Endothelial dysfunction induces atherosclerosis: increased aggrecan expression promotes apoptosis in vascular smooth muscle cells.

Endothelial dysfunction-induced lipid retention is an early feature of atherosclerotic lesion formation. Apoptosis of vascular smooth muscle cells (VSMCs) is one of the major modulating factors of atherogenesis, which accelerates atherosclerosis progression by causing plaque destabilization and rupture. However, the mechanism underlying VSMC apoptosis mediated by endothelial dysfunction in relation to atherosclerosis remains elusive. In this study, we reveal differential expression of several genes related to lipid retention and apoptosis, in conjunction with atherosclerosis, by utilizing a genetic mouse model of endothelial nitric oxide synthase (eNOS) deficiency manifesting endothelial dysfunction. Moreover, eNOS deficiency led to the enhanced susceptibility against pro-apoptotic insult in VSMCs. In particular, the expression of aggrecan, a major proteoglycan, was elevated in aortic tissue of eNOS deficient mice compared to wild type mice, and administration of aggrecan induced apoptosis in VSMCs. This suggests that eNOS deficiency may elevate aggrecan expression, which promotes apoptosis in VSMC, thereby contributing to atherosclerosis progression. These results may facilitate the development of novel approaches for improving the diagnosis or treatment of atherosclerosis. [BMB Reports 2019; 52(2): 145-150].

Enhancement of liposome mediated gene transfer by adding cholesterol and cholesterol modulating drugs.

Cholesterol is an important cell membrane component and has been used as co-lipid for cationic liposome to enhance gene delivery. However, the role of cholesterol in transfection efficiency has not been fully understood. In this study, transfection efficiency of liposome was measured after cholesterol was added to the cell culture medium. As a result, addition of cholesterol increased transfection efficiency of several liposomes consisting of different lipid components in various cells (AGS, CHO, COS7 and, MCF7). Furthermore, treatment of cells with cholesterol modulating drugs, imipramine and U18666A, also increased transfection efficiency of liposomes. To elucidate the role of added cholesterol in gene transfer, endocytotic mechanism was studied and also revealed that adding cholesterol in culture media induced participation of caveolae-mediated endocytosis and micropinocytosis in CHO cell. Therefore, the results of this work suggest that modulation of intracellular cholesterol can be an important method to enhance gene delivery.

Perfusion Assessment Using Intravoxel Incoherent Motion-Based Analysis of Diffusion-Weighted Magnetic Resonance Imaging: Validation Through Phantom Experiments.

OBJECTIVES: The aims of this study were to demonstrate the theoretical meaning of intravoxel incoherent motion (IVIM) parameters and to compare the robustness of 2 biexponential fitting methods through magnetic resonance experiments using IVIM phantoms. MATERIALS AND METHODS: Intravoxel incoherent motion imaging was performed on a 3 T magnetic resonance imaging scanner using 15 b values (0-800 s/mm) for 4 phantoms with different area fractions of the flowing water compartment (FWC%), at the infusion flow rates of 0, 1, 2, and 3 mL/min. Images were quantitatively analyzed using monoexponential free biexponential, and segmented biexponential fitting models. RESULTS: There were some inconsistent variations in Dslow with changing flow rates. The perfusion fraction, f, showed a significant positive correlation with the flow rate for both the free and segmented fitting methods (ρ = 0.838 to 0.969; P < 0.001). The fast diffusion coefficient, Dfast, had a significant positive correlation with the flow rate for segmented fitting (ρ = 0.745 to 0.969; P < 0.001), although it showed an inverse correlation with the flow rate for free fitting (ρ = -0.527 to -0.791; P ≤ 0.017). Significant positive correlations with the FWC% of the phantoms were noted for f (P = 0.510 for free fitting and P = 0.545 for segmented fitting, P < 0.001). CONCLUSIONS: The IVIM model allows for an approximate segmentation of molecular diffusion and perfusion, with a minor contribution of the perfusion effect on Dslow. The f and Dfast can provide a rough estimation of the flow fraction and flow velocity. Segmented fitting may be a more robust method than free fitting for calculating the IVIM parameters, especially for Dfast.

Multivalent di-nucleosome recognition enables the Rpd3S histone deacetylase complex to tolerate decreased H3K36 methylation levels.

The Rpd3S histone deacetylase complex represses cryptic transcription initiation within coding regions by maintaining the hypo-acetylated state of transcribed chromatin. Rpd3S recognizes methylation of histone H3 at lysine 36 (H3K36me), which is required for its deacetylation activity. Rpd3S is able to function over a wide range of H3K36me levels, making this a unique system to examine how chromatin regulators tolerate the reduction of their recognition signal. Here, we demonstrated that Rpd3S makes histone modification-independent contacts with nucleosomes, and that Rpd3S prefers di-nucleosome templates since two binding surfaces can be readily accessed simultaneously. Importantly, this multivalent mode of interaction across two linked nucleosomes allows Rpd3S to tolerate a two-fold intramolecular reduction of H3K36me. Our data suggest that chromatin regulators utilize an intrinsic di-nucleosome-recognition mechanism to prevent compromised function when their primary recognition modifications are diluted.

Sitagliptin increases tau phosphorylation in the hippocampus of rats with type 2 diabetes and in primary neuron cultures.

Increasing evidence supports an association between Alzheimer's disease (AD) and diabetes. In this context, anti-diabetic agents such as rosiglitazone and glucagon-like peptide (GLP)-1 have been reported to reduce pathologies associated with AD, including tau hyperphosphorylation, suggesting that such agents might be used to treat AD. One such anti-diabetic agent is sitagliptin, which acts through inhibition of dipeptidyl peptidase (DPP)-IV to increase GLP-1 levels. Given this action, sitagliptin would be predicted to reduce AD pathology. Accordingly, we investigated whether sitagliptin is effective in attenuating AD pathologies, focusing on tau phosphorylation in the OLETF type 2 diabetic rat model. Unexpectedly, we found that sitagliptin was not effective against pathological tau phosphorylation in the hippocampus of OLETF type 2 diabetes rats, and instead aggravated it. This paradoxically increased tau phosphorylation was attributed to activation of the tau kinase, GSK3ß (glycogen synthase kinase 3ß). Sitagliptin also increased ser-616 phosphorylation of the insulin receptor substrate (IRS)-1, suggesting increased insulin resistance in the brain. These phenomena were recapitulated in primary rat cortical neurons treated with sitagliptin, further confirming sitagliptin's effects on AD-related pathologies in neurons. These results highlight the need for caution in considering the use of sitagliptin in AD therapy.

Reconstitution of modified chromatin templates for in vitro functional assays.

To study the functions of histone modifications in the context of chromatin, it is necessary to be able to prepare nucleosomal templates that carry specific posttranslational modifications in a defined biochemical system. Here, we describe two sets of protocols for reconstituting designer nucleosomes that contain specifically modified histones. The resulting nucleosomes are suitable for electromobility shift assays, chromatin remodeling assays, and other functional and structural studies.

Structural basis of cooperativity in human UDP-glucose dehydrogenase.

BACKGROUND: UDP-glucose dehydrogenase (UGDH) is the sole enzyme that catalyzes the conversion of UDP-glucose to UDP-glucuronic acid. The product is used in xenobiotic glucuronidation in hepatocytes and in the production of proteoglycans that are involved in promoting normal cellular growth and migration. Overproduction of proteoglycans has been implicated in the progression of certain epithelial cancers, while inhibition of UGDH diminished tumor angiogenesis in vivo. A better understanding of the conformational changes occurring during the UGDH reaction cycle will pave the way for inhibitor design and potential cancer therapeutics. METHODOLOGY: Previously, the substrate-bound of UGDH was determined to be a symmetrical hexamer and this regular symmetry is disrupted on binding the inhibitor, UDP-α-D-xylose. Here, we have solved an alternate crystal structure of human UGDH (hUGDH) in complex with UDP-glucose at 2.8 Å resolution. Surprisingly, the quaternary structure of this substrate-bound protein complex consists of the open homohexamer that was previously observed for inhibitor-bound hUGDH, indicating that this conformation is relevant for deciphering elements of the normal reaction cycle. CONCLUSION: In all subunits of the present open structure, Thr131 has translocated into the active site occupying the volume vacated by the absent active water and partially disordered NAD+ molecule. This conformation suggests a mechanism by which the enzyme may exchange NADH for NAD+ and repolarize the catalytic water bound to Asp280 while protecting the reaction intermediates. The structure also indicates how the subunits may communicate with each other through two reaction state sensors in this highly cooperative enzyme.

PICH and BLM limit histone association with anaphase centromeric DNA threads and promote their resolution.

Centromeres nucleate the formation of kinetochores and are vital for chromosome segregation during mitosis. The SNF2 family helicase PICH (Plk1-interacting checkpoint helicase) and the BLM (the Bloom's syndrome protein) helicase decorate ultrafine histone-negative DNA threads that link the segregating sister centromeres during anaphase. The functions of PICH and BLM at these threads are not understood, however. Here, we show that PICH binds to BLM and enables BLM localization to anaphase centromeric threads. PICH- or BLM-RNAi cells fail to resolve these threads in anaphase. The fragmented threads form centromeric-chromatin-containing micronuclei in daughter cells. Anaphase threads in PICH- and BLM-RNAi cells contain histones and centromere markers. Recombinant purified PICH has nucleosome remodelling activities in vitro. We propose that PICH and BLM unravel centromeric chromatin and keep anaphase DNA threads mostly free of nucleosomes, thus allowing these threads to span long distances between rapidly segregating centromeres without breakage and providing a spatiotemporal window for their resolution.

Inhibitory effects of gallic acid and quercetin on UDP-glucose dehydrogenase activity.

We have examined polyphenols as potential inhibitors of UDP-glucose dehydrogenase (UGDH) activity. Gallic acid and quercetin decreased specific activities of UGDH and inhibited the proliferation of MCF-7 human breast cancer cells. Western blot analysis showed that gallic acid and quercetin did not affect UGDH protein expression, suggesting that UGDH activity is inhibited by polyphenols at the post-translational level. Kinetics studies using human UGDH revealed that gallic acid was a non-competitive inhibitor with respect to UDP-glucose and NAD+. In contrast, quercetin showed a competitive inhibition and a mixed-type inhibition with respect to UDP-glucose and NAD+, respectively. These results indicate that gallic acid and quercetin are effective inhibitors of UGDH that exert strong antiproliferative activity in breast cancer cells.

Soluble expression and purification of synthetic human bone morphogenetic protein-2 in Escherichia coli.

A 345-bp gene that encodes human bone morphogenetic protein-2 (hBMP-2) has been synthesized. The codon usage of the resulting gene was modified to include those triplets that are utilized in highly expressed Escherichia coli genes. The hBMP-2 gene was efficiently expressed in E. coli as a soluble and active protein. Since the recombinant hBMP-2 was readily solublized, no further solublization steps were required throughout purification. No additional tagging residues were introduced into the synthetic hBMP-2 gene product. The developed synthetic gene is a promising approach for scaling-up the soluble expression of hBMP-2.

Small-interfering-RNA-mediated silencing of human glutamate dehydrogenase induces apoptosis in neuroblastoma cells.

In the nervous system, GDH (glutamate dehydrogenase) is enriched in astrocytes and is important for recycling glutamate, a major excitatory neurotransmitter. The function of hGDH (human GDH) may be important in neurodegenerative diseases such as Parkinson's disease. To test the effect of decreased hGDH expression, several vector-based plasmidlinked hGDH siRNAs (small interfering RNAs) were expressed intracellularly in BE(2)C human neuroblastoma cells. Immunoblotting and reverse-transcription-PCR confirmed that expression of hGDH protein and mRNA was knocked down by co-transfection with phGDH-siRNA vectors in BE(2)C human neuroblastoma cells. TUNEL (terminal uridine deoxynucleotidyl transferase dUTP nick-end labelling) and DNA fragmentation assays 48 h after transfection of phGDH-siRNAs revealed that inhibition of hGDH expression induced cellular apoptosis and activated phospho-ERK1/2 (phospho-extracellular-signal-regulated kinase 1/2). These findings show that inhibition of hGDH expression by siRNA is related to apoptosis in neuronal cells.

Identification of amino acid residues responsible for different GTP preferences of human glutamate dehydrogenase isozymes.

Human glutamate dehydrogenase isozymes (hGDH1 and hGDH2) differ markedly in their inhibition by GTP. These regulatory preferences must arise from amino acid residues that are not common between hGDH isozymes. We have constructed chimeric enzymes by reciprocally switching the corresponding amino acid segments 390-465 in hGDH isozymes that are located within or near the C-terminal 48-residue antenna helix, which is thought to be part of the regulatory domain of mammalian GDHs. These resulted in triple mutations in amino acid sequences at 415, 443, and 456 sites that are not common between hGDH1 and hGDH2. The chimeric enzymes did not change their enzyme efficiency (k(cat)/K(m)) and expression level. Functional analyses, however, revealed that the chimeric mutants almost completely acquired the different GTP regulatory preference between hGDH isozymes. These results suggest that the 415, 443, and 456 residues acting in concert are responsible for the GTP inhibitory properties of hGDH isozymes.

Alteration of the quaternary structure of human UDP-glucose dehydrogenase by a double mutation.

There are conflicting views for the polymerization process of human UDP-glucose dehydrogenase (UGDH) and no clear evidence has been reported yet. Based on crystal coordinates for Streptococcus pyogenes UGDH, we made double mutant A222Q/S233G. The double mutagenesis had no effects on expression, stability, and secondary structure. Interestingly, A222Q/S233G was a dimeric form and showed an UGDH activity, although it showed increased Km values for substrates. These results suggest that Ala222 and Ser233 play an important role in maintaining the hexameric structure and the reduced binding affinities for substrates are attributable to its altered subunit communication although quaternary structure may not be critical for catalysis.

Protective effect of benzothiazole derivative KHG21834 on amyloid beta-induced neurotoxicity in PC12 cells and cortical and mesencephalic neurons.

We have investigated the effect of KHG21834, a benzothiazole derivative, on the amyloid beta protein (Abeta)-induced cell death in rat pheochromocytoma (PC12) cells and rat cortical and mesencephalic neuron-glia cultures. KHG21834 attenuated the Abeta(25-35)-induced apoptotic death in PC12 cells determined by characteristic morphological alterations and positive in situ terminal end-labeling (TUNEL). In the cortical neuron-glia cultures, KHG21834 reduced the Abeta(25-35)-induced apoptosis determined by TUNEL staining. Immunocytochemical analysis and Western blot analysis of Abeta(25-35)-induced neurotoxicity in mesencephalic neuron-glia cultures with anti-tyrosine hydroxylase (TH) antibody showed that Abeta(25-35) decreased the expression of TH protein by 60% and KHG21834 significantly attenuated the Abeta(25-35)-induced reduction in the expression of TH. Moreover, KHG21834 attenuates Abeta(25-35)-induced toxicity concomitant with the reduction of activation of extracellular signal-regulated kinase (ERK)1/2 to a lesser extent. ERK1 was more sensitively affected than ERK2 in attenuation of Abeta(25-35)-induced phosphorylation by KHG21834. These results demonstrated that KHG21834 was capable of protecting neuronal cells from Abeta(25-35)-induced degeneration.

Amino acid changes within antenna helix are responsible for different regulatory preferences of human glutamate dehydrogenase isozymes.

Human glutamate dehydrogenase (hGDH) exists in hGDH1 (housekeeping isozyme) and in hGDH2 (nerve-specific isozyme), which differ markedly in their allosteric regulation. Because they differ in only 16 of their 505 amino acids, the regulatory preferences must arise from amino acid residues that are not common between hGDH1 and hGDH2. To our knowledge none of the mutagenesis studies on the hGDH isozymes to date have identified the amino acid residues fully responsible for the different regulatory preferences between hGDH1 and hGDH2. In this study we constructed hGDH1(hGDH2(390-448))hGDH1 (amino acid segment 390-448 of hGDH1 replaced by the corresponding hGDH2 segment) and hGDH2(hGDH1(390-448))hGDH2 (amino acid segment 390-448 of hGDH2 replaced by the corresponding hGDH1 segment) by swapping the corresponding amino acid segments in hGDH1 and hGDH2. The chimeric enzymes by reciprocal swapping resulted in double mutations in amino acid sequences at 415 and 443 residues that are not common between hGDH1 and hGDH2 and are located in the C-terminal 48-residue "antenna" helix, which is thought to be part of the regulatory domain of mammalian GDHs. Functional analyses revealed that the doubly mutated chimeric enzymes almost completely acquired most of the different regulatory preferences between hGDH1 and hGDH2 for electrophoretic mobility, heat-stability, ADP activation, palmitoyl-CoA inhibition, and l-leucine activation, except for GTP inhibition. Our results indicate that substitutions of the residues in the antenna region may be important evolutionary changes that led to the adaptation of hGDH2 to the unique metabolic needs of the nerve tissue.

Expression, purification, crystallization, and preliminary X-Ray analysis of the human UDP-glucose dehydrogenase.

UDP-glucose dehydrogenase (UGDH) catalyzes the synthesis of UDP-glucuronic acid from UDP-glucose resulting in the formation of proteoglycans that are involved in promoting normal cellular growth and migration. Overproduction of proteoglycans has been implicated in the progression of certain epithelial cancers. Here, human UGDH (hUGDH) was purified and crystallized from a solution of 0.2 M ammonium sulfate, 0.1 M Na cacodylate, pH 6.5, and 21% PEG 8000. Diffraction data were collected to a resolution of 2.8 A. The crystal belongs to the orthorhombic space group P2(1)2(1)2(1) with unit-cell parameters a = 173.25, b = 191.16, c = 225.94 A, and alpha = beta = gamma = 90.0 degrees. Based on preliminary analysis of the diffraction data, we propose that the biological unit of hUGDH is a tetramer.

Calpain activation in okadaic-acid-induced neurodegeneration.

Calpain activation has been implicated in the pathogenesis of Alzheimer's disease. Okadaic acid, a protein phosphatase-2A inhibitor, has been used in Alzheimer's disease research models to increase tau phosphorylation and induce neuronal death. We previously reported that okadaic acid induced predominant activation of caspase-3 in immature neurons, but less activation in mature neurons. We found here that, in okadaic-acid-treated mature neurons, levels of an inactive form of m-calpain decreased and levels of calpain-cleaved spectrin and synapsin-I fragments increased, suggestive of calpain activation. Pretreatment with calpain inhibitor decreased lactate dehydrogenase release by 20% and increased average dendritic branch length by 50% compared with neurons treated with okadaic acid alone. These findings suggest that calpain is activated during okadaic-acid-induced neurodegeneration and calpain inhibition can be protective against it.

BACE inhibitor reduces APP-beta-C-terminal fragment accumulation in axonal swellings of okadaic acid-induced neurodegeneration.

Emerging evidence suggests that not only beta-amyloid but also other amyloid precursor protein (APP) fragments, such as the beta-C-terminal fragment (betaCTF), might be involved in Alzheimer's disease (AD). Treatment of neurons with okadaic acid (OA), a protein phosphatase-2A inhibitor, has been used to induce tau phosphorylation and neuronal death to create a research model of AD. In this study, we analyzed axonopathy and APP regulation in cultured rat neurons treated with OA. After OA treatment, the neurons presented with axonal swellings filled with vesicles, microtubule fragments, and transport molecules such as kinesin and synapsin-I. Western blotting showed that intracellular APP levels were increased and immunocytochemistry using antibodies against the APP C-terminus showed that APP accumulated in the axonal swellings. This APP C-terminus immunoreactivity disappeared when neurons were cotreated with a beta-secretase inhibitor, but not with alpha- or gamma-secretase inhibitors, indicating that the accumulation was primarily composed of APP-betaCTF. These findings provide the first evidence that APP-betaCTF can accumulate in the axons of OA-treated neurons, and may suggest that APP-betaCTF is involved in the pathogenesis of AD.

Okadaic acid induces JNK activation, bim overexpression and mitochondrial dysfunction in cultured rat cortical neurons.

Apoptosis via tau phosphorylation has been implicated in the selective neuronal losses seen in Alzheimer's disease (AD). Previous studies in vivo and in cultured neurons have shown that okadaic acid (OA) evokes tau phosphorylation to initiate a neurodegeneration that resembles the pathogenesis of AD. In an effort to identify additional key molecules in this neurodegeneration, we treated cultured rat neurons with OA and examined the apoptosis-related effects, such as changes in mitochondrial activity and expression levels of JNK, Bim, Bad, Bax and caspase-3. Western blotting revealed that phosphorylation of JNK and c-jun occurred first, followed by increased expression of Bim and subsequent caspase-3 activation in OA-treated neurons. In contrast, Bad levels decreased as early as 4 h after OA treatment. Immunocytochemistry showed that the increased phospho-JNK immunoreactivity was localized in the cytosol of degenerating neurons, while increased phospho-c-jun was localized in the nucleus. The mitochondria showed decreased membrane potential and increased swelling after OA treatment. Collectively, these data suggest that JNK- and Bim-related mitochondrial dysfunction is involved in OA-induced neurodegeneration.

Regulation of glutamate level in rat brain through activation of glutamate dehydrogenase by Corydalis ternata.

When treated with protopine and alkalized extracts of the tuber of Corydalis ternata for one year, significant decrease in glutamate level and increase in glutamate dehydrogenase (GDH) activity was observed in rat brains. The expression of GDH between the two groups remained unchanged as determined by Western and Northern blot analysis, suggesting a post-translational regulation of GDH activity in alkalized extracts treated rat brains. The stimulatory effects of alkalized extracts and protopine on the GDH activity was further examined in vitro with two types of human GDH isozymes, hGDH1 (house-keeping GDH) and hGDH2 (nerve-specific GDH). Alkalized extracts and protopine activated the human GDH isozymes up to 4.8-fold. hGDH2 (nerve- specific GDH) was more sensitively affected by 1 mM ADP than hGDH1 (house-keeping GDH) on the activation by alkalized extracts. Studies with cassette mutagenesis at ADP-binding site showed that hGDH2 was more sensitively regulated by ADP than hGDH1 on the activation by Corydalis ternata. Our results suggest that prolonged exposure to Corydalis ternata may be one of the ways to regulate glutamate concentration in brain through the activation of GDH.