Key gene and protein changes in the beta-amyloid pathway following Longyanshen polysaccharides treatment in a mouse model of Alzheimer's disease

BACKGROUND： During onset and development of Alzheimer＇s disease, β-amyloid （Aβ） precursor protein （APP）, β-site amyloid precursor protein cleaving enzyme （BACE）, and β-amyloid are key genes and proteins in the Aβ pathway, and over-expression of these genes can lead to Aβ deposit/on in the brain. OBJECTIVE： To observe the influence of Longyanshen polysaccharides on expression of BACE, APP, and Aβ in the senescence-accelerated mouse prone/8 （SAMP8） brain, and to compare these effects with huperzine A treatment. DESIGN, TIME AND SETTING： A randomized, controlled, neurobiochemical experiment was performed at the Department of Pharmacology and Scientific Experimental Center of Guangxi Medical University from September 2005 to January 2008. MATERIALS： Longyanshen polysaccharfdes powder was extracted from the dried slices of the medicinal plant Longyanshen. The active component, Longyanshen polysaccharides, was provided by the Department of Pharmacology, Guangxi Medical University; huperzine A was purchased from Yuzhong Drug Manufactory, China. METHODS： Healthy SAMP8 mice were used to establish a model of Alzheimer＇s disease. A total of 50 SAMP8 mice were randomly assigned to 5 groups （n = 10）： SAMP8, huperzine A, low-, middle-, and high-dose polysaccharides. In addition, 10 senescence-accelerated mouse resistant 1 （SAMR1） mice were selected as normal controls. SAMP8 and SAMR1 mice were administered 30 mL/kg normal saline; the huperzine A group was administered 0.02 mg/kg huperzine A; the low-, middle-, and high-dose polysaccharides groups were respectively administered 45, 90, and 180 mg/kg Longyanshen polysaccharides. Each group was treated by intragastric administration, once per day, for 50 consecutive days. MAIN OUTCOME MEASURES： One hour after the final administration, immunohistochemical analysis was used to determine Aβ expression in the cortex and hippocampus of SAMP8 mice. Reverse-transcription polymerase chain reaction was used to determine mRNA levels of BACE and APP in SAMP8 brain tissue. RESULTS： Compared with the SAMR1 group, Aβ expression in the cerebral cortex and hippocampus, as well as expression of BACE, APP mRNA in the brain was significantly increased in the SAMP8 group （P 〈 0.05-0.01）. Compared with the SAMP8 group, Aβ expression, as well as BACE and APP mRNA expression, were significantly decreased in the cerebral cortex and hippocampus of huperzine A and low-, middle-, and high-dose polysaccharides groups （P 〈 0.05-0.01）. In particular, the effect of high-dose polysaccharides was the most significant （P 〈 0.05-0.01 ）. CONCLUSION： Longyanshen polysaccharides reduced or inhibited over-expression of BACE, APP, and Aβ in SAMP8 mice in a dose-dependent manner, and the effect was not worse than huperzine A.

Inhibition of beta-site amyloid precursor protein-cleaving enzyme and beta-amyloid precursor protein genes in SK-N-SH cells

BACKGROUND： Previous studies have demonstrated that Piper futokadsura stem selectively inhibits expression of amyloid precursor protein （APP） at the mRNA level. In addition, the piperlonguminine （A） and dihydropiperlonguminine （B） components （1 ： 0.8）, which can be separated from Futokadsura stem, selectively inhibit expression of the APP at mRNA and protein levels. OBJECTIVE： Based on previous findings, the present study investigated the effects of β-site amyloid precursor protein cleaving enzyme （BACE1） and APP genes on the production of β-amyloid peptide 42 （Aβ42） in human neuroblastoma cells （SK-N-SH cells） using small interfering RNAs （siRNAs） and A/B components separated from Futokadsura stem, respectively. DESIGN, TIME AND SETTING： A gene interference-based randomized, controlled, in vitro experiment was performed at the Key Laboratory of Cardiovascular Remodeling and Function Research, Ministries of Education and Public Health, and Institute of Pharmacologic Research, School of Pharmaceutical Science ＆ Department of Biochemistry, School of Medicine, Shandong University between July 2006 and December 2007. MATERIALS： SK-N-SH cells were provided by Shanghai Institutes of Biological Sciences, Chinese Academy of Sciences, Shanghai, China; mouse anti-human BACE1 monoclonal antibody was purchased from R＆D Systems, USA; mouse anti-human APP monoclonal antibody was purchased from Cell Signaling Technology, USA; and horseradish peroxidase （HRP）-conjugated goat anti-mouse IgG was provided by Sigma, USA. METHODS： The human BACE1 cDNA sequence was obtained from NCBI website （www.ncbi.nlm.nih.gov/sites/entrez）. Three pairs of siRNAs, specific to human BACE1 gene, were synthesized through the use of Silencer pre-designed siRNA specification, and were transfected into SK-N-SH cells with siPORT NeoFX transfection agent to compare the effects of different concentrations of siRNAs （10-50 nmol/L） on SK-N-SH cells. Futokadsura stem was separated and purified with chemical methods, and the crystal was composed of A/B components, with an A to B ratio of 1：0.8. The A/B （1 ： 0.8） components were added to the SK-N-SH cells at different concentrations （13.13, 6.56, and 3.28 mg/mL）. MAIN OUTCOME MEASURES： Using RT-PCR and Western blot methods, BACE1 and APP expression at mRNA and protein levels was detected in SK-N-SH cells following treatment with different siRNAs and concentrations of Futokadsura stem-separated A/B components, respectively. Altered Aβ42 secretion by SK-N-SH cells was determined by ELISA. RESULTS： BACE1 mRNA and protein levels were significantly suppressed by 40 and 50 nmol/L siRNAs at 48 hours post-transfection. A/B components （1 ： 0.8）, which were separated from Futokadsura stem, selectively inhibited mRNA and protein expression of APP in SK-N-SH cells. Aβ42 secretion by SK-N-SH cells was significantly decreased following treatment with siRNAs or A/B components. CONCLUSION： Inhibition of BACE1 and APP genes by various materials and methods efficiently decreased production of Aβ42.

Effects of soil salinity on rhizosphere soil microbes in transgenic Bt cotton fields

With increased cultivation of transgenic Bacillus thuringiensis （Bt） cotton in the saline alkaline soil of China, assessments of transgenic crop biosafety have focused on the effects of soil salinity on rhizosphere microbes and Bt protein residues. In 2013 and 2014, investigations were conducted on the rhizosphere microbial biomass, soil enzyme activities and Bt protein contents of the soil under transgenic Bt cotton （variety GK19） and its parental non-transgenic cotton （Simian 3） cultivated at various salinity levels （1.15, 6.00 and 11.46 dS m-1）. Under soil salinity stress, trace amounts of Bt proteins were ob- served in the Bt cotton GK19 rhizosphere soil, although the protein content increased with cotton growth and increased soil salinity levels. The populations of slight halophilic bacteria, phosphate solubilizing bacteria, ammonifying bacteria, nitrifying bacteria and denitrifying bacteria decreased with increased soil salinity in the Bt and non-Bt cotton rhizosphere soil, and the microbial biomass carbon, microbial respiration and soil catalase, urease and alkaline phosphatase activity also decreased. Correlation analyses showed that the increased Bt protein content in the Bt cotton rhizosphere soil may have been caused by the slower decomposition of soil microorganisms, which suggests that salinity was the main factor influencing the relevant activities of the soil microorganisms and indicates that Bt proteins had no clear adverse effects on the soil microorganisms. The results of this study may provide a theoretical basis for risk assessments of genetically modified cotton in saline alkaline soil.

Association between heat stress and oxidative stress in poultry;mitochondrial dysfunction and dietary interventions with phytochemicals

Heat as a stressor of poultry has been studied extensively for many decades; it affects poultry production on a worldwide basis and has significant impact on well-being and production. More recently, the involvement of heat stress in inducing oxidative stress has received much interest. Oxidative stress is defined as the presence of reactive species in excess of the available antioxidant capacity of animal cells. Reactive species can modify several biologically cellular macromolecules and can interfere with cell signaling pathways. Furthermore, during the last decade, there has been an ever-increasing interest in the use of a wide array of natural feed-delivered phytochemicals that have potential antioxidant properties for poultry. In light of this, the current review aims to（1） summarize the mechanisms through which heat stress triggers excessive superoxide radical production in the mitochondrion and progresses into oxidative stress,（2） illustrate that this pathophysiology is dependent on the intensity and duration of heat stress,（3） present different nutritional strategies for mitigation of mitochondrial dysfunction, with particular focus on antioxidant phytochemicals.Oxidative stress that occurs with heat exposure can be manifest in all parts of the body; however, mitochondrial dysfunction underlies oxidative stress. In the initial phase of acute heat stress, mitochondrial substrate oxidation and electron transport chain activity are increased resulting in excessive superoxide production. During the later stage of acute heat stress, down-regulation of avian uncoupling protein worsens the oxidative stress situation causing mitochondrial dysfunction and tissue damage. Typically, antioxidant enzyme activities are upregulated. Chronic heat stress, however, leads to downsizing of mitochondrial metabolic oxidative capacity, up-regulation of avian uncoupling protein, a clear alteration in the pattern of antioxidant enzyme activities, and depletion of antioxidant reserves.Some phytochemicals, such as various types of flavonoids and related compounds, were shown to be beneficial in chronic heat-stressed poultry, but were less or not effective in non-heat-stressed counterparts. This supports the contention that antioxidant phytochemicals have potential under challenging conditions. Though substantial progress has been made in our understanding of the association between heat stress and oxidative stress, the means by which phytochemicals can alleviate oxidative stress have been sparsely explored.

BACE1 in the retina:a sensitive biomarker for monitoring early pathological changes in Alzheimer's disease

Because of a lack of sensitive biomarkers,the diagnosis of Alzheimer＇s disease（AD） cannot be made prior to symptom manifestation.Therefore,it is crucial to identify novel biomarkers for the presymptomatic diagnosis of AD.While brain lesions are a major feature of AD,retinal pathological changes also occur in patients.In this study,we investigated the temporal changes in β-site APP-cleaving enzyme 1（BACE1） expression in the retina and brain to determine whether it could serve as a suitable biomarker for early monitoring of AD.APP/PS-1 transgenic mice,3,6 and 8 months of age,were used as an experimental group,and age-matched C57/BL6 wild-type mice served as the control group.In the Morris water maze test,there were no significant differences in escape latency or in the number of crossings in the target area among mice of different ages.Compared with wild-type mice,no changes in learning or memory abilities were detected in transgenic mice at 3 months of age.However,compared with wild-type mice,the escape latency was significantly increased in transgenic mice at 6 months,starting on day 3,and at 8 months,starting on day 2,during Morris water maze training.In addition,the number of crossings of the target area was significantly decreased in transgenic mice.The learning and memory abilities of transgenic mice were further worsened at 8 months of age.Immunohistochemical staining revealed no BACE1 plaques in wild-type mice at 3,6 or 8 months or in transgenic mice at 3 months,but they were clearly found in the entorhinal cortex,hippocampus and prefrontal cortex of transgenic mice at 6 and 8 months.BACE1 expression was not detected in the retina of wild-type mice at 3 months,but weak BACE1 expression was detected in the ganglion cell layer,inner plexiform layer and outer plexiform layer at 6 and 8 months.In transgenic mice,BACE1 expression in the ganglion cell layer was increased at 3 months,and BACE1 expression in the ganglion cell layer,inner plexiform layer and outer plexiform layer was significantly increased at 6 and 8 months,compared with age-matched wild-type mice.Taken together,these results indicate that changes in BACE1 expression appear earlier in the retina than in the brain and precede behavioral deficits.Our findings suggest that abnormal expression of BACE1 in the retina is an early pathological change in APP/PS-1 transgenic mice,and that BACE1 might have potential as a biomarker for the early diagnosis of AD in humans.

Antibody-Like Phosphorylation Sites in Focus of Statistically Based Bilingual Approach

In accordance with previous reports, the sequences related to phosporylated protein segments occur in conserved variable domains of immunoglobulins including first of all certain N-terminally located segments. Consequently, we look here for the sequences 1) composing human and mouse proteins different from antigen receptors, 2) identical with or highly similar to nucleotide sequence representatives of conserved variable immunoglobulin segments and 3) identical with or closely related to phosphorylation sites. More precisely, we searched for the corresponding actual pairs of DNA and protein sequence segments using five-step bilingual approach employing among others a) different types of BLAST searches, b) two in-principle-different machine-learning methods predicting phosphorylated sites and c) two large databases recording existing phosphorylation sites. The approach identified seven existing phosphorylation sites and thirty-seven related human and mouse segments achieving limits for several predictions or phylogenic parameters. Mostly serines phosporylated with ataxia-telangiectasia-related kinase (involved in regulation of DNA-double-strand-break repair) were indicated or predicted in this study. Hypermutation motifs, located in effective positions of the selected sequence segments, occurred significantly less frequently in transcribed than non-transcribed DNA strands suggesting thus the incidence of mutation events. In addition, marked differences between the numbers and proportions of human and mouse cancer-related sequence items were found in different steps of selection process. The possible role of hypermutation changes within the selected segments and the observed structural relationships are discussed here with respect to DNA damage, carcinogenesis, cancer vaccination, ageing and evolution. Taken together, our data represent additional and sometimes perhaps complementary information to the existing databases of empirically proven phosphorylation sites or pathogenically important spots.

Effect of angiotensin converting enzyme inhibitor on the calcium transients and calcium handling proteins in ventricular myocytes from rats with heart failure

Background Chronic heart failure (CHF) is associated with calcium transients and calcium handling proteins. Angiotensin converting enzyme (ACE) inhibitor has been demonstrated to have beneficial effect on CHF. Yet studies addressed to the relationship between ACE inhibitor and calcium transients in CHF are rare. The aim of this study was to investigate the influence of ACE inhibitor (perindopril) on the contractility and calcium transients and calcium handling proteins in ventricular myocytes from rats with experimental heart failure.Methods Male Wistar rats were randomized to heart failure group treated with perindopril (CHF-T, 3 mg·kg -1 ·d -1 ), heart failure group without treatment (CHF-C) and sham-operated group (PS). Heart failure was induced by abdominal aortic constriction. All groups were further followed up for 12 weeks. Left ventricular myocytes were then isolated. Single cell shortening fraction and [Ca 2+ ]_i were simultaneously measured by laser scanning confocal microscope under the field stimulation (1.0 Hz). Reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blot were performed to evaluate the changes of mRNA and protein of Na +-Ca 2+ exchanger (NCX_1), sarcoplasmic reticulum Ca 2+ -ATPase (SERCA_2) and phospholamban (PLB).Results The fraction of cell shortening (FS%) and [Ca 2+ ]_ imax (nmol/L) were significantly reduced in group CHF-C compared with group PS (FS%: 7.51±1.15 vs 13.21±1.49;[Ca 2+ ]_ imax :330.85±50.05 vs 498.16±14.07; both P <0.01), and restored at least partially in CHF-T group. In CHF-C group, the left ventricular mRNA of NCX_1 and PLB were significantly upregulated in comparing with PS group (R_ NCX1/β-Actin : 0.51±0.12 vs 0.19±0.06, P <0.01; R_ PLB/β-Actin : 0.26±0.12 vs 0.20±0.08, P <0.05), while SERCA_2 mRNA was downregulated (0.48±0.10 vs 0.80±0.11, P <0.01). The mRNA levels of NCX_1 and SERCA_2 in CHF-T group were between the CHF-C and PS group, and the differences of the latter two groups were significant (all P <0.05). In CHF-C and CHF-T groups, the protein expression of NCX_1 were 1.141±0.047 and 1.074±0.081 times of that in PS group respectively (both P <0.05), and SERCA_2 protein levels were 0.803±0.100 and 0.893±0.084 times of that in PS group respectively (both P <0.05). The protein expression of NCX_1 and SERCA_2 in the CHF-C and CHF-T groups is significantly different (both P <0.05).ConclusionACE inhibitor could improve cardiac function of failing heart through directly enhancing the contractility of single cardiomyocyte, and these effects are probably mediated by its roles in preventing the deleterious changes of calcium transients and calcium handling proteins in CHF.

Nanocapsules of oxalate oxidase for hyperoxaluria treatment

Enzyme therapeutics have great potential for the treatment of systemic disorders such as urolithiasis and nephrocalcinosis, which are caused by the excessive accumulation of oxalate. However, exogenous enzymes have short half-lives in vivo and elicit high immunogenicity, which largely limit the therapeutic outcomes. Herein, we report a delivery strategy whereby therapeutic enzymes are encapsulated within a thin zwitterionic polymer shell to form enzyme nanocapsules. The strategy is exemplified by the encapsulation of oxalate oxidase （OxO） for the treatment of hyperoxaluria, because as-synthesized OxO nanocapsules have a prolonged blood circulation half-life and elicit reduced immunogenicity. Our design of enzyme nanocapsules that enable the systemic delivery of therapeutic enzymes can be extended to various biomedical applications.