Down-regulation of trypsinogen expression is associated with growth retardation in alpha1,6-fucosyltransferase-deficient mice: attenuation of proteinase-activated receptor 2 activity

Alpha1,6-fucosyltransferase (Fut8) plays important roles inphysiological and pathological conditions. Fut8-deficient (Fut8^–/–)mice exhibit growth retardation, earlier postnatal death, andemphysema-like phenotype. To investigate the underlying molecularmechanism by which growth retardation occurs, we examined themRNA expression levels of Fut8^–/– embryos (18.5days postcoitum [dpc]) using a cDNA microarray. The DNA microarrayand real-time polymerase chain reaction (PCR) analysis showedthat a group of genes, including trypsinogens 4, 7, 8, 11, 16,and 20, were down-regulated in Fut8^–/– embryos.Consistently, the expression of trypsinogen proteins was foundto be lower in Fut8^–/– mice in the duodenum, smallintestine, and pancreas. Trypsin, an active form of trypsinogen,regulates cell growth through a G-protein-coupled receptor,the proteinase-activated receptor 2 (PAR-2). In a cell culturesystem, a Fut8 knockdown mouse pancreatic acinar cell carcinoma,TGP49-Fut8-KDs, showed decreased growth rate, similar to thatseen in Fut8^–/– mice, and the decreased growth ratewas rescued by the application of the PAR-2-activating peptide(SLIGRL-NH₂). Moreover, epidermal growth factor (EGF)-inducedreceptor phosphorylation was attenuated in TGP49-Fut8-KDs, whichwas highly associated with a reduction of trypsinogens mRNAlevels. The addition of exogenous EGF recovered c-fos, c-jun,and trypsinogen mRNA expression in TGP49-Fut8-KDs. Again, theEGF-induced up-regulation of c-fos and c-jun mRNA expressionwas significantly blocked by the protein kinase C (PKC) inhibitor.Our findings clearly demonstrate a relationship between Fut8and the regulation of EGF receptor (EGFR)-trypsin-PAR-2 pathwayin controlling cell growth and that the EGFR-trypsin-PAR-2 pathwayis suppressed in TGP49-Fut8-KDs as well as in Fut8^–/–mice.     

Regeneration of transgenic Cryptomeria japonica D. Don after Agrobacterium tumefaciens-mediated transformation of embryogenic tissue

A genetic transformation procedure for Cryptomeria japonica was developed after co-cultivation of embryogenic tissues with the disarmed Agrobacterium tumefaciens strain C58/pMP90, which harbours the visual reporter gene sgfp and two selectable marker genes, hpt and nptII. We were able to generate eight and three independent transgenic lines per gram of embryogenic tissue after selection on hygromycin and kanamycin medium, respectively. Transgenic plants were regenerated through somatic embryogenesis in 4 lines out of these 11 lines. Green fluorescent protein fluorescence was observed under fluorescent microscopy. Integration of the genes into the genome was confirmed by polymerase chain reaction analysis of embryogenic tissues and Southern blot analysis of regenerated plantlets.     

Flocculation and pentadecane production of a novel filamentous cyanobacterium <Emphasis Type="Italic">Limnothrix</Emphasis> sp. strain SK1-2-1

Objective

A novel filamentous cyanobacterium, a photosynthesizing microorganism, was isolated from a river, and its unique features of flocculation and pentadecane production were characterized.

Results

Microscopic observations and a phylogenetic analysis with 16S rDNA revealed that this strain was a Limnothrix species denoted as the SK1-2-1 strain. Auto cell-flocculation was observed when this strain was exposed to a two-step incubation involving a standing cultivation following a shaking preincubation. Flocculation was enhanced by blue light at a wavelength at 470 nm and irradiation for several hours to 1 day. Moreover, the strain exhibiting exponential cell growth may preferentially accumulate alkanes as pentadecane C₁₅H₃₂ alkane, which may be used as jet fuel, at a range of approximately 1% in the dry cell weight of flocculated cells.

Conclusion

This is the first study on biofuel production using flocculated cells in which the specific manner of production may be regulated by cultivation conditions.

     

Factors associated with a purine-rich exonic splicing enhancer sequence in Xenopus oocyte nucleus

Purine-rich exonic splicing enhancers (ESEs) stimulate splicing of the adjacent introns with suboptimal splice sites. To elucidate the mechanism regarding ESEs, factors specifically associated with ESEs in HeLa cell nuclear extracts were previously investigated, and shown to include SR (serine/arginine-rich) proteins. However, factors associated with ESEs in vivo have not yet been explored. Here we show that a GAA repeat RNA sequence, a typical ESE, is associated in Xenopus oocyte nuclei with at least one SR protein, SF2/ASF, as was expected. Moreover, components of SF3a/b complexes, U2 snRNA, and U2AF(65) were also found to be associated with the ESE in the nucleus. Since SF3a/b complexes are the constituents of the 17S U2 snRNP, these results suggest that the 17S U2 snRNP is associated with the ESE in the nucleus, probably through bridging interactions of U2AF and SR proteins. The identified factors may represent a functional splicing enhancer complex in vivo.     

Aldehyde reductase gene expression by lipid peroxidation end products, MDA and HNE

Membrane lipid peroxidation results in the production of a variety of aldehydic compounds that play a significant role in aging, drug toxicity and the pathogenesis of a number of human diseases, such as atherosclerosis and cancer. Increased lipid peroxidation and reduced antioxidant status may also contribute to the development of diabetic complications. This study reports that lipid peroxidation end products such as malondialdehyde (MDA) and 4-hydroxynonenal (HNE) induce aldehyde reductase (ALR) gene expression. MDA and HNE induce an increase in intracellular peroxide levels; N-Acetyl-L-cysteine (NAC) suppressed MDA- and HNE-induced ALR gene expression. These results indicate that increased levels of intracellular peroxides by MDA and HNE might be involved in the upregulation of ALR.     

Antiparallel pleated beta-sheets observed in crystal structures of N,N-bis(trichloroacetyl) and N,N-bis(m-bromobenzoyl) gramicidin S.

Despite intensive efforts, the structures of gramicidin S (GS) [cyclo(-Val-Orn-Leu-d-Phe-Pro-)(2)] and its analogues have not been elucidated by the X-ray diffraction method, except for the GS-urea complex (Hull et al., Nature 275, 206-207, 1978; Tishchenko et al., Acta Cryst. D53, 151-159, 1997). We focused on the acetylation of GS to obtain suitable crystals for X-ray diffraction. The amino groups of Orn residues were capped with trichloroacetic and m-bromobenzoic acids. Both trichloroacetyl and m-bromobenzoyl GSs (TcGS and BzGS, respectively) are hydrophobic and their properties are similar to those of acetyl-GS (AcGS). Although it is well known that AcGS yields hexagonal crystals, TcGS and BzGS yield monoclinic and orthorhombic crystals in aqueous dimethylformamide solution, respectively. Their cell volumes were approximately one-fourth or one-eighth of the hexagonal cell volume. The crystal structures of TcGS and BzGS were determined as the first examples of acetylated GS analogues: TcGS, C(64)H(90)N(12)O(12)Cl(6). 3(C(3)H(7)NO), M(r) = 1651.47, monoclinic, P2(1), a = 15.4366(6) A, b = 18.5312(4) A, c = 16.4774(6) A, beta = 14.160(2) degrees, V = 4300.6(2) A(3), Z = 2; and BzGS, C(64)H(98)N(12)O(12)Br(2). 1.54(H(2)O), M(r) = 1535.21, orthorhombic, P2(1)2(1)2(1), a = 16.748(10) A, b = 18.834(5) A, c = 28.558(10) A, V = 9008(7) A(3), Z = 4. Both these peptide molecules formed an antiparallel pleated beta-sheet, and pseudo twofold symmetries existed in the repeated sequence. beta-Turns formed at the fragments of d-Phe-Pro were classified into type II' based on their characteristics. The peptide conformations of TcGS and BzGS were similar to each other, and these structural features agreed with those of structures proposed by the previous studies.     

Molecular oxygen controls nitrate transport of Escherichia coli nitrate-respiring cells

Escherichia coli cells grown anaerobically in the presence of nitrate reduce the nitrate as a terminal electron acceptor in place of molecular oxygen by an induced respiratory-type electron transferring system residing in the inner membrane structure. When oxygen is introduced to a suspension of nitrate-respiring cells, the oxygen is immediately reduced preferentially and the cellular uptake of nitrate ceases abruptly. In contrast, we found that the cells exhibited no oxygen control on uptake of chlorate, a competitive substrate analogue, indicating operation of an oxygen-sensitive transport system specific to nitrate. This was further evidenced by the fact that chlorate inhibition of reduction of nitrate was brought about only when the transport of both chlorate and nitrate was facilitated by the aid of carrier-type chlorate (or nitrate) ionophore. We demonstrated that oxygen inhibition on reduction of nitrate was abolished within the cells treated by octyl glucoside resulting in a removal of permeability barrier specific to nitrate. We conclude that the transient control by molecular oxygen is primarily due to the inhibition of nitrate transport into the cytoplasmic side. Since nitrate induces the nitrate-respiring system, the repression of the nitrate reductase operon by molecular oxygen is consistently interpreted on the basis of the "inducer exclusion mechanism."     

Polysaccharide-polynucleotide complexes (15): thermal stability of schizophyllan (SPG)/poly(C) triple strands is controllable by alpha-amino acid modification

Schizophyllan (SPG), a beta-1,3-glucan polysaccharide which is known to form macromolecular complexes with certain polynucleotides, was modified by a reductive amination method with alpha-amino acids (Arg, Lys, and Ser). The thermal stability of the complexes as estimated by T(m) was enhanced in SPG-Arg and SPG-Lys conjugates which have pI values higher than the pH of the medium (8.0). The T(m) shift increased with the increase in the percentage of alpha-amino acid introduced and the highest T(m) values attained were 64 degrees C for SPG-Arg conjugate and 62 degrees C for SPG-Lys conjugate, which are higher by 13 and 11 degrees C, respectively, than those of the unmodified SPG+poly(C) complex. In the SPG-Ser conjugate with a pI lower than the medium pH (8.0), the T(m) values decreased with an increase in the percentage of Ser. Formation of the macromolecular complex was no longer detected above 13.2% Ser. The findings indicate that the T(m) values are easily controllable by the type and percentage of the introduced alpha-amino acids. We believe, therefore, that the present conjugates, consisting of naturally originated SPG and alpha-amino acids, provide an important lead for developing nontoxic artificial vectors and to control the affinity with polynucleotides in response to medium pH and temperature.     

8-Nitroguanine formation in oral leukoplakia, a premalignant lesion.

Oral leukoplakia is a premalignant lesion associated with development of oral cancer. To clarify the mechanism of development of oral carcinogenesis from leukoplakia, we examined DNA damage in oral epithelium of biopsy specimens of patients with leukoplakia by immunohistochemical methods. Histological changes, such as epithelial dysplasia and infiltration of inflammatory cells were observed in oral tissues of leukoplakia patients. A double immunofluorescence labeling study demonstrated that the accumulation of mutagenic 8-nitroguanine, an indicator of nitrative DNA damage, and 8-oxo-7,8-dihydro-2'-deoxyguanosine, an indicator of oxidative DNA damage, was apparently observed in the oral epithelium of patients with leukoplakia, whereas little or no immunoreactivity was observed in normal oral mucosa. Expression of inducible nitric oxide synthase (iNOS) was also observed in oral epithelium of leukoplakia patients. Immunoreactivity of 3-nitrotyrosine, an indicator of nitrative stress, was observed in oral epithelial cells and colocalized with 8-nitroguanine. Moreover, proliferating cell nuclear antigen and p53 were expressed in 8-nitroguanine-positive epithelial cells in the basal layer. These results suggest that iNOS-mediated nitrative stress contributes to development of oral carcinogenesis from leukoplakia through DNA damage as well as oxidative stress.