Effect of temperature on permeation of low‐density lipoprotein particles through human carotid artery tissues

Quantification of the diffusion of small molecules and large lipid transporting lipoproteins across arterial tissues could be useful in elucidating the mechanism(s) of atherosclerosis. Optical coherence tomography (OCT) was used to determine the effect of temperature on the rate of diffusion of glucose and low‐density lipoproteins (LDL) in human carotid endarterectomy tissue in vitro. The permeability rate for glucose was calculated to be (3.51 ± 0.27) × 10^–5 cm/s (n = 13) at 20 °C, and (3.70 ± 0.44) × 10^–5 cm/s (n = 5) at 37 °C; for LDL the rate was (2.42 ± 0.33) × 10^–5 cm/s (n = 5) at 20 °C and (4.77 ± 0.48) × 10^–5 cm/s (n = 7) at 37 °C, where n is the number of samples. These results demonstrate that temperature does not significantly influence the permeation of small molecules (e.g. glucose), however, raising the temperature does significantly increase the permeation of LDL. These results provide new information about the capacity of an atherogenic lipoprotein to traverse the intimal layer of the artery. These results also demonstrate the potential of OCT for elucidating the dynamics of lipoprotein perfusion across the arterial wall. (© 2009 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Small-angle X-ray scattering studies of the intact eye lens: Effect of crystallin composition and concentration on microstructure

Background

The cortex and nucleus of eye lenses are differentiated by both crystallin protein concentration and relative distribution of three major crystallins (α, β, and γ). Here, we explore the effects of composition and concentration of crystallins on the microstructure of the intact bovine lens (37 °C) along with several lenses from Antarctic fish (− 2 °C) and subtropical bigeye tuna (18 °C).

Methods

Our studies are based on small-angle X-ray scattering (SAXS) investigations of the intact lens slices where we study the effect of crystallin composition and concentration on microstructure.

Results

We are able to distinguish the nuclear and cortical regions by the development of a characteristic peak in the intensity of scattered X-rays. For both the bovine and fish lenses, the peak corresponds to that expected for dense suspensions of α-crystallins.

Conclusions

The absence of the scattering peak in the nucleus indicates that there is no characteristic wavelength for density fluctuations in the nucleus although there is liquid-like order in the packing of the different crystallins. The loss in peak is due to increased polydispersity in the sizes of the crystallins and due to the packing of the smaller γ-crystallins in the void space of α-crystallins.

General significance

Our results provide an understanding for the low turbidity of the eye lens that is a mixture of different proteins. This will inform design of optically transparent suspensions that can be used in a number of applications (e.g., artificial liquid lenses) or to better understand human diseases pathologies such as cataract.     

Acute and chronic leptin treatment mediate contrasting effects on signaling,glucose uptake,and GLUT4 translocation in L6-GLUT4myc myotubes

We have previously shown that in L6-GLUT4myc rat skeletal muscle cells, acute treatment with leptin reduced insulin-stimulated glucose uptake without altering insulin-stimulated GLUT4 translocation. In contrast, we show here that the ability of leptin to increase phosphorylation of its receptor and to reduce insulin-stimulated glucose uptake was lost in cells that were continuously exposed to leptin for 24 h. This desensitization correlated with an increase in expression of suppressor of cytokine signaling-3 (SOCS-3). Time course analysis demonstrated that the transition from acute to chronic effects of leptin occurs after 2 h. The desensitization of leptin action at 24 h was not reversed by 30 min washout period prior to re-exposing cells to leptin. However, despite insulin-stimulated glucose uptake being unaffected upon 24 h preincubation with leptin, a small but significant decrease (37%) in insulin-stimulated GLUT4 translocation and phosphorylation of Akt on T308 was detected. Insulin-stimulated phosphorylation of Akt on S473 or of p38 MAPK were unaffected. These results suggest that the chronic leptin treatment leads to desensitization of leptin signaling yet can simultaneously decrease the ability of insulin to phosphorylate Akt on T308 and translocate GLUT4. However, this does not manifest as a reduction in total glucose uptake into L6 myotubes.     

Photo-induced unfolding and inactivation of bovine carbonic anhydrase in the presence of a photoresponsive surfactant

Photoreversible changes in the conformation and enzymatic activity of bovine carbonic anhydrase have been investigated as a function of photoresponsive surfactant concentration and light conditions. The light-responsive surfactant undergoes a photoisomerization from the relatively hydrophobic trans isomer under visible light to the relatively hydrophilic cis isomer upon UV illumination, providing a means to photoreversibly control enzyme–surfactant interactions. Small-angle neutron scattering and dynamic light scattering measurements, along with fluorescence spectroscopy, indicate that carbonic anhydrase unfolds upon addition of the surfactant under visible light, while only a small degree of unfolding is observed under UV light. Therefore, the enzyme is completely inactivated in the presence of the trans surfactant, while 40% of the native activity is preserved under UV light, providing a photoreversible “on/off switch” of enzyme activity. Small-angle neutron scattering data provide details of the in vitro conformational changes of the enzyme in response to the photosurfactant and light, with the enzyme found to aggregate as a result of photosurfactant-induced unfolding. Fourier transform infrared (FT-IR) spectroscopy further provides information on the secondary structure changes of the protein in the presence of photosurfactant.     

Reversible control of enzyme-inhibitor interactions with light illumination using a photoresponsive surfactant

The effects of a photoresponsive surfactant and light illumination on the complex formed between ribonuclease A (RNase A) and a protein ribonuclease inhibitor (RI) have been investigated to develop a light-based technique to reactivate an enzyme through surfactant-induced dissociation of the enzyme-inhibitor complex. The photoresponsive surfactant undergoes a photoisomerization from the relatively hydrophobic trans isomer under visible light to the relatively hydrophilic cis isomer upon UV illumination, providing a means to reversibly control protein-inhibitor interactions. In the absence of surfactant, RI binds tightly to RNase A through noncovalent interactions, which inhibits the enzyme activity. Upon addition of the surfactant under visible light, RNase A is reactivated, regaining ~75% of the native activity in the absence of RI. In the presence of the surfactant under UV light, however, the enzyme remains inhibited. Fluorescence spectroscopy, dynamic light scattering, and circular dichroism spectroscopy reveal that RI dramatically unfolds upon addition of the trans form of the surfactant, while RNase A does not undergo noticeable structural changes under the same conditions. This indicates that RNase A reactivation occurs through dissociation of the enzyme-inhibitor complex arising from surfactant-induced unfolding of the inhibitor. As a result, photoresponsive surfactant and light illumination can be used as a novel light-based technique to dissociate enzyme-inhibitor complexes and, thus, reactivate an inhibited enzyme.     

Light-controlled protein dynamics observed with neutron spin echo measurements

A photoresponsive surfactant has been used as a means to control protein structure and dynamics with light illumination. This cationic azobenzene surfactant, azoTAB, which undergoes a reversible photoisomerization upon exposure to the appropriate wavelength of light, adopts a relatively hydrophobic, trans structure under visible light illumination and a relatively hydrophilic cis structure under UV light illumination. Small-angle neutron scattering (SANS) and neutron spin echo (NSE) spectroscopy were used to measure the tertiary structure and internal dynamics of lysozyme in the presence of the photosurfactant, respectively. The SANS-based in vitro structures indicate that under visible light the photosurfactant induces partial unfolding that principally occurs away from the active site near the hinge region connecting the α and β domains. Upon UV exposure, however, the protein refolds to a nativelike structure. At the same time, enhanced internal dynamics of lysozyme were detected with the surfactant in the trans form through NSE measurements of the Q-dependent effective diffusion coefficient (D(eff)) of the protein. In contrast, the D(eff) values of lysozyme in the presence of cis azoTAB largely agree with the rigid-body calculation as well as those measured for pure lysozyme, suggesting that the native protein is dormant on the nanosecond time and nanometer length scales. Lysozyme internal motions were modeled by assuming a protein of two (α and β domains) or three (α and β domains and the hinge region) domains connects by either soft linkers or rigid, freely rotating bonds. Protein dynamics were also tracked with Fourier transform infrared spectroscopy through hydrogen-deuterium exchange kinetics, which further demonstrated enhanced protein flexibility induced by the trans form of the surfactant relative to the native protein. Ensemble-averaged intramolecular fluorescent resonance energy transfer measurements similarly demonstrated the enhanced dynamics of lysozyme with the trans form of the photosurfactant. Previous results have shown a significant increase in protein activity in the presence of azoTAB in the trans conformation. Combined, these results provide insight into a unique light-based method of controlling protein structure, dynamics, and function and strongly support the relevance of large domain motions for the activity of proteins.     

Zellweger spectrum disorder patient–derived fibroblasts with the PEX1-Gly843Asp allele recover peroxisome functions in response to flavonoids

Zellweger spectrum disorder (ZSD) results from biallelic mutations in PEX genes required for peroxisome biogenesis. PEX1-G843D is a common hypomorphic allele in the patient population that is associated with milder disease. In prior work using a PEX1-G843D/null patient fibroblast line expressing a green fluorescent protein (GFP) reporter with a peroxisome-targeting signal (GFP-PTS1), we demonstrated that treatments with the chemical chaperone betaine and flavonoid acacetin diacetate recovered peroxisome functions. To identify more effective compounds for preclinical investigation, we evaluated 54 flavonoids using this cell-based phenotype assay. Diosmetin showed the most promising combination of potency and efficacy (EC50 2.5 µM). All active 5′,7′-dihydroxyflavones showed greater average efficacy than their corresponding flavonols, whereas the corresponding flavanones, isoflavones, and chalcones tested were inactive. Additional treatment with the proteostasis regulator bortezomib increased the percentage of import-rescued cells over treatment with flavonoids alone. Cotreatments of diosmetin and betaine showed the most robust additive effects, as confirmed by three independent functional assays in primary PEX1-G843D patient cells, but neither agent was active alone or in combination in patient cells homozygous for the PEX1 c.2097_2098insT null allele. Moreover, diosmetin treatment increased PEX1, PEX6, and PEX5 protein levels in PEX1-G843D patient cells, but none of these proteins increased in PEX1 null cells. We propose that diosmetin acts as a pharmacological chaperone that improves the stability, conformation, and functions of PEX1/PEX6 exportomer complexes required for peroxisome assembly. We suggest that diosmetin, in clinical use for chronic venous disease, and related flavonoids warrant further preclinical investigation for the treatment of PEX1-G843D–associated ZSD.     

Inhibition of glucose uptake in murine cardiomyocyte cell line HL-1 by cardioprotective drugs dilazep and dipyridamole

Inhibition of adenosine reuptake by nucleoside transport inhibitors, such as dipyridamole and dilazep, is proposed to increase extracellular levels of adenosine and thereby potentiate adenosine receptor-dependent pathways that promote cardiovascular health. Thus adenosine can act as a paracrine and/or autocrine hormone, which has been shown to regulate glucose uptake in some cell types. However, the role of adenosine in modulating glucose transport in cardiomyocytes is not clear. Therefore, we investigated whether exogenously applied adenosine or inhibition of adenosine transport by S-(4-nitrobenzyl)-6-thioinosine (NBTI), dipyridamole, or dilazep modulated basal and insulin-stimulated glucose uptake in the murine cardiomyocyte cell line HL-1. HL-1 cell lysates were subjected to SDS-PAGE and immunoblotting to determine which GLUT isoforms are present. Glucose uptake was measured in the presence of dipyridamole (3-300 microM), dilazep (1-100 microM), NBTI (10-500 nM), and adenosine (50-250 microM) or the nonmetabolizable adenosine analog 2-chloro-adenosine (250 microM). Our results demonstrated that HL-1 cells possess GLUT1 and GLUT4, the isoforms typically present in cardiomyocytes. We found no evidence for adenosine-dependent regulation of basal or insulin-stimulated glucose transport in HL-1 cardiomyocytes. However, we did observe a dose-dependent inhibition of glucose transport by dipyridamole (basal, IC(50) = 12.2 microM, insulin stimulated, IC(50) = 13.09 microM) and dilazep (basal, IC(50) = 5.7 microM, insulin stimulated, IC(50) = 19 microM) but not NBTI. Thus our data suggest that dipyridamole and dilazep, which are widely used to specifically inhibit nucleoside transport, have a broader spectrum of transport inhibition than previously described. Moreover, these data may explain previous observations, in which dipyridamole was noted to be proischemic at high doses.