Journal: The Journal of Biological Chemistry

Article Title: Mesotrypsin and Caspase-14 Participate in Prosaposin Processing

doi: 10.1074/jbc.M113.543421

Figure Lengend Snippet: Effect of epidermal proteases on prosaposin processing. A, Coomassie Brilliant Blue staining of GST-prosaposin. Recombinant GST-prosaposin was subjected to SDS-PAGE and stained with Coomassie Brilliant Blue. The band of the recombinant protein can be seen at ∼75 kDa. B, Western blot analysis of prosaposin degradation products. Extracts from differentiated keratinocytes containing prosaposin were incubated with revC14. Western blot analysis were carried out using antibodies to prosaposin (anti-Prosap), saposin A (anti-SapA), sapoins B (anti-SapB), saposin C (anti-SapC), and saposin D (anti-SapD). During incubation with active caspase-14, prosaposin in the extract was degraded into multiple intermediate products. The glycosylated form of saposin A (15 kDa) was detected. Arrowhead, GST-prosaposin; arrow, saposin A. C, co-transfection of pCMV-HA-Prosap, pCMV-HA-revC14, and pCMV-HA-mesotrypsin in growth and differentiated phases. The Western blot was carried out using a specific antibody to each molecule. D, detection of the active form of mesotrypsin in revC14-transfected keratinocytes. Keratinocytes were transfected with pCMV-HA-vector (control), pCMV-HA-revC14, or pCMV-HA-mesotrypsin and further incubated for 24 h in the presence or absence of protease inhibitors. Cell extracts were subjected to SDS-polyacrylamide gel electrophoresis and the presence of mesotrypsin was analyzed by Western blotting using anti-mesotrypsin antibody. Lane 1, pCMV-HA-vector; lane 2, pCMV-HA-revC14; lane 3, pCMV-HA-revC14 + Z-VAD-fmk; lane 4, pCMV-HA-revC14 + leupeptin; lane 5, pCMV-HA-revC14 + Z-VAD-fmk + leupeptin; lane 6, pCMV-HA-mesotrypsin. E, effect of caspase-14 on mesotrypsinogen activation. Enzymatic activity of mesotrypsin was measured using Boc-Gln-Ala-Arg-methylcoumarin amide as a substrate after incubation with caspase-14. To evaluate the direct hydrolytic activity of caspase-14 on this substrate, the same concentration of caspase-14 was incubated without mesotrypsinogen. Amounts of enzymes used in each assay (ng) are listed in parentheses. For comparison, enterokinase was also used. Results are shown as the mean of duplicate experiments. F, Western blot analysis of prosaposin degradation products by mesotrypsin, KLK5, and KLK7. After incubation with each protease, prosaposin degradation products were detected with antibodies to GST, prosaposin, saposin A, saposin B, saposin C, and saposin D. Asterisks indicate the presence of each saposin protein band. Lane 1, prosaposin control; lane 2, prosaposin + mesotrypsin; lane 3, prosaposin + KLK5; lane 4, prosaposin + KLK7.

Article Snippet: Control siRNA and siRNAs to caspase-14 and mesotrypsin were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).

Techniques: Staining, Recombinant, SDS Page, Western Blot, Incubation, Cotransfection, Transfection, Plasmid Preparation, Polyacrylamide Gel Electrophoresis, Activation Assay, Activity Assay, Concentration Assay

Journal: The Journal of Biological Chemistry

Article Title: Mesotrypsin and Caspase-14 Participate in Prosaposin Processing

doi: 10.1074/jbc.M113.543421

Figure Lengend Snippet: Expression and localization of prosaposin and saposin A. A, expression of prosaposin gene transcript in cultured keratinocytes. cDNAs were prepared from cultured keratinocytes at 80% confluence, 100% confluence, 2 days after confluence (120%), 2 days after confluence in the presence of 1.2 mm CaCl2, and 2 days after confluence together with air exposure. The prosaposin mRNA levels were determined using real-time RT-PCR. Data were normalized to the GAPDH gene. B, immunohistochemical localization of mesotrypsin, active caspase-14, prosaposin, and saposin A. Nuclei were counterstained with DAPI. Merged figures of antibody staining images with nuclear staining images are also shown. The dotted line shows the edge of the cornified layer. The broken line shows the epidermal-dermal junction. Scale bars: 50 μm. C, demonstration of interaction by PLA. Interaction between prosaposin, active caspase-14, and mesotypsin was investigated in vivo using the PLA method. Detection was carried out using the following antibody combinations: normal rabbit IgG/anti-Prosap Ab (negative control), anti-mesotrypsin Ab/h14D146, anti-Prosap Ab/anti-mesotrypsin Ab, and anti-Prosap Ab/h14D146. Merged figures of antibody staining images with nuclear staining images are also shown. Scale bars, 50 μm.

Article Snippet: Control siRNA and siRNAs to caspase-14 and mesotrypsin were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).

Techniques: Expressing, Cell Culture, Quantitative RT-PCR, Immunohistochemical staining, Staining, In Vivo, Negative Control

Journal: The Journal of Biological Chemistry

Article Title: Mesotrypsin and Caspase-14 Participate in Prosaposin Processing

doi: 10.1074/jbc.M113.543421

Figure Lengend Snippet: Effects of knockdown of caspase-14 and mesotrypsin on skin equivalent models. A, immunohistochemical analysis of the expression of caspase-14 and mesotrypsin. After treatment of keratinocytes with nonspecific control siRNA, caspase-14 siRNA, mesotrypsin siRNA, or the combination of both, keratinocytes were seeded on dermal components and skin equivalent models were constructed. Thin sections were stained with caspase-14 mAb and anti-mesotrypsin Ab. HE, hematoxylin and eosin stain. B, down-regulation of saposin A in the skin equivalent models after knockdown of caspase-14 and mesotrypsin. Sections from skin equivalent models treated with appropriate siRNAs were stained with anti-Prosap Ab and anti-SapA Ab. Merged figures of antibody staining images with nuclear staining images are also shown. Scale bars, 50 μm.

Article Snippet: Control siRNA and siRNAs to caspase-14 and mesotrypsin were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).

Techniques: Immunohistochemical staining, Expressing, Construct, Staining, H&E Stain

Journal: The Journal of Biological Chemistry

Article Title: Mesotrypsin and Caspase-14 Participate in Prosaposin Processing

doi: 10.1074/jbc.M113.543421

Figure Lengend Snippet: Prosaposin and saposin A deficiency affects lipid content in the cornified layer. A, immunohistochemical analysis of filaggrin, active caspase-14, and mesotrypsin expression in wild-type and prosaposin-deficient mice. Merged figures of antibody staining images with nuclear staining images are also shown. Scale bars, 50 μm. B, Oil Red staining of skin of prosaposin-deficient and saposin A-deficient mice. Two skin examples each are shown for wild-type mice, prosaposin-deficient mice, and saposin A-deficient mice. Scale bars, 50 μm.

Article Snippet: Control siRNA and siRNAs to caspase-14 and mesotrypsin were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).

Techniques: Immunohistochemical staining, Expressing, Staining

Journal: Emerging Infectious Diseases

Article Title: Human Exposure to Novel Bartonella Species from Contact with Fruit Bats

doi: 10.3201/eid2412.181204

Figure Lengend Snippet: Phylogenetic relationships of Bartonella rousetti (proposed name) obtained from Egyptian fruit bats ( Rousettus aegyptiacus ) collected in Nigeria, 2010 and 2013, and other Bartonella species and bat-associated Bartonella based on internal transcribed spacer sequences. The neighbor-joining method by the Kimura 2-parameter distance method and bootstrap calculation was conducted with 1,000 replicates for phylogenetic analysis. The internal transcribed spacer sequence obtained from the bat flies was closely clustered with B. rousetti . GenBank accession numbers are provided for the B. rousetti sequence and the comparison sequences.

Article Snippet: The positive control against the Egyptian fruit bat–associated Bartonella species was produced in laboratory mice via mouse immunization with heat-inactivated bacterium (ProSci Incorporated, Poway, CA, USA).

Techniques: Sequencing

Journal: Emerging Infectious Diseases

Article Title: Human Exposure to Novel Bartonella Species from Contact with Fruit Bats

doi: 10.3201/eid2412.181204

Figure Lengend Snippet: Epidemiologic data for persons with antibodies to Bartonella rousetti detected in study of human exposure to a novel Bartonella species from contact with fruit bats, Nigeria, 2013*

Article Snippet: The positive control against the Egyptian fruit bat–associated Bartonella species was produced in laboratory mice via mouse immunization with heat-inactivated bacterium (ProSci Incorporated, Poway, CA, USA).

Techniques: