Journal: Biomacromolecules

Article Title: Combining Biologically Active β-Lactams Integrin Agonists with Poly( l -lactic acid) Nanofibers: Enhancement of Human Mesenchymal Stem Cell Adhesion

doi: 10.1021/acs.biomac.9b01550

Figure Lengend Snippet: Qualitative and quantitative analysis of specific proteins involved in the cell adhesive process to plain PLLA and SR610-PLLA (6.18 wt %), GM18-PLLA (7.48 wt %), and LT25-PLLA (6.31 wt %) after 2 h from seeding. (A) Western blotting analysis. Bar graphs show β 1 integrin and vinculin expression level obtained normalizing to the β-actin housekeeping protein signal. The activation level of FAK was presented as a ratio between the phosphorylated and total FAK protein after normalization to β-actin. Bars represent the mean values ± SD of results from three experiments ( n = 3). Statistical significance values are indicated as *** p < 0.001 and * p < 0.05. (B) CLSM images, showing the expression of focal adhesion β 1 integrin (green, 488 Alexa Fluor), vinculin (red, 633 Alexa Fluor), and p-FAK (green, 488 Alexa Fluor) on different PLLA scaffolds, were acquired at 40× magnification. Nuclei were stained with Hoechst 33342 (blue). Scale bars: 50 μm. Yellow arrows indicated protein distribution at cellular level. The insets display a protein staining with false coloring from dark purple to bright yellow by use of the fire lookup table (LUT) scheme to highlight differences in the intensities of the signals obtained with ImageJ software. Graphs show the correct total cell fluorescence intensity (CTCF) measured in each sample ( n = 3, *** p < 0.001 and * p < 0.05).

Article Snippet: For focal adhesion detection, cells were incubated with primary mouse anti-α-vinculin antibody (1:500 in 1% bovine serum albumin, BSA, BosterBio, Pleasanton, CA, USA), anti-β 1 -integrin (1:100 in 1% BSA, NSJ Bioreagents, San Diego, CA, USA), or anti-p-FAK (pY397, 1:250, Santa Cruz, USA).

Techniques: Western Blot, Expressing, Activation Assay, Staining, Software, Fluorescence

Journal: Biomacromolecules

Article Title: Combining Biologically Active β-Lactams Integrin Agonists with Poly( l -lactic acid) Nanofibers: Enhancement of Human Mesenchymal Stem Cell Adhesion

doi: 10.1021/acs.biomac.9b01550

Figure Lengend Snippet: hBM-MSCs viability and morphology on PLLA and GM18-PLLA (7.48 wt %) scaffolds after prewetting treatment. (A) Cell viability was evaluated at day 3 and 7, respectively. Cell viability was plotted as the percentage of viable cells in comparison with initial state (day 0 = T 0 ) set as 100% cell viability. Bars indicate mean values ± SD of the mean of results from three experiments. (B) Representative SEM images of hBM-MSCs cultured on PLLA and GM18-PLLA at day 7 of culture. (C) Gene expression of the indicated integrin subunits. The graphs show the fold increase of gene expression related to cells during the initial state (day 0), set equal to 1 ( n = 3). Statistical significance values are indicated as ** p < 0.01; *** p < 0.001.

Article Snippet: For focal adhesion detection, cells were incubated with primary mouse anti-α-vinculin antibody (1:500 in 1% bovine serum albumin, BSA, BosterBio, Pleasanton, CA, USA), anti-β 1 -integrin (1:100 in 1% BSA, NSJ Bioreagents, San Diego, CA, USA), or anti-p-FAK (pY397, 1:250, Santa Cruz, USA).

Techniques: Cell Culture, Expressing

Journal: The Journal of Biological Chemistry

Article Title: Human Cathepsin V Protease Participates in Production of Enkephalin and NPY Neuropeptide Neurotransmitters *

doi: 10.1074/jbc.M111.310607

Figure Lengend Snippet: Cellular expression of cathepsin V with PE results in (Met)enkephalin production in PC12 cells. a, expression of cathepsin V with PE results in ME production. The human cathepsin V cDNA in expression plasmid vector was transfected into PC12 neuroendocrine cells, and measurement of ME by RIA was conducted 3 days after transfection of PE. The coexpression of cathepsin V with PE resulted in increased cellular levels of (Met)enkephalin, expressed as x ± S.E. *, statistically significant (n = 3 for each experiment, p < 0.05 by Student's t test, repeated twice). b, shown is control expression of PE alone. Transfection of the PE cDNA in PC12 cells results in expression of PE shown as a band of ∼31 kDa (lane 2). Control experiments show that PE is absent in cells when transfected with vector alone (pcDNA3.1 without PE cDNA) (lane 1). c, shown is control expression of cathepsin V alone. Transfection of the cathepsin V cDNA results in expression of this protease, shown as a main band of ∼24-kDa cathepsin V (lane 2), likely corresponding to the mature form of the enzyme of 23,999 daltons (∼24 kDa) calculated molecular mass (31). Cell extract (15 μg protein) contained ∼10–25 ng of cathepsin V, based on the high sensitivity of the Western blot with standard cathepsin V (shown in supplemental Fig. S1). The Western blot detects low levels of cathepsin V, illustrated by detection of 25 ng of purified cathepsin V. Furthermore, the anti-cathepsin V Western blot shows specificity for detection of cathepsin V but does not detect the related cysteine cathepsins L, B, and H (supplemental Fig. S1).

Article Snippet: The human cathepsin V cDNA (encoding preprocathepsin V) in pCMV6-XL5 plasmid expression vector was obtained from Origene (Rockville, MD).

Techniques: Expressing, Plasmid Preparation, Transfection, Western Blot, Purification

Journal: The Journal of Biological Chemistry

Article Title: Human Cathepsin V Protease Participates in Production of Enkephalin and NPY Neuropeptide Neurotransmitters *

doi: 10.1074/jbc.M111.310607

Figure Lengend Snippet: Expression of cathepsin V in human neuroblastoma SK-N-MC cells results in (Met)enkephalin production. a, cathepsin V expression mediates enkephalin production. Human cathepsin V was transfected in human SK-N-MC neuroblastoma cells, and cells were harvested 2 days later. (Met)enkephalin levels in cell extracts were measured by RIA, expressed as x ± S.E. *. statistically significant (n = 6 for each experiment, p < 0.01, Student's t test, repeated twice). b, shown is control expression of cathepsin V. Transfection of the cathepsin V cDNA results in expression of the protease, shown as an ∼24-kDa band, likely corresponding to the mature form of the enzyme. The two band areas of ∼38–40 and ∼40–45 kDa in b are consistent with preprocathepsin V and procathepsin V, respectively (8, 10, 31).

Article Snippet: The human cathepsin V cDNA (encoding preprocathepsin V) in pCMV6-XL5 plasmid expression vector was obtained from Origene (Rockville, MD).

Techniques: Expressing, Transfection

Journal: The Journal of Biological Chemistry

Article Title: Human Cathepsin V Protease Participates in Production of Enkephalin and NPY Neuropeptide Neurotransmitters *

doi: 10.1074/jbc.M111.310607

Figure Lengend Snippet: Gene silencing of cathepsin V reduces (Met)enkephalin in human neuroblastoma SK-N-MC cells. a, gene silencing of cathepsin V by siRNA reduces (Met)enkephalin production. Human neuroblastoma SK-N-MC cells were transfected with siRNA to cathepsin V as described under “Experimental Procedures.” As controls, transfection of scrambled sequences of siRNA was conducted as well as no siRNA. One day after transfection cells were harvested, and cell levels of (Met)enkephalin were measured by radioimmunoassay. A significant decrease in (Met)enkephalin occurred after transfection of cells with siRNA to cathepsin V (p < 0.05 by Student's t test) compared with the controls of scrambled siRNA or no siRNA. b, cathepsin V protein expression is substantially reduced by siRNA gene silencing. Silencing of cathepsin V expression was assessed by Western blots after transfection of cathepsin V siRNA into SK-N-MC cells. Cathepsin V was substantially reduced with 50 nm siRNA (lane 2) and partially reduced with 25 nm siRNA (lane 3) compared with control transfection with scrambled siRNA (lane 1). These data show decreased cathepsin V expression when cathepsin V siRNA is transfected in SK-N-MC cells.

Article Snippet: The human cathepsin V cDNA (encoding preprocathepsin V) in pCMV6-XL5 plasmid expression vector was obtained from Origene (Rockville, MD).

Techniques: Transfection, RIA Assay, Expressing, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Human Cathepsin V Protease Participates in Production of Enkephalin and NPY Neuropeptide Neurotransmitters *

doi: 10.1074/jbc.M111.310607

Figure Lengend Snippet: In vitro processing of PE by cathepsin V generates an intermediate present in human brain. a, in vitro processing of PE by cathepsin V is shown. PE was incubated with or without human cathepsin V for 30 min and subjected to SDS-PAGE and Western blot using anti-ME (panel i) or LE (panel ii). Results show the presence of an ∼24-kDa PE-derived band recognized by both anti-ME and anti-LE antisera. b, shown is the PE-derived ∼24-kDa intermediate in human brain cortex and hippocampus. Western blots of human brain cortex (panel i) and hippocampus (panel ii) with anti-ME and anti-LE are shown. Results show the presence of an ∼24-kDa band that is recognized by both antisera, indicating the presence of a PE-derived ∼24-kDa intermediate.

Article Snippet: The human cathepsin V cDNA (encoding preprocathepsin V) in pCMV6-XL5 plasmid expression vector was obtained from Origene (Rockville, MD).

Techniques: In Vitro, Incubation, SDS Page, Western Blot, Derivative Assay

Journal: The Journal of Biological Chemistry

Article Title: Human Cathepsin V Protease Participates in Production of Enkephalin and NPY Neuropeptide Neurotransmitters *

doi: 10.1074/jbc.M111.310607

Figure Lengend Snippet: In vitro processing of PE by cathepsin V via cleavage at dibasic residues generates enkephalin peptides. a, shown is the PE precursor. The PE precursor contains several copies of enkephalin-related peptides consisting of ME, LE, ME-Arg-Gly-Leu (O), and ME-Arg-Phe (H), flanked by dibasic residues (KR, KK) that are known to be processed to generate mature enkephalin. b, shown are PE-derived products. Results of recombinant human cathepsin V cleavage of recombinant PE resulted in production of a ∼24-kDa (from Fig. 4). Peptide products identified by mass spectrometry are indicated by Lys-Arg-ME consisting of the KRYGGFM sequence (with the ME sequence underlined) and C-terminal peptides consisting of the sequence RFAEALPSDEEGESYSKEVPEMEKRYGGFMRF and KRFAEALPSDEEGESYSKEVPEMEKRYGGFMRF that contain the heptapeptide ME-Arg-Phe (underlined). Peptide products result from cleavage at the N-terminal side and between dibasic residues. Mass spectrometry data of peptide products are provided in supplemental Table S1, and MS/MS spectra of identified peptide products are shown in supplemental Fig. S2.

Article Snippet: The human cathepsin V cDNA (encoding preprocathepsin V) in pCMV6-XL5 plasmid expression vector was obtained from Origene (Rockville, MD).

Techniques: In Vitro, Derivative Assay, Recombinant, Mass Spectrometry, Sequencing, Tandem Mass Spectroscopy

Journal: The Journal of Biological Chemistry

Article Title: Human Cathepsin V Protease Participates in Production of Enkephalin and NPY Neuropeptide Neurotransmitters *

doi: 10.1074/jbc.M111.310607

Figure Lengend Snippet: Cathepsin V proteolytic activity with dibasic and monobasic peptide-MCA substrates Cathepsin V was incubated with peptide-MCA, and proteolytic activity of cathepsin V was measured as μmol of fluorescent AMC/h/mg of cathepsin V. All assays were performed in quadruplicate with each substrate; replicate values varied by less than 10%. These results indicate that cathepsin V cleaves at dibasic and monobasic residues. Boc, t -butoxycarbonyl.

Article Snippet: The human cathepsin V cDNA (encoding preprocathepsin V) in pCMV6-XL5 plasmid expression vector was obtained from Origene (Rockville, MD).

Techniques: Activity Assay, Incubation

Journal: The Journal of Biological Chemistry

Article Title: Human Cathepsin V Protease Participates in Production of Enkephalin and NPY Neuropeptide Neurotransmitters *

doi: 10.1074/jbc.M111.310607

Figure Lengend Snippet: Cathepsin V in human secretory vesicles and neuronal tissues. a, cathepsin V in isolated human secretory vesicles from sympathoadrenal pheochromocytoma. Western blot with anti-cathepsin V was assessed in soluble and membrane fractions (lanes 1 and 2, respectively) of secretory vesicles isolated from human pheochromocytoma of sympathoadrenal tissue. Blots illustrate the presence of mature cathepsin V of an ∼24-kDa band (31). b–d, shown is cathepsin V in human brain cortex and hippocampus. Cathepsin V in human neuronal tissues was analyzed by Western blots after immunoprecipitation of cathepsin V from human brain cortex (C) and hippocampus (H) (panel b, lanes 1 and 2, respectively), human SH-SY-5Y neuroblastoma cells (panel c), and human SK-N-MC neuroblastoma cells (panel d). Cathepsin V was observed as its mature form of ∼24 kDa (31). The band in panels b–d of ∼40–45 kDa are consistent to that of preprocathepsin V (8, 10, 31). The specificity of the anti-cathepsin V Western blot shows its sensitive detection of 25 ng or lower levels of standard cathepsin V, with no detection of the cysteine cathepsins L, B, or H (supplemental Fig. 1). In addition, control Western blot without primary antibody resulted in a lack of immunopositive bands.

Article Snippet: The human cathepsin V cDNA (encoding preprocathepsin V) in pCMV6-XL5 plasmid expression vector was obtained from Origene (Rockville, MD).

Techniques: Isolation, Western Blot, Immunoprecipitation

Journal: The Journal of Biological Chemistry

Article Title: Human Cathepsin V Protease Participates in Production of Enkephalin and NPY Neuropeptide Neurotransmitters *

doi: 10.1074/jbc.M111.310607

Figure Lengend Snippet: Neuronal localization of cathepsin V with enkephalin in secretory vesicles assessed by immunofluorescent confocal microscopy in human SK-N-MC neuroblastoma cells. a, localization of cathepsin V with enkephalin in secretory vesicles is shown. Cathepsin V was observed (red fluorescence) as a punctate, discrete pattern of localization like that of (Met)enkephalin (green fluorescence). The colocalization of the protease with enkephalin was illustrated by the merged yellow fluorescence. Quantitation of the relative amount of cellular cathepsin V that is colocalized with enkephalin was assessed with the Pearson correlation coefficient (Rr value) (Table 2). Measurement of the Rr value as 0.45 indicates partial colocalization of cathepsin V with enkephalin. It is noted that cathepsin V is also observed in nuclei; this is consistent with other reports demonstrating nuclear functions of cathepsin V (52, 53). b, shown is an enlarged view of secretory vesicles containing cathepsin V and enkephalin. An enlarged image more clearly shows the secretory vesicle colocalization of cathepsin V (red immunofluorescence) with enkephalin (green immunofluorescence) as indicated by the merged yellow immunofluorescence; arrows indicate the secretory vesicles containing both cathepsin V and enkephalin. Controls using normal mouse IgG instead of mouse anti-cathepsin V resulted in markedly reduced immunofluorescence (supplemental Fig. 3), illustrating the specificity of the antibody to cathepsin V.

Article Snippet: The human cathepsin V cDNA (encoding preprocathepsin V) in pCMV6-XL5 plasmid expression vector was obtained from Origene (Rockville, MD).

Techniques: Confocal Microscopy, Fluorescence, Quantitation Assay, Immunofluorescence

Journal: The Journal of Biological Chemistry

Article Title: Human Cathepsin V Protease Participates in Production of Enkephalin and NPY Neuropeptide Neurotransmitters *

doi: 10.1074/jbc.M111.310607

Figure Lengend Snippet: Quantitation of cathepsin V localization with enkephalin and NPY in human neuroblastoma cells The colocalization of cathepsin V immunofluorescence with (Met)enkephalin in human neuroblastoma SK-N-MC cells and with NPY in human neuroblastoma SH-SY-5Y cells was quantitated by measuring the Pearson correlation coefficient (Rr) with the Velocity software as described under “Experimental Procedures.” A Pearson correlation of 1 indicates complete colocalization, and a value of 0 indicates no specific colocalization. Measurements of the Pearson correlation coefficient of 0.448 ± 0.018 for cathepsin V and (Met)enkephalin indicate their partial colocalization. The Pearson correlation coefficient of 0.736 ± 0.030 for cathepsin V and NPY in SH-SY-5Y cells indicates a reasonable degree of partial colocalization. Additional evaluation of cathepsin V localization with Lamp-1, a marker for lysosomes (54), indicated the Rr value of 0.535 ± 0.036, indicating partial colocalization. The Pearson correlation coefficient was measured with n = 5 cells. The mean ± S.E. is shown, and statistical significance of colocalization ( p < 0.0001 by Student's t test) was compared to the null hypothesis of no specific colocalization (Pearson correlation coefficient value of 0).

Article Snippet: The human cathepsin V cDNA (encoding preprocathepsin V) in pCMV6-XL5 plasmid expression vector was obtained from Origene (Rockville, MD).

Techniques: Quantitation Assay, Immunofluorescence, Software, Marker

Journal: The Journal of Biological Chemistry

Article Title: Human Cathepsin V Protease Participates in Production of Enkephalin and NPY Neuropeptide Neurotransmitters *

doi: 10.1074/jbc.M111.310607

Figure Lengend Snippet: Localization of cathepsin V with Lamp-1 marker of lysosomes in human SK-N-MC cells. a, shown is cathepsin V localization assessed with Lamp-1. Cathepsin V subcellular distribution was evaluated with Lamp-1, a marker for lysosomes (32). Cathepsin V (red) and Lamp-1 (green) display partial colocalization (merged yellow immunofluorescence), shown by arrows. Quantitative analyses of their partial colocalization was conducted with the Pearson correlation coefficient, indicated as 0.535 ± 0.036 (see Table 2, legend). b, shown is an enlarged view of cathepsin V localization with Lamp-1. An enlarged image shows the distinct colocalization of cathepsin V (red) with Lamp-1 (green) shown by the merged yellow immunofluorescence.

Article Snippet: The human cathepsin V cDNA (encoding preprocathepsin V) in pCMV6-XL5 plasmid expression vector was obtained from Origene (Rockville, MD).

Techniques: Marker, Immunofluorescence

Journal: The Journal of Biological Chemistry

Article Title: Human Cathepsin V Protease Participates in Production of Enkephalin and NPY Neuropeptide Neurotransmitters *

doi: 10.1074/jbc.M111.310607

Figure Lengend Snippet: Cellular production of NPY by cathepsin V. a, localization of cathepsin V and NPY in human SH-SY-5Y neuroblastoma cells is shown. Cathepsin V localization in human SH-SY-5Y neuroblastoma cells was observed by immunofluorescent confocal microscopy. Cathepsin V (red fluorescence) and NPY (green fluorescence) were discretely localized, with colocalization among many secretory vesicles (yellow immunofluorescence, indicated by arrows). Quantitation of the relative cathepsin V colocalization with NPY was assessed by the Pearson correlation coefficient (Rr value) (Table 2). The measured Rr value of 0.74 indicates that cathepsin V is partially colocalized with NPY. Controls using normal mouse IgG instead of mouse anti-cathepsin V resulted in markedly reduced immunofluorescence (supplemental Fig. S3), illustrating the specificity of the antibody to cathepsin V. b, NPY production by cathepsin V in PC12 cells is shown. PC12 cells were cotransfected with proNPY cDNA (in pcDNA3.1 vector) and cathepsin V cDNA (in pCMV6-XL5 vector), and cells were harvested 3 days later for analyses of cellular levels of NPY (by RIA). Results are shown as x ± S.E. *, statistically significant (n = 6, p < 0.001, Student's t test).

Article Snippet: The human cathepsin V cDNA (encoding preprocathepsin V) in pCMV6-XL5 plasmid expression vector was obtained from Origene (Rockville, MD).

Techniques: Confocal Microscopy, Fluorescence, Immunofluorescence, Quantitation Assay, Plasmid Preparation