Slc1a5 plays an important role in osteoclast formation.

Osteoclasts are giant multinucleated cells of hematopoietic origin that are responsible for bone resorption. The differentiation of osteoclasts can be induced by treating bone marrow macrophages with RANKL.² After stimulation, bone marrow macrophages mature and then fuse to become multinucleated osteoclasts. The processes of osteoclastogenesis and bone resorption are known to be energy-demanding,⁸ but little is known about the amino acid requirements of osteoclasts. In this study, we investigated the role of glutamine in osteoclastogenesis. Glutamine was selected for this work because it provides an excellent example of amino acid metabolism.Although glutamine acts as an essential amino acid in some specific physiologic situations, it is classified as a nonessential amino acid.⁵ The need for the biosynthesis and metabolism of amino acids is significantly increased in cells with high rates of proliferation, such as functionally active cells and cancer cells. The activity of amino acid synthetases such as glutamine synthetase is increased in these cells. In addition, glutamine transporters on the plasma membrane are important, because they mediate glutamine uptake to meet the intracellular glutamine demand. The transporter Slc1a5, also known as ASCT2, is particularly important for glutaminolysis and mTOR signaling.^14,16Glutamine concentrations in tissue and blood are regulated by the activities of glutamine synthetase and glutaminase. ­Endogenous synthesis cannot meet the cell’s demands for glutamine in conditions including cancer, infections, and intense physical exercise. Glutamine is released into the blood from the lungs, adipocytes, and skeletal muscles and is transported into the cytoplasm via glutamine acid transporter molecules on the cell membrane. Glutamine is required for the growth of cancer cells; upregulation of the expression of the proteins involved in glutamine transport has been observed in tumor cells.⁴ Slc1a5 (ASCT2) is a small neutral amino acid exchanger that is overexpressed in many cancers and is the most well-described glutamine transporter in cancer cells.⁹ However, previous studies^1,10,22,23 have reported that silencing, deletion, and amino-acid analog substitution of Slc1a5 in cancer cells generated different results for mTORC1 signaling, proliferation, and cell migration.^{1,3,4,10,22,23} Additional work^3,4 has shown that Slc1a5 is indispensable for tumor growth and mTORC1 signaling. Slc1a5 is important in accumulating nonessential amino acids to quickly restore amino acid composition during imbalanced amino acid usage,⁴ whereas Slc38a1 (SNAT1) and Slc38a2 (SNAT2) mediate the net import of glutamine.In bone homeostasis, glutamine is a critical regulator of energy for protein and nucleic acid synthesis via the tricarboxylic acid cycle. Active glutamine metabolism stimulates the proliferation and differentiation of osteoblasts, chondrocytes, and osteoclasts. The enzyme glutaminase deaminates glutamine to form glutamate. Glutaminase deficiency in osteoblasts and chondrocytes leads to reduced osteoblast formation and decreased bone mass, resulting in potentially dangerous conditions, such as osteoporosis.²⁴ In osteoclasts, glutamine is an important source of fuel for protein and nucleic acid biosynthesis. Therefore, Slc1a5 deficiency in mice may influence bone homeostasis, including osteoclastogenesis. We therefore created Slc1a5-deficient mice to investigate the contribution of Slc1a5 to the development and functional properties of osteoclasts.