Multipotent cells from the human third molar: feasibility of cell-based therapy for liver disease

Abstract Adult stem cells have been reported to exist in various tissues. The isolation of high-quality human stem cells that can be used for regeneration of fatal deseases from accessible resources is an important advance in stem cell research. In the present study, we identified a novel stem cell, which we named tooth germ progenitor cells (TGPCs), from discarded third molar, commonly called as wisdom teeth. We demonstrated the characterization and distinctiveness of the TGPCs, and found that TGPCs showed high proliferation activity and capability to differentiate in vitro into cells of three germ layers including osteoblasts, neural cells, and hepatocytes. TGPCs were examined by the transplantation into a carbon tetrachloride (CCl₄)-treated liver injured rat to determine whether this novel cell source might be useful for cell-based therapy to treat liver diseases. The successful engraftment of the TGPCs was demonstrated by PKH26 fluorescence in the recipient's rat as to liver at 4 weeks after transplantation. The TGPCs prevented the progression of liver fibrosis in the liver of CCl₄-treated rats and contributed to the restoration of liver function, as assessed by the measurement of hepatic serum markers aspartate aminotransferase and alanine aminotransferase. Furthermore, the liver functions, observed by the levels of serum bilirubin and albumin, appeared to be improved following transplantation of TGPCs. These findings suggest that multipotent TGPCs are one of the candidates for cell-based therapy to treat liver diseases and offer unprecedented opportunities for developing therapies in treating tissue repair and regeneration.     

ATP depletion alters the mode of cell death induced by benzyl isothiocyanate

Pro-inflammatory death is presumably an undesirable event in cancer prevention process, thus biochemical comprehension and molecular definition of this process could have important clinical implications. In the present study, we examined the cytophysiological conversion of cell death mode by benzyl isothiocyanate (BITC) in human cervical cancer HeLa cells. The detailed studies using flow cytometric and morphological analyses demonstrated that the cells treated with appropriate concentration (25 microM) of BITC showed apoptotic feature, such as chromatin condensation, DNA fragmentation, and preserved plasma membrane integrity, whereas these features were disappeared by treatment with higher concentration (100 microM). The treatment with 2-deoxyglucose, an inhibitor of ATP synthesis, drastically increased in the ratio of necrotic dead cells, while it influences little that of apoptotic cells. Moreover, an analysis using the mitochondrial DNA-deficient HeLa cells demonstrated that the rho degrees cells were more susceptible to the BITC-induced necrosis-like cell death compared to the wild-type (rho(+)) cells, whereas the ROS production was significantly inhibited in the rho degrees cells. It is likely that the BITC-induced ROS is derived from mitochondrial respiratory chain and ruled out the contribution to the mechanism of cell death mode switching. In addition, the BITC treatment resulted in a more rapid depletion of ATP in the rho degrees cells than in the rho(+) cells. Furthermore, a caspase inhibitor, Z-VAD-fmk counteracted not only apoptosis, but also necrosis-like cell death induced by BITC, suggesting that increment in this cell death pattern might be due to the interruption of events downstream of a caspase-dependent pathway. The obtained data suggested that the decline in the intracellular ATP level plays an important role in tuning the mode of cell death by BITC.     

Synthetic studies on glycosphingolipids from Protostomia phyla: syntheses and biological activities of amphoteric glycolipids containing a phosphocholine residue from the earthworm Pheretima hilgendorfi

Two types of amphoteric glycosphingolipid found in the earthworm Pheretima hilgendorfi, PC(-->6)-beta-d-Galp-(1-->6)-beta-d-Galp-(1-->1)Cer (1) and PC(-->6)-beta-d-Galp-(1-->6)-beta-d-Galp-(1-->6)-beta-d-Galp-(1-->1)Cer (2), and their derivatives (4, 5) were synthesized. These were examined for their ability to enhance production of interleukin-8 (IL-8), a potent inflammatory cytokine involved in neutrophil chemotaxis, in a TNFalpha-stimulated granulocytic HL-60 cells. Compounds 1 and 2 were found to be potent enhancers of IL-8 production.     

Structural characterization of glycosylinositolphospholipids with a blood group type B sugar unit from the edible mushroom, Hypsizygus marmoreus

Edible fungi, mushrooms, are a popular food in Japan and over 15 cultured mushroom species are available at the food markets. Recently, constituents or ingredients of edible mushrooms have drawn attention because possibilities have been seen for their medical usage. Mycoglycolipids (basidiolipids) of higher mushrooms have been characterized as glycosylinositolphosphoceramides, having a common core structure of Manalpha1-2Ins1-[PO(4)]-Cer and extensions of Man, Gal, and/or Fuc sugar moieties. Seven mycoglycolipids were purified from the edible mushroom Hypsizygus marmoreus by successive column chromatography on ion exchange Sephadex (DEAE-Sephadex) and silicic acid (Iatrobeads). Their structures were characterized to be Ins1-[PO(4)]-Cer (AGL0), Manalpha1-2Ins1-[PO(4)]-Cer (AGL1), Galbeta1-6Manalpha1-2Ins1-[PO(4)]-Cer (AGL2), Fucalpha1- 2Galbeta1-6Manalpha1-2Ins1-[PO(4)]-Cer (AGL3), Galalpha1-3(Fucalpha1-2)Galbeta1-6Manalpha1-2Ins1-[PO(4)]-Cer (AGL4), Galalpha1-2Galalpha1-3(Fucalpha1-2)Galbeta1-6Manalpha1-2Ins1-[PO(4)]-Cer (AGL5), and Galalpha1-2Galalpha1-2Galalpha1-3(Fucalpha1-2)Galbeta1-6Manalpha1-2Ins1-[PO(4)]-Cer (AGL6) by sugar compositional analysis, methylation analysis, periodate oxidation, partial acid hydrolysis, enzymatic hydrolysis, immunochemical analysis, gas-liquid chromatography (GC), gas chromatography-mass spectrometry (GC-MS), matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS), and (1)H-nuclear magnetic resonance spectroscopy (NMR). Ceramide constituents of their mycoglycolipids were composed of phytosphingosine as the sole sphingoid, and mainly 2-hydroxy C22:0 and C24:0 acids as the fatty acids. By immunochemical detection, the terminal structure of AGL4, Galalpha1-3(Fucalpha1-2)Galbeta-, was shown to have blood group type B activity. Galalpha1-2 and its repeating sequence in AGL5 and AGL6 are novel structures on the nonreducing sugar end in mycoglycolipids. These two mycoglycolipids in H. marmoreus distinguish it from other basidiomycetes.     

Mechanisms for the proliferation of eosinophilic leukemia cells by FIP1L1-PDGFRalpha

The constitutively activated tyrosine kinase Fip1-like 1 (FIP1L1)-platelet-derived growth factor receptor α (PDGFRα) causes eosinophilic leukemia EoL-1 cells to proliferate. Recently, we demonstrated that histone deacetylase inhibitors suppressed this proliferation and induced the differentiation of EoL-1 cells into eosinophils in parallel with a decrease in the level of FIP1L1-PDGFRα. In this study, we analyzed the mechanism by which FIP1L1-PDGFRα induces the proliferation and whether the suppression of cell proliferation triggers the differentiation into eosinophils. The FIP1L1-PDGFRα inhibitor imatinib inhibited the proliferation of EoL-1 cells and decreased the level of the oncoprotein c-Myc as well as the phosphorylation of extracellular signal-regulated kinase and c-Jun N-terminal kinase (JNK). The proliferation of EoL-1 cells and expression of c-Myc were also inhibited by the MEK inhibitor U0126 and JNK inhibitor SP600125. The expression of the eosinophilic differentiation marker CCR3 was not induced by imatinib. These findings suggest that FIP1L1-PDGFRα induces the proliferation of EoL-1 cells through the induction of c-Myc expression via ERK and JNK signaling pathways, but is not involved in the inhibition of differentiation toward mature eosinophils.     

Benefits of mass reduction for commuting flight with heavy food load in Leach’s storm-petrel,Oceanodroma leucorhoa

When rearing chicks, Leach’s storm-petrels (Oceanodroma leucorhoa) commute between foraging areas and breeding colonies with heavy food loads. At this time they should maximize the size of energy-supplying organs in response to increased energy expenditure but minimize total body mass to decrease the energetic cost of flight. Nineteen storm-petrels were killed to examine the changes in body composition and the masses of energy-supplying organs in birds that were incubating and rearing chicks. Parents lost a mean of 7.95 g in body mass between the stages of incubation and chick-rearing mainly via a loss of skin including subcutaneous adipose tissue, and a small fraction of heart and digestive organs, which are considered energy-supplying organs. This mass loss actually enables them to decrease flight cost by 14.4%. The benefits of decreasing flight costs by reducing total body mass are greater than if the energy-supplying organs of birds are enlarged only.

     

Synthesis of chimeric RNAs between U6 small nuclear RNA and (-)sTRSV and analysis of their cleavage activities against the substrate RNA.

U6 small nuclear RNA (U6 snRNA) is one of the spliceosomal RNAs essential for pre-mRNA splicing. Highly conserved region of U6 snRNA shows a structural similarity with the catalytic center of the negative strand of the satellite RNA of tobacco ring spot virus [(-)sTRSV], supporting the hypothesis that U6 snRNA has a catalytic role in pre-mRNA splicing. To test this hypothesis, we examined in vitro whether synthetic RNAs consisting of the sequence of the highly conserved region of U6 snRNA or various chimeric RNAs between the U6 region and the catalytic center of (-)sTRSV could cleave a substrate RNA that can partially base-pair with them and has a GU sequence between the pairing regions. Chimeric RNAs with 70 to 83% sequence identity with the conserved region of S. pombe U6 snRNA cleaved the substrate RNA at the 5' side of the GU sequence. In addition, we found that the highly conserved region of U6 snRNA is similar in structure to the catalytic core region of the group I self-splicing intron in cyanobacteria. These results support the hypothesis that U6 snRNA catalyzes the pre-mRNA splicing reaction and U6 snRNA may originate from the catalytic domain of an ancient self-splicing intron.     

Electrostatic interaction between ion-penetrable multi-layered membranes

A theory of the double layer interaction regulated by the Donnan potential between two ion-penetrable membranes in an electrolyte solution developed previously by Ohshima and Kondo is extended to the case in which the membranes consist of many layers having different thickness and densities of membrane-fixed charges. The interaction force is found to be determined mainly by the contributions from layers located within the depth of 1/kappa (kappa, Debye-Hückel parameter) from the membrane surface. It is also predicted that the interaction force may alter its sign with changing electrolyte concentration.     

Thermostable phenylalanine dehydrogenase of Thermoactinomyces intermedius: cloning, expression, and sequencing of its gene

The gene encoding the thermostable phenylalanine dehydrogenase [EC 1.4.1.-] of a thermophile, Thermoactinomyces intermedius, was cloned and its complete DNA sequence was determined. The phenylalanine dehydrogenase gene (pdh) consists of 1,098 nucleotides and encodes 366 amino acid residues corresponding to the subunit (Mr 41,000) of the hexameric enzyme. The amino acid sequence deduced from the nucleotide sequence of the pdh gene of T. intermedius was 56.0 and 42.1% homologous to those of the phenylalanine dehydrogenases of Bacillus sphaericus and Sporosarcina ureae, respectively. It shows 47.5% homology to that of the thermostable leucine dehydrogenase from B. stearothermophilus. The pdh gene was highly expressed in E. coli JM109, the amount of phenylalanine dehydrogenase produced amounting up to about 8.3% of that of the total soluble protein. We purified the enzyme to homogeneity from transformant cells in a day, with a 58% recovery.     

Azoreductase from alkaliphilic Bacillus sp. AO1 catalyzes indigo reduction

Indigo is an insoluble blue dye historically used for dyeing textiles. A traditional approach for indigo dyeing involves microbial reduction of polygonum indigo to solubilize it under alkaline conditions; however, the mechanism by which microorganisms reduce indigo remains poorly understood. Here, we aimed to identify an enzyme that catalyzes indigo reduction; for this purpose, from alkaline liquor that performed microbial reduction of polygonum indigo, we isolated indigo carmine-reducing microorganisms. All isolates were facultative anaerobic and alkali-tolerant Bacillus spp. An isolate termed AO1 was found to be an alkaliphile that preferentially grows at pH 9.0–11.0 and at 30–35 °C. We focused on flavin-dependent azoreductase as a possible enzyme for indigo carmine reduction and identified its gene (azoA) in Bacillus sp. AO1 using homology-based strategies. azoA was monocistronic but clustered with ABC transporter genes. Primary sequence identities were < 50% between the azoA product (AzoA) and previously characterized flavin-dependent azoreductases. AzoA was heterologously produced as a flavoprotein tolerant to alkaline and organic solvents. The enzyme efficiently reduced indigo carmine in an NADH-dependent manner and showed strict specificity for electron acceptors. Notably, AzoA oxidized NADH in the presence, but not the absence, of indigo. The reaction rate was enhanced by adding organic solvents to solubilize indigo. Absorption spectrum analysis showed that indigo absorption decreased during the reaction. These observations suggest that AzoA can reduce indigo in vitro and potentially in Bacillus sp. AO1. This is the first study that identified an indigo reductase, providing a new insight into a traditional approach for indigo dyeing.