Regulation of CCAAT/Enhancer-binding Protein Homologous Protein (CHOP) Expression by Interleukin-1β in Pancreatic β Cells

Apoptosis contributes to immune-mediated pancreatic β cell destruction in type I diabetes. Exposure of β cells to interleukin-1β (IL-1β) causes endoplasmic reticulum stress and activates proapoptotic networks. Here, we show that nuclear factor κB (NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathways regulate the expression of CCAAT/enhancer-binding protein homologous protein (CHOP), which mediates endoplasmic reticulum stress-induced apoptosis. Both CHOP mRNA and protein increase in β cells treated with IL-1β. In addition, prolonged exposure to high glucose further increases IL-1β-triggered CHOP expression. IL-1β also causes increased expression of C/EBP-β and a reduction of MafA, NFATc2, and Pdx-1 expression in β cells. Inhibition of the NF-κB and MAPK signaling pathways differentially attenuates CHOP expression. Knocking down CHOP by RNA interference protects β cells from IL-1β-induced apoptosis. These studies provide direct mechanistic links between cytokine-induced signaling pathways and CHOP-mediated apoptosis of β cells.     

Specific Deletion of AMP-Activated Protein Kinase (α1AMPK) in Murine Oocytes Alters Junctional Protein Expression and Mitochondrial Physiology

Oogenesis and folliculogenesis are dynamic processes that are regulated by endocrine, paracrine and autocrine signals. These signals are exchanged between the oocyte and the somatic cells of the follicle. Here we analyzed the role of AMP-activated protein kinase (AMPK), an important regulator of cellular energy homeostasis, by using transgenic mice deficient in α1AMPK specifically in the oocyte. We found a decrease of 27% in litter size was observed in ZP3-α1AMPK^-/- (ZP3-KO) female mice. Following in vitro fertilization, where conditions are stressful for the oocyte and embryo, ZP3-KO oocytes were 68% less likely to pass the 2-cell stage. In vivo and in cumulus-oocyte complexes, several proteins involved in junctional communication, such as connexin37 and N-cadherin were down-regulated in the absence of α1AMPK. While the two signalling pathways (PKA and MAPK) involved in the junctional communication between the cumulus/granulosa cells and the oocyte were stimulated in control oocytes, ZP3-KO oocytes exhibited only low phosphorylation of MAPK or CREB proteins. In addition, MII oocytes deficient in α1AMPK had a 3-fold lower ATP concentration, an increase in abnormal mitochondria, and a decrease in cytochrome C and PGC1α levels, suggesting perturbed energy production by mitochondria. The absence of α1AMPK also induced a reduction in histone deacetylase activity, which was associated with an increase in histone H3 acetylation (K9/K14 residues). Together, the results of the present study suggest that absence of AMPK, modifies oocyte quality through energy processes and oocyte/somatic cell communication. The limited effect observed in vivo could be partly due to a favourable follicle microenvironment where nutrients, growth factors, and adequate cell interaction were present. Whereas in a challenging environment such as that of in vitro culture following IVF, the phenotype is revealed.     

Integration of G Protein α (Gα) Signaling by the Regulator of G Protein Signaling 14 (RGS14)

RGS14 contains distinct binding sites for both active (GTP-bound) and inactive (GDP-bound) forms of Gα subunits. The N-terminal regulator of G protein signaling (RGS) domain binds active Gα_i/o-GTP, whereas the C-terminal G protein regulatory (GPR) motif binds inactive Gα_i1/3-GDP. The molecular basis for how RGS14 binds different activation states of Gα proteins to integrate G protein signaling is unknown. Here we explored the intramolecular communication between the GPR motif and the RGS domain upon G protein binding and examined whether RGS14 can functionally interact with two distinct forms of Gα subunits simultaneously. Using complementary cellular and biochemical approaches, we demonstrate that RGS14 forms a stable complex with inactive Gα_i1-GDP at the plasma membrane and that free cytosolic RGS14 is recruited to the plasma membrane by activated Gα_o-AlF₄⁻. Bioluminescence resonance energy transfer studies showed that RGS14 adopts different conformations in live cells when bound to Gα in different activation states. Hydrogen/deuterium exchange mass spectrometry revealed that RGS14 is a very dynamic protein that undergoes allosteric conformational changes when inactive Gα_i1-GDP binds the GPR motif. Pure RGS14 forms a ternary complex with Gα_o-AlF₄⁻ and an AlF₄⁻-insensitive mutant (G42R) of Gα_i1-GDP, as observed by size exclusion chromatography and differential hydrogen/deuterium exchange. Finally, a preformed RGS14·Gα_i1-GDP complex exhibits full capacity to stimulate the GTPase activity of Gα_o-GTP, demonstrating that RGS14 can functionally engage two distinct forms of Gα subunits simultaneously. Based on these findings, we propose a working model for how RGS14 integrates multiple G protein signals in host CA2 hippocampal neurons to modulate synaptic plasticity.     

The Effects of Non-Synonymous Single Nucleotide Polymorphisms (nsSNPs) on Protein–Protein Interactions

Non-synonymous single nucleotide polymorphisms (nsSNPs) are single base changes leading to a change to the amino acid sequence of the encoded protein. Many of these variants are associated with disease, so nsSNPs have been well studied, with studies looking at the effects of nsSNPs on individual proteins, for example, on stability and enzyme active sites. In recent years, the impact of nsSNPs upon protein–protein interactions has also been investigated, giving a greater insight into the mechanisms by which nsSNPs can lead to disease.     

Cloning-Independent Expression and Analysis of ω-Transaminases by Use of a Cell-Free Protein Synthesis System

Herewith we report the expression and screening of microbial enzymes without involving cloning procedures. Computationally predicted putative ω-transaminase (ω-TA) genes were PCR amplified from the bacterial colonies and expressed in a cell-free protein synthesis system for subsequent analysis of their enzymatic activity and substrate specificity. Through the cell-free expression analysis of the putative ω-TA genes, a number of enzyme-substrate pairs were identified in a matter of hours. We expect that the proposed strategy will provide a universal platform for bridging the information gap between nucleotide sequence and protein function to accelerate the discovery of novel enzymes.Recent advances in genome sequencing technology have accumulated enormous amounts of sequence information (12). Although protein function encoded in nucleotide sequences can be annotated using computational alignment tools, in many cases, significant similarity to proteins with known function is hard to establish (5, 18). To understand the biological function of these unknown proteins, as well as to validate the computer-annotated results, efficient methods that enable rapid translation of genetic information into protein function are in high demand. The availability of high-throughput method for protein generation is also essential for accelerating the discovery and evolution of biocatalysts (3, 4, 6, 14, 22, 23) used in industry. While gene cloning and cultivation of transformed cells have long been used as standard methods for production of recombinant proteins, the vast amount of sequence information from various genome sequencing projects is now demanding a throughput of protein expression that exceeds that of the present in vivo expression techniques.Compared to cell-based gene expression, cell-free protein synthesis offers substantial advantages in speed and flexibility for the simultaneous expression of multiple proteins (7, 9, 13, 16, 19, 21). As a part of our efforts to extend the application of cell-free protein synthesis into the field of enzyme technology, we report in this paper an integrated methodology for fast expression screening of enzymes using ω-transaminases (ω-TAs) as a model target. Transaminases are pyridoxal-5′-phosphate (PLP)-dependent enzymes that catalyze reversible transfer of amine groups to keto acids, producing diverse proteogenic or nonproteogenic amino acids (1).In this work, ω-TA genes from microbial colonies were amplified by PCR and directly expressed in a cell-free protein synthesis system. Expressed enzymes were then screened for their activity toward different amine donors by colorimetric measurement of the changes in the concentration of pyruvate, which was used as a common amine acceptor. As a result, analysis of the substrate specificities of the enzymes encoded by 11 ω-TA genes toward 16 amine-donating compounds were completed within a matter of hours, identifying a number of enzyme-substrate matches.We started by examining whether sufficient amount of proteins could be generated for enzymatic analysis of ω-TAs when the PCR products amplified from the bacterial colonies were used as the template for cell-free synthesis reactions. The efficiency of protein synthesis was compared for reactions programmed with a plasmid-cloned ω-TA gene from Vibrio fluvialis (Vfω-TA) (pIVEX2.3d ω-TA Vf) and reactions programmed with the same gene prepared by two-step PCR from a bacterial colony (Vibrio fluvialis JS17 [20]), as depicted in Fig. ​Fig.1.1. The ω-TA genes examined in this study are listed in Table S1 in the supplemental material along with their bacterial sources.Open in a separate windowFIG. 1.Experimental scheme for cloning-independent cell-free expression screening of ω-transaminases. The expression templates for cell-free synthesis were prepared through two-step amplification of the target open reading frame (ORF) from bacterial genomes. PCR products were translated into corresponding enzymes in microtiter plates as described in the text. Upon completion of the synthesis reaction, the reaction mixture was sequentially supplied with the assay mixture and chromogenic compound to determine the residual pyruvate concentration after the amine transfer reaction. Abbreviations: For and Rev Primer, forward and reverse primers, respectively; 5′-UTR, 5′ UTR; T7P, T7 promoter; RBS, ribosome binding site; T7T, T7 terminator; ω-TAp, putative ω-transaminase; ω-TA Rs, ω-transaminase from Rhodobacter sphaeroides; ω-TA At, ω-transaminase from Agrobacterium tumefaciens.The templates for cell-free synthesis of ω-TA were prepared by colony PCR and subsequent second-round PCR using the MEGA primers flanking the T7 promoter, ribosome binding site, polyhistidine tag, and the T7 terminator. All of the PCRs were carried out using LA Taq DNA polymerase (Takara Bio Inc., Otsu, Japan). PCR products were directly used as the template for protein synthesis without purification. The standard cell-free reaction mixture consisted of the following components in a final volume of 50 μl: 57 mM HEPES-KOH (pH 7.5); 1.2 mM ATP; 0.85 mM (each) CTP, GTP, and UTP; 1.7 mM dithiothreitol; 0.17 mg/ml Escherichia coli total tRNA mixture (from strain MRE600); 90 mM potassium glutamate; 80 mM ammonium acetate; 12 mM magnesium acetate; 34 μg/ml l-5-formyl-5,6,7,8-tetrahydrofolic acid (folinic acid); 1.5 mM (each) 20 amino acids; 2% polyethylene glycol 8000 (PEG 8000); 67 mM creatine phosphate; 3.2 μg/ml creatine kinase; 10 μM l-[U-¹⁴C]leucine (11.3 GBq/mmol); 0.5 μg/ml PCR-amplified DNA; and 14 μl of the S12 extract (11). Cell-free synthesized proteins were quantified by measuring trichloroacetic acid (TCA)-insoluble radioactivity (10), and the size and relative solubility of the synthesized protein were determined by Western blot analysis on a 12% Tricine-SDS-polyacrylamide gel (17). Mouse anti-histidine-tagged IgG antibody (Merck KGaA, Darmstadt, Germany) and rabbit anti-mouse IgG conjugated to horseradish peroxidase (HRP) (Sigma, St. Louis, MO) were used as the primary and secondary antibodies, respectively. The PCR products served as translation substrates appropriate for producing as much protein as the corresponding plasmid-cloned gene when expressed in the reaction mixture (see Fig. S1 in the supplemental material).We next proceeded to amplify 11 ω-TA genes, including 9 putative ones from the colonies of bacterial origins (see Table S2 in the supplemental material for the list of primers used in this study), and express them in the reaction mixture prepared in two microtiter plates. Each of the 11 target genes was added to the plates by column on the plate (columns 2 through 12). Column 1 was used for negative-control reactions without any template DNAs (Fig. ​(Fig.1).1). After the PCR-amplified template DNAs were added to the plates, the plates were sealed with a plastic film to prevent evaporation and incubated at 37°C. From the measurements of TCA-insoluble radioactivity, it was estimated that 301 (±13) to 501 (±9) μg/ml of the encoded enzymes were produced after 90 min of incubation (see Fig. S2A in the supplemental material). Although most of the cell-free synthesized ω-TAs was highly insoluble in the initial experiments conducted under standard reaction conditions, the relative amounts of the soluble enzymes were markedly improved by using the GroEL/ES-enriched S12 extract (8) (Fig. S2B).Instead of the conventional high-performance liquid chromatography (HPLC) methods which have limited throughput for handling many reaction samples from different enzyme-substrate combinations, in this study, we used a simple colorimetric method for combinatorial analysis of the cell-free synthesized ω-TAs with different amine-donating substrates. Using Vfω-TA as a model ω-TA, it was first examined whether the progress of amine transfer reaction can be assayed quantitatively by colorimetric measurement of the residual pyruvate concentration. On the basis of our previous finding that Vfω-TA takes amine donors containing aryl groups as effective substrates (20), cell-free synthesized Vfω-TA was incubated with α-methylbenzylamine or benzylamine in the presence of pyruvate, and the residual pyruvate concentration in the assay mixture was determined. In brief, the amine transfer reaction was initiated by adding 50 μl of assay mixture (50 mM Tris-HCl buffer [pH 7.2], 10 mM sodium pyruvate, 10 mM [each] amine donors, and 20 μM pyridoxal-5′-phosphate) to the completed cell-free synthesis reaction mixtures (50 μl) in a 96-well plate. After 3 h of incubation at 37°C, the assay mixture was diluted with an equal volume of distilled water. When 100 μl of the diluted solution was transferred to a fresh plate containing 50 μl of 0.5 mM 2,4-dinitrophenylhydrazine (DNP), a yellow precipitate of pyruvate-dinitrophenylhydrazone (PA-DNPH) derivative was formed instantly. The absorbance at 450 nm was measured in a microplate reader (Bio-Rad, Hercules, CA) and compared with a standard curve to determine the amount of residual pyruvate in each well. Although the sensitivity of the residual pyruvate assay was as low as 0.01 mM, considering the error range, it was determined that a sensitivity of 0.1 mM should be used instead. When the optical density at 450 nm (OD₄₅₀) was measured after the addition of DNP and referred to a standard curve, the conversion yield based on the amount of residual pyruvate concentration showed good correlation with the results from standard HPLC assay (Table ​(Table11 and Fig. ​Fig.2A,2A, insets) where acetophenone or benzaldehyde generated from the corresponding substrates was separated in the Discovery HS F5 (5-μm particle size; 150- by 4.6-mm inner diameter [i.d.]; Supelco, Bellefonte, PA) column and measured at 254 nm. In addition, the relative amount of pyruvate was also able to be compared visually by adding 100 μl of 4 N NaOH solution, which turned the color of PA-DNPH to dark red (2, 15).Open in a separate windowFIG. 2.(A) Reactivity of 16 amine donors toward Vibrio fluvialis ω-TA. Reduced amounts of pyruvate concentration after the amine transfer reactions are plotted. Substrate number abbreviations: S01, α-methylbenzylamine; S02, α-ethylbenzylamine; S03, benzylamine; S04, 3-phenyl-1-propylamine; S05, phenylbutylamine; S06, 1-aminoindan; S07, ethylamine; S08, propylamine; S09, butylamine; S10, amylamine; S11, isopropylamine; S12, sec-butylamine; S13, β-alanine; S14, 3-amino-n-butyric acid; S15, phenylalanine; S16, 3-amino-3-phenylpropionic acid. The insets show HPLC traces of acetophenone and benzaldehyde after termination of transamination reaction by use of Vibrio fluvialis ω-TA against α-methylbenzylamine and benzylamine. mAU, milliabsorbance units. (B) Photo image of the assay plate after the addition of DNP and NaOH. ω-TA Vf, ω-TA from Vibrio fluvialis.

TABLE 1.

Activity comparison of Vibrio fluvialis ω-TA by HPLC and colorimetric method

Receptor Activator of Nuclear Factor κB Ligand (RANKL) Protein Expression by B Lymphocytes Contributes to Ovariectomy-induced Bone Loss

Production of the cytokine receptor activator of NFκB ligand (RANKL) by lymphocytes has been proposed as a mechanism by which sex steroid deficiency causes bone loss. However, there have been no studies that functionally link RANKL expression in lymphocytes with bone loss in this condition. Herein, we examined whether RANKL expression in either B or T lymphocytes contributes to ovariectomy-induced bone loss in mice. Mice harboring a conditional RANKL allele were crossed with CD19-Cre or Lck-Cre mice to delete RANKL in B or T lymphocytes, respectively. Deletion of RANKL from either cell type had no impact on bone mass in estrogen-replete mice up to 7 months of age. However, mice lacking RANKL in B lymphocytes were partially protected from the bone loss caused by ovariectomy. This protection occurred in cancellous, but not cortical, bone and was associated with a failure to increase osteoclast numbers in the conditional knock-out mice. Deletion of RANKL from T lymphocytes had no impact on ovariectomy-induced bone loss. These results demonstrate that lymphocyte RANKL is not involved in basal bone remodeling, but B cell RANKL does contribute to the increase in osteoclasts and cancellous bone loss that occurs after loss of estrogen.     

β-Transducin Repeat-containing Protein 1 (β-TrCP1)-mediated Silencing Mediator of Retinoic Acid and Thyroid Hormone Receptor (SMRT) Protein Degradation Promotes Tumor Necrosis Factor α (TNFα)-induced Inflammatory Gene Expression

Cytokine modulation of the endothelium is considered an important contributor to the inflammation response. TNFα is an early response gene during the initiation of inflammation. However, the detailed mechanism by which TNFα induces proinflammatory gene expression is not completely understood. In this report, we demonstrate that silencing mediator of retinoic acid and thyroid hormone receptor (SMRT) represses the expression of a subset of TNFα target genes in human umbilical vein endothelial cells. Upon TNFα stimulation, we observed an increase in the E3 ubiquitin ligase β-TrCP1 and a decrease in SMRT protein levels. We show that β-TrCP1 interacts with SMRT in a phosphorylation-independent manner and cooperates with the E2 ubiquitin-conjugating enzyme E2D2 to promote ubiquitination-dependent SMRT degradation. Knockdown of β-TrCP1 increases SMRT protein accumulation, increases SMRT association with its targeted promoters, and decreases SMRT target gene expression. Taken together, our results support a model in which TNFα-induced β-TrCP1 accumulation promotes SMRT degradation and the subsequent induction of proinflammatory gene expression.     

Characterization of Protein–Protein Interfaces

We analyze the characteristics of protein–protein interfaces using the largest datasets available from the Protein Data Bank (PDB). We start with a comparison of interfaces with protein cores and non-interface surfaces. The results show that interfaces differ from protein cores and non-interface surfaces in residue composition, sequence entropy, and secondary structure. Since interfaces, protein cores, and non-interface surfaces have different solvent accessibilities, it is important to investigate whether the observed differences are due to the differences in solvent accessibility or differences in functionality. We separate out the effect of solvent accessibility by comparing interfaces with a set of residues having the same solvent accessibility as the interfaces. This strategy reveals residue distribution propensities that are not observable by comparing interfaces with protein cores and non-interface surfaces. Our conclusions are that there are larger numbers of hydrophobic residues, particularly aromatic residues, in interfaces, and the interactions apparently favored in interfaces include the opposite charge pairs and hydrophobic pairs. Surprisingly, Pro-Trp pairs are over represented in interfaces, presumably because of favorable geometries. The analysis is repeated using three datasets having different constraints on sequence similarity and structure quality. Consistent results are obtained across these datasets. We have also investigated separately the characteristics of heteromeric interfaces and homomeric interfaces.     

Splicing Factor Transformer-2β (Tra2β) Regulates the Expression of Regulator of G Protein Signaling 4 (RGS4) Gene and Is Induced by Morphine

Regulator of G protein signaling 4 (RGS4) is a critical modulator of G protein-coupled receptor (GPCR)-mediated signaling and plays important roles in many neural process and diseases. Particularly, drug-induced alteration in RGS4 protein levels is associated with acute and chronic effects of drugs of abuse. However, the precise mechanism underlying the regulation of RGS4 expression is largely unknown. Here, we demonstrated that the expression of RGS4 gene was subject to regulation by alternative splicing of the exon 6. Transformer-2β (Tra2β), an important splicing factor, bound to RGS4 mRNA and increased the relative level of RGS4-1 mRNA isoform by enhancing the inclusion of exon 6. Meanwhile, Tra2β increased the expression of full-length RGS4 protein. In rat brain, Tra2β was co-localized with RGS4 in multiple opioid action-related brain regions. In addition, the acute and chronic morphine treatment induced alteration in the expression level of Tra2β in rat locus coerulus (LC) in parallel to that of RGS4 proteins. It suggests that induction of this splicing factor may contribute to the change of RGS4 level elicited by morphine. Taken together, the results provide the evidence demonstrating the function of Tra2β as a new mediator in opioid-induced signaling pathway via regulating RGS4 expression.     

Expression of von Hippel–Lindau Protein in Normal and Pathological Human Tissues

To examine the localization of von Hippel–Lindau (VHL) protein in human tissues, we produced four novel monoclonal antibodies against human VHL protein. Western blot analysis revealed that two of these antibodies recognized the epitope in amino acid sequence 60–89 of the VHL protein and the others recognized sequences 54–60 and 189–213. In a wild-type VHL gene-transfected cell line, immunocytochemistry and immunoelectron microscopy demonstrated the intracytoplasmic localization of VHL protein, particularly in mitotic cells. In normal human tissues, VHL protein was detected immunohistochemically in epithelial cells covering the body surface and the alimentary, respiratory, and genitourinary tracts; in secretory epithelial cells of exocrine and endocrine organs; in parenchymal cells of visceral organs; in cardiomyocytes; in neurons in nervous tissue; in lymphocytes in lymphoid tissue; and in macrophages. In pathological specimens, VHL protein was expressed in VHL-related tumor, as well as in endothelial cells, fibroblasts, and pericytes, all of which are involved in active angiogenesis. These findings suggest that these monoclonal antibodies can be useful for various immunological assays and that the VHL protein plays fundamental roles in physiological and pathological situations, especially in neovascularization.     

Computational Prediction of Protein–Protein Interactions

Recently a number of computational approaches have been developed for the prediction of protein–protein interactions. Complete genome sequencing projects have provided the vast amount of information needed for these analyses. These methods utilize the structural, genomic, and biological context of proteins and genes in complete genomes to predict protein interaction networks and functional linkages between proteins. Given that experimental techniques remain expensive, time-consuming, and labor-intensive, these methods represent an important advance in proteomics. Some of these approaches utilize sequence data alone to predict interactions, while others combine multiple computational and experimental datasets to accurately build protein interaction maps for complete genomes. These methods represent a complementary approach to current high-throughput projects whose aim is to delineate protein interaction maps in complete genomes. We will describe a number of computational protocols for protein interaction prediction based on the structural, genomic, and biological context of proteins in complete genomes, and detail methods for protein interaction network visualization and analysis.     

Transient Spinal Cord Ischemia in Rat: The Time Course of Spinal FOS Protein Expression and the Effect of Intraischemic Hypothermia (27°C)

1. In the present study, we characterize the time course of spinal FOS protein expression after transient noninjurious (6-min) or injurious (12-min) spinal ischemia induced by inflation of a balloon catheter placed into the descending thoracic aorta. In addition, this work examined the effects of spinal hypothermia on FOS expression induced either by ischemia or by potassium-evoked depolarization (intrathecal KCl).2. Short-lasting (6-min) spinal ischemia evoked a transient FOS protein expression. The peak expression was seen 2 hr after reperfusion in all laminar levels in lumbosacral segments. At 4 hr of reperfusion, more selective FOS expression in spinal interneurons localized in the central part of laminae V–VII was seen. At 24 hr no significant increase in FOS protein was detected.3. After 12 min of ischemia and 2 hr of reflow, nonspecific FOS expression was seen in both white and gray matter, predominantly in nonneuronal elements. Intrathecal KCl-induced FOS expression in spinal neurons in the dorsal horn and in the intermediate zone. Spinal hypothermia (27°C) significantly suppressed FOS expression after 6 or 12 min of ischemia but not after KCl-evoked depolarization.4. Data from the present study show that an injurious (but not noninjurious) interval of spinal ischemia evokes spinal FOS protein expression in glial cells 2 hr after reflow. The lack of neuronal FOS expression corresponds with extensive neuronal degeneration seen in this region 24 hr after reflow. Noninjurious (6-min) ischemia induced a transient, but typically neuronal FOS expression. The significant blocking effect of hypothermia (27°C) on the FOS induction after ischemia but not after potassium-evoked depolarization also suggests that simple neuronal depolarization is a key trigger in FOS induction.     

Are Rod Outer Segment ATP-ase and ATP-Synthase Activity Expression of the Same Protein?

Vertebrate retinal rod outer segments (OS) consist of a stack of disks surrounded by the plasma membrane, where phototransduction takes place. Energetic metabolism in rod OS remains obscure. Literature described a so-called Mg²⁺-dependent ATPase activity, while our previous results demonstrated the presence of oxidative phosphorylation (OXPHOS) in OS, sustained by an ATP synthetic activity. Here we propose that the OS ATPase and ATP synthase are the expression of the same protein, i.e., of F₁F_o-ATP synthase. Imaging on bovine retinal sections showed that some OXPHOS proteins are expressed in the OS. Biochemical data on bovine purified rod OS, characterized for purity, show an ATP synthase activity, inhibited by classical F₁F_o-ATP synthase inhibitors. Moreover, OS possess a pH-dependent ATP hydrolysis, inhibited by pH values below 7, suggestive of the functioning of the inhibitor of F1 (IF1) protein. WB confirmed the presence of IF1 in OS, substantiating the expression of F₁F_o ATP synthase in OS. Data suggest that the OS F₁Fo ATP synthase is able to hydrolyze or synthesize ATP, depending on in vitro or in vivo conditions and that the role of IF1 would be pivotal in the prevention of the reversal of ATP synthase in OS, for example during hypoxia, granting photoreceptor survival.     

Alzheimer’s Disease-Related Protein Expression in the Retina of Octodon degus

New studies show that the retina also undergoes pathological changes during the development of Alzheimer’s disease (AD). While transgenic mouse models used in these previous studies have offered insight into this phenomenon, they do not model human sporadic AD, which is the most common form. Recently, the Octodon degus has been established as a sporadic model of AD. Degus display age-related cognitive impairment associated with Aβ aggregates and phosphorylated tau in the brain. Our aim for this study was to examine the expression of AD-related proteins in young, adult and old degus retina using enzyme-linked or fluorescence immunohistochemistry and to quantify the expression using slot blot and western blot assays. Aβ4G8 and Aβ6E10 detected Aβ peptides in some of the young animals but the expression was higher in the adults. Aβ peptides were observed in the inner and outer segment of the photoreceptors, the nerve fiber layer (NFL) and ganglion cell layer (GCL). Expression was higher in the central retinal region than in the retinal periphery. Using an anti-oligomer antibody we detected Aβ oligomer expression in the young, adult and old retina. Immunohistochemical labeling showed small discrete labeling of oligomers in the GCL that did not resemble plaques. Congo red staining did not result in green birefringence in any of the animals analyzed except for one old (84 months) animal. We also investigated expression of tau and phosphorylated tau. Expression was seen at all ages studied and in adults it was more consistently observed in the NFL-GCL. Hyperphosphorylated tau detected with AT8 antibody was significantly higher in the adult retina and it was localized to the GCL. We confirm for the first time that Aβ peptides and phosphorylated tau are expressed in the retina of degus. This is consistent with the proposal that AD biomarkers are present in the eye.     

Hypoxia-inducible Factor-1α (HIF-1α) Protein Diminishes Sodium Glucose Transport 1 (SGLT1) and SGLT2 Protein Expression in Renal Epithelial Tubular Cells (LLC-PK1) under Hypoxia

In this work, we demonstrated the regulation of glucose transporters by hypoxia inducible factor-1α (HIF-1α) activation in renal epithelial cells. LLC-PK₁ monolayers were incubated for 1, 3, 6, or 12 h with 0% or 5% O₂ or 300 μm cobalt (CoCl₂). We evaluated the effects of hypoxia on the mRNA and protein expression of HIF-1α and of the glucose transporters SGLT1, SGLT2, and GLUT1. The data showed an increase in HIF-1α mRNA and protein expression under the three evaluated conditions (p < 0.05 versus t = 0). An increase in GLUT1 mRNA (12 h) and protein expression (at 3, 6, and 12 h) was observed (p < 0.05 versus t = 0). SGLT1 and SGLT2 mRNA and protein expression decreased under the three evaluated conditions (p < 0.05 versus t = 0). In conclusion, our results suggest a clear decrease in the expression of the glucose transporters SGLT1 and SGLT2 under hypoxic conditions which implies a possible correlation with increased expression of HIF-1α.