Self‐assembling amyloid‐like peptides as exogenous second harmonic probes for bioimaging applications

Amyloid‐like peptides are an ideal model for the mechanistic study of amyloidosis, which may lead to many human diseases, such as Alzheimer disease. This study reports a strong second harmonic generation (SHG) effect of amyloid‐like peptides, having a signal equivalent to or even higher than those of endogenous collagen fibers. Several amyloid‐like peptides (both synthetic and natural) were examined under SHG microscopy and shown they are SHG‐active. These peptides can also be observed inside cells (in vitro). This interesting property can make these amyloid‐like peptides second harmonic probes for bioimaging applications. Furthermore, SHG microscopy can provide a simple and label‐free approach to detect amyloidosis. Lattice corneal dystrophy was chosen as a model disease of amyloidosis. Morphological difference between normal and diseased human corneal biopsy samples can be easily recognized, proving that SHG can be a useful tool for disease diagnosis.     

The gap junction protein connexin32 interacts with the Src homology 3/hook domain of discs large homolog 1

Scaffolding of membrane proteins is a common strategy for forming complexes of proteins, including some connexins, within membrane microdomains. Here we describe studies indicating that Cx32 interacts with a PDZ-containing scaffolding protein, Dlgh1 (Discs Large homolog 1). Initial screens of liver lysates using antibody arrays indicated an interaction between Cx32 and Dlgh1 that was confirmed using coimmunoprecipitation studies. Yeast two-hybrid complementation determined that the Cx32 bound via interaction with the SH3/Hook domain of Dlgh1. Confocal microscopy of liver sections revealed that Cx32 and Dlgh1 could colocalize in hepatocyte membranes in wild type mice. Examination of levels and localization of Dlgh1 in livers from Cx32 null mice indicate that, in the absence of Cx32, Dlgh1 was decreased, and the remainder was translocated from the hepatocyte membrane to the nucleus with some remaining in cytoplasmic compartments. This translocation was confirmed by Western blots comparing Dlgh1 levels in nuclear extracts from wild type and Cx32 null murine livers. Using SKHep cells stably transfected with Cx32 under the control of a tet-off promoter, we found that acute removal of Cx32 led to a decrease of membrane-localized Dlgh1 and an increase in the nuclear localization of this tumor suppressor protein. Together, these results suggest that loss of Cx32 alters the levels, localization, and interactions of the tumor suppressor protein Dlgh1, events known in other systems to alter cell cycle and increase tumorigenicity.     

Expression of aromatase,androgen and estrogen receptors in peripheral target tissues in diabetes

Our previous studies have shown that diabetes in the male streptozotocin (STZ)-induced diabetic rat is characterized by a decrease in circulating testosterone and concomitant increase in estradiol levels. Interestingly, this increase in estradiol levels persists even after castration, suggesting extra-testicular origins of estradiol in diabetes. The aim of the present study was to examine whether other target organs of diabetes may be sources of estradiol. The study was performed in male Sprague–Dawley non-diabetic (ND), STZ-induced diabetic (D) and STZ-induced diabetic castrated (Dcas) rats (n = 8–9/group). 14 weeks of diabetes was associated with decreased testicular (ND, 26.3 ± 4.19; D, 18.4 ± 1.54; P < 0.05), but increased renal (ND, 1.83 ± 0.92; D, 7.85 ± 1.38; P < 0.05) and ocular (D, 23.4 ± 3.66; D, 87.1 ± 28.1; P < 0.05) aromatase activity. This increase in renal (Dcas, 6.30 ± 1.25) and ocular (Dcas, 62.7 ± 11.9) aromatase activity persisted after castration. The diabetic kidney also had increased levels of tissue estrogen (ND, 0.31 ± 0.01; D, 0.51 ± 0.11; Dcas, 0.45 ± 0.08) as well as estrogen receptor alpha protein expression (ND, 0.63 ± 0.09; D, 1.62 ± 0.28; Dcas, 1.38 ± 0.20). These data suggest that in male STZ-induced diabetic rats, tissues other than the testis may become sources of estradiol. In particular, the diabetic kidney appears to produce estradiol following castration, a state that is associated with a high degree or renal injury. Overall, our data provides evidence for the extra-testicular source of estradiol that in males, through an intracrine mechanism, may contribute to the development and/or progression of end-organ damage associated with diabetes.     

Photoreactions of metarhodopsin III

Meta III is an inactive intermediate thermally formed following light activation of the visual pigment rhodopsin. It is produced from the Meta I/Meta II photoproduct equilibrium of rhodopsin by a thermal isomerization of the protonated Schiff base C=N bond of Meta I, and its chromophore configuration is therefore all-trans 15-syn. In contrast to the dark state of rhodopsin, which catalyzes exclusively the cis to trans isomerization of the C11=C12 bond of its 11-cis 15-anti chromophore, Meta III does not acquire this photoreaction specificity. Instead, it allows for light-dependent syn to anti isomerization of the C15=N bond of the protonated Schiff base, yielding Meta II, and for trans to cis isomerizations of C11=C12 and C9=C10 of the retinal polyene, as shown by FTIR spectroscopy. The 11-cis and 9-cis 15-syn isomers produced by the latter two reactions are not stable, decaying on the time scale of few seconds to dark state rhodopsin and isorhodopsin by thermal C15=N isomerization, as indicated by time-resolved FTIR methods. Flash photolysis of Meta III produces therefore Meta II, dark state rhodopsin, and isorhodopsin. Under continuous illumination, the latter two (or its unstable precursors) are converted as well to Meta II by presumably two different mechanisms.     

Consequences of counterion mutation in sensory rhodopsin II of Natronobacterium pharaonis for photoreaction and receptor activation: an FTIR study

In many retinal proteins the proton transfer from the Schiff base to the counterion represents a functionally important step of the photoreaction. In the signaling state of sensory rhodopsin II from Natronobacterium pharaonis this transfer has already occurred, but in the counterion mutant Asp75Asn it is blocked during all steps of the photocycle. Therefore, the study of the molecular changes during the photoreaction of this mutant should provide a deeper understanding of the activation mechanism, and for this, we have applied time-resolved step-scan FTIR spectroscopy. The photoreaction is drastically altered; only red-shifted intermediates are formed with a chromophore strongly twisted around the 14-15 single bond. In addition, the photocycle is shortened by 2 orders of magnitude. Nevertheless, a transition involving only protein changes similar to that of the wild type is observed, which has been correlated with the formation of the signaling state. However, whereas in the wild type this transition occurs in the millisecond range, it is shortened to 200 micros in the mutant. The results are discussed with respect to the altered electrostatic interactions, role of proton transfer, the published 3D structure, and physiological activity.     

Sexual dimorphism in the renin-angiotensin system in aging spontaneously hypertensive rats

In young adult spontaneously hypertensive rats (SHR), mean arterial pressure (MAP) is higher in males than in females and inhibition of the renin-angiotensin system (RAS) eliminates this sex difference. After cessation of estrous cycling in female SHR, MAP is similar to that in male SHR. The purpose of this study was to determine the role of the RAS in maintenance of hypertension in aging male and female SHR. At 16 mo of age, MAP was similar in male and female SHR (183+/-5 vs. 193+/-8 mmHg), and chronic losartan (40 mg.kg-1.day-1 po for 3 wk) reduced MAP by 52% (to 90+/-8 mmHg, P<0.05 vs. control) in males and 37% (to 123+/-11 mmHg, P<0.05 vs. control) in females (P<0.05, females vs. males). The effect of losartan on angiotensin type 1 (AT1) receptor blockade was similar: MAP responses to acute doses of ANG II (62.5-250 ng/kg) were blocked to a similar extent in losartan-treated males and females. F2-isoprostane excretion was reduced with losartan more in males than in females. There were no sex differences in plasma renin activity, plasma angiotensinogen or ANG II, or renal expression of AT1 receptors, angiotensin-converting enzyme, or renin. However, renal angiotensinogen mRNA and protein expression was higher in old males than females, whereas renal ANG II was higher in old females than males. The data show that, in aging SHR, when blood pressures are similar, there remains a sexual dimorphism in the response to AT1 receptor antagonism, and the differences may involve sex differences in mechanisms responsible for oxidative stress with aging.     

Kinetics of proton release and uptake by channelrhodopsin-2

Electrophysiological experiments showed that the light-activated cation channel channelrhodopsin-2 (ChR2) pumps protons in the absence of a membrane potential. We determined here the kinetics of transient pH change using a water-soluble pH-indicator. It is shown that ChR2 released protons prior to uptake with a stoichiometry of 0.3 protons per ChR2. Comparison to the photocycle kinetics revealed that proton release and uptake match rise and decay of the P(3)(520) intermediate. As the P(3)(520) state also represents the conductive state of cation channeling, the concurrence of proton pumping and channel gating implies an intimate mechanistic link of the two functional modes. Studies on the E123T and S245E mutants show that these residues are not critically involved in proton translocation.     

Quercetin and epigallocatechin gallate effects on the cell membranes biophysical properties correlate with their antioxidant potential

Quercetin and epigallocatechin gallate are two of the most abundant polyphenols in dietary plants, including apples, onions, red wine and green tea. The bioactivity of polyphenols is linked to their ability to interact with cell membranes without being internalized. The aim of the present study was to assess the short-time effect of these polyphenols on membrane anisotropy and transmembrane potential of U937 monocytes and Jurkat T lymphoblasts. Results showed that quercetin and epigallocatechin gallate induced, after 20 minutes cell exposure, a dose-dependent increase of membrane anisotropy and polarization. Anisotropy increase was correlated with the reduction of lipid peroxidation. Our results could indicate that the antioxidant capacity of the tested polyphenols is due to their stabilizing effect on the cell membranes, thus contributing to cell protection in various pathologies and as adjuvant therapy in highly toxic treatment regimens.     

Dual effect of methylglyoxal on the intracellular Ca2+ signaling and neurite outgrowth in mouse sensory neurons

The formation of advanced glycation end products is one of the major factors involved in diabetic neuropathy, aging, and neurodegenerative diseases. Reactive carbonyl compounds, such as methylglyoxal (MG), play a key role in cross-linking to various proteins in the extracellular matrix, especially in neurons, which have a high rate of oxidative metabolism. The MG effect was tested on dorsal root ganglia primary neurons in cultures from adult male Balb/c mice. Lower MG doses contribute to an increased adherence of neurons on their support and an increased glia proliferation, as proved by MTS assay and bright-field microscopy. Time-lapse fluorescence microscopy by Fura-2 was performed for monitoring the relative fluorescence ratio changes (ΔR/R(0)) upon depolarization and immunofluorescence staining for quantifying the degree of neurites extension. The relative change in fluorescence ratio modifies the amplitude and dispersion depending on the subtype of sensory neurons, the medium-sized neurons are more sensitive to MG treatment when compared to small ones. Low MG concentrations (0-150 μM) increase neuronal viability, excitability, and the capacity of neurite extension, while higher concentrations (250-750 μM) are cytotoxic in a dose-dependent manner. In our opinion, MG could be metabolized by the glyoxalase system inside sensory neurons up to a threshold concentration, afterwards disturbing the cell equilibrium. Our study points out that MG has a dual effect concentration dependent on the neuronal viability, excitability, and neurite outgrowth, but only the excitability changes are soma-sized dependent. In conclusion, our data may partially explain the distinct neuronal modifications in various neurodegenerative pathologies.