Coupling of proadipocyte growth arrest and differentiation. II. A cell cycle model for the physiological control of cell proliferation

Experimental evidence is presented that supports a cell cycle model showing that there are five distinct biological processes involved in proadipocyte differentiation. These include: (a) growth arrest at a distinct state in the G1 phase of the cell cycle; (b) nonterminal differentiation; (c) terminal differentiation; (d) loss of the differentiated phenotype; and (e) reinitiation of cell proliferation. Each of these events is shown to be regulated by specific human plasma components or other physiological factors. At two states designated GD and GD', coupling of growth arrest and differentiation is shown to occur. We propose that these mechanisms for the coupling of growth arrest and differentiation are physiologically significant and mimic the regulatory processes that control stem cell proliferation in vivo.     

Spectrophotometric determination of bromopyruvate by reaction with 2-nitro-5-thiobenzoic acid

A spectrophotometric method for determination of bromopyruvate, based on the reaction with 2-nitro-5-thiobenzoic acid, is described. The reaction is complete in 30 min at room temperature in 0.1 m Tris-MES, 1 mm EDTA, pH 8.0. The method is sensitive to at least 1 × 10^?5m bromopyruvate. Reagents are stable, easy to prepare, and specific for β-halopyruvate. Bromopyruvate solutions must be prepared fresh daily. Solutions of bromopyruvate at pH 8.0 and 23°C have a half-life of 3 hr.     

花尾榛鸡冬季活动区及社群行为

无线电遥测结果表明,长白山冬季花尾榛鸡的月活动区大小为22.5～6.52hm~2。11月至1月,随着天气变冷,花尾榛鸡的活动区面积明显减小(P<0.001)。冬季花尾榛鸡的日活动范围很小,平均466±127m~2。整个冬季花尾榛鸡的活动中心区出现阶段性改变。花角榛鸡对其活动区有一定的依赖性。花尾榛鸡冬季不存在明显的领域,出现集群行为,这与其栖息地食物丰富与抵御天敌有关。花尾榛鸡集群的组织结构是松散的,缺乏义务性,集群中的个体关系有亲疏,存在2只或2只以上个体组成的小组,同组个体之间的距离大多数情况在150～200m以内的联系范围内。推测花尾榛鸡集群时的活动区面积增大。     

Possible anti-obesity therapeutics from nature – A review

Obesity is associated with many diseases, particularly diabetes, hypertension, osteoarthritis, and heart disease. The obesity incidence has increased at an alarming rate in recent years, becoming a worldwide health problem, with incalculable social costs. Two different obesity-treatment drugs are currently on the market: orlistat, which reduces intestinal fat absorption via inhibiting pancreatic lipase; and sibutramine, an anorectic or appetite suppressant. Both drugs have hazardous side-effects, including increased blood pressure, dry mouth, constipation, headache, and insomnia. For this reason, a wide variety of natural materials have been explored for their obesity treatment potential. These are mainly complex products having several components with different chemical and pharmacological features. This review aimed to survey the literature covering natural products with anti-obesity activity and to review the scientific data, including experimental methodologies, active components, and mechanisms of action against obesity.     

Spectrum structures and biological functions of 8-mers in the human genome

The spectra of k-mer frequencies can reveal the structures and evolution of genome sequences. We confirmed that the trimodal spectrum of 8-mers in human genome sequences is distinguished only by CG2, CG1 and CG0 8-mer sets, containing 2,1 or 0 CpG, respectively. This phenomenon is called independent selection law. The three types of CG 8-mers were considered as different functional elements. We conjectured that (1) nucleosome binding motifs are mainly characterized by CG1 8-mers and (2) the core structural units of CpG island sequences are predominantly characterized by CG2 8-mers. To validate our conjectures, nucleosome occupied sequences and CGI sequences were extracted, then the sequence parameters were constructed through the information of the three CG 8-mer sets respectively. ROC analysis showed that CG1 8-mers are more preference in nucleosome occupied segments (AUC > 0.7) and CG2 8-mers are more preference in CGI sequences (AUC > 0.99). This validates our conjecture in principle.     

Bacterial Artificial Chromosomes: A Functional Genomics Tool for the Study of Positive-strand RNA Viruses

Reverse genetics, an approach to rescue infectious virus entirely from a cloned cDNA, has revolutionized the field of positive-strand RNA viruses, whose genomes have the same polarity as cellular mRNA. The cDNA-based reverse genetics system is a seminal method that enables direct manipulation of the viral genomic RNA, thereby generating recombinant viruses for molecular and genetic studies of both viral RNA elements and gene products in viral replication and pathogenesis. It also provides a valuable platform that allows the development of genetically defined vaccines and viral vectors for the delivery of foreign genes. For many positive-strand RNA viruses such as Japanese encephalitis virus (JEV), however, the cloned cDNAs are unstable, posing a major obstacle to the construction and propagation of the functional cDNA. Here, the present report describes the strategic considerations in creating and amplifying a genetically stable full-length infectious JEV cDNA as a bacterial artificial chromosome (BAC) using the following general experimental procedures: viral RNA isolation, cDNA synthesis, cDNA subcloning and modification, assembly of a full-length cDNA, cDNA linearization, in vitro RNA synthesis, and virus recovery. This protocol provides a general methodology applicable to cloning full-length cDNA for a range of positive-strand RNA viruses, particularly those with a genome of >10 kb in length, into a BAC vector, from which infectious RNAs can be transcribed in vitro with a bacteriophage RNA polymerase.     

A Novel Electrochemically Active and Fe(III)-reducing Bacterium Phylogenetically Related to Clostridium butyricum Isolated from a Microbial Fuel Cell

An obligatory anaerobic bacterium was isolated from a mediator-less microbial fuel cell using starch processing wastewater as the fuel and designated as EG3. The isolate was Gram-positive, motile and rod (2.8–3.0 μm long, 0.5–0.6 μm wide). The partial 16S rRNA gene sequence and analysis of the cellular fatty acids profile suggested that EG3 clusters with Clostridium sub-phylum and exhibited the highest similarity (98%) with Clostridium butyricum. The temperature and pH optimum for growth were 37°C and 7.0, respectively. The major products of glucose and glucose/Fe(O)OH metabolism were lactate, formate, butyrate, acetate, CO₂and H₂. Growth was faster at the initial phase and the cell yield was higher when the medium was supplemented with Fe(O)OH than without Fe(O)OH. These results suggest that Fe(III) ion is utilised as an electron sink. Cyclic voltammetry showed that Clostridium butyricum EG3 cells were electrochemically active. It is a novel characteristic of strict anaerobic Gram-positive bacteria.     

Slit2 Inactivates GSK3β to Signal Neurite Outgrowth Inhibition

Slit molecules comprise one of the four canonical families of axon guidance cues that steer the growth cone in the developing nervous system. Apart from their role in axon pathfinding, emerging lines of evidence suggest that a wide range of cellular processes are regulated by Slit, ranging from branch formation and fasciculation during neurite outgrowth to tumor progression and to angiogenesis. However, the molecular and cellular mechanisms downstream of Slit remain largely unknown, in part, because of a lack of a readily manipulatable system that produces easily identifiable traits in response to Slit. The present study demonstrates the feasibility of using the cell line CAD as an assay system to dissect the signaling pathways triggered by Slit. Here, we show that CAD cells express receptors for Slit (Robo1 and Robo2) and that CAD cells respond to nanomolar concentrations of Slit2 by markedly decelerating the rate of process extension. Using this system, we reveal that Slit2 inactivates GSK3β and that inhibition of GSK3β is required for Slit2 to inhibit process outgrowth. Furthermore, we show that Slit2 induces GSK3β phosphorylation and inhibits neurite outgrowth in adult dorsal root ganglion neurons, validating Slit2 signaling in primary neurons. Given that CAD cells can be conveniently manipulated using standard molecular biological methods and that the process extension phenotype regulated by Slit2 can be readily traced and quantified, the use of a cell line CAD will facilitate the identification of downstream effectors and elucidation of signaling cascade triggered by Slit.     

Neurogranin Regulates Alcohol Sensitivity through AKT Pathway in the Nucleus Accumbens

Dysfunction of glutamate neurotransmission in the nucleus accumbens (NAc) has been implicated in the pathophysiology of alcohol use disorders (AUD). Neurogranin (Ng) is exclusively expressed in the brain and mediates N‐methyl‐d ‐aspartate receptor (NMDAR) hypo‐function by regulating the intracellular calcium‐calmodulin (Ca²⁺‐CaM) pathway. Ng null mice (Ng^–/– mice) demonstrate increased alcohol drinking compared to wild‐type mice, while also showing less tolerance to the effect of alcohol. To identify the molecular mechanism related to alcohol seeking, both in vivo microdialysis and label‐free quantification proteomics comparing Ng genotype and effects of alcohol treatment on the NAc are utilized. There is significant difference in glutamate and gamma‐aminobutyric acid (GABA) neurotransmission between genotypes; however, alcohol administration normalizes both glutamate and GABA levels in the NAc. Using label‐free proteomics, 427 protein expression changes are identified against alcohol treatment in the NAc among 4347 total proteins detected. Bioinformatics analyses reveal significant molecular differences in Ng null mice in response to acute alcohol treatment. Ingenuity pathway analysis found that the AKT network is altered significantly between genotypes, which may increase the sensitivity of alcohol in Ng null mice. The pharmacoproteomics results presented here illustrate a possible molecular basis of the alcohol sensitivity through Ng signaling in the NAc.     

Classification of the Gut Microbiota of Patients in Intensive Care Units During Development of Sepsis and Septic Shock

The gut microbiota of intensive care unit (ICU) patients displays extreme dysbiosis associated with increased susceptibility to organ failure, sepsis, and septic shock. However, such dysbiosis is difficult to characterize owing to the high dimensional complexity of the gut microbiota. We tested whether the concept of enterotype can be applied to the gut microbiota of ICU patients to describe the dysbiosis. We collected 131 fecal samples from 64 ICU patients diagnosed with sepsis or septic shock and performed 16S rRNA gene sequencing to dissect their gut microbiota compositions. During the development of sepsis or septic shock and during various medical treatments, the ICU patients always exhibited two dysbiotic microbiota patterns, or ICU-enterotypes, which could not be explained by host properties such as age, sex, and body mass index, or external stressors such as infection site and antibiotic use. ICU-enterotype I (ICU E1) comprised predominantly Bacteroides and an unclassified genus of Enterobacteriaceae, while ICU-enterotype II (ICU E2) comprised predominantly Enterococcus. Among more critically ill patients with Acute Physiology and Chronic Health Evaluation II (APACHE II) scores > 18, septic shock was more likely to occur with ICU E1 (P = 0.041). Additionally, ICU E1 was correlated with high serum lactate levels (P = 0.007). Therefore, different patterns of dysbiosis were correlated with different clinical outcomes, suggesting that ICU-enterotypes should be diagnosed as independent clinical indices. Thus, the microbial-based human index classifier we propose is precise and effective for timely monitoring of ICU-enterotypes of individual patients. This work is a first step toward precision medicine for septic patients based on their gut microbiota profiles.