Journal: European journal of biochemistry

Article Title: The N-glycans of jack bean alpha-mannosidase. Structure, topology and function.

doi: 10.1046/j.1432-1327.1999.00598.x

Figure Lengend Snippet: Fig. 1. HPLC profiles of PA-sugar chains from jack bean a-man- nosidase. Panel A: RP-HPLC of PA-derivatives from jack bean a-man- nosidase. The PA-derivatives were applied onto a Cosmosil 5C18-AR (6.0 250 mm) column and eluted as described in the text. The run- through-fraction (around 9 min) contained some PA-derivatives bearing only one GlcNAc residue at their reducing-ends, which are known to be derived from released intact N-glycans as by-products of hydrazinolysis [7,12]. Panel B: Size-fractionation HPLC of the partially purified PA-sugar chains shown in Panel A (Peaks I and II) by RP-HPLC.

Article Snippet: The two glycopeptide fractions [elution volume from 244±292 mL (high-mannose-type glycan) and from 408±500 mL (complex-type glycan)] were concentrated in a rotary evaporator and the former glycopeptide-fraction was applied to a Jasco 880-PU HPLC apparatus with a Poros R1 column (4.6 100 mm).

Techniques: Residue, Derivative Assay, Fractionation, Purification

Journal: European journal of biochemistry

Article Title: The N-glycans of jack bean alpha-mannosidase. Structure, topology and function.

doi: 10.1046/j.1432-1327.1999.00598.x

Figure Lengend Snippet: Fig. 3. RP-HPLC of a lysylendopeptidase digest of alkylated jack bean a-mannosidase. The lysylendopeptidase digest of alkylated jack bean a- mannosidase (100 mg) was applied onto a Vydac C8 column. The column was eluted by a linear gradient of 2±40% acetonitrile in 0.1% trifluoroacetic acid. Panel A: Total ion monitoring chromatogram (TIC). `RT: 28.2 min' indicates the position of glycopeptide (GP) which eluted after 28.2 min. Panel B: Precursor ion (m/z 366.0; Hex-HexNAc) monitoring. Panel C: Precursor ion (m/z 204.1; HexNAc) monitoring. Panel D: ESI-MS spectrum of the glycopeptide GP (complex-type) from Panel A.

Article Snippet: The two glycopeptide fractions [elution volume from 244±292 mL (high-mannose-type glycan) and from 408±500 mL (complex-type glycan)] were concentrated in a rotary evaporator and the former glycopeptide-fraction was applied to a Jasco 880-PU HPLC apparatus with a Poros R1 column (4.6 100 mm).

Techniques: Glycoproteomics

Journal: European journal of biochemistry

Article Title: The N-glycans of jack bean alpha-mannosidase. Structure, topology and function.

doi: 10.1046/j.1432-1327.1999.00598.x

Figure Lengend Snippet: Fig. 5. RP-HPLC of a glycopeptide-fraction obtained by gel-filtration of a lysylendopeptidase-digest of alkylated jack bean a-mannosidase. The glycopeptide fraction obtained by Sephadex G-25 gel-filtration (fraction volume from 244 to 292 mL) was applied onto a Poros R1 column. The column was eluted with 5% acetonitrile in 0.1% trifluoroacetic acid for 5 min, followed by a linear gradient of 5±60% acetonitrile in 0.1% trifluoroacetic acid. Only the peak indicated by an arrow was found to contain neutral sugars. GP (high-mannose-type) indicates a glycopeptide bearing the high-mannose-type N-glycan.

Article Snippet: The two glycopeptide fractions [elution volume from 244±292 mL (high-mannose-type glycan) and from 408±500 mL (complex-type glycan)] were concentrated in a rotary evaporator and the former glycopeptide-fraction was applied to a Jasco 880-PU HPLC apparatus with a Poros R1 column (4.6 100 mm).

Techniques: Glycoproteomics, Filtration

Journal: Genetics in Medicine

Article Title: High-affinity FcγRIIIa genetic variants and potent NK cell-mediated antibody-dependent cellular cytotoxicity (ADCC) responses contributing to severe COVID-19

doi: 10.1016/j.gim.2022.04.005

Figure Lengend Snippet: Analysis of the SARS-CoV-2-specific ADCC responses. Analysis of the kinetics of SARS-CoV-2-specific and NK cell-mediated antibody-mediated cellular cytotoxicity (ADCC) response in plasma obtained from hospitalized patients with COVID-19 ( n = 19) collected at different time points: 3 ±1 ( n = 7), 6 ±1 ( n = 14), 9 ±1 ( n = 15), 12 ±1 ( n = 18), 15 ±1 ( n = 18), 18 ±1 ( n = 18), 21 ±1 ( n = 15), 24 ±1 ( n = 11), and 27 ±1 ( n = 7) days after symptoms onset or nonhospitalized, mildly ill patients with COVID-19 ( n = 9) 28 to 31 (median: 30) days after symptom onset. Each plasma sample as well as each of 6 control samples from SARS-CoV-2 seronegative persons was individually tested against 26 different CD56 + CD16 + NK cell preparations from SARS-CoV-2 seronegative healthy blood donors and the percentage of CD107a (A), perforin (B), IFNγ (C), or TNFα (D) positive cells was assessed using flow cytometry. Percentage of all CD107a (A), IFNγ (C), or TNFα (D) positive cells, as well as only highly perforin-expressing cells (B) was assessed. All samples were normalized to the same SARS-CoV-2 seronegative control. From each sample, the median of independent experiments was calculated. Data are shown as mean values (±95% CI). ADCC positive plasma samples were identified using 6 SARS-CoV-2 seronegative controls. Fold change in positive cells at each time point were compared either between hospitalized patients with COVID-19 (indicated as a red bar) or mildly diseased nonhospitalized patients with COVID-19 (indicated as a blue bar) and seronegative controls (indicated as a black bar) using analysis of variance and Dunn’s post test (hospitalized patients with COVID-19) or Mann-Whitney test (nonhospitalized patients with COVID-19). P < .05 was considered significant. IFNγ, interferon gamma; TNFα, tumor necrosis factor α.

Article Snippet: The CD56 + CD16 + NK cell subset was then enriched via 2-step magnetic labeling using human CD56 + CD16 + NK Cell Isolation Kit (Miltenyi Biotec) according to the manufacturer’s instruction.

Techniques: Clinical Proteomics, Control, Flow Cytometry, Expressing, MANN-WHITNEY

Journal: Genetics in Medicine

Article Title: High-affinity FcγRIIIa genetic variants and potent NK cell-mediated antibody-dependent cellular cytotoxicity (ADCC) responses contributing to severe COVID-19

doi: 10.1016/j.gim.2022.04.005

Figure Lengend Snippet: Impact of the FcγRIIIa-158-V/F variants on the ADCC responses. Analysis of the extent of SARS-CoV-2-specific and NK cell-mediated antibody-mediated cellular cytotoxicity (ADCC) response with plasma obtained from hospitalized patients with COVID-19 ( n = 19) 6 ±1 ( n = 10), 9 ±1 ( n = 14), 12 ±1 ( n = 14), 15 ±1 ( n = 18), 18 ±1 ( n = 18), 21 ±1 ( n = 15), 24 ±1 ( n = 11), and 27 ±1 ( n = 7) days after symptom onset (A-D) or nonhospitalized patients with COVID-19 ( n = 9) 28 to 31 (median: 30 days) days after symptom onset (E-H). Each plasma sample from hospitalized and nonhospitalized patients with COVID-19 was stimulated with CD56 + CD16 + NK cells from 26 healthy blood donors (FcγRIIIa-158-V/V: n = 5, FcγRIIIa-158-V/F: n = 12, FcγRIIIa-158-F/F: n = 9) and the MFI of CD107a (A,E), perforin (B,F), IFNγ (C,G), or TNFα (D,H) positive cells were assessed using flow cytometry. All samples were normalized to the same nonhospitalized SARS-CoV-2 seropositive control. MFI of all CD107a (A), IFNγ (C) or TNFα (D) positive cells, as well as of only high perforin-expressing cells (B) was assessed. Data are shown as mean values (±95% CI). Fold change MFI at each time point was compared between assays using FcγRIIIa-158-V/V, FcγRIIIa-158-V/F, and FcγRIIIa-158-F/F variant expressing NK cells, respectively using a ANOVA (A-D) or a paired t test (E-H). P < .05 was considered significant. ANOVA, analysis of variance; d.p.s.o, days post symptom onset; IFNγ, interferon gamma; MFI, mean fluorescence intensity; TNFα, tumor necrosis factor α.

Article Snippet: The CD56 + CD16 + NK cell subset was then enriched via 2-step magnetic labeling using human CD56 + CD16 + NK Cell Isolation Kit (Miltenyi Biotec) according to the manufacturer’s instruction.

Techniques: Clinical Proteomics, Flow Cytometry, Control, Expressing, Variant Assay, Fluorescence