Inhibiting β-secretase activity in Alzheimer's disease cell models with single-chain antibodies specifically targeting APP

The Amyloid-β (Aβ) peptide is produced from the amyloid precursor protein (APP) by sequential proteolytic cleavage of APP first by β-secretase and then by γ-secretase. β-Site APP cleaving enzyme-1 (BACE-1) is the predominant enzyme involved in β-secretase processing of APP and is a primary therapeutic target for treatment of Alzheimer's disease. While inhibiting BACE-1 activity has obvious therapeutic advantages, BACE-1 also cleaves numerous other substrates with important physiological activity. Thus, blanket inhibition of BACE-1 function may have adverse side effects. We isolated a single chain variable fragment (scFv) from a human-based scFv yeast display library that selectively inhibits BACE-1 activity toward APP by binding the APP substrate at the proteolytic site. We selected the iBSEC1 scFv, since it recognizes the BACE-1 cleavage site on APP but does not bind the adjacent highly antigenic N-terminal of Aβ, and thus it will target APP but not soluble Aβ. When added to 7PA2 cells, a mammalian cell line that overexpresses APP, the iBSEC1 scFv binds APP on the cell surface, reduces toxicity induced by APP overexpression, and reduces both intracellular and extracellular Aβ levels by around 50%. Since the iBSEC1 scFv does not contain the antibody F_c region, this construct does not pose the risk of exacerbating inflammation in the brain as faced with full-length monoclonal antibodies for potential therapeutic applications.     

Interferon-β-armed oncolytic adenovirus induces both apoptosis and necroptosis in cancer cells

Interferon-β (IFN-β) has been widely used in cancer therapy, but the clinical trial results are generally disappointing. Our previous studies have shown that an oncolytic adenovirus carrying IFN-β (ZD55-IFN-β) exhibits significant anti-tumor activities. However, the underlying mechanisms are not clear. Here we showed that ZD55-IFN-β infection-induced S-phase cell cycle arrest in a p53-dependent manner by activating the ataxia telangiectasia mutated-dependent DNA damage pathway. In addition, ZD55-IFN-β infection could initiate both caspase-dependent apoptosis and necroptosis in cancer cells. More importantly, ZD55-IFN-β showed a synergistic effect on cancer cells when combined with doxorubicin. These results suggest that the combination of ZD55-IFN-β with doxorubicin may represent a promising clinical strategy in cancer therapy.     

Exogenous seeding of cerebral β-amyloid deposition in βAPP-transgenic rats

Deposition of the amyloid-β (Aβ) peptide in senile plaques and cerebral Aβ angiopathy (CAA) can be stimulated in Aβ-precursor protein (APP)-transgenic mice by the intracerebral injection of dilute brain extracts containing aggregated Aβ seeds. Growing evidence implicates a prion-like mechanism of corruptive protein templating in this phenomenon, in which aggregated Aβ itself is the seed. Unlike prion disease, which can be induced de novo in animals that are unlikely to spontaneously develop the disease, previous experiments with Aβ seeding have employed animal models that, as they age, eventually will generate Aβ lesions in the absence of seeding. In the present study, we first established that a transgenic rat model expressing human APP (APP21 line) does not manifest endogenous deposits of Aβ within the course of its median lifespan (30?months). Next, we injected 3-month-old APP21 rats intrahippocampally with dilute Alzheimer brain extracts containing aggregated Aβ. After a 9-month incubation period, these rats had developed senile plaques and CAA in the injected hippocampus, whereas control rats remained free of such lesions. These findings underscore the co-dependence of agent and host in governing seeded protein aggregation, and show that cerebral Aβ-amyloidosis can be induced even in animals that are relatively refractory to the spontaneous origination of parenchymal and vascular deposits of Aβ.     

Dynorphin-(1–13) induces spontaneous feeding in rats

Dynorphin-(1–13), a recently isolated opioid peptide stimulates food ingestion in rats after intracerebroventricular administration at doses of 1 and 10 μg. The latency until food ingestion is 22.4 ± 1.9 min. The ability of dynorphin-(1–13) to initiate food ingestion is antagonized by the opiate antagonist, naloxone. The food ingestion is accompanied by excessive grooming behavior.     

Double diastereoselection explains limitations in synthesizing mannose-containing β-(1,3)-glucans

It is known that 3-O-glycosylation of glucosidic acceptors bearing acyl groups in the 4 and 6 positions instead of a 4,6-O-benzylidene ring mainly affords α-glycosides. Described here is an unexpected stereochemical outcome for elongation at glucose O-3 of a β-d-Glcp-(1→3)-α-d-Manp disaccharide using peracetylated ethyl thioglucoside as a donor. This unexpected reaction was correlated with match-mismatch effects, as shown by efficient coupling of the same acceptor by a donor of l-configuration.     

Cimicifuga racemosa impairs fatty acid β-oxidation and induces oxidative stress in livers of ovariectomized rats with renovascular hypertension

The aim of this work was to evaluate the effects of therapeutic doses of Cimicifuga racemosa on cardiovascular parameters and on liver lipid metabolism and redox status in an animal model of estrogen deficiency associated with hypertension, a condition that could make the liver more vulnerable to drug-induced injuries. Female Wistar rats were subjected to the surgical procedures of bilateral ovariectomy (OVX) and induction of renovascular hypertension (two-kidneys, one-clip; 2K1C). These animals (OVX + 2K1C) were treated with daily doses of a C. racemosa extract, using a dose that is similar to that recommended to postmenopausal women (0.6mg/kg), over a period of 15 days. The results were compared to those of untreated OVX + 2K1C, OVX, and control rats. The treatment with C. racemosa caused a significant reduction in blood pressure. In the liver, treatment did not prevent the development of steatosis, and it reduced the mitochondrial and peroxisomal capacity to oxidize octanoyl-CoA compared to the untreated animals. In addition, C. racemosa caused numerous undesirable effects on the liver redox status: it increased the mitochondrial reactive oxygen species generation, an event that was not accompanied by an increase in the activity of superoxide dismutase, and it induced a decrease in peroxisomal catalase activity. Although the reduced glutathione content had not been affected, a phenomenon that probably reflected the restoration of glucose-6-phosphate dehydrogenase activity by C. racemosa, oxidative damage was evidenced by the elevated level of thiobarbituric acid-reactive substances found in the liver of treated animals.     

Increase in Thermostability of Recombinant Barley β-Amylase by Random Mutagenesis

Random mutations were introduced into recombinant barley β-amylase by modified PCR to increase its thermostability. Two clones were obtained. One was found to have a change of Ser-351 to Pro and another, a change of Ala-376 to Ser, and 2.3°C and 1.0°C increases, respectively, in the thermostabilities compared with that of native recombinant β-amylase.     

12α-Hydroxylated bile acid induces hepatic steatosis with dysbiosis in rats

There is an increasing need to explore the mechanism of the progression of non-alcoholic fatty liver disease. Steroid metabolism is closely linked to hepatic steatosis and steroids are excreted as bile acids (BAs). Here, we demonstrated that feeding WKAH/HkmSlc inbred rats a diet supplemented with cholic acid (CA) at 0.5 g/kg for 13 weeks induced simple steatosis without obesity. Liver triglyceride and cholesterol levels were increased accompanied by mild elevation of aminotransferase activities. There were no signs of inflammation, insulin resistance, oxidative stress, or fibrosis. CA supplementation increased levels of CA and taurocholic acid (TCA) in enterohepatic circulation and deoxycholic acid (DCA) levels in cecum with an increased ratio of 12α-hydroxylated BAs to non-12α-hydroxylated BAs. Analyses of hepatic gene expression revealed no apparent feedback control of BA and cholesterol biosynthesis. CA feeding induced dysbiosis in cecal microbiota with enrichment of DCA producers, which underlines the increased cecal DCA levels. The mechanism of steatosis was increased expression of Srebp1 (positive regulator of liver lipogenesis) through activation of the liver X receptor by increased oxysterols in the CA-fed rats, especially 4β-hydroxycholesterol (4βOH) formed by upregulated expression of hepatic Cyp3a2, responsible for 4βOH formation. Multiple regression analyses identified portal TCA and cecal DCA as positive predictors for liver 4βOH levels. The possible mechanisms linking these predictors and upregulated expression of Cyp3a2 are discussed. Overall, our observations highlight the role of 12α-hydroxylated BAs in triggering liver lipogenesis and allow us to explore the mechanisms of hepatic steatosis onset, focusing on cholesterol and BA metabolism.     

NO donor induces Nec-1-inhibitable, but RIP1-independent, necrotic cell death in pancreatic β-cells

Nitric oxide (NO) has been implicated in pancreatic β-cell death in the development of diabetes. The mechanisms underlying NO-induced β-cell death have not been clearly defined. Recently, receptor-interacting protein-1 (RIP1)-dependent necrosis, which is inhibited by necrostatin-1, an inhibitor of RIP1, has emerged as a form of regulated necrosis. Here, we show that NO donor-induced β-cell death was inhibited by necrostatin-1. Unexpectedly, however, RIP1 knockdown neither inhibited cell death nor altered the protective effects of necrostatin-1 in NO donor-treated β-cells. These results indicate that NO donor induces necrostatin-1-inhibitable necrotic β-cell death independent of RIP1. Our findings raise the possibility that NO-mediated β-cell necrosis may be a novel form of signal-regulated necrosis, which play a role in the progression of diabetes.     

Detection of SHV β-lactamases in Gram-negative bacilli using fluorescein-labeled antibodies

Background  

β-lactam resistance in Gram-negative bacteria is a significant clinical problem in the community, long-term care facilities, and hospitals. In these organisms, β-lactam resistance most commonly results from the production of β-lactamases. In Gram-negative bacilli, TEM-, SHV-, and CTX-M-type β-lactamases predominate. Therefore, new and accurate detection methods for these β-lactamase producing isolates are needed.     

Expression in Escherichia coli of cDNA Encoding Barley β-Amylase and Properties of Recombinant β-Amylase

To express the cloned β-amylase cDNA in Escherichia coli under control of the tac promoter, a plasmid pBETA92 was constructed. The plasmid consisted of 6312 bp. An extract of E. coli JM109 harboring pBETA92 had β-amylase activity that produced β-maltose from soluble starch. The enzyme production started in the logarithmic phase, increased linearly, and reached a maximum after 12 h. The recombinant barley β-amylase gave two major (pI 5.43 and 5.63) and four minor (pI 5.20, 5.36, 5.80, and 6.13) activity bands on isoelectric focusing, and their pIs didn’t change throughout the incubation. But Western blot analysis found that one β-amylase having a molecular weight of about 56,000 was synthesized. The recombinant β-amylase was purified from the cells by consecutive column chromatography. The purified enzyme gave a single band of protein on SDS–PAGE but showed heterogeneity on isoelectric focusing. The N-terminal amino acid sequence showed that the recombinant β-amylase lacked four amino acids at positions 2–5 (Glu-Val-Asn-Val) when compared with the presumed amino acid sequence of barley β-amylase. Therefore, the recombiant β-amylase consisted of 531 amino acids, and its molecular weight was calculated to be 59,169. The N-terminal amino acid sequence of the recombinant β-amylase and the nucleotide sequence of the junction position in plasmid pBETA92 indicated that GTG (Val-5 in the case of barley β-amylase) at positions 27–29 from the SD sequence (AGGA) was the translation initiation codon. The properties of the recombinant β-amylase were almost the same as those of barley β-amylase except for the pI and the K_m values for maltohexaose and maltoheptaose. The pI of recombiant barley β-amylase calculated by Genetyx Version 9 based on the presumed amino acid sequence was 5.60, but the real pIs were 5.20–6.13. Therefore, some post-translational reaction(s) might happen after protein synthesis in E. coli cells, and this modification might cause the differences in the pI and the K_m values for maltohexaose and maltoheptaose between the barley and the recombinant β-amylases.     

Impairment of mitochondrial β-oxidation in rats under cold-hypoxic environment

Mitochondrial ß-oxidation of fatty acid provides a major source of energy in mammals. High altitude (HA), characterized by hypobaric hypoxia and low ambient temperatures, causes alteration in metabolic homeostasis. Several studies have depicted that hypoxic exposure in small mammals causes hypothermia due to hypometabolic state. Moreover, cold exposure along with hypoxia reduces hypoxia tolerance in animals. The present study investigated the rate of β-oxidation and key enzymes, carnitine palmitoyl transferase-I (CPT-I) and hydroxyacyl CoA dehydrogenase (HAD), in rats exposed to cold-hypobaric hypoxic environment. Male Sprague Dawley rats (190–220 g) were randomly divided into eight groups (n?=?6 rats in each group): 1 day hypoxia (H1); 7  days hypoxia (H7); 1 day cold (C1); 7 days cold (C7); 1 day cold-hypoxia (CH1); 7 days cold-hypoxia (CH7) exposed; and unexposed control for 1 and 7 days (UC1 and UC7). After exposure, animals were anaesthetized with ketamine (50 mg/kg body weight) and xylazine (10 mg/kg body weight) intraperitonialy and sacrificed. Mitochondrial CPT-I, HAD, ¹⁴C-palmitate oxidation in gastrocnemius muscle and liver, and plasma leptin were measured. Mitochondrial CPT-I was significantly reduced in muscle and liver in CH1 and CH7 as compared to respective controls. HAD activity was significantly reduced in H1 and CH7, and in H1, H7, CH1, and CH7 as compared to unexposed controls in muscle and liver, respectively. A concomitant decrease in ¹⁴C-palmitate oxidation was found. Significant reduction in plasma leptin in hypoxia and cold-hypoxia suggested hypometabolic state. It can be concluded that ß-oxidation of fatty acids is reduced in rats exposed to cold-hypoxic environment due to the persisting hypometabolic state in cold-hypoxia exposure.     

Age-related up-regulation of β3-adrenergic receptor in heart-failure rats

Stimulation of β1- and β2-adrenergic receptors (ARs) in the heart results in positive inotropy. In contrast, it has been reported that the β3-AR is also expressed in the heart and that its stimulation leads to negative inotropic effects. The aim of this study was to investigate the expression of β3-AR in age-related heart-failure rats and its relevance to left ventricular dysfunction. Aging male Wistar rats were divided into young and aging groups according to age, and each group included sham-operation and heart-failure subgroups. Left ventricular end-diastolic pressure (LVEDP) and the ratio of left ventricular weight to body weight (LV/BW) were significantly higher for the aging heart-failure versus young heart-failure and the heart-failure versus sham-operation groups (P?<?0.01, respectively). However, the left ventricular end-systolic pressure (LVESP) and the maximal rate of rise or fall of left ventricular pressure were all significantly lower for the aging heart-failure versus young heart-failure and the heart-failure versus sham-operation groups (P?<?0.01, respectively). β3-AR protein levels increased significantly when heart failure worsened in aging rats. These results suggest that β3-AR expression in age-related heart-failure rats and left ventricular function were highly correlated.     

Changes of red cell phospholipids in β-thalassemia minor

Lipid and phospholipid concentrations were determined in the red cells of β-thalassemia minor patients. A reduced concentration of total lipids and total phospholipids in red cells was found. Phosphatidylcholine and sphingomyelin were increased while phosphatidylethanolamine, phosphatidylserine, and phosphatidylinositol concentrations were decreased. These changes are similar to, though less pronounced, than those seen in β-thalassemia major. The relation of these lipids changes, free hemoglobin chains which damage the red cell membrane, and the increased rate of hemolysis is unclear.     

Production of anti-amyloid β antibodies in mice fed rice expressing amyloid β

The main signs of Alzheimer's disease (AD) are cognitive impairment and senile plaques composed of amyloid beta (Aβ) observed in patients' brains. Therefore, therapy for AD focuses on the removal of Aβ. We developed an "edible vaccine" that employs intestinal immunity with little to no side effects. Rice was utilized as an edible vaccine. It expressed GFP-Aβ42. Aβ rice was administered orally to wild-type (WT) mice causing production of anti-Aβ antibodies. Since Aβ rice was mixed with the cholera toxin B subunit (CTB), antibody against the rice seed protein was also produced. Then, mice were caused to develop immune tolerance against the rice seed protein by oral administration of Aβ rice mixed with CTB. The results indicated that only anti-Aβ antibodies were produced.     

Constitutive activation of β-catenin in odontoblasts induces aberrant pulp calcification in mouse incisors

Journal of Molecular Histology - During dentin formation, odontoblast polarization ensures that odontoblasts directionally secrete dentin matrix protein, leading to tubular dentin formation;...     

Overexpression of phytosulfokine-α induces male sterility and cell growth by regulating cell wall development in Arabidopsis

Key message

Over-production of functional PSK-α in Arabidopsis caused increases in both plant cell growth and biomass and induced male sterility by regulating cell wall development.

Abstract

Phytosulfokine-α (PSK-α) is a novel disulfated pentapeptide hormone that is involved in promoting plant cell growth. Although a role for PSK-α in stimulating protoplast expansion has been suggested, how PSK-α regulates cell growth in planta remains poorly understood. In this study, we found that overexpression of the normal PSK-α precursor gene AtPSK4, which resulted in high levels of PSK-α, caused longer roots and larger leaves with enlarged cells. As expected, these changes were not observed in transgenic plants overexpressing mutated AtPSK4, which generated unsulfated PSK-α. These findings confirmed the role of PSK-α in promoting plant cell growth. Furthermore, we found that overexpressing AtPSK4, but not mutated AtPSK4, induced a phenotype of male sterility that resulted from the failure of fibrous cell wall development in the endothecium. In addition, overexpressing AtPSK4 enhanced expression of a number of genes encoding expansins, which are involved in cell wall loosening. Accordingly, in addition to its role in cell growth, we propose a novel function for PSK-α signaling in the modulation of plant male sterility via regulation of cell wall development.

     

α2 and β-adrenergic stimulation of corticosterone secretion in rats

Bilateral injection of 6-hydroxydopamine into the medial forebrain bundle (MFB) significantly decreased monoamine concentrations in the hypothalamus. The noradrenaline and serotonin content of the paraventricular nucleus (PVN) was also significantly reduced. These drastic decreases in neurotransmitter concentration did not alter basal secretion of corticosterone. Isoproterenol. a -adrenoceptor agonist (1 mg/kg, i.p.), significantly stimulated corticosterone release in saline and MFB lesioned rats. This stimulation did not differ significantly between the two groups. Clonidine, an ₂-adreceptor agonist, injected either intraperitoneally or intracerebrally just dorsal to the PVN, caused a dose-dependent increase in corticosterone secretion. The stimulation of corticosterone release by clonidine (250 g/kg, i.p.) was antagonised by the selective ₂-adreceptors antagonist, yohimbine (1 mg/kg, i.p.) and significantly reduced by the MFB lesion. These results suggest that corticosterone secretion is stimulated by activation of ₂-adreceptors which occur on noradrenergic nerve terminals in the PVN.     

Soybean β-conglycinin improves carbohydrate and lipid metabolism in Wistar rats

The effects of dietary soybean β-conglycinin on lipid metabolism and energy consumption were studied in Wistar adult rats. Rats were fed, a diet containing casein (control group) or β-conglycinin (β-conglycinin group), for 4 weeks. Carbohydrate consumption was higher and fat consumption was lower in the β-conglycinin group than in the control group, whereas the total energy consumption was the same between the two groups. Serum adiponectin was higher in the β-conglycinin group than in the control group. Serum triacylglycerol levels in the β-conglycinin group were significantly lower than those in the control group. The secretion rate of triacylglycerols from the liver after the administration of tyloxapol, an inhibitor of lipolysis, was significantly lower in the β-conglycinin group than in the control group. These results suggest the possibility that β-conglycinin exerts hypolipidemic effects through an acceleration in carbohydrate consumption associated with an increase in adiponectin in rats.