Hepatocytes maintain their function on basement membrane formed by epithelial cells

To establish liver tissue engineering, the effective substratum for hepatocytes culture should be developed. Up to now, it is believed that Matrigel, which contains several basement membrane proteins produced by sarcoma cells, is the most effective substratum. Matrigel does not contain extracellular matrix molecules derived from epithelial cells although the space of Disse contains the molecules such as laminin-511/521 (laminin-10/11). Therefore, the basement membrane formed by epithelial cells can be more effective substratum than Matrigel. In this study, we evaluated hepatocytes behavior on basement membrane (rBM) formed by alveolar epithelial cells. The viability of hepatocytes on rBM is higher than that of Matrigel within 5 days. Also, the expression of Cyp1a2 induced by beta-naphthoflavone can be observed in hepatocytes on rBM but not in Matrigel. These results indicate that rBM is a more effective substratum for hepatocyte culture than Matrigel.     

Cloning and analysis of methanol oxidation genes in the methylotroph Hyphomicrobium methylovorum GM2

The gene encoding the α-subunit of methanol dehydrogenase (mxaF) and its flanking region was isolated from a methylotrophic bacterium, Hyphomicrobium methylovorum GM2. The deduced amino acid sequence of MxaF showed 80, 80, 74 and 66% identity with those of Methylobacterium extorquens AM1, M. organophilum XX, Paracoccus denitrificans and Methylophilus methylotrophus, respectively. The putative mxaF promoter sequence (−35 -AAAGACA-, −10 -TAGAA-) observed in other methylotrophs was not found in the region 5′ of H. methylovorum GM2 mxaF. Downstream of mxaF, five open reading frames and one partial open reading frame were detected which had high identity with the genes mxaJ, mxaG, mxaI, mxaR, mxaS and mxaA. This indicated the existence of a mxaFJGIRSA gene cluster in H. methylovorum GM2, as previously observed in M. extorquens AM1.     

Isolation and characterization of osteoclast precursors that differentiate into osteoclasts on calvarial cells within a short period of time

Osteoclasts are formed in cocultures of mouse calvarial cells and hematopoietic cells in the presence of osteotropic factors such as 1α,25-dihydroxyvitamin D₃ [1α,25(OH)₂D₃], parathyroid hormone (PTH) and prostaglandin E₂ (PGE₂). We isolated osteoclast precursors (OCPs) from the coculture and examined their characteristics. After coculture for 7 days of mouse calvarial cells and bone marrow cells in the absence of osteotropic factors, hematopoietic cells were recovered and applied to a Sephadex G-10 column. Cells which passed through the column were collected as OCPs. When OCPs were cultured on calvarial cell layers in the presence of 1α,25(OH)₂D₃, tartrate-resistant acid phosphatase (TRAP)–positive cells first appeared within 24 h, and their number increased thereafter. OCPs also differentiated into TRAP-positive cells within 48 h on the calvarial cell layer which had been pretreated with either 1α,25(OH)₂D₃, PTH, or PGE₂. Autoradiography using [¹²⁵I]-labeled calcitonin showed that TRAP-positive cells formed on the calvarial cell layer expressed calcitonin receptors. Direct contact between OCPs and calvarial cells was required for the differentiation of OCPs into TRAP-positive cells. Flow cytometric analysis revealed that OCPs were positive for Mac-1, Mac-2, and Gr-1 but negative for F4/80, B220 and CD3e. Calvarial cells obtained from macrophage-colony stimulating factor (M-CSF)–deficient osteopetrotic (op/op) mice did not support OCP formation. A cell preparation disaggregated from long bones of newborn mice contained OCPs that differentiated into TRAP-positive cells on calvarial cells within 48 h, but cell preparations of freshly isolated bone marrow cells and alveolar macrophages did not. These results suggest that OCPs are specific cells which are formed only in the bone microenvironment and that OCPs recognize a signal(s) expressed by stromal cells in response to osteotropic factors and differentiate into osteoclasts. J. Cell. Physiol. 177:26–35, 1998. © 1998 Wiley-Liss, Inc.     

Cryo-EM of the ATP11C flippase reconstituted in Nanodiscs shows a distended phospholipid bilayer inner membrane around transmembrane helix 2

ATP11C is a member of the P4-ATPase flippase family that mediates translocation of phosphatidylserine (PtdSer) across the lipid bilayer. In order to characterize the structure and function of ATP11C in a model natural lipid environment, we revisited and optimized a quick procedure for reconstituting ATP11C into Nanodiscs using methyl-β-cyclodextrin as a reagent for the detergent removal. ATP11C was efficiently reconstituted with the endogenous lipid, or the mixture of endogenous lipid and synthetic dioleoylphosphatidylcholine (DOPC)/dioleoylphosphatidylserine (DOPS), all of which retained the ATPase activity. We obtained 3.4 Å and 3.9 Å structures using single-particle cryo-electron microscopy (cryo-EM) of AlF- and BeF-stabilized ATP11C transport intermediates, respectively, in a bilayer containing DOPS. We show that the latter exhibited a distended inner membrane around ATP11C transmembrane helix 2, possibly reflecting the perturbation needed for phospholipid release to the lipid bilayer. Our structures of ATP11C in the lipid membrane indicate that the membrane boundary varies upon conformational changes of the enzyme and is no longer flat around the protein, a change that likely contributes to phospholipid translocation across the membrane leaflets.     

Effects of ethidium bromide and chloramphenicol on the mitochondrial nucleoids in Euglena gracilis

The effects of ethidium bromide (EB) at 0.13 m M and of chloramphenicol (CAP) at 46 m M on the mitochondria and mitochondrial nucleoids in Euglena gracilis . Z strain, were examined by fluorescence microscopy and by electron microscopy. Ethidium bromide stopped the multiplication of cells and decreased their respiratory activity by 55% after treatment for 10 days. Most of the mitochondria became slender with few cristae and some became cup-shaped with stacked cristac. Mitochondrial nucleoids decreased markedly in number after treatment with EB for more than 2 days. After treatment for 3 days with EB, mitochondrial nucleoids could not be detected in about half of all cells examined. Treatment with CAP for 10 days reduced the respiratory activity by 47%. Chloramphenicol did not decrease the number of mitochondrial nucleoids but it increased the number of cristae and the volume of mitochondria.     

Stress Tolerance in Doughs of Saccharomyces cerevisiae Trehalase Mutants Derived from Commercial Baker’s Yeast

Accumulation of trehalose is widely believed to be a critical determinant in improving the stress tolerance of the yeast Saccharomyces cerevisiae, which is commonly used in commercial bread dough. To retain the accumulation of trehalose in yeast cells, we constructed, for the first time, diploid homozygous neutral trehalase mutants (Δnth1), acid trehalase mutants (Δath1), and double mutants (Δnth1 ath1) by using commercial baker’s yeast strains as the parent strains and the gene disruption method. During fermentation in a liquid fermentation medium, degradation of intracellular trehalose was inhibited with all of the trehalase mutants. The gassing power of frozen doughs made with these mutants was greater than the gassing power of doughs made with the parent strains. The Δnth1 and Δath1 strains also exhibited higher levels of tolerance of dry conditions than the parent strains exhibited; however, the Δnth1 ath1 strain exhibited lower tolerance of dry conditions than the parent strain exhibited. The improved freeze tolerance exhibited by all of the trehalase mutants may make these strains useful in frozen dough.     

Multifunctions of the upper lip and a ventral reflecting organ in a bioluminescent ostracod Vargula hilgendorfii (Müller, 1890)

Multifunctions of the upper lip in a bioluminescent myodocopid Vargula hilgendorfii were studied by video observation and histological method. The localization of luciferin and luciferase gland cells within the upper lip was partly successful. Two long protrusions of the upper lip, both of V. hilgendorfii and a non-luminescent species of the same family, immediately anterior to the mouth, were found to show very flexible movement especially while eating, as if smearing on the food surface a secretion from the protrusions (glands), which may support the hypothesized secretion of digestive enzymes from the upper lip. This hypothesis is further supported by the new finding of a pair of ducts which connect the basal part of the upper lip with the posterior digestive duct (stomach). Comparative studies of V. hilgendorfii with several sympatric non-luminescent species of the same family have also revealed that it has a characteristic reflecting organ immediately posterior to the anus. It is a conical small protrusion, as if dangling from the ventral edge of the abdomen at the apex of the cone. It is observable only in live specimens, when the furca, which is located outwardly to the organ, is sufficiently transparent. When illuminated, the reflecting organ reflects the distinct light. The diameter of the mirror (chemical composition provisionally analyzed) is about 6–8% of the carapace length. The organ develops from the very first stage of its ontogeny without reference to sex, which suggests that the function may be related to intraspecific signaling or predatory deterrence.     

Improved ethanol tolerance of Saccharomyces cerevisiae strains by increases in fatty acid unsaturation via metabolic engineering

To enhance the ethanol tolerance of Saccharomyces cerevisiae, the Arabidopsis thaliana FAD2 gene and/or the S. cerevisiae OLE1 gene were over-expressed in this yeast. The transformant over-expressing both these genes could not only synthesize dienoic fatty acids but also increased the unsaturated fatty acid content of membrane lipid and then showed the highest viability in the presence of 15% (v/v) ethanol.     

Effects of saline and osmotic stress on proline and sugar accumulation in Populus euphratica in vitro

The use of in vitro shoot cultures to evaluate osmotic and salt tolerance and the effects of salt and mannitol in the medium on proline and sugar accumulation were investigated in two poplar species, P. euphratica and P. alba cv. Pyramidalis × P. tomentosa. Shoot length, leaf number, whole plant dry weight, and the accumulation of proline and total soluble sugars in leaves were quantified after 2 weeks. All P. euphratica plantlets survived at all levels of mannitol and NaCl, while the mortality of P. alba cv. Pyramidalis × P. tomentosa increased both at the mannitol and the NaCl treatments. A significant increase in proline accumulation was observed in both young and mature P. euphratica leaves at 200 mM mannitol and above, and at 150 mM NaCl and above. The total soluble sugar content increased in young P. euphratica leaves at 250 mM NaCl; however, it decreased in the mature leaves. Similar increases of the total soluble sugar content were not seen in P. alba cv. Pyramidalis × P. tomentosa plants in response to either mannitol or NaCl treatment. Our results suggest that accumulated proline and sugars promote osmotic and salt tolerance. The effects of accumulated proline and total soluble sugars on leaves are discussed in relation to growth and osmotic adjustment. This revised version was published online in August 2006 with corrections to the Cover Date.