Isolation of Aspergillus caninus (Synonym: Phialosimplex caninus) from a Canine Iliac Lymph node

Mycopathologia - Aspergillus caninus (synonym: Phialosimplex caninus) is an anamorphic fungus species associated with systemic infections in dogs that has been transferred from the genus...     

Translocation and activation of sphingosine kinase 1 by ceramide-1-phosphate

Sphingosine kinases (SphKs) and ceramide kinase (CerK) phosphorylate sphingosine to sphingosine-1-phosphate (S1P) and ceramide to ceramide-1-phosphate (C1P), respectively. S1P and C1P are bioactive lipids that regulate cell fate/function and human health/diseases. The translocation and activity of SphK1 are regulated by its phosphorylation of Ser ²²⁵ and by anionic lipids such as phosphatidic acid and phosphatidylserine. However, the roles of another anionic lipid C1P on SphK1 functions have not yet been elucidated, thus, we here investigated the regulation of SphK1 by CerK/C1P. C1P concentration dependently bound with and activated recombinant human SphK1. The inhibition of CerK reduced the phorbol 12-myristate 13-acetate-induced translocation of SphK1 to the plasma membrane (PM) and activation of the enzyme in membrane fractions of cells. A treatment with C1P translocated wild-type SphK1, but not the SphK1-S225A mutant, to the PM without affecting phosphorylation signaling. A cationic RxRH sequence is proposed to be a C1P-binding motif in α-type cytosolic phospholipase A ₂ and tumor necrosis factor α-converting enzyme. The mutation of four cationic amino acids to Ala in the 56-RRNHAR-61 domain in SphK1 reduced the phorbol 12-myristate 13-acetate- and C1P-induced translocation of SphK1 to the PM, however, the capacity of C1P to bind with and activate SphK1 was not affected by this mutation. In conclusion, C1P modulates SphK1 functions by interacting with multiple sites in SphK1.     

Periosteum-derived podoplanin-expressing stromal cells regulate nascent vascularization during epiphyseal marrow development

Bone marrow development and endochondral bone formation occur simultaneously. During endochondral ossification, periosteal vasculatures and stromal progenitors invade the primary avascular cartilaginous anlage, which induces primitive marrow development. We previously determined that bone marrow podoplanin (PDPN)-expressing stromal cells exist in the perivascular microenvironment and promote megakaryopoiesis and erythropoiesis. In this study, we aimed to examine the involvement of PDPN-expressing stromal cells in postnatal bone marrow generation. Using histological analysis, we observed that periosteum-derived PDPN-expressing stromal cells infiltrated the cartilaginous anlage of the postnatal epiphysis and populated on the primitive vasculature of secondary ossification center. Furthermore, immunophenotyping and cellular characteristic analyses indicated that the PDPN-expressing stromal cells constituted a subpopulation of the skeletal stem cell lineage. In vitro xenovascular model cocultured with human umbilical vein endothelial cells and PDPN-expressing skeletal stem cell progenies showed that PDPN-expressing stromal cells maintained vascular integrity via the release of angiogenic factors and vascular basement membrane-related extracellular matrices. We show that in this process, Notch signal activation committed the PDPN-expressing stromal cells into a dominant state with basement membrane-related extracellular matrices, especially type IV collagens. Our findings suggest that the PDPN-expressing stromal cells regulate the integrity of the primitive vasculatures in the epiphyseal nascent marrow. To the best of our knowledge, this is the first study to comprehensively examine how PDPN-expressing stromal cells contribute to marrow development and homeostasis.     

Structural basis of the strict specificity of a bacterial GH31 α-1,3-glucosidase for nigerooligosaccharides

Carbohydrate-active enzymes are involved in the degradation, biosynthesis, and modification of carbohydrates and vary with the diversity of carbohydrates. The glycoside hydrolase (GH) family 31 is one of the most diverse families of carbohydrate-active enzymes, containing various enzymes that act on α-glycosides. However, the function of some GH31 groups remains unknown, as their enzymatic activity is difficult to estimate due to the low amino acid sequence similarity between characterized and uncharacterized members. Here, we performed a phylogenetic analysis and discovered a protein cluster (GH31_u1) sharing low sequence similarity with the reported GH31 enzymes. Within this cluster, we showed that a GH31_u1 protein from Lactococcus lactis (LlGH31_u1) and its fungal homolog demonstrated hydrolytic activities against nigerose [α-D-Glcp-(1→3)-D-Glc]. The k_cat/K_m values of LlGH31_u1 against kojibiose and maltose were 13% and 2.1% of that against nigerose, indicating that LlGH31_u1 has a higher specificity to the α-1,3 linkage of nigerose than other characterized GH31 enzymes, including eukaryotic enzymes. Furthermore, the three-dimensional structures of LlGH31_u1 determined using X-ray crystallography and cryogenic electron microscopy revealed that LlGH31_u1 forms a hexamer and has a C-terminal domain comprising four α-helices, suggesting that it contributes to hexamerization. Finally, crystal structures in complex with nigerooligosaccharides and kojibiose along with mutational analysis revealed the active site residues involved in substrate recognition in this enzyme. This study reports the first structure of a bacterial GH31 α-1,3-glucosidase and provides new insight into the substrate specificity of GH31 enzymes and the physiological functions of bacterial and fungal GH31_u1 members.     

Sugar transport systems in Corynebacterium glutamicum: features and applications to strain development

Corynebacterium glutamicum uses the phosphoenolpyruvate-dependent sugar phosphotransferase system (PTS) to take up and phosphorylate glucose, fructose, and sucrose, the major sugars from agricultural crops that are used as the primary feedstocks for industrial amino acid fermentation. This means that worldwide amino acid production using this organism has depended exclusively on the PTS. Recently, a better understanding not only of PTS-mediated sugar uptake but also of global regulation associated with the PTS has permitted the correction of certain negative aspects of this sugar transport system for amino acid production. In addition, the recent identification of different glucose uptake systems in this organism has led to a strategy for the generation of C. glutamicum strains that express non-PTS routes instead of the original PTS. The potential practical advantages of the development of such strains are discussed.     

Tumor T1 Relaxation Time for Assessing Response to Bevacizumab Anti-Angiogenic Therapy in a Mouse Ovarian Cancer Model

Purpose

To assess whether T₁ relaxation time of tumors may be used to assess response to bevacizumab anti-angiogenic therapy. Procedures: 12 female nude mice bearing subcutaneous SKOV3ip1-LC ovarian tumors were administered bevacizumab (6.25ug/g, n=6) or PBS (control, n=6) therapy twice a week for two weeks. T₁ maps of tumors were generated before, two days, and 2 weeks after initiating therapy. Tumor weight was assessed by MR and at necropsy. Histology for microvessel density, proliferation, and apoptosis was performed.

Results

Bevacizumab treatment resulted in tumor growth inhibition (p<0.04, n=6), confirming therapeutic efficacy. Tumor T₁ relaxation times increased in bevacizumab treated mice 2 days and 2 weeks after initiating therapy (p<.05, n=6). Microvessel density decreased 59% and cell proliferation (Ki67+) decreased 50% in the bevacizumab treatment group (p<.001, n=6), but not apoptosis.

Conclusions

Findings suggest that increased tumor T₁ relaxation time is associated with response to bevacizumab therapy in ovarian cancer model and might serve as an early indicator of response.     

A Novel Peroxisome Proliferator-activated Receptor (PPAR)α Agonist and PPARγ Antagonist,Z-551, Ameliorates High-fat Diet-induced Obesity and Metabolic Disorders in Mice

A novel peroxisome proliferator-activated receptor (PPAR) modulator, Z-551, having both PPARα agonistic and PPARγ antagonistic activities, has been developed for the treatment of obesity and obesity-related metabolic disorders. We examined the effects of Z-551 on obesity and the metabolic disorders in wild-type mice on the high-fat diet (HFD). In mice on the HFD, Z-551 significantly suppressed body weight gain and ameliorated insulin resistance and abnormal glucose and lipid metabolisms. Z-551 inhibited visceral fat mass gain and adipocyte hypertrophy, and reduced molecules involved in fatty acid uptake and synthesis, macrophage infiltration, and inflammation in adipose tissue. Z-551 increased molecules involved in fatty acid combustion, while reduced molecules associated with gluconeogenesis in the liver. Furthermore, Z-551 significantly reduced fasting plasma levels of glucose, triglyceride, free fatty acid, insulin, and leptin. To elucidate the significance of the PPAR combination, we examined the effects of Z-551 in PPARα-deficient mice and those of a synthetic PPARγ antagonist in wild-type mice on the HFD. Both drugs showed similar, but weaker effects on body weight, insulin resistance and specific events provoked in adipose tissue compared with those of Z-551 as described above, except for lack of effects on fasting plasma triglyceride and free fatty acid levels. These findings suggest that Z-551 ameliorates HFD-induced obesity, insulin resistance, and impairment of glucose and lipid metabolisms by PPARα agonistic and PPARγ antagonistic activities, and therefore, might be clinically useful for preventing or treating obesity and obesity-related metabolic disorders such as insulin resistance, type 2 diabetes, and dyslipidemia.