Neuron-specific recombination by Cre recombinase inserted into the murine tau locus

To determine the neuronal function of genes in vivo, the neuron-specific deletion of a target gene in animals is required. Tau, a microtubule-associated protein, is expressed abundantly in neurons but scarcely in glias and other tissues. Therefore, to generate mice that express Cre recombinase in neurons, we inserted Cre recombinase into the tau locus. By crossing these tau-Cre mice with ROSA26 lacZ reporter mice, we observed Cre recombinase activity in the neurons from most of the central nervous system, but not in glias nor in non-neuronal tissues. This neuronal-specific activity appeared during embryogenesis. We further crossed tau-Cre mice with rab8 ‘floxed’ mice, and showed that the recombination was nearly complete in the brain, but incomplete or non-detectable in other tissues. Thus, tau-Cre knockin mouse is a useful tool for studying the neuronal function of a gene in vivo.     

The incorporation of 32Pi into intramitochondrial ADP fraction dependent on the substrate-level phosphorylation

1. A significant amount of ³²P_i is incorporated into ADP fraction if mitochondrial phosphorylation is allowed to proceed solely dependent on the endogenous adenine nucleotides even in the absence of uncouplers or inhibitors of oxidative phosphorylation. This formation of [³²P]ADP is accompanied by a significant labelling of the GTP fraction as well as by a decrease in mitochondrial AMP.2. A good correlation, highly significant on a statistical basis, is obtained between the incorporation of ³²P_i into ADP on the one hand and the oxidation of [1-¹⁴C]glutamate to ¹⁴CO₂ on the other, under a wide variety of conditions of respiration, suggesting that the substrate-level phosphorylation linked to the oxidation of 2-oxoglutarate leads to the phosphorylation of AMP in rat liver mitochondria.3. Since intramitochondrial GTP is not directly labelled by the [³²P]ATP added, it is concluded that neither nucleoside diphosphokinase (ATP:nucleoside diphosphate phosphotransferase, EC 2.7.4.6) nor adenylate kinase (ATP:AMP phosphotransferase, EC 2.7.4.3) is functioning in such an EDTA-containing medium as employed in the present study because of lack of the enzymes inside the inner membrane. This not only indicates that ATP never serves as a phosphate donor for the observed phosphorylation of AMP, but also, along with several other lines of evidence, lends strong support to the view that [³²P]GTP generated as a result of the substrate-level phosphorylation is a direct precursor of [³²P]ADP through the mediation of GTP:AMP phosphotransferase, which has been verified to be located inside the inner membrane by the significant labelling of GTP by [³²P]ADP.     

The ABC subfamily B auxin transporter AtABCB19 is involved in the inhibitory effects of N-1-naphthyphthalamic acid on the phototropic and gravitropic responses of Arabidopsis hypocotyls

N-1-Naphthylphthalamic acid (NPA) causes the abnormal growth and development of plants by suppressing polar auxin transport. The mechanisms underlying this inhibition, however, have remained elusive. In Arabidopsis, we show that a defect in the ABC subfamily B auxin transporter AtABCB19 suppresses the inhibitory effects of NPA on hypocotyl phototropism and gravitropism, but not on hypocotyl elongation. Expression analysis using the auxin reporter gene DR5:GUS further suggests that NPA partially inhibits the asymmetric distribution of auxin in an AtABCB19-dependent manner. These data thus suggest that AtABCB19 plays an important role in the inhibitory effects of NPA on hypocotyl tropism induced by auxin.     

Lipophagy maintains energy homeostasis in the kidney proximal tubule during prolonged starvation

Macroautophagy/autophagy is a self-degradation process that combats starvation. Lipids are the main energy source in kidney proximal tubular cells (PTCs). During starvation, PTCs increase fatty acid (FA) uptake, form intracellular lipid droplets (LDs), and hydrolyze them for use. The involvement of autophagy in lipid metabolism in the kidney remains largely unknown. Here, we investigated the autophagy-mediated regulation of renal lipid metabolism during prolonged starvation using PTC-specific Atg5-deficient (atg5-TSKO) mice and an in vitro serum starvation model. Twenty-four h of starvation comparably induced LD formation in the PTCs of control and atg5-TSKO mice; however, additional 24 h of starvation reduced the number of LDs in control mice, whereas increases were observed in atg5-TSKO mice. Autophagic degradation of LDs (lipophagy) in PTCs was demonstrated by electron microscopic observation and biochemical analysis. In vitro pulse-chase assays demonstrated that lipophagy mobilizes FAs from LDs to mitochondria during starvation, whereas impaired LD degradation in autophagy-deficient PTCs led to decreased ATP production and subsequent cell death. In contrast to the in vitro assay, despite impaired LD degradation, kidney ATP content was preserved in 48-h starved atg5-TSKO mice, probably due to increased utilization of ketone bodies. This compensatory mechanism was accompanied by a higher plasma FGF21 (fibroblast growth factor 21) level and its expression in the PTCs; however, this was not essential for the production of ketone bodies in the liver during prolonged starvation. In conclusion, lipophagy combats prolonged starvation in PTCs to avoid cellular energy depletion.     

Epitope Mapping for Monoclonal Antibody Reveals the Activation Mechanism for αVβ3 Integrin

Epitopes for a panel of anti-αVβ3 monoclonal antibodies (mAbs) were investigated to explore the activation mechanism of αVβ3 integrin. Experiments utilizing αV/αIIb domain-swapping chimeras revealed that among the nine mAbs tested, five recognized the ligand-binding β-propeller domain and four recognized the thigh domain, which is the upper leg of the αV chain. Interestingly, the four mAbs included function-blocking as well as non-functional mAbs, although they bound at a distance from the ligand-binding site. The epitopes for these four mAbs were further determined using human-to-mouse αV chimeras. Among the four, P3G8 recognized an amino acid residue, Ser-528, located on the side of the thigh domain, while AMF-7, M9, and P2W7 all recognized a common epitope, Ser-462, that was located close to the α-genu, where integrin makes a sharp bend in the crystal structure. Fibrinogen binding studies for cells expressing wild-type αVβ3 confirmed that AMF-7, M9, and P2W7 were inhibitory, while P3G8 was non-functional. However, these mAbs were all unable to block binding when αVβ3 was constrained in its extended conformation. These results suggest that AMF-7, M9, and P2W7 block ligand binding allosterically by stabilizing the angle of the bend in the bent conformation. Thus, a switchblade-like movement of the integrin leg is indispensable for the affinity regulation of αVβ3 integrin.     

Multiple roles of the mitogen-activated protein kinase kinase/mitogen-activated protein kinase cascade in Xenopus laevis

Mitogen-activated protein kinase (MAPK) was originally identified as a serine/threonine protein kinase that is rapidly activated in response to various growth factors and tumor promoters in mammalian cultured cells. The kinase cascade including MAPK and its direct activator, MAPK kinase (MAPKK), is now believed to transmit various extracellular signals into their intracellular targets in eukaryotic cells. It has been reported that activation of MAPKK and MAPK occurs during the meiotic maturation of oocytes in several species, including Xenopus laevis . Studies with neutralizing antibodies against MAPKK, MAPK phosphatases and constitutively active MAPKK or MAPK have revealed a crucial role of the MAPKK/MAPK cascade in a number of developmental processes in Xenopus oocytes and embryos.     

Two genes controlling acute phase responses by the antitumor polysaccharide,lentinan

Lentinan, a -1,6;1,3-glucan, is tumor-specific for transplantable mouse solid-type tumors and it also stimulates the production of acute phase proteins (APPs). The APP response to lentinan is of the delayed type (DT-APR) and differs from that to lipopolysaccharide, which is acute. We found that the responses were genetically controlled in mice and that low responsiveness is dominant (Maeda et al. 1991). Using 123 segregants of crosses between SWR/J (a high responder) andMus spretus (a low responder), we analyzed the linkage between DT-APR responsiveness and the DNA polymerase chain reaction-simple sequence lenght polymorphism (PCR-SSLP) phenotype using 80 chromosome-specific microsatellite markers. We identified two loci (ltn1.1 andltn1.2) responsible for DT-APR.ltn1.1 is closely linked toD3Mit11 on chromosome 3 andltn1.2 toD11Nds9 on chromosome 11 (P<0.001). The linkage analysis also suggested thatltn1.2 is the major determinant for DT-APR. Correlation between lentinan-specific IL-6 mRNA expression (the late expression) controlled recessively and DT-APR induction suggests that theltn1 loci control some process(es) of IL-6 expression in the regulation step before NF-IL6.     

Flavor Constituents of Pouchong Tea and a Comparison of the Aroma Pattern with Jasmine Tea

The aroma constituents of the highest quality pouchong tea were characterized by GC–MS. Forty-eight components, including five newly identified compounds were characterized. GC peak area percentages of the main components in pouchong tea were compared with those of jasmine tea to differentiate compounds contributing to the aroma characteristics of pouchong tea with superior floral elegant flavor. Nerolidol, jasmine lactone, methyl jasmonate, indole, benzyl cyanide and linalool oxides were found in much higher concentration in pouchong tea than in jasmine tea. These compounds seemed to contribute to the aroma characteristics of the highest quality pouchong tea.     

in Vitro Effect of Deoxynivalenol on the Differentiation of Human Colonic Cell Lines Caco-2 and t84

The effects of low concentrations of deoxynivalenol (DON) on structural and functional characteristics of human colonic adenocarcinoma cell lines Caco-2 and T84 were examined. Scanning electron microscopic (SEM) analysis of the apical surfaces of Caco-2 cells revealed reduction or abnormal formation of brush borders in the presence of 50, 100 and 200 ng/ml of DON. Monolayer integrity of Caco-2 and T84 cells was studied using cells which were cultured on permeable membranes. The transepithelial electrical resistance (TEER) of Caco-2 cells was significantly reduced at 50, 100 and 200 ng/ml of DON, significant increase in lucifer yellow (LY) permeability was also observed in these cells at 100 ng/ml of DON. The TEER of T84 cells was significantly reduced at 100 and 200 ng/ml of DON. LY permeability significantly increased at 200 ng/ml of DON in T84 cells. Enzyme activities in Caco-2 cells were also examined. Alkaline phosphatase activity was reduced from the 6th to 15th day of culture in the presense of 100 or 200 ng/ml of DON, whereas sucrase- isomaltase activity was significantly decreased by adding 50 or 100 ng/ml of DON for 15 or 20 days. Protein content was attenuated only by treatment with 200 ng/ml of DON thoughout the experimental period. The results indicate that DON interferes with structural and functional characteristics of differentiation in enterocytes at low doses. This revised version was published online in June 2006 with corrections to the Cover Date.