Suppression of Grb2 expression improved hepatic steatosis, oxidative stress, and apoptosis induced by palmitic acid in vitro partly through insulin signaling alteration

In this study, we aimed to study the role of growth factor receptor-bound protein 2 (Grb2) in palmitic acid-induced steatosis and other “fatty liver” symptoms in vitro. HepG2 cells, with or without stably suppressed Grb2 expression, were incubated with palmitic acid for 24 h to induce typical clinical “fatty liver” features, including steatosis, impaired glucose metabolism, oxidative stress, and apoptosis. MTT and Oil Red O assays were applied to test cell viability and fat deposition, respectively. Glucose uptake assay was used to evaluate the glucose utilization of cells. Quantitative polymerase chain reaction and Western blot were used to measure expressional changes of key markers of insulin signaling, lipid/glucose metabolism, oxidative stress, and apoptosis. After 24-h palmitic acid induction, increased fat accumulation, reduced glucose uptake, impaired insulin signaling, enhanced oxidative stress, and increased apoptosis were observed in HepG2 cells. Suppression of Grb2 in HepG2 significantly reduced fat accumulation, improved glucose metabolism, ameliorated oxidative stress, and restored the activity of insulin receptor substrate-1/Akt and MEK/ERK pathways. In addition, Grb2 deficiency attenuated hepatic apoptosis shown by reduced activation of caspase-3 and fluorescent staining. Modulation of Bcl-2 and Bak1 also contributed to reduced apoptosis. In conclusion, suppression of Grb2 expression in HepG2 cells improved hepatic steatosis, glucose metabolism, oxidative stress, and apoptosis induced by palmitic acid incubation partly though modulating the insulin signaling pathway.     

Comparative studies of products obtained from solvolysis liquefaction of oil palm empty fruit bunch fibres using different solvents

Solvolysis of oil palm empty fruit bunches (EFB) fibres using different solvents (acetone, ethylene glycol (EG), ethanol, water and toluene) were carried out using an autoclave at 275°C for 60 min. The solvent efficiency in term of conversion yield was found to be: EG>water>ethanol>acetone>toluene. The liquid products and residue obtained were analyzed using Fourier transform infrared spectroscopy (FTIR) and gas chromatography/mass selectivity. The obtained results showed that the chemical properties of the oil product were significantly affected by the type of solvent used for the solvolysis process. The higher heating value (HHV) of oil products obtained using ethanol is ～29.42 MJ/kg, which is the highest among the oil products produced using different solvents. Water, ethanol and toluene yield major phenolic compounds. While EG favors the formation of alcohol compounds and acetone yields ketone and aldehyde compounds.     

Mutagenesis of D80-82 and G83 residues in West Nile Virus NS2B: Effects on NS2B-NS3 activity and viral replication

Flaviviral NS2B is a required cofactor for NS3 serine protease activity and plays an important role in promoting functional NS2B-NS3 protease configuration and maintaining critical interactions with protease catalysis substrates. The residues D⁸⁰DDG in West Nile virus (WNV) NS2B are important for protease activity. To investigate the effects of D⁸⁰DDG in NS2B on protease activity and viral replication, the negatively charged region D⁸⁰DD and the conserved residue G83 of NS2B were mutated (D⁸⁰DD/E⁸⁰EE, D⁸⁰DD/K⁸⁰KK, D⁸⁰DD/A⁸⁰AA, G83F, G83S, G83D, G83K, and G83A), and NS3 D75A was designated as the negative control. The effects of the mutations on NS2B-NS3 activity, viral translation, and viral RNA replication were analyzed using kinetic analysis of site-directed enzymes and a transient replicon assay. All substitutions resulted in significantly decreased enzyme activity and blocked RNA replication. The negative charge of D⁸⁰DD is not important for maintaining NS2B function, but side chain changes in G83 have dramatic effects on protease activity and RNA replication. These results demonstrate that NS2B is important for viral replication and that D⁸⁰DD and G83 substitutions prevent replication; they will be useful for understanding the relationship between NS2B and NS3.     

Epidermal growth factor receptor and notch pathways participate in the tumor suppressor function of gamma-secretase

Gamma-secretase, a unique aspartyl protease, is required for the regulated intramembrane proteolysis of Notch and APP, pathways that are implicated, respectively, in the pathogenesis of cancer and Alzheimer disease. However, the mechanism whereby reduction of gamma-secretase causes tumors such as squamous cell carcinoma (SCC) remains poorly understood. Here, we demonstrate that gamma-secretase functions in epithelia as a tumor suppressor in an enzyme activity-dependent manner. Notch signaling is down-regulated and epidermal growth factor receptor (EGFR) is activated in SCC caused by genetic reduction of gamma-secretase. Moreover, the level of EGFR is inversely correlated with the level of gamma-secretase in fibroblasts, suggesting that the up-regulation of EGFR stimulates hyperproliferation in epithelia of mice with genetic reduction of gamma-secretase. Supporting this notion is our finding that the proliferative response of fibroblasts lacking gamma-secretase activity is more sensitive when challenged by either EGF or an inhibitor of EGFR as ompared with wild type cells. Interestingly, the up-regulation of EGFR is independent of Notch signaling, suggesting that the EGFR pathway functions in parallel with Notch in the tumorigenesis of SCC. Collectively, our results establish a novel mechanism linking the EGFR pathway to the tumor suppressor role of gamma-secretase and that mice with genetic reduction of gamma-secretase represent an excellent rodent model for clarifying pathogenesis of SCC and for testing therapeutic strategy to ameliorate this type of human cancer.     

A dye mixture (Neurobiotin and Alexa 488) reveals extensive dye-coupling among neurons in leeches; physiology confirms the connections

Although the neuronal circuits that generate leech movements have been studied for over 30 years, the list of interneurons (INs) in these circuits remains incomplete. Previous studies showed that some motor neurons (MNs) are electrically coupled to swim-related INs, e.g., rectifying junctions connect IN 28 to MN DI-1 (dorsal inhibitor), so we searched for additional neurons in these behavioral circuits by co-injecting Neurobiotin and Alexa Fluor 488 into segmental MNs DI–1, VI–2, DE–3 and VE–4. The high molecular weight Alexa dye is confined to the injected cell, whereas the smaller Neurobiotin molecules diffuse through gap junctions to reveal electrical coupling. We found that MNs were each dye-coupled to approximately 25 neurons, about half of which are likely to be INs. We also found that (1) dye-coupling was reliably correlated with physiologically confirmed electrical connections, (2) dye-coupling is unidirectional between MNs that are linked by rectifying connections, and (3) there are novel electrical connections between excitatory and inhibitory MNs, e.g. between excitatory MN VE-4 and inhibitory MN DI-1. The INs found in this study provide a pool of novel candidate neurons for future studies of behavioral circuits, including those underlying swimming, crawling, shortening, and bending movements.     

小麦TaMlo基因的原核表达、抗体制备及细胞化学分析(简报)

白粉病菌(Blumeria graminis)是一类高度专化性的寄生真菌,可侵染650多种单子叶植物和 9000多种双子叶植物,能够引起多种麦类作物的白粉病,给农业生产带来巨大的损失。由于白粉病菌生理小种多、变异快,所以利用专化性抗病基因难以解决植物的持久抗病性问题。人们在研究大麦白粉病时．发现大麦Mlo基因的隐性突变可导致大麦对绝大多数白粉病菌生理小种的高效持久的广谱抗病性。Schulze-Lefert等多家实验室合作于1997年成功克隆了野生的 Mlo基因。进一步研究表明．该基因编码一种植物特有的具有7个跨膜区和羧基端长尾的膜蛋白(Mlo),它可能对植物细胞的坏死起负调控作用。但Mlo基因如何表达及其在白粉病菌发育中的作用机制尚不清楚。     

Targeted overexpression of inducible 6-phosphofructo-2-kinase in adipose tissue increases fat deposition but protects against diet-induced insulin resistance and inflammatory responses

Increasing evidence demonstrates the dissociation of fat deposition, the inflammatory response, and insulin resistance in the development of obesity-related metabolic diseases. As a regulatory enzyme of glycolysis, inducible 6-phosphofructo-2-kinase (iPFK2, encoded by PFKFB3) protects against diet-induced adipose tissue inflammatory response and systemic insulin resistance independently of adiposity. Using aP2-PFKFB3 transgenic (Tg) mice, we explored the ability of targeted adipocyte PFKFB3/iPFK2 overexpression to modulate diet-induced inflammatory responses and insulin resistance arising from fat deposition in both adipose and liver tissues. Compared with wild-type littermates (controls) on a high fat diet (HFD), Tg mice exhibited increased adiposity, decreased adipose inflammatory response, and improved insulin sensitivity. In a parallel pattern, HFD-fed Tg mice showed increased hepatic steatosis, decreased liver inflammatory response, and improved liver insulin sensitivity compared with controls. In both adipose and liver tissues, increased fat deposition was associated with lipid profile alterations characterized by an increase in palmitoleate. Additionally, plasma lipid profiles also displayed an increase in palmitoleate in HFD-Tg mice compared with controls. In cultured 3T3-L1 adipocytes, overexpression of PFKFB3/iPFK2 recapitulated metabolic and inflammatory changes observed in adipose tissue of Tg mice. Upon treatment with conditioned medium from iPFK2-overexpressing adipocytes, mouse primary hepatocytes displayed metabolic and inflammatory responses that were similar to those observed in livers of Tg mice. Together, these data demonstrate a unique role for PFKFB3/iPFK2 in adipocytes with regard to diet-induced inflammatory responses in both adipose and liver tissues.     

Effect of luteal-phase support on endometrial microRNA expression following controlled ovarian stimulation

ABSTRACT: BACKGROUND: Studies suggested that microRNAs influence cellular activities in the uterus including cell differentiation and embryo implantation. In assisted reproduction cycles, luteal phase support, given to improve endometrial characteristics and to facilitate the implantation process, has been a standard practice. The effect of different types of luteal phase support using steroid hormones in relation to endometrial miRNA profiles during the peri-implantation period has not seen described. This study was designed to evaluate the expression of miRNAs during the luteal phase following controlled ovarian stimulation for IVF and the influence of different luteal phase support protocols on miRNA profiles. METHODS: The study was approved by the Johns Hopkins Hospital Institutional Review Board. Endometrial biopsies were obtained on the day of oocyte retrieval from 9 oocyte donors (group I). An additional endometrial biopsy was obtained 3-5 days later (Group II) after the donors were randomized into three groups. Group IIa had no luteal-phase support, group IIb had luteal support with micronized progesterone (P), and Group IIc had luteal support with progesterone plus 17-beta-estradiol (P+E). Total RNA was isolated and microarray analysis was performed using an Illumina miRNA expression panel. RESULTS: A total of 526 miRNAs were identified. Out of those, 216 miRNAs were differentially regulated (p<0.05) between the comparison groups. As compared to the day of retrieval, 19, 11 and 6 miRNAs were differentially regulated more than 2 fold in the groups of no support, in the P support only, and in the P+E support respectively, 3-5 days after retrieval. During the peri-implantation period (3-5 days after retrieval) the expression of 33 and 6 miRNAs increased, while the expression of 3 and 0 miRNAs decreased, in the P alone and in the P+E group respectively as compared to the no steroid supplementation group. CONCLUSION: Luteal support following COS has a profound influence on miRNA profiles. Up or down regulation of miRNAs after P or P+E support suggest a role(s) of luteal support in the peri-implantation uterus in IVF cycles through the regulation of associated target genes.     

BALB/c mice produce blister-causing antibodies upon immunization with a recombinant human desmoglein 3

Pemphigus vulgaris (PV) is an Ab-mediated autoimmune blistering disease of mucotaneous surfaces. Over 95% of the patients with PV express DR4 or DRw6, and the disease is characterized by the presence of autoantibodies directed against desmoglein 3 (Dsg 3), a protein expressed on keratinocytes. An appropriate animal model is required to understand immunoregulation and to address the role of immunogenetic components in the production of pathogenic Abs that are characteristic of PV. Therefore, we turned to the development of a mouse model. Four strains of female mice (BALB/c, DBA/1, SJL/J, and HRS/J) were screened for their ability to produce pathogenic anti-Dsg 3 Abs. We demonstrated that only BALB/c mice immunized with a full-length Dsg 3 can produce pathogenic Abs capable of causing acantholysis of human foreskin in culture and blistering in neonatal mice. This observation suggested that either H-2d or the BALB background contains the immunogenetic makeup necessary for the production of pathogenic anti-Dsg 3 Abs. No correlation was noted between a given isotype and the pathogenic potential of autoantibodies from different strains of mice. Similarly, the pattern of reactivity of Abs with a panel of 46 synthetic peptides that span the entire Dsg 3 failed to reveal any association between binding specificity and the pathogenic potential, and suggested that pathogenic Abs might recognize conformational epitopes. Moreover, our studies showed that the epitopes recognized by pathogenic Abs are contained within the extracellular Dsg 3.