Proteomic analysis on insulin signaling in human hematopoietic cells: identification of CLIC1 and SRp20 as novel downstream effectors of insulin

Insulin/IGF-I-dependent signals play important roles for the regulation of proliferation, differentiation, metabolism, and autophagy in various cells, including hematopoietic cells. Although the early protein kinase activation cascade has been intensively studied, the whole picture of intracellular signaling events has not yet been clarified. To identify novel downstream effectors of insulin-dependent signals in relatively early phases, we performed high-resolution two-dimensional electrophoresis (2-DE)-based proteomic analysis using human hematopoietic cells 1 h after insulin stimulation. We identified SRp20, a splicing factor, and CLIC1, an intracellular chloride ion channel, as novel downstream effectors besides previously reported effectors of Rho-guanine nucleotide dissociation inhibitor 2 and glutathione S-transferase-pi. Reduction in SRp20 was confirmed by one-dimensional Western blotting. Moreover, MG-132, a proteasome inhibitor, prevented this reduction. By contrast, upregulation of CLIC1 was not observed in one-dimensional Western blotting, unlike the 2-DE results. As hydrophilic proteins were predominantly recovered in 2-DE, the discrepancy between the 1-DE and 2-DE results may indicate a certain qualitative change of the protein. Indeed, the nuclear localization pattern of CLIC1 was remarkably changed by insulin stimulation. Thus insulin induces the proteasome-dependent degradation of SRp20 as well as the subnuclear relocalization of CLIC1.     

Human pluripotent stem cells:Towards therapeutic development for the treatment of lifestyle diseases

There are two types of human pluripotent stem cells: Embryonic stem cells(ESCs) and induced pluripotent stem cells(iPSCs),both of which launched themselves on clinical trials after having taken measures to overcome problems: Blocking rejections by immunosuppressants regarding ESCs and minimizing the risk of tumorigenicity by depleting exogenous gene components regarding iP SCs.It is generally assumed that clinical applications of human pluripotent stem cells should be limited to those cases where there are no alternative measures for treatments because of the risk in transplanting those cells to living bodies.Regarding lifestyle diseases,we have already several therapeutic options,and thus,development of human pluripotent stem cell-based therapeutics tends to be avoided.Nevertheless,human pluripotent stem cells can contribute to the development of new therapeutics in this field.As we will show,there is a case where only a short-term presence of human pluripotent stem-derived cells can exert long-term therapeutic effects even after they are rejected.In those cases,immunologically rejections of ESC-or allogenic iP SC-derived cells may produce beneficial outcomes by nullifying the risk of tumorigenesis without deterioration of therapeutic effects.Another utility of human pluripotent stem cells is the provision of an innovative tool for drug discovery that are otherwise unavailable.For example,clinical specimens of human classical brown adipocytes(BAs),which has been attracting a great deal of attention as a new target of drug discovery for the treatment of metabolic disorders,are unobtainable from living individuals due to scarcity,fragility and ethical problems.However,BA can easily be produced from human pluripotent stem cells.In this review,we will contemplate potential contribution of human pluripotent stem cells to therapeutic development for lifestyle diseases.     

A Guyon's canal ganglion presenting as occupational overuse syndrome: A case report

We report a case of severe weight loss secondary to anorexia nervosa causing bilateral superficial peroneal nerve entrapment in a young female patient who was treated successfully by bilateral surgical decompression.     

High Expression Level of Tra2-β1 Is Responsible for Increased SMN2 Exon 7 Inclusion in the Testis of SMA Mice

Spinal muscular atrophy (SMA) is an inherited neuromuscular disease caused by deletion or mutation of SMN1 gene. All SMA patients carry a nearly identical SMN2 gene, which produces low level of SMN protein due to mRNA exon 7 exclusion. Previously, we found that the testis of SMA mice (smn^−/− SMN2) expresses high level of SMN2 full-length mRNA, indicating a testis-specific mechanism for SMN2 exon 7 inclusion. To elucidate the underlying mechanism, we established primary cultures of testis cells from SMA mice and analyzed them for SMN2 exon 7 splicing. We found that primary testis cells after a 2-hour culture still expressed high level of SMN2 full-length mRNA, but the level decreased after longer cultures. We then compared the protein levels of relevant splicing factors, and found that the level of Tra2-β1 also decreased during testis cell culture, correlated with SMN2 full-length mRNA downregulation. In addition, the testis of SMA mice expressed the highest level of Tra2-β1 among the many tissues examined. Furthermore, overexpression of Tra2-β1, but not ASF/SF2, increased SMN2 minigene exon 7 inclusion in primary testis cells and spinal cord neurons, whereas knockdown of Tra2-β1 decreased SMN2 exon 7 inclusion in primary testis cells of SMA mice. Therefore, our results indicate that high expression level of Tra2-β1 is responsible for increased SMN2 exon 7 inclusion in the testis of SMA mice. This study also suggests that the expression level of Tra2-β1 may be a modifying factor of SMA disease and a potential target for SMA treatment.     

Transport genes and chemotaxis in Laribacter hongkongensis: a genome-wide analysis

Background

Laribacter hongkongensis is a Gram-negative, sea gull-shaped rod associated with community-acquired gastroenteritis. The bacterium has been found in diverse freshwater environments including fish, frogs and drinking water reservoirs. Using the complete genome sequence data of L. hongkongensis, we performed a comprehensive analysis of putative transport-related genes and genes related to chemotaxis, motility and quorum sensing, which may help the bacterium adapt to the changing environments and combat harmful substances.

Results

A genome-wide analysis using Transport Classification Database TCDB, similarity and keyword searches revealed the presence of a large diversity of transporters (n = 457) and genes related to chemotaxis (n = 52) and flagellar biosynthesis (n = 40) in the L. hongkongensis genome. The transporters included those from all seven major transporter categories, which may allow the uptake of essential nutrients or ions, and extrusion of metabolic end products and hazardous substances. L. hongkongensis is unique among closely related members of Neisseriaceae family in possessing higher number of proteins related to transport of ammonium, urea and dicarboxylate, which may reflect the importance of nitrogen and dicarboxylate metabolism in this assacharolytic bacterium. Structural modeling of two C⁴-dicarboxylate transporters showed that they possessed similar structures to the determined structures of other DctP-TRAP transporters, with one having an unusual disulfide bond. Diverse mechanisms for iron transport, including hemin transporters for iron acquisition from host proteins, were also identified. In addition to the chemotaxis and flagella-related genes, the L. hongkongensis genome also contained two copies of qseB/qseC homologues of the AI-3 quorum sensing system.

Conclusions

The large number of diverse transporters and genes involved in chemotaxis, motility and quorum sensing suggested that the bacterium may utilize a complex system to adapt to different environments. Structural modeling will provide useful insights on the transporters in L. hongkongensis.     

Technology platform development for targeted plasma metabolites in human heart failure

Background

Heart failure is a multifactorial disease associated with staggeringly high morbidity and motility. Recently, alterations of multiple metabolites have been implicated in heart failure; however, the lack of an effective technology platform to assess these metabolites has limited our understanding on how they contribute to this disease phenotype. We have successfully developed a new workflow combining specific sample preparation with tandem mass spectrometry that enables us to extract most of the targeted metabolites. 19 metabolites were chosen ascribing to their biological relevance to heart failure, including extracellular matrix remodeling, inflammation, insulin resistance, renal dysfunction, and cardioprotection against ischemic injury.

Results

In this report, we systematically engineered, optimized and refined a protocol applicable to human plasma samples; this study contributes to the methodology development with respect to deproteinization, incubation, reconstitution, and detection with mass spectrometry. The deproteinization step was optimized with 20% methanol/ethanol at a plasma:solvent ratio of 1:3. Subsequently, an incubation step was implemented which remarkably enhanced the metabolite signals and the number of metabolite peaks detected by mass spectrometry in both positive and negative modes. With respect to the step of reconstitution, 0.1% formic acid was designated as the reconstitution solvent vs. 6.5 mM ammonium bicarbonate, based on the comparable number of metabolite peaks detected in both solvents, and yet the signal detected in the former was higher. By adapting this finalized protocol, we were able to retrieve 13 out of 19 targeted metabolites from human plasma.

Conclusions

We have successfully devised a simple albeit effective workflow for the targeted plasma metabolites relevant to human heart failure. This will be employed in tandem with high throughput liquid chromatography mass spectrometry platform to validate and characterize these potential metabolic biomarkers for diagnostic and therapeutic development of heart failure patients.     

福建淡水枝角类及其区系

本文记录了福建淡水枝角类7科25属71种,其中25种为福建新纪录.文章分析了福建淡水枝角类的区系特点,并与邻近地区进行了比较.     

Stimulation and priming of human neutrophils by granulocyte colony-stimulating factor and granulocyte-macrophage colony-stimulating factor: qualitative and quantitative differences.

Granulocyte colony-stimulating factor(G-CSF) and granulocyte-macrophage colony-stimulating factor (GM-CSF) increased neutrophil C3bi-receptor expression and adherence and rapidly (less than 10 min) primed neutrophils to enhanced O2- release and membrane depolarization stimulated by chemotactic peptide. Direct triggering of O2- release in suspended neutrophils was also provoked by GM-CSF but not by G-CSF. GM-CSF-induced O2- release was inhibited by cyclic AMP agonists and cytochalasin B. The biological activity was greater in non-glycosylated GM-CSF than in glycosylated GM-CSF, whereas it was identical in glycosylated and non-glycosylated G-CSFs. Direct stimulation and priming by GM-CSF were consistently greater than those by G-CSF and the combined addition of the optimal concentrations of G-CSF and GM-CSF resulted in the effects of GM-CSF alone. These findings indicate that the effects of G-CSF and GM-CSF on neutrophil functions are qualitatively and quantitatively different from each other.     

A feeder-free hematopoietic differentiation system with generation of functional neutrophils from feeder- and cytokine-free primate embryonic stem cells

We have established a novel feeder- and recombinant cytokine-free culture system for the maintenance of primate embryonic stem (ES) cells along with a feeder-free hematopoietic differentiation protocol for high efficiency CD45-positive cell production. In our system, cynomolgus monkey ES cells were properly maintained in an undifferentiated state with high immature marker expressions and teratoma-producing activities. Embryoid bodies (EBs) were generated in the presence of serum and cytokine cocktail and subjected to attachment culture on gelatin-coated plates. After about 2 weeks, a sac-like structure filled with abundant round cells emerged at the center of flattened EB. Then total cells were collected and transferred onto new gelatin-coated plates, where cells were firmly attached and actively proliferated to confluence. After another few days culture, abundant floating cells were detected in the culture supernatant. These cells expressed high levels of CD45 (>90%), while adherent cells expressed low levels of CD45 (<10%). The former consisted of various differentiated stages of myeloid cells from immature myeloblasts to mature polymorphonuclear neutrophils and macrophages. Although the percentages of neutrophils varied between 10 to 20 depending on experiments, their mature phenotype was reproducibly confirmed by specific staining and functional assays. Our protocol provides the minimum essence for primate ES cell maintenance and hematopoietic differentiation that is beneficial from economical and clinical points of view.