Phosphatidylinositol 4-phosphate 5-kinase is indispensable for mouse spermatogenesis

The lipid kinase phosphatidylinositol 4-phosphate 5-kinase (PIP5K) produces a versatile signaling phospholipid, phosphatidylinositol 4,5-bisphosphate. Three PIP5K isozymes, PIP5K1A, PIP5K1B, and PIP5K1C, have been identified in mammals so far. Although the functions of these three PIP5K isozymes have been extensively studied in vitro, the in vivo physiological roles of these PIP5K isozymes remain largely unknown. In this study, we examined the functions of PIP5K1A and PIP5K1B in spermatogenesis, using Pip5k1a-knockout (KO), Pip5k1b-KO, and Pip5k1a/Pip5k1b double (D)-KO mice. Pip5k1a-KO and D-KO males were subfertile and completely sterile, respectively. F-actin in the seminiferous epithelium was disorganized in the D-KO mice, although F-actin bundles at the apical ectoplasmic specialization was not affected. D-KO seminiferous tubules contained a greatly decreased number of elongated spermatids. Flagella of sperm from Pip5k1a-KO and D-KO mice remarkably underwent morphological change, whereas Pip5k1b-KO sperm were morphologically normal. Notably, the flagellar shape of D-KO sperm was more severely impaired than that of Pip5k1a-KO sperm. These results suggest that PIP5K1A and PIP5K1B may coordinately and/or redundantly function in the maintenance of sperm number and morphology during spermatogenesis.     

Escape of leukemia blasts from HLA-specific CTL pressure in a recipient of HLA one locus-mismatched bone marrow transplantation

A case of leukemia escape from an HLA-specific cytotoxic T lymphocyte (CTL) response in a recipient of bone marrow transplantation is presented. Only the expression of HLA-B51, which was a mismatched HLA locus in the graft-versus-host direction, was down-regulated in post-transplant leukemia blasts compared with that in pre-transplant blasts. All CTL clones, that were isolated from the recipient's blood when acute graft-versus-host disease developed, recognized the mismatched B(?)51:01 molecule in a peptide-dependent manner. The pre-transplant leukemia blasts were lysed by CTL clones, whereas the post-transplant leukemia blasts were not lysed by any CTL clones. The IFN-γ ELISPOT assay revealed that B(?)51:01-reactive T lymphocytes accounted for the majority of the total alloreactive T lymphocytes in the blood just before leukemia relapse. These data suggest that immune escape of leukemia blasts from CTL pressure toward a certain HLA molecule can lead to clinical relapse after bone marrow transplantation.     

Purification and characterization of two phospho-β-galactosidases, LacG1 and LacG2, from Lactobacillus gasseri ATCC33323(T)

Lactobacillus gasseri ATCC33323(T) expresses four enzymes showing phospho-β-galactosidase activity (LacG1, LacG2, Pbg1 and Pbg2). We previously reported the purification and characterization of two phospho-β-galactosidases (Pbg1 and Pbg2) from Lactobacillus gasseri JCM1031 cultured in lactose medium. Here we aimed to characterize LacG1 and LacG2, and classify the four enzymes into 'phospho-β-galactosidase' or 'phospho-β-glucosidase.' LacG1 and recombinant LacG2 (rLacG2), from Lb. gasseri ATCC33323(T), were purified to homogeneity using column chromatography. Kinetic experiments were performed using sugar substrates, o-nitrophenyl-β-D-galactopyranoside 6-phosphate (ONPGal-6P) and o-nitrophenyl-β-D-glucopyranoside 6-phosphate (ONPGlc-6P), synthesized in our laboratory. LacG1 and rLacG2 exhibited high k(cat)/K(m) values for ONPGal-6P as compared with Pbg1 and Pbg2. The V(max) values for ONPGal-6P were higher than phospho-β-galactosidases previously purified and characterized from several lactic acid bacteria. A phylogenetic tree analysis showed that LacG1 and LacG2 belong to the phospho-β-galactosidase cluster and Pbg1 and Pbg2 belong to the phospho-β-glucosidase cluster. Our data suggest two phospho-β-galactosidase, LacG1 and LacG2, are the primary enzymes for lactose utilization in Lb. gasseri ATCC33323(T). We propose a reclassification of Pbg1 and Pbg2 as phospho-β-glucosidase.     

Design and synthesis of novel opioid ligands with an azabicyclo[2.2.2]octane skeleton and their pharmacologies

A novel opioid ligand possessing a stable and cyclic imine 16 and its derivatives with an azabicyclo[2.2.2]octane skeleton were synthesized. The imine 16 showed higher affinity for the μ receptor than compound 21 with an oxabicyclo[2.2.2]octane skeleton. Azabicyclo[2.2.2]octane derivative 18d with a cyclopropylmethyl group on the 8-nitrogen showed the highest affinity for the μ receptor among the synthesized derivatives.     

Effects of a novel Y5 antagonist in obese mice: combination with food restriction or sibutramine

Objective: To further address the function of the Y5 receptor in energy homeostasis, we investigated the effects of a novel spironolactone Y5 antagonist in diet-induced obese (DIO) mice. Methods and Procedures: Male C57BL/6 or Npy5r^−/− mice were adapted to high-fat (HF) diet for 6–10 months and were submitted to three experimental treatments. First, the Y5 antagonist at a dose of 10 or 30 mg/kg was administered for 1 month to DIO C57BL/6 or Npy5r^−/− mice. Second, the Y5 antagonist at 30 mg/kg was administered for 1.5 months to DIO C57BL/6 mice, and insulin sensitivity was evaluated using an insulin tolerance test. After a recovery period, nuclear magnetic resonance measurement was performed to evaluate body composition. Third, DIO mice were treated with the Y5 antagonist alone, or in combination with 10% food restriction, or with another anorectic agent, sibutramine at 10 mg/kg, for 1.5 months. Plasma glucose, insulin, and leptin levels, and adipose tissue weights were quantified. Results: The spironolactone Y5 antagonist significantly reduced body weight in C57BL DIO mice, but not in Npy5r^−/− DIO mice. The Y5 antagonist produced a fat-selective loss of body weight, and ameliorated obesity-associated insulin resistance in DIO mice. In addition, the Y5 antagonist combined with either food restriction or sibutramine tended to produce greater body weight loss, as compared with single treatment. Discussion: These findings demonstrate that the Y5 receptor is an important mediator of energy homeostasis in rodents.     

Toll-like receptor 4 and cytokine expression involved in functional immune response in an originally established porcine intestinal epitheliocyte cell line

To study the immune responses of porcine intestinal epithelial cells to gram-negative bacteria via toll-like receptors (TLRs), originally established porcine intestinal epitheliocyte (PIE) cells were treated with lipopolysaccharide (LPS) or swine-specific enterotoxigenic Escherichia coli (ETEC). Real-time quantitative PCR revealed that PIE cells expressed TLR1-9 and MD-2 mRNAs, preferentially expressed TLR4/MD-2. Immunostaining of PIE cells revealed that TLR4 was precisely expressed in PIE cells at the protein level. PIE cells treated with LPS had up-regulated expression of several TLRs (TLR2, 3, 4, 5 and 8), type 1 helper T (Th1) cytokines (interleukin (IL)-1alpha, IL-1beta, IL-6, IL-15, 18, leukemia inhibitory factor (LIF), and interferon (IFN)-beta), and chemokines (monocyte chemoattractant protein (MCP)-1 and IL-8). ETEC enhanced the expression of TLR2, Th1 type cytokines (IL-1alpha, IL-12p35 and IL-6) and chemokines (MCP-1 and IL-8). These results indicate that PIE induces inflammatory responses by up-regulating Th1 cytokines and chemokines in response to LPS or ETEC, suggesting that PIE is a useful cell line for studying inflammatory responses via TLR4/MD-2 in intestinal epithelial cells.     

Plant Callus: Mechanisms of Induction and Repression

Plants develop unorganized cell masses like callus and tumors in response to various biotic and abiotic stimuli. Since the historical discovery that the combination of two growth-promoting hormones, auxin and cytokinin, induces callus from plant explants in vitro, this experimental system has been used extensively in both basic research and horticultural applications. The molecular basis of callus formation has long been obscure, but we are finally beginning to understand how unscheduled cell proliferation is suppressed during normal plant development and how genetic and environmental cues override these repressions to induce callus formation. In this review, we will first provide a brief overview of callus development in nature and in vitro and then describe our current knowledge of genetic and epigenetic mechanisms underlying callus formation.