Production of l-lactic acid by electrodialysis fermentation (EDF)

An efficient process for producing l-lactic acid using an, EDF method is described. The results showed that intermittent EDF with continuous medium feed was the best one among the experiment methods employed. Comparing with the conventional EDF, intermittent EDF (seven on–off) with continuous medium feed indicated that the maximum value of o.d.₆₆₀ was not increased, but productivity was 1.5 times higher. The yield increased by above 30% and glucose transport decreased to 1/10 (from 0.46 to 0.05).     

Circadian rhythm of circumnutation in inflorescence stems of Arabidopsis

We investigate the modulation of circumnutation in inflorescence stems of Arabidopsis to determine the circadian regulation of circumnutation. Under constant light conditions (LL), circumnutation speed in wild-type plants fluctuates, with the phase of the highest speed at subjective dawn; the period length is close to 24 h. toc1 appears to shorten the period and elf3 causes an arrhythmic phenotype in circumnutation speed in LL, suggesting that a common circadian clock may control both circumnutation speed and other circadian outputs. These results highlight for the first time a role for a circadian clock in the regulation of circumnutation based on genetic analysis of Arabidopsis.     

Simultaneous measurement of F2-isoprostane, hydroxyoctadecadienoic acid, hydroxyeicosatetraenoic acid, and hydroxycholesterols from physiological samples

Oxidative stress induced by various oxidants in a random and destructive manner is considered to play an important role in the pathophysiology of a number of human disorders and diseases. It is important to assess the oxidative injury in vivo accurately and inclusively. We have developed an improved method for the measurement of in vivo lipid peroxidation by using a single plasma or liver sample, where total 8-iso-prostaglandin F(2alpha) (t8-iso-PGF(2alpha)), total hydroxyoctadecadienoic acids (tHODEs), total hydroxyeicosatetraenoic acids (tHETEs), and total 7-hydroxycholesterol (t7-OHCh), as well as their parent molecules linoleic acid (t18:2) and cholesterol (tCh), are determined by LC-MS/MS (for t8-iso-PGF(2alpha), tHODE, and tHETE) and GC-MS (for t7-OHCh, t18:2, and tCh) analyses. The plasma and liver samples from human are reduced with sodium borohydride and saponified by potassium hydroxide after the addition of heavy isotopic standards. After extraction by chloroform/ethyl acetate (CHCl(3)/CH(3)COOC(2)H(5), 4:1), they are analyzed without any further sample processing. We applied this method to hepatitis C virus-infected patients (n=8, plasma and liver), hepatitis B virus-infected patients (n=2, plasma and liver), and controls (virus free, n=8, plasma and liver). It was found that in the plasma of patients and controls, the concentrations of oxidized lipids decreased in the following order: tHODE tHETE t7-OHCh > t8-iso-PGF(2alpha). As expected, the virus clearly increased these concentrations. The ratio of stereoisomers of HODE [(E,E)-HODE/(E,Z)-HODE], which reflects the antioxidant capacity in vivo, can also be determined by this method. A significant decrease in the stereoisomer ratio for the liver of patients was observed, indicating liver dysfunction. t8-iso-PGF(2alpha), tHODE, tHETE, and t7-OHCh are measured satisfactorily and inclusively by the current method from biological fluids and tissues, and they can account for a large portion of oxidized lipids in vivo.     

Identification and characterization of the ER/lipid droplet-targeting sequence in 17beta-hydroxysteroid dehydrogenase type 11

17β-Hydroxysteroid dehydrogenase type 11 (17βHSD11) is mostly localized on the endoplasmic reticulum (ER) membrane under normal conditions and redistributes to lipid droplets (LDs) when the formation of LDs is induced. In this study, confocal microscopy analyses of the subcellular localization of the mutated 17βHSD11 proteins in cells with or without LDs revealed that both an N-terminal hydrophobic sequence and an adjacent sequence that has a weak homology with the PAT motif are independently necessary and both parts together (28 amino acid residues in total) are sufficient for the dual localization of 17βHSD11. Mutation analyses suggest that the PAT-like motif in 17βHSD11 will not be functionally similar to the canonical PAT motif. Hsp60 was identified as a possibly interacting protein with the PAT-like motif, and biochemical and microscopic analyses suggest that Hsp60 may be partly, but not necessarily involved in recognition of the PAT-like part of the targeting sequence of 17βHSD11.     

Dysfunction of the ER chaperone BiP accelerates the renal tubular injury

Tubular-interstitial injury plays a key role in the progression of chronic kidney disease. Although endoplasmic reticulum (ER) stress plays significant roles in the development of chronic diseases such as neurodegenerative disease, cardiomyopathy and diabetes mellitus, its pathophysiological role in chronic renal tubular cell injury remains unknown. BiP is an essential chaperone molecule that helps with proper protein folding in the ER. Recently, we have produced a knock-in mouse that expresses a mutant-BiP in which the retrieval sequence to the ER is deleted in order to elucidate physiological processes that are sensitive to ER functions in adulthood. The heterozygous mutant-BiP mice showed significant tubular-interstitial lesions with aging. Furthermore, proteinuria induced by chronic protein overload accelerated the tubular-interstitial lesions in the mutant mice, accompanying caspase-12 activation and tubular cell apoptosis. These results suggest that the ER stress pathway is significantly involved in the pathophysiology of chronic renal tubular-interstitial injury in vivo.     

Staphylococcus aureus surface protein SasG contributes to intercellular autoaggregation of Staphylococcus aureus

Staphylococcus aureus surface protein G (SasG) is one of cell surface proteins with cell-wall sorting motif. The sasG mutant showed significantly reduced cell aggregation and biofilm formation. SasG is comprised of variable A domain and multiple tandem repeats of B domain, native-PAGE and in vitro formaldehyde cross-linking experiments revealed that the recombinant protein of the A domain showed homo-oligomerization as an octamer, but B domain did not. This study shows that SasG-A domain contributes to intercellular autoaggregation by homo-oligomerization, and that may facilitate the adherence to host-tissues in the infection of S. aureus.     

Production of human erythropoietin by chimeric chickens

The use of transgenic avian allows cost effective and safe production of pharmaceutical proteins. Here, we report the successful production of chimeric chickens expressing human erythropoietin (hEpo) using a high-titer retroviral vector. The hEpo expressed by transgenic hens accumulated abundantly in egg white and had N- and O-linked carbohydrates. While attachment of terminal sialic acid and galactose was incomplete, portions of N- and O-linked carbohydrates were present. In vitro biological activity of egg white-hEpo was comparable to that produced by recombinant CHO cells.     

Molecular cloning, expression, and characterization of a novel endo-alpha-N-acetylgalactosaminidase from Enterococcus faecalis

We report here the molecular cloning, expression and characterization of a novel endo-alpha-N-acetylgalactosaminidase, classified into the GH101 family, from Enterococcus faecalis (endo-EF). The recombinant endo-EF was found to catalyze the liberation of core1-disaccharides (Galbeta1-3GalNAc) from core1-pNP (Galbeta1-3GalNAcalpha-pNP) like other GH101 family enzymes. However, endo-EF seems to differ in specificity from the GH101 enzymes reported to date, because it was able to release trisaccharides from core2-pNP (Galbeta1-3[GlcNAcbeta1-6]GalNAcalpha-pNP) and tetrasaccharides from Gal-core2-pNP (Galbeta1-3[Galbeta1-3GlcNAcbeta1-6]GalNAcalpha-pNP). Interestingly, the enzyme could transfer not only core1-disaccharides but also core2-trisaccharides to alkanols generating alkyl-oligosaccharides. Endo-EF should facilitate O-glycoprotein research.     

Physical interaction between Tbx6 and mespb is indispensable for the activation of bowline expression during Xenopus somitogenesis

Crk is a member of a family of adaptor proteins that are involved in intracellular signal pathways altering cell adhesion, proliferation, and migration. Increased expression of Crk has been described in lung cancer and associated with increased tumor invasiveness. MicroRNAs (miRNAs) are a family of small non-coding RNAs (approximately 21–25 nt long) that are capable of targeting genes for either degradation of mRNA or inhibition of translation. Crk is a predicted putative target gene for miR-126. Over-expression of miR126 in a lung cancer cell line resulted in a decrease in Crk protein without any alteration in the associated mRNA. These lung cancer cells exhibit a decrease in adhesion, migration, and invasion. Decreased cancer cell invasion was also evident following targeted knockdown of Crk. MiR-126 alters lung cancer cell phenotype by inhibiting adhesion, migration, and invasion and the effects on invasion may be partially mediated through Crk regulation.     

Changes in cell morphology and cytoskeletal organization are induced by human mitotic checkpoint gene, Bub1

The mitotic spindle checkpoint prevents the onset of anaphase and subsequent cell division until chromosomes are properly aligned on a bipolar spindle. Thus, it regulates the cell division cycle by keeping cells with defective spindles from leaving mitosis. The budding uninhibited by benzimidazole (Bub1) is a key component of mitotic checkpoint. Bub1 encodes a serine/threonine kinase required for mitotic spindle checkpoint function. The regulation of cell morphology in eukaryotic cells is a complex process involving major components of the cytoskeleton including actin microfilaments, microtubules, and intermediate filaments (IFs). Here we show that Bub1 directly affects the structural integrity of IFs. Constitutive expression of Bub1 caused disappearance of filamentous vimentin, a type III IF, and consequently changed cell morphology. Expression of kinase domain—deleted Bub1 induced neither morphological change nor disappearance of vimentin. These observations suggest that Bub1 not only regulates the cell cycle, but also may be involved in the cytoskeletal control in interphase cells.