Journal: bioRxiv

Article Title: Syncytial coupling of mid-capillary pericytes underlies seizure-associated electro-metabolic signaling

doi: 10.64898/2026.03.16.711912

Figure Lengend Snippet: (A) Changes in pericyte membrane potential after norepinephrine and UDP-glucose application. (B) The thromboxane analogue U46619 depolarizes and constricts pericytes. Upper left corner: The amplitude of the depolarization induced by U46619 depended on the intracellular chloride concentration. See the orange trace (34 mM Cl⁻) and the light purple trace (8 mM Cl⁻). Lower left and right panel: The plateau phase was reversed by the TMEM16 inhibitor Ani9 (blue trace), indicating the involvement of Ca 2+ -activated Cl - channels (Kruskal-Wallis test: p = 0.00016, post-hoc pairwise Dunn test: significant differences between group 1 and 3, p = 0.0003). (C) Upper panel: Recording of the resting membrane potential (Vm) of a pericyte and a neuron immediately after establishing the whole cell configuration with a CsCl-containing intracellular solution. In contrast to neurons, pericytic depolarization is moderate even after 10 min. Lower panel: Quantification of pericyte membrane potential immediately after rupturing the membrane and after 10 min using CsCl intracellular solution. (D) Representative recording of a voltage ramp (-100 mV to 60 mV) of a neuron (turquoise) and pericyte (red), indicating the presence of voltage gated inward currents in neurons but not in pericytes. (E) Relative fluorescence change of OGB-1 in response to depolarization steps starting from a holding potential of -100 mV. Neurons showed a marked increase in fluorescence for steps above -50 mV while pericytes solely presented with continuous slight baseline increase. (F) Comparison of the percentage change in vessel diameter, pericyte length, and fluorescence change upon i. control (n = 11), ii. different depolarizing current injection protocols (depolarization to 0 mV (n = 6), depolarization to -20mV (n = 13), recurrent depolarization steps (n = 11) or iii. 200 nM U46619 (n = 14)). Pericyte length, vessel diameter and Ca 2+ concentration changed significantly upon U46619 (OGB-1 fluorescence increase: 42.77 ± 7.33 %, p = 0.0001**, pericyte length change: -7.15 ± 1.61 %, p = 0.0002**, vessel diameter change: -33.92 ± 5.54 %, p = 0.0001**) but remained unchanged upon depolarization (current injection to 0 mV: p = 0.44, p = 0.56, p = 0.31; current injection to -20 mV: p = 0.86, p = 0.68, p = 0.19; recurrent depolarization steps: p = 0.12, p = 0.97, p = 0.03). Bonf.:*=significant. Scale bar: 10 µm. (G) Representative recording of a pericyte exhibiting spontaneous Ca 2+ fluctuations in the presence of the VGCC activator BAY-K-8644 (100 nM). (H) Change in OGB-1 fluorescence upon depolarization of the pericyte (on the excerpt) to 0 mV under BAY-K-8644. Slow increase in fluorescence was observed in 7 out of 9 cells.

Article Snippet: To constrict capillaries UDP-glucose (100 μM, Merck, CAS-Nr. 117756-22-6), norepinephrine (10 μM Sigma A7256) and the thromboxane agonist, U46619 (Stock solution: 2 mM, Tocris bioscience, diluted in DMSO) were applied via the perfusion.

Techniques: Membrane, Concentration Assay, Fluorescence, Comparison, Control, Injection

Journal: ACS Omega

Article Title: Characterizing Family 1 Glycosyltransferases (GT1, UGT) by Reverse Glycosylation: Fast Determination of Acceptor Specificity, Donor Specificity, Hydrolysis, and Enzyme Stability

doi: 10.1021/acsomega.5c04757

Figure Lengend Snippet: UGT catalyzed hydrolysis. (a) Proposed reaction mechanism for the overnight glycosylation reaction with TCP with residue numeration according to UGT72G3. TCP is glycosylated until reverse glycosylation of TCP-glycoside is induced by UDP released from TCP glycosylation and hydrolysis of UDP-α-glucose, ultimately leading to full depletion of the glycoside product and hydrolysis of the UDP-α-glucose donor, which is irreversible. (b) TCP-glycoside yields of the 25 UGTs from the 24 h screening conducted in reverse-phase HPLC. (c) Reaction mechanism in the chromogenic hydrolysis assay with CNP-β-glucose. In the event of hydrolysis of UDP-α-glucose, UDP catalyzes the reverse glycosylation reaction and CNP will be detached and measured spectrophotometrically. (d) Table of the measured rates of hydrolysis at 125, 250, and 500 μg/mL enzyme and the apparent k cat of hydrolysis.

Article Snippet: 2-Chloro-4-nitrophenyl β- d -glucopyranoside (CNP-β-glucose), 2-chloro-4-nitrophenyl β- d -galactopyranoside (CNP-β-galactose), and UDP-α-glucose were purchased from Biosynth.

Techniques: Glycoproteomics, Residue, Hydrolysis Assay