In vitro trans-differentiation of rat mesenchymal cells into insulin-producing cells by rat pancreatic extract

Recent reports have suggested that mesenchymal cells derived from bone marrow may differentiate into not only mesenchymal lineage cells but also other lineage cells. There is possibility for insulin-producing cells (IPCs) to be differentiated from mesenchymal cells. We used self-functional repair stimuli of stem cells by partial injury. Rat pancreatic extract (RPE) from the regenerating pancreas (2 days after 60% pancreatectomy) was treated to rat mesenchymal cells. After the treatment of RPE, they made clusters like islet of Langerhans within a week and expressed four pancreatic endocrine hormones; insulin, glucagon, pancreatic polypeptide, and somatostatin. Moreover, IPCs released insulin in response to normal glucose challenge. Here we demonstrate that the treatment of RPE can differentiate rat mesenchymal cells into IPCs which can be a potential source for the therapy of diabetes.     

Proteomic profiling of brain cortex tissues in a Tau transgenic mouse model of Alzheimer’s disease

Alzheimer’s disease (AD) involves regionalized neuronal death, synaptic loss, and an accumulation of intracellular neurofibrillary tangles and extracellular senile plaques. Although there have been numerous studies on tau proteins and AD in various stages of neurodegenerative disease pathology, the relationship between tau and AD is not yet fully understood. A transgenic mouse model expressing neuron-specific enolase (NSE)-controlled human wild-type tau (NSE-htau23), which displays some of the typical Alzheimer-associated pathological features, was used to analyze the brain proteome associated with tau tangle deposition. Two-dimensional electrophoresis was performed to compare the cortex proteins of transgenic mice (6- and 12-month-old) with those of control mice. Differentially expressed spots in different stages of AD were identified with ESI-Q-TOF (electrospray ionization quadruple time-of-flight) mass spectrometry and liquid chromatography/tandem mass spectrometry. Among the identified proteins, glutathione S-transferase P 1 (GSTP1) and carbonic anhydrase II (CAII) were down-regulated with the progression of AD, and secerin-1 (SCRN1) and V-type proton ATPase subunit E 1 (ATP6VE1) were up-regulated only in the early stages, and down-regulated in the later stages of AD. The proteins, which were further confirmed by RT-PCR at the mRNA level and with western blotting at the protein level, are expected to be good candidates as drug targets for AD. The study of up- and down-regulation of proteins during the progression of AD helps to explain the mechanisms associated with neuronal degeneration in AD.     

Enhanced transfection rates of small-interfering RNA using dioleylglutamide-based magnetic lipoplexes

Magnetic force-guided delivery (magnetofection) has been studied as a new modality for introducing small-interfering RNA (siRNA) into target cells, but its delivery efficiency needs to be improved. Here, we report that magnetofection of N,N'-dioleylglutamide (DG)-based magnetic lipoplexes can substantially enhance the cellular delivery rates of siRNA. The siRNA was triply complexed with DG-based cationic liposomes and cationic iron-oxide nanoparticles. The formation of siRNA-containing magnetic lipoplexes was confirmed by gel retardation, sizes, and zeta potential values. Fluorescence microscopy and flow cytometry of fluorescent marker-labeled siRNA revealed that the DG-based magnetic lipoplexes conferred a higher cellular delivery rate of siRNA than DG-based lipoplexes or Lipofectamine 2000. In addition to the enhanced delivery of siRNA, the DG-based magnetic lipoplexes showed lack of cytotoxicity. We then tested the application of these magnetic lipoplexes for the cellular delivery of anticancer siRNA. Cancer cell lines magnetofected with DG-based magnetic lipoplexes containing Mcl1-specific siRNA (siMcl1) showed much lower viability than the groups treated with DG-based lipoplexes or Lipofectamine 2000, indicating that our magnetofection strategy conferred greater siMcl1-induced anticancer activity. These results suggest that DG-based magnetic lipoplexes are promising candidates for enhancing the efficiency of magnetic field-guided siRNA delivery.     

Crystallization of antiangiogenic Kringle V derived from human apolipoprotein A: crystallization applied to purification and formulation

In this study, the Kringle V domain (Glu4225-Ser4310) of human apolipoprotein A, an antiangiogenic polypeptide, was expressed as a secreted form in Pichia pastoris, and was purified via a process consisting of three chromatographic steps. The chromatographically purified kringle V domain contained a C-terminal serine-deleted form and several high-molecular-weight forms, which were suspected to represent glycosylated derivatives. In order to remove these derivatives, we employed a crystallization process. The crystallization of kringle V resulted in an 85% recovery yield, and also resulted in the complete removal of the aforementioned high-molecular-weight forms. However, we were still able to detect a trace of the C-terminal serine-deleted form. The prepared Kringle V crystals were stable within a pH range of 7.0 to 8.0, and were completely dissolved by dilution, which is a crucial factor in the preparation of a highly concentrated formulation. The chromatogram of the crystallized kringle V on reversed-phase HPLC analysis was identical to that observed without crystallization. Also, we noted that the original anti-wound migration activities of the molecule toward human umbilical vein endothelial cells were completely retained.     

Sample preparation method for plasma membrane proteome analysis

The preparation of plasma membrane (PM) proteome samples is seriously difficult and time-consuming, owing to their profound hydrophobicity and low abundance. We have developed an efficient PM sample preparation method using Ultracentrifugation with Percoll and an aqueous two-phase extraction. The developed method was rapid (3h) and provided high purities (26-fold of cell lysate) with a high yield (2.6% of whole cell lysate proteins). This method is especially useful for PM proteome studies using 2D gel electrophoresis.     

Differentially expressed proteins of mesenchymal stem cells derived from human cord blood (hUCB) during osteogenic differentiation

Multipotent mesenchymal stem cells (MSCs) derived from human umbilical cord blood (hUCB) represent promising candidates for the development of future cellular therapy strategies. MSCs have been found to be able to differentiate into various tissues. One of the primary limitations in our understanding of the biology of human MSCs is the absence of prospective markers required for the monitoring of lineage-specific differentiation. hUCB-derived MSCs have been found to have significantly greater osteogenic potential. In this study, we focused on proteins that were differentially expressed during osteogenic differentiation of hUCB-MSCs. And we analyzed the protein expression inherent to osteogenic differentiation by two-dimensional gel electrophoresis, ESI-Q-TOF, and Western blotting. Eleven differentially expressed spots were observed between the two groups (before and after differentiation) on the 2-DE map. These might also be proved as useful cytosolic biomarker proteins for osteogenesis, and might be employed in quality control of osteoblasts in cell-therapy applications.     

Optimization of culture condition for the production of D-amino acid oxidase in a recombinant Escherichia coli

The gene encoding D-amino acid oxidase (DAAO) from Trigonopsis variabilis CBS 4095 has been cloned and expressed in Escherichia coli BL21 (DE3). Unfortunately, it was observed that the host cell was negatively affected by the expressed DAAO, resulting in a remarkable decrease in cell growth. To overcome this problem, we investigated several factors that affect cell growth rate and DAAO production such as addition time of inducer and dissolved oxygen (DO) concentration. The addition time of lactose, which was used as an inducer, and DO concentration appeared to be critical for the cell growth of E. coli BL21 (DE3)/pET-DAAO. A two-stage DO control strategy was developed, in which the DO concentration was controlled above 50% until specific stage of bacterial growth (OD₆₀₀ 30–40) and then downshifted to 30% by changing the agitation speed and aeration rate, and they remained at these rates until the end of fermentation. With this strategy, the maximum DAAO activity and cell growth reached 18.5 U/mL and OD₆₀₀ 81, respectively. By reproducing these optimized conditions in a 12-m³ fermentor, we were able to produce DAAO at a productivity of 19 U/mL with a cell growth of OD₆₀₀ 80.     

Protein biomarkers in the plasma of workers occupationally exposed to polycyclic aromatic hydrocarbons

Polycyclic aromatic hydrocarbons (PAHs) form a chemical family containing several hundred compounds, including benzo[a]pyrene and pyrene. They are usually produced by the incomplete burning of coal, oil, gas, garbage, or other organic substances like tobacco or charbroiled meat. Exposure to PAH causes tumors, primarily in the lung, the bladder, and the skin. To investigate the differentially expressed proteins resultant from PAH exposure, the protein expression in human plasma was analyzed using two-dimensional electrophoresis (2-DE). The plasma exposed to PAH was obtained from 48 waste gas pollution measurers working at an automobile emission inspection center. The 1-hydroxypryene (1-OHP) level, which is the urinary PAH metabolite used for evaluation of PAH exposure, was 0.28 micromol/mol creatinine in PAH exposure groups, and 0.078 micromol/mol creatinine in unexposed groups (control, n = 33). A protein upregulated by PAH (putative capacitative calcium entry channel) and five overexpressed proteins (two fibrinogen gamma-A chain precursors, a hemopexin precursor, an albumin precursor, and T-cell receptor beta chain C region) were identified with matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) and confirmed with tandem MS (MS/MS) and Western blotting. The putative capacitative calcium entry channel was partially validated with a laboratory made antibody of a representative peptide fragment in PAH-exposed human plasma samples.     

Proteomics of Halophilic archaea

Halophilic archaea is a member of the Halobacteriacea family, the only family in the Halobacteriales order. Most Halophilic archaea require 1.5M NaCl both to grow and retain the structural integrity of the cells. The proteins of these organisms have thus been adapted to be active and stable in the hypersaline condition. Consequently, the unique properties of these biocatalysts have resulted in several novel applications in industrial processes. Halophilic archaea are also to be useful for bioremediation of hypersaline environment. Proteome data have expended enormously with the significant advance recently achieved in two-dimensional gel electrophoresis (2-DE) and mass spectrometry (MS). The whole genome sequencing of Halobacterium species NRC-1 was completed and this would also provide tremendous help to analyze the protein mass data from the similar strain Halobacterium salinarum. Proteomics coupled with genomic databases now has become a basic tool to understand or identify the function of genes and proteins. In addition, the bioinformatics approach will facilitate to predict the function of novel proteins of Halophilic archaea. This review will discuss current proteome study of Halophilic archaea and introduce the efficient procedures for screening, predicting, and confirming the function of novel halophilic enzymes.