A new acid carboxypeptidase,O-1, fromAspergillus oryzae

A new acid carboxypeptidase was purified fromAspergillus oryzae grown on solid bran culture medium. The purified enzyme was found to be homogeneous by disc gel electrophoresis at pH 9.4 and isoelectric focusing. The enzyme was termedA. oryzae acid carboxypeptidase O-1 with isoelectric point 4.08. The substrate specificity of the new enzyme was investigated with proangiotensin, angiotensin, and bradykinin. Even when the proline was present at the penultimate position of the peptide, the enzyme rapidly hydrolyzed the carboxyterminal Pro-X (X=amino acid) peptide bond. TheK _m andk _cat values for angiotension (–Pro⁷–Phe⁸) at pH 3.7 and 30°C were 0.2 mM and 1.7 sec^–1, respectively.     

Identification and characterization of RPK118, a novel sphingosine kinase-1-binding protein

Sphingosine kinase (SPHK) is a key enzyme catalyzing the formation of sphingosine 1 phosphate (SPP), a lipid messenger that is implicated in the regulation of a wide variety of important cellular events through intracellular as well as extracellular mechanisms. However, the molecular mechanism of the intracellular actions of SPP remains unclear. Here we have cloned a novel sphingosine kinase-1 (SPHK1)-binding protein, RPK118, by yeast two-hybrid screening. RPK118 contains several functional domains whose sequences are homologous to other known proteins including the phox homology domain and pseudokinase 1 and 2 domains and is shown to be a member of an evolutionarily highly conserved gene family. The pseudokinase 2 domain of RPK118 is responsible for SPHK1 binding as judged by yeast two-hybrid screening and immunoprecipitation studies. RPK118 is also shown to co-localize with SPHK1 on early endosomes in COS7 cells expressing both recombinant proteins. Furthermore, RPK118 specifically binds to phosphatidylinositol 3-phosphate. These results strongly suggest that RPK118 is a novel SPHK1-binding protein that may be involved in transmitting SPP-mediated signaling into the cell.     

Apoptotic activities of C2-ceramide and C2-dihydroceramide homologues against HL-60 cells

The apoptotic activities of non-natural ceramide homologues, C2-homo-ceramide, C2-homo-dihydroceramide, C2-bishomo-ceramide and C2-bishomo-dihydroceramide, were examined using human leukemia HL-60 cells. The apoptotic activity was in order of C2-ceramide>C2-homo-ceramide approximately C2-bishomo-ceramide and the activities of the L-erythro- and D-erythro-ceramide homologues were similar. The morphological features of the cells, DNA fragmentations, proteolytic processing of pro-caspase-3 and the cleavage of PARP as the result of treatments with these homologues indicated that cell death was induced by apoptosis.     

Plant growth regulators and Axl and immune checkpoint inhibitors from the edible mushroom Leucopaxillus giganteus

ABSTRACT

A novel compound, (R)-4-ethoxy-2-hydroxy-4-oxobutanoic acid (1), and six known compounds (2–7) were isolated from the fruiting bodies of the wild edible mushroom Leucopaxillus giganteus. The planar structure of 1 was determined by the interpretation of spectroscopic data analysis. The absolute configuration of 1 was determined by comparing specific rotation of the synthetic compounds. In the plant regulatory assay, the isolated compounds (1–7) and the chemically prepared compounds (8–10) were evaluated their biological activity against the lettuce (Lactuca sativa) growth. Compounds 1 and 3–10 showed the significant regulatory activity of lettuce growth. 1 showed the strongest inhibition activity among the all the compounds tested. In the lung cancer assay, all the compounds were assessed the mRNA expression of Axl and immune checkpoints (PD-L1, PD-L2) in the human A549 alveolar epithelial cell line by RT-PCR. Compounds 1–10 showed significant inhibition activity against Axl and/or immune checkpoint.     

Novel Strain of Spiroplasma Found in Flower Bugs of the Genus Orius (Hemiptera: Anthocoridae): Transovarial Transmission,Coexistence with Wolbachia and Varied Population Density

Spiroplasma, a group of small, wall-less, helical, and motile bacteria belonging to the Mollicutes, contains species with diverse life histories. To date, all the Spiroplasma strains that are known to be transmitted vertically in arthropod lineages belong to either the Spiroplasma ixodetis group or the Spiroplasma poulsonii group. Here, we found that a unique strain of Spiroplasma vertically transmitted in predatory flower bugs of the genus Orius belongs to the Spiroplasma insolitum group, which is a group of bacteria phylogenetically closely related to S. insolitum derived from the tickseed sunflower, Bidens sp. (Asterales: Asteraceae). The infection frequencies in natural populations were16.0 % in Orius sauteri (n?=?75), 40.5 % in Orius nagaii (n?=?37), and 8.0 % in Orius minutus (n?=?87). Orius strigicollis was not infected with Spiroplasma (n?=?147). In the early stage of oogenesis (i.e., within the germarium), a large number of bacteria with the typical morphology of Spiroplasma existed, keeping a distance from Wolbachia bacteria. The Spiroplasma population seemed to increase during host development but Wolbachia population did not.     

In vivo delivery of small interfering RNA targeting brain capillary endothelial cells

Brain capillary endothelial cells (BCECs) play an important role in blood-brain barrier (BBB) functions and pathophysiologic mechanisms in brain ischemia and inflammation. We try to suppress gene expression in BCECs by intravenous application of small interfering RNA (siRNA). After injection of large dose siRNA with hydrodynamic technique to mouse, suppression of endogenous protein and the BBB function of BCECs was investigated. The brain-to-blood transport function of organic anion transporter 3 (OAT3) that expressed in BCECs was evaluated by Brain Efflux Index method in mouse. The siRNA could be delivered to BCECs and efficiently inhibited endogenously expressed protein of BCECs. The suppression effect of siRNA to OAT3 is enough to reduce the brain-to-blood transport of OAT3 substrate, benzylpenicillin at BBB. The in vivo siRNA-silencing method with hydrodynamic technique may be useful for the study of BBB function and gene therapy targeting BCECs.     

Specific binding of Autographa californica M nucleopolyhedrovirus occlusion-derived virus to midgut cells of Heliothis virescens larvae is mediated by products of pif genes Ac119 and Ac022 but not by Ac115

Per os infectivity factors PIF1 (Ac119) and PIF2 (Ac022), like P74, are essential for oral infection of lepidopteran larval hosts of Autographa californica M nucleopolyhedrovirus (AcMNPV). Here we show that Ac115 also is a PIF (PIF3) and that, unlike PIF1 and PIF2, it does not mediate specific binding of AcMNPV occlusion-derived virus (ODV) to midgut target cells. We used an improved in vivo fluorescence dequenching assay to compare binding, fusion, and competition among control AcMNPV ODV and the ODVs of AcMNPV PIF1, PIF2, and PIF3 deletion mutants. Our results showed that binding and fusion of PIF1 and PIF2 mutants, but not the PIF3 mutant, were both qualitatively and quantitatively different from those of control ODV. Unlike control and PIF3-deficient ODV, an excess of PIF1- or PIF2-deficient ODV failed to compete effectively with control ODV's binding to specific receptors on midgut epithelial cells. Moreover, the levels of PIF1- and PIF2-deficient ODV binding were depressed threefold compared to control levels. Binding, fusion, and competition by PIF3-deficient ODV, however, were all indistinguishable from those of control ODV. These results implicated PIF1 and PIF2 as ODV envelope attachment proteins that mediate specific binding to primary target cells within the midgut. In contrast, PIF3 mediates another unidentified, but critical, early event during primary infection.     

Design and synthesis of a new class of malonyl-CoA decarboxylase inhibitors with anti-obesity and anti-diabetic activities

A new series of thiazole-substituted 1,1,1,3,3,3-hexafluoro-2-propanols were prepared and evaluated as malonyl-CoA decarboxylase (MCD) inhibitors. Key analogs caused dose-dependent decreases in food intake and body weight in obese mice. Acute treatment with these compounds also led to a drop in elevated blood glucose in a murine model of type II diabetes.     

Calpastatin, an endogenous calpain-inhibitor protein, regulates the cleavage of the Cdk5 activator p35 to p25

Cyclin-dependent kinase 5 (Cdk5) is a Ser/Thr kinase that is activated by binding to its regulatory subunit, p35. The calpain-mediated cleavage of p35 to p25 and the resulting aberrant activity and neurotoxicity of Cdk5 have been implicated in neurological disorders, such as Alzheimer's disease. To gain further insight into the molecular mechanisms underlying the pathological function of Cdk5, we investigated the role of the calpain inhibitor protein calpastatin (CAST), in controlling the aberrant production of p25. For this purpose, brain tissue from wild-type, CAST-over-expressing (transgenic), and CAST knockout mice were analyzed. Cleavage of p35 to p25 was increased in extracts from CAST knockout mice, compared with wild-type. Conversely, generation of p25 was not detected in brain lysates from CAST-over-expressing mice. CAST expression was 5-fold higher in mouse cerebellum than cerebral cortex. Accordingly, p25 production was lower in the cerebellum than the cerebral cortex. Furthermore, the Ca(2+) -dependent degradation of p35 by proteasome was evident when calpain was inhibited. Taken together, these results suggest that CAST is a crucial regulator of calpain activity, the production of p25, and, hence, the deregulation of Cdk5. Therefore, impairment of CAST expression and its associated mechanisms may contribute to the pathogenesis of neurodegenerative disorders.     

H-Ferritin Is Preferentially Incorporated by Human Erythroid Cells through Transferrin Receptor 1 in a Threshold-Dependent Manner

Ferritin is an iron-storage protein composed of different ratios of 24 light (L) and heavy (H) subunits. The serum level of ferritin is a clinical marker of the body’s iron level. Transferrin receptor (TFR)1 is the receptor not only for transferrin but also for H-ferritin, but how it binds two different ligands and the blood cell types that preferentially incorporate H-ferritin remain unknown. To address these questions, we investigated hematopoietic cell-specific ferritin uptake by flow cytometry. Alexa Fluor 488-labeled H-ferritin was preferentially incorporated by erythroid cells among various hematopoietic cell lines examined, and was almost exclusively incorporated by bone marrow erythroblasts among human primary hematopoietic cells of various lineages. H-ferritin uptake by erythroid cells was strongly inhibited by unlabeled H-ferritin but was only partially inhibited by a large excess of holo-transferrin. On the other hand, internalization of labeled holo-transferrin by these cells was not inhibited by H-ferritin. Chinese hamster ovary cells lacking functional endogenous TFR1 but expressing human TFR1 with a mutated RGD sequence, which is required for transferrin binding, efficiently incorporated H-ferritin, indicating that TFR1 has distinct binding sites for H-ferritin and holo-transferrin. H-ferritin uptake by these cells required a threshold level of cell surface TFR1 expression, whereas there was no threshold for holo-transferrin uptake. The requirement for a threshold level of TFR1 expression can explain why among primary human hematopoietic cells, only erythroblasts efficiently take up H-ferritin.