Journal: The Journal of biological chemistry

Article Title: Molecular studies define the primary structure of alpha1-antichymotrypsin (ACT) protease inhibitor in Alzheimer's disease brains. Comparison of act in hippocampus and liver.

doi: 10.1074/jbc.274.3.1821

Figure Lengend Snippet: FIG. 2. RT-PCR of 5*and 3* domains of ACT cDNA from hip- pocampus of AD and normal brains. A, RT-PCR of the 59 domain of ACT cDNA, detected by DNA agarose gels. RT-PCR with primers 1 and 2 generated a 790-bp DNA band from poly(A)1 RNA isolated from hippocampus (H) of AD and normal (N) brains (lanes 1 and 2, respec- tively) as well as from human liver (L) (lane 3). B, RT-PCR of the 39 domain of ACT cDNA. RT-PCR with primers 3 and 4 generated a DNA band of approximately 500 bp from poly(A)1 RNA isolated from hip- pocampus (H) of AD and normal (N) brains (lanes 1 and 2, respectively) as well as from human liver (L) (lane 3).

Article Snippet: For deglycosylation of protein extracts from hippocampus and of human liver ACT (from Athens Research and Technology Biochemicals), ACT samples were incubated with N-glycosidase F (0.2 units, Boehringer Mannheim) at 37 °C for 18 h in buffer (25 ml total volume) consisting of 20 mM sodium phosphate, pH 7.2, 10 mM sodium azide, 50 mM EDTA, and 0.5% (w/v) octyloglucoside.

Techniques: Reverse Transcription Polymerase Chain Reaction, Generated, Isolation

Journal: The Journal of biological chemistry

Article Title: Molecular studies define the primary structure of alpha1-antichymotrypsin (ACT) protease inhibitor in Alzheimer's disease brains. Comparison of act in hippocampus and liver.

doi: 10.1074/jbc.274.3.1821

Figure Lengend Snippet: FIG. 1. Strategy for RT-PCR of ACT cDNA from hippocampus. Primers 1 and 2 and primers 3 and 4 were designed to amplify 59 and 39domains of the hip- pocampus ACT cDNA, respectively. The overlapping 59 and 39 domains were pre- dicted to include the RSL and the NH2 terminus of mature, processed ACT, which begins at the COOH terminus of the signal peptide sequence. Primers 5 and 6 allowed amplification of the 39-UTR of the cDNA. Primer A was used in primer exten- sion analyses of ACT gene transcripts.

Article Snippet: For deglycosylation of protein extracts from hippocampus and of human liver ACT (from Athens Research and Technology Biochemicals), ACT samples were incubated with N-glycosidase F (0.2 units, Boehringer Mannheim) at 37 °C for 18 h in buffer (25 ml total volume) consisting of 20 mM sodium phosphate, pH 7.2, 10 mM sodium azide, 50 mM EDTA, and 0.5% (w/v) octyloglucoside.

Techniques: Reverse Transcription Polymerase Chain Reaction, Sequencing, Amplification

Journal: The Journal of biological chemistry

Article Title: Molecular studies define the primary structure of alpha1-antichymotrypsin (ACT) protease inhibitor in Alzheimer's disease brains. Comparison of act in hippocampus and liver.

doi: 10.1074/jbc.274.3.1821

Figure Lengend Snippet: FIG. 3. Complementary DNA sequence of human hippocampus ACT. The ACT cDNAs obtained from hippocampus of normal brains is illustrated. The open reading frame domains of ACT cDNAs from normal and Alzheimer’s disease brains were identical. The DNA sequence of the 39-UTR domain of the hippocampus ACT cDNA (from normal brain) was also determined. Alignment of the DNA sequences determined for overlapping 59 and 39 PCR fragments indicates the human hippocampus ACT cDNA (shown in bold for nucleotides 111 to 1576), whose deduced primary sequence (shown in bold for residues 27 to 398) corresponds to the mature ACT protein. The positions of primers 1–6 used in RT-PCRs are shown by dotted lines with arrows. Arrows above the His (11) and Asn (13) residues indicate the predicted NH2 terminus of the mature ACT, which lacks the signal sequence. The RSL domain is boxed, with the predicted P1 residue as Leu underlined. Consensus glycosylation sites are indicated by asterisks under the Asn residues as possible sites of glycosylation. The predicted 59-region analyzed by primer extension is shown (not bold) for nucleotides 1–110 (6, 17).

Article Snippet: For deglycosylation of protein extracts from hippocampus and of human liver ACT (from Athens Research and Technology Biochemicals), ACT samples were incubated with N-glycosidase F (0.2 units, Boehringer Mannheim) at 37 °C for 18 h in buffer (25 ml total volume) consisting of 20 mM sodium phosphate, pH 7.2, 10 mM sodium azide, 50 mM EDTA, and 0.5% (w/v) octyloglucoside.

Techniques: Sequencing, Residue, Glycoproteomics

Journal: The Journal of biological chemistry

Article Title: Molecular studies define the primary structure of alpha1-antichymotrypsin (ACT) protease inhibitor in Alzheimer's disease brains. Comparison of act in hippocampus and liver.

doi: 10.1074/jbc.274.3.1821

Figure Lengend Snippet: FIG. 4. Northern blot of ACT mRNA in hippocampus from AD and normal hippocampus as well as liver. Northern blots of total RNA isolated from hippocampus of normal (N) and AD brains (10 mg of RNA each, lanes 1 and 2, respectively) and liver poly(A)1 RNA (1 mg, lane 3) probed with the human ACT cDNA (6), as described under “Experimental Procedures.” Autoradiography of Northern blots (15 h exposure to x-ray film) (lanes 1–3) showed ACT mRNA in hippocampus and a high level of ACT mRNA in liver. Intact ribosomal RNAs were detected by ethidium bromide staining of the RNA samples on dena- turing formaldehyde gels (data not shown). kb, kilobases.

Article Snippet: For deglycosylation of protein extracts from hippocampus and of human liver ACT (from Athens Research and Technology Biochemicals), ACT samples were incubated with N-glycosidase F (0.2 units, Boehringer Mannheim) at 37 °C for 18 h in buffer (25 ml total volume) consisting of 20 mM sodium phosphate, pH 7.2, 10 mM sodium azide, 50 mM EDTA, and 0.5% (w/v) octyloglucoside.

Techniques: Northern Blot, Isolation, Autoradiography, Staining

Journal: The Journal of biological chemistry

Article Title: Molecular studies define the primary structure of alpha1-antichymotrypsin (ACT) protease inhibitor in Alzheimer's disease brains. Comparison of act in hippocampus and liver.

doi: 10.1074/jbc.274.3.1821

Figure Lengend Snippet: FIG. 5. Slot blot of ACT mRNA in hippocampus and liver. Slot blots of poly(A)1 RNA (with the indicated amounts of RNA) from hip- pocampus and liver were performed to compare ACT mRNA levels in these two tissues. Hybridization of slot blots with ACT cDNA as probe was performed identically as described for Northern blots of ACT mRNA (Fig. 4). Northern blots were subjected to autoradiography (15 h exposure to x-ray film) for detection of ACT mRNA.

Article Snippet: For deglycosylation of protein extracts from hippocampus and of human liver ACT (from Athens Research and Technology Biochemicals), ACT samples were incubated with N-glycosidase F (0.2 units, Boehringer Mannheim) at 37 °C for 18 h in buffer (25 ml total volume) consisting of 20 mM sodium phosphate, pH 7.2, 10 mM sodium azide, 50 mM EDTA, and 0.5% (w/v) octyloglucoside.

Techniques: Dot Blot, Hybridization, Northern Blot, Autoradiography

Journal: The Journal of biological chemistry

Article Title: Molecular studies define the primary structure of alpha1-antichymotrypsin (ACT) protease inhibitor in Alzheimer's disease brains. Comparison of act in hippocampus and liver.

doi: 10.1074/jbc.274.3.1821

Figure Lengend Snippet: FIG. 6. Genomic blot of human ACT. DNA agarose (0.8%) gel electrophoresis of KpnI-, HindIII-, or EcoRI- (lanes 1–4 respectively) digested human genomic DNA (10 mg) as well as undigested control DNA (lane 5) was subjected to in situ hybridization with the human liver ACT cDNA as probe (6), as described under “Experimental Proce- dures.” kb, kilobases.

Article Snippet: For deglycosylation of protein extracts from hippocampus and of human liver ACT (from Athens Research and Technology Biochemicals), ACT samples were incubated with N-glycosidase F (0.2 units, Boehringer Mannheim) at 37 °C for 18 h in buffer (25 ml total volume) consisting of 20 mM sodium phosphate, pH 7.2, 10 mM sodium azide, 50 mM EDTA, and 0.5% (w/v) octyloglucoside.

Techniques: Nucleic Acid Electrophoresis, Control, In Situ Hybridization

Journal: The Journal of biological chemistry

Article Title: Molecular studies define the primary structure of alpha1-antichymotrypsin (ACT) protease inhibitor in Alzheimer's disease brains. Comparison of act in hippocampus and liver.

doi: 10.1074/jbc.274.3.1821

Figure Lengend Snippet: FIG. 7. Primer extension of ACT mRNA from hippocampus and liver. Primer extension of poly(A)1 RNA from hippocampus and liver (lanes 1 and 2, respectively) was conducted with 32P-labeled primer 59-CTGCCTCAGGGAGCTGGA-39. The 32P-extended cDNA was ana- lyzed on 8% acrylamide, bis-acrylamide, 7 M urea DNA sequencing gels, with detection of the extended cDNA by autoradiography, as described under “Experimental Procedures.” Arrows indicate the radiolabeled cDNAs of 58 bp obtained by primer extension.

Article Snippet: For deglycosylation of protein extracts from hippocampus and of human liver ACT (from Athens Research and Technology Biochemicals), ACT samples were incubated with N-glycosidase F (0.2 units, Boehringer Mannheim) at 37 °C for 18 h in buffer (25 ml total volume) consisting of 20 mM sodium phosphate, pH 7.2, 10 mM sodium azide, 50 mM EDTA, and 0.5% (w/v) octyloglucoside.

Techniques: Labeling, DNA Sequencing, Autoradiography

Journal: The Journal of biological chemistry

Article Title: Molecular studies define the primary structure of alpha1-antichymotrypsin (ACT) protease inhibitor in Alzheimer's disease brains. Comparison of act in hippocampus and liver.

doi: 10.1074/jbc.274.3.1821

Figure Lengend Snippet: FIG. 8. Deglycosylation of ACT in hippocampus of Alzheimer’s disease and normal brains as well as in liver. Deglycosylation by N-glycosidase F of ACT in tissue extracts from hippocampus of AD and normal brains as well as hu- man liver ACT was assessed by Western blots with anti-ACT serum. Panel A shows ACT in normal (N) and AD hip- pocampus (lanes 1 and 3, respectively). ACT in these tissues was also incubated with (1) N-glycosidase F (lanes 2 and 4, respectively). Panel B shows human liver ACT without and with N-glycosidase F treatment (lanes 1 and 2, respectively).

Article Snippet: For deglycosylation of protein extracts from hippocampus and of human liver ACT (from Athens Research and Technology Biochemicals), ACT samples were incubated with N-glycosidase F (0.2 units, Boehringer Mannheim) at 37 °C for 18 h in buffer (25 ml total volume) consisting of 20 mM sodium phosphate, pH 7.2, 10 mM sodium azide, 50 mM EDTA, and 0.5% (w/v) octyloglucoside.

Techniques: Western Blot, Incubation

Journal: Neuron

Article Title: Alzheimer’s risk factors age, APOE genotype, and sex drive distinct molecular pathways

doi: 10.1016/j.neuron.2020.02.034

Figure Lengend Snippet: (A, D and G) Module preservation in the ROSMAP human prefrontal cortex (A, n = 313), Mayo clinic human temporal cortex (D, n = 159), and Mayo clinic human cerebellum (G, n = 158) datasets. Preservation Z summary between 2 and 10 indicates moderate preservation. Z summary >10 indicates strong preservation. (B and C) Blue (B) and pink (C) MEs in human control, MCI and AD samples in the ROSMAP prefrontal cortex dataset (n = 85 Ctrl, n = 78 MCI, and n = 150 AD). (E and F) Blue (E) and pink (F) MEs in human AD and controls samples in the Mayo Clinic temporal cortex dataset (n = 77 Ctrl and n = 82 AD). (H and I) Blue (H) and pink (I) MEs in human AD and controls samples in the Mayo Clinic cerebellum dataset (n = 76 Ctrl and n = 82 AD). (J-M) The gene expression levels (log2 transformed RPKM) of SERPINA3 and ATF4, two lightcyan module genes in the Mayo Clinic human temporal cortex dataset. (J, L) The expression levels of SERPINA3 (J) and ATF4 (L) between APOE4− (n = 108, including 69 Ctrl and 39 AD) and APOE4+ (n = 51, including 8 Ctrl and 43 AD) samples: left panel: expression values not adjusted by AD status; right panel: expression values adjusted by AD status. (K, M) The expression levels of SERPINA3 (K) and ATF4 (M) between AD (n = 82, including 39 APOE4− and 43 APOE4+) and Ctrl (n = 77, including 69 APOE4− and 8 APOE4+) samples: left panel: expression values not adjusted by APOE4 status; right panel: expression values adjusted by APOE4 status. In all box plots, the upper and lower lines in the boxplots represent the maximum and minimum values after Tukey’s test. The center line represents the median. P values were calculated by Mann-Whitney U tests.

Article Snippet: The Serpina3 level in mouse serum was measured using a commercial ELISA kit (Cat# CSBE13727m, Cusabio) according to the instructions provided by the manufacturer.

Techniques: Preserving, Control, Gene Expression, Transformation Assay, Expressing, MANN-WHITNEY

Journal: Neuron

Article Title: Alzheimer’s risk factors age, APOE genotype, and sex drive distinct molecular pathways

doi: 10.1016/j.neuron.2020.02.034

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: The Serpina3 level in mouse serum was measured using a commercial ELISA kit (Cat# CSBE13727m, Cusabio) according to the instructions provided by the manufacturer.

Techniques: Sample Prep, Sequencing, Software, Enzyme-linked Immunosorbent Assay, RNAscope, Multiplex Assay