Direct Targeting of Proteins from the Cytosol to Organelles: The ER versus Endosymbiotic Organelles

In eukaryotic cells consisting of many different types of organelles, targeting of organellar proteins is one of the most fundamental cellular processes. Proteins belonging to the endoplasmic reticulum (ER), chloroplasts and mitochondria are targeted individually from the cytosol to their cognate organelles. As the targeting to these organelles occurs in the cytosol during or after translation, the most crucial aspect is how specific targeting to these three organelles can be achieved without interfering with other targeting pathways. For these organelles, multiple mechanisms are used for targeting proteins, but the exact mechanism used depends on the type of protein and organelle, the location of targeting signals in the protein and the location of the protein in the organelle. In this review, we discuss the various mechanisms involved in protein targeting to the ER, chloroplasts and mitochondria, and how the targeting specificity is determined for these organelles in plant cells .     

The role of disulfide bond in hyperthermophilic endocellulase

The hyperthermophilic endocellulase, EGPh (glycosyl hydrolase family 5) from Pyrococcus horikoshii possesses 4 cysteine residues forming 2 disulfide bonds, as identified by structural analysis. One of the disulfide bonds is located at the proximal region of the active site in EGPh, which exhibits a distinct pattern from that of the thermophilic endocellulase EGAc (glycosyl hydrolase family 5) of Acidothermus cellulolyticus despite the structural similarity between the two endocellulases. The structural similarity between EGPh and EGAc suggests that EGPh possesses a structure suitable for changing the position of the disulfide bond corresponding to that in EGAc. Introduction of this alternative disulfide bond in EGPh, while removing the original disulfide bond, did not result in a loss of enzymatic activity but the EGPh was no longer hyperthermostable. These results suggest that the contribution of disulfide bond to hyperthermostability at temperature higher than 100 °C is restrictive, and that its impact is dependent on the specific structural environment of the hyperthermophilic proteins. The data suggest that the structural position and environment of the disulfide bond has a greater effect on high-temperature thermostability of the enzyme than on the potential energy of the dihedral angle that contributes to disulfide bond cleavage.     

A pilot study of autologous CD34-depleted bone marrow mononuclear cell transplantation via the hepatic artery in five patients with liver failure

Background aimsMany rodent experiments and human studies on stem cell therapy have shown promising therapeutic approaches to liver diseases. We investigated the clinical outcomes of five patients with liver failure of various causes who received autologous CD34-depleted bone marrow-derived mononuclear cell (BM-MNC) transplantation, including mesenchymal stromal cells, through the hepatic artery.MethodsCD34-depleted BM-MNCs were obtained from five patients waiting for liver transplantation by bone marrow aspiration and using the CliniMACS CD34 Reagent System (Miltenyi Biotech, Bergisch Gladbach, Germany), and autologous hepatic artery infusion was performed. The causes of hepatic decompensation were hepatitis B virus (HBV), hepatitis C virus (HCV), propylthiouracil-induced toxic hepatitis and Wilson disease.ResultsSerum albumin levels improved 1 week after transplantation from 2.8 g/dL, 2.4 g/dL, 2.7 g/dL and 1.9 g/dL to 3.3 g/dL, 3.1 g/dL, 2.8 g/dL and 2.6 g/dL. Transient liver elastography data showed some change from 65 kPa, 33 kPa, 34.8 kPa and undetectable to 46.4 kPa, 19.8 kPa, 29.1 kPa and 67.8 kPa at 4 weeks after transplantation in a patient with Wilson disease, a patient with HCV, and two patients with HBV. Ascites decreased in two patients. One of the patients with HBV underwent liver transplantation 4 months after the infusion, and the hepatic progenitor markers (cytokeratin [CD]-7, CD-8, CD-9, CD-18, CD-19, c-Kit and epithelial cell adhesion molecule [EpCAM]) were highly expressed in the explanted liver.ConclusionsSerum albumin levels, liver stiffness, liver volume, subjective healthiness and quality of life improved in the study patients. Although these findings were observed in a small population, the results may suggest a promising future for autologous CD34-depleted BM-MNC transplantation as a bridge to liver transplantation in patients with liver failure.     

Acute exacerbations in patients with idiopathic pulmonary fibrosis

Idiopathic pulmonary fibrosis (IPF) is a chronic, fibrosing interstitial lung disease that primarily affects older adults. Median survival after diagnosis is 2–3 years. The clinical course of IPF may include periods of acute deterioration in respiratory function, which are termed acute exacerbations of IPF (AEx-IPF) when a cause cannot be identified. AEx-IPF may represent a sudden acceleration of the underlying disease process of IPF, or a biologically distinct pathological process that is clinically undiagnosed. An AEx-IPF can occur at any time during the course of IPF and may be the presenting manifestation. The incidence of AEx-IPF is hard to establish due to variation in the methodology used to assess AEx-IPF in different studies, but AEx-IPF are believed to occur in between 5 and 10% of patients with IPF every year. Risk factors for AEx-IPF are unclear, but there is evidence that poorer lung function increases the risk of an AEx-IPF and reduces the chances of a patient surviving an AEx-IPF. The presence of comorbidities such as gastroesophageal reflux disease (GERD) and pulmonary hypertension may also increase the risk of an AEx-IPF. AEx-IPF are associated with high morbidity and mortality. Patients who experience an AEx-IPF show a worsened prognosis and AEx-IPF are believed to reflect disease progression in IPF. Current treatments for AEx-IPF have only limited data to support their effectiveness. The latest international treatment guidelines state that supportive care remains the mainstay of treatment for AEx-IPF, but also give a weak recommendation for the treatment of the majority of patients with AEx-IPF with corticosteroids. There is emerging evidence from clinical trials of investigational therapies that chronic treatment of IPF may reduce the incidence of AEx-IPF. Additional clinical trials investigating this are underway.     

Therapeutic angiogenesis of three-dimensionally cultured adipose-derived stem cells in rat infarcted hearts

Background aimsTo successfully treat myocardial infarction (MI), blood must be resupplied to the ischemic myocardium by inducing angiogenesis. Many studies report enhanced angiogenesis using stem cells; however, the therapeutic efficacy of cell transplant remains low because transplanted cells may not survive, be retained at the site of transplant, or develop into vascular tissue. In this study, we assessed the therapeutic potential of three-dimensional cell masses (3DCM) composed of human adipose-derived stem cells (hASC) in a rat MI model.MethodsFor formation of 3DCM, hASC were cultured on a substrate with immobilized fibroblast growth factor 2. The morphology and phenotypes of 3DCM were analyzed 1 day after culture. The cells (hASC and 3DCM, 5 × 10⁵ cells) were injected into ischemic regions after ligation of the left coronary artery (n = 6 in each group). Cell retention ratio, therapeutic efficacy and vascularization were evaluated 4 weeks after transplant.ResultsA spheroid-type 3DCM, which included vascular cells (CD34⁺/CD31⁺/KDR⁺/α-SMA⁺) with high production of human vascular endothelial growth factor, was obtained. Infarct size and cardiomyocyte apoptosis were reduced in the 3DCM-injected group compared with the hASC-injected group. The retention ratio of hASC was 14-fold higher in the 3DCM-injected group. Many transplanted cells differentiated into endothelial and smooth muscle cells and formed vascular networks incorporated into host vessels.ConclusionsTransplant of 3DCM may be useful for angiogenic cell therapy to treat MI.     

Virmid: accurate detection of somatic mutations with sample impurity inference

Detection of somatic variation using sequence from disease-control matched data sets is a critical first step. In many cases including cancer, however, it is hard to isolate pure disease tissue, and the impurity hinders accurate mutation analysis by disrupting overall allele frequencies. Here, we propose a new method, Virmid, that explicitly determines the level of impurity in the sample, and uses it for improved detection of somatic variation. Extensive tests on simulated and real sequencing data from breast cancer and hemimegalencephaly demonstrate the power of our model. A software implementation of our method is available at http://sourceforge.net/projects/virmid/.     

Cyclin-dependent kinase 4 signaling acts as a molecular switch between syngenic differentiation and neural transdifferentiation in human mesenchymal stem cells

Multipotent mesenchymal stem/stromal cells (MSCs) are capable of differentiating into a variety of cell types from different germ layers. However, the molecular and biochemical mechanisms underlying the transdifferentiation of MSCs into specific cell types still need to be elucidated. In this study, we unexpectedly found that treatment of human adipose- and bone marrow-derived MSCs with cyclin-dependent kinase (CDK) inhibitor, in particular CDK4 inhibitor, selectively led to transdifferentiation into neural cells with a high frequency. Specifically, targeted inhibition of CDK4 expression using recombinant adenovial shRNA induced the neural transdifferentiation of human MSCs. However, the inhibition of CDK4 activity attenuated the syngenic differentiation of human adipose-derived MSCs. Importantly, the forced regulation of CDK4 activity showed reciprocal reversibility between neural differentiation and dedifferentiation of human MSCs. Together, these results provide novel molecular evidence underlying the neural transdifferentiation of human MSCs; in addition, CDK4 signaling appears to act as a molecular switch from syngenic differentiation to neural transdifferentiation of human MSCs.     

Proteomic analysis of kidneys from selenoprotein M transgenic rats in response to increased bioability of selenium

Background

To characterize changes in global protein expression in kidneys of transgenic rats overexpressing human selenoprotein M (SelM) in response to increased bioabivility of selenium (Sel), total proteins extracted from kidneys of 10-week-old CMV/hSelM Tg and wild-type rats were separated by 2-dimensional gel electrophoresis and measured for changes in expression.

Results

Ten and three proteins showing high antioxidant enzymatic activity were up- and down-regulated, respectively, in SelM-overexpressing CMV/hSelM Tg rats compared to controls based on an arbitrary 2-fold difference. Up-regulated proteins included LAP3, BAIAP2L1, CRP2, CD73 antigen, PDGF D, KIAA143 homolog, PRPPS-AP2, ZFP313, HSP-60, and N-WASP, whereas down-regulated proteins included ALKDH3, rMCP-3, and STC-1. After Sel treatment, five of the up-regulated proteins were significantly increased in expression in wild-type rats, whereas there were no changes in CMV/hSelM Tg rats. Only two of the down-regulated proteins showed reduced expression in wild-type and Tg rats after Sel treatment.

Conclusions

These results show the primary novel biological evidences that new functional protein groups and individual proteins in kidneys of Tg rats relate to Sel biology including the response to Sel treatment and SelM expression.