The role of the sucrose transporter, OsSUT1, in germination and early seedling growth and development of rice plants

Using expression analysis, the role of the sucrose transporter OsSUT1 during germination and early growth of rice seedlings has been examined in detail, over a time-course ranging from 1 d to 7 d post-imbibition. Unlike the wheat orthologue, TaSUT1, which is thought to be directly involved in sugar transfer across the scutellar epithelium, OsSUT1 is not expressed in the scutellar epithelial cell layer of germinating rice and is, therefore, not involved in transport of sugars across the symplastic discontinuity between the endosperm and the embryo. OsSUT1 expression was also absent from the aleurone cells, indicating it is not involved in the transport of sucrose in this cell layer during germination. However, by 3 d post-imbibition, OsSUT1 was present in the companion cells and sieve elements of the scutellar vascular bundle, where it may play a role in phloem loading of sucrose for transport to the developing shoot and roots. This sucrose is most likely sourced from hexoses imported from the endosperm. In addition, sucrose may be remobilized from starch granules which are present at a high density in the scutellar ground tissues surrounding the vasculature and at the base of the shoot. OsSUT1 was also present in the coleoptile and the first and second leaf blades, where it was localized to the phloem along the entire length of these tissues, and was also present within the phloem of the primary roots. OsSUT1 may be involved in retrieval of sugars from the apoplasm in these tissues.     

peg10, an imprinted gene, plays a crucial role in adipocyte differentiation

An imprinted gene, paternally expressed gene (peg) 10, was isolated as one of the genes expressed early in adipogenesis. The expression of peg10 was elevated after the addition of inducers, and was detected in adipocyte differentiable 3T3-L1 cells, but not observed in the non-adipogenic cell line NIH-3T3. Moreover, the knockdown of peg10 by RNA interference (RNAi) inhibited the differentiation of 3T3-L1 cells into lipid-laden adipocytes. Interestingly, peg10 RNAi-treatment reduced the expressions of C/EBPbeta and C/EBPdelta, and inhibited mitotic clonal expansion. These findings strongly indicate that peg10 plays a crucial role at the immediate early stage of adipocyte differentiation.     

Inferring the evolutionary history of vibrios by means of multilocus sequence analysis

We performed the first broad study aiming at the reconstruction of the evolutionary history of vibrios by means of multilocus sequence analysis of nine genes. Overall, 14 distinct clades were recognized using the SplitsTree decomposition method. Some of these clades may correspond to families, e.g., the clades Salinivibrio and Photobacteria, while other clades, e.g., Splendidus and Harveyi, correspond to genera. The common ancestor of all vibrios was estimated to have been present 600 million years ago. We can define species of vibrios as groups of strains that share >95% gene sequence similarity and >99.4% amino acid identity based on the eight protein-coding housekeeping genes. The gene sequence data were used to refine the standard online electronic taxonomic scheme for vibrios (http://www.taxvibrio.lncc.br).     

Gemin2 plays an important role in stabilizing the survival of motor neuron complex

The survival of motor neuron (SMN) protein, responsible for the neurodegenerative disease spinal muscular atrophy (SMA), oligomerizes and forms a stable complex with seven other major components, the Gemin proteins. Besides the SMN protein, Gemin2 is a core protein that is essential for the formation of the SMN complex, although the mechanism by which it drives formation is unclear. We have found a novel interaction, a Gemin2 self-association, using the mammalian two-hybrid system and the in vitro pull-down assays. Using in vitro dissociation assays, we also found that the self-interaction of the amino-terminal SMN protein, which was confirmed in this study, became stable in the presence of Gemin2. In addition, Gemin2 knockdown using small interference RNA treatment revealed a drastic decrease in SMN oligomer formation and in the assembly activity of spliceosomal small nuclear ribonucleoprotein (snRNP). Taken together, these results indicate that Gemin2 plays an important role in snRNP assembly through the stabilization of the SMN oligomer/complex via novel self-interaction. Applying the results/techniques to amino-terminal SMN missense mutants that were recently identified from SMA patients, we successfully showed that amino-terminal self-association, Gemin2 binding, the stabilization effect of Gemin2, and snRNP assembly activity were all lowered in the mutant SMN(D44V), suggesting that instability of the amino-terminal SMN self-association may cause SMA in patients carrying this allele.     

Cyclooxygenase-2 is required for activated pancreatic stellate cells to respond to proinflammatory cytokines

Cyclooxygenase-2 (COX-2) mediates various inflammatory responses and is expressed in pancreatic tissue from patients with chronic pancreatitis. To examine the role of COX-2 in chronic pancreatitis, we investigated its participation in regulating functions of pancreatic stellate cells (PSCs), using isolated rat PSCs. COX-2 was expressed in culture-activated PSCs but not in freshly isolated quiescent PSCs. TGF-1, IL-1, and IL-6 enhanced COX-2 expression in activated PSCs, concomitantly increasing the expression of -smooth muscle actin (-SMA), a parameter of PSC activation. The COX-2 inhibitor NS-398 blocked culture activation of freshly isolated quiescent PSCs. NS-398 also inhibited the enhancement of -SMA expression by TGF-1, IL-1, and IL-6 in activated PSCs. These data indicate that COX-2 is required for the initiation and promotion of PSC activation. We further investigated the mechanism by which cytokines enhance COX-2 expression in PSCs. Adenovirus-mediated expression of dominant negative Smad2/3 inhibited the increase in expression of COX-2, -SMA, and collagen-1 mediated by TGF-1 in activated PSCs. Moreover, dominant negative Smad2/3 expression attenuated the expression of COX-2 and -SMA enhanced by IL-1 and IL-6. Anti-TGF- neutralizing antibody also attenuated the increase in COX-2 and -SMA expression caused by IL-1 and IL-6. IL-6 as well as IL-1 enhanced TGF-1 secretion from PSCs. These data indicate that Smad2/3-dependent pathway plays a central role in COX-2 induction by TGF-1, IL-1, and IL-6. Furthermore, IL-1 and IL-6 promote PSC activation by enhancing COX-2 expression indirectly through Smad2/3-dependent pathway by increasing TGF-1 secretion from PSCs. transforming growth factor-; interleukin; Smad; autocrine; pancreatic fibrosis     

Development of yeast cells displaying <Emphasis Type="Italic">Candida antarctica</Emphasis> lipase B and their application to ester synthesis reaction

We isolated the lipase B from Candida antarctica CBS 6678 (CALB CBS6678) and successfully constructed CALB-displaying yeast whole-cell biocatalysts using the Flo1p short (FS) anchor system. For the display of CALB on a yeast cell surface, the newly isolated CALB CBS6678 exhibited higher hydrolytic and ester synthesis activities than the well-known CALB, which is registered in GenBank (Z30645). A protease accessibility assay using papain as a protease showed that a large part of CALB, approximately 75%, was localized on an easily accessible part of the yeast cell surface. A comparison of the lipase hydrolytic activities of yeast whole cells displaying only mature CALB (CALB) and those displaying mature CALB with a Pro region (ProCALB) revealed that mature CALB is preferable for yeast cell surface display using the Flo1p anchor system. Lyophilized yeast whole cells displaying CALB were applied to an ester synthesis reaction at 60°C using adipic acid and n-butanol as substrates. The amount of dibutyl adipate (DBA) produced increased with the reaction time until 144 h. This indicated that CALB displayed on the yeast cell surface retained activity under the reaction conditions.     

Adipocytes and preadipocytes promote the proliferation of colon cancer cells in vitro

Obesity, a risk factor for colon cancer, is associated with elevated serum levels of leptin, a protein produced by adipocytes. The aim of the present study was to clarify the effects of adipose tissue on colon cancer proliferation by using cultured cell lines. To achieve this, colon cancer cells (CACO-2, T84, and HT29) were cocultured with adipose tissue, isolated mature adipocytes, and isolated preadipocytes in a three-dimensional collagen gel culture system. The adipocytes and preadipocytes used were isolated from C57BL/6J and leptin-deficient ob/ob mice. Proliferation of the cancer cells was evaluated by nuclear bromodeoxyuridine uptake. The adipose tissue, mature adipocytes, and preadipocytes isolated from C57BL/6J mice significantly increased the proliferation of the colon cancer cells. This trophic effect of mature adipocytes on the cancer cell lines was observed only for cells from lean littermates and not for those from ob/ob mice. In contrast, the trophic effect of preadipocytes was not abolished in ob/ob mice, and this finding was supported by the result that leptin had a trophic effect on cancer cells. In conclusion, adipocytes were able to enhance the proliferation of colon cancer cells in vitro, partly via leptin, suggesting that adipose tissues, including mature adipocytes and preadipocytes, may promote the growth of colorectal cancer.     

Mutation and polymorphism analyses of INSL3 and LGR8/GREAT in 62 Japanese patients with cryptorchidism

BACKGROUND/AIMS: Although insulin-like factor 3 (INSL3) and its receptor leucine-rich repeat-containing G protein-coupled receptor 8/G protein-coupled receptor affecting testis descent (LGR8/GREAT) are essential for the gubernacular development, mutations of INSL3 and LGR8/GREAT are infrequent in patients with cryptorchidism (CO), and there is no report documenting a positive association of CO with a polymorphism in INSL3 or LGR8/GREAT. Here, we further examined the relevance of INSL3 and LGR8/GREAT mutations and polymorphisms to the development of CO. METHODS: Sixty-two Japanese CO patients and 60 fertile males were studied. INSL3 was analyzed by direct sequencing and restriction enzyme digestion, and LGR8/GREAT was examined by denaturing high-performance liquid chromatography followed by direct sequencing for exons with abnormal chromatogram patterns. RESULTS: No definitive mutation was identified in both genes. Six polymorphisms were detected in INSL3 or LGR8/GREAT and Thr/Thr genotype of Ala60Thr polymorphism in INSL3 was strongly associated with CO (p=0.0024, odds ratio=5.3, 95% confidence interval=1.7-17). CONCLUSION: The results, in conjunction with the previous data, suggest that mutations of INSL3 and LGR8/GREAT remain rare, and that the Thr/Thr genotype of Ala60Thr polymorphism in INSL3 may constitute a susceptibility factor for the development of CO.     

Insulin-induced impairment via peroxynitrite production of endothelium-dependent relaxation and sarco/endoplasmic reticulum Ca(2+)-ATPase function in aortas from diabetic rats

We designed this study to determine whether a high insulin level and a diabetic state need to exist together to cause an impairment of endothelium-dependent relaxation. In diabetic rat aortas organ-cultured with insulin [vs both control rat aortas cultured with insulin and diabetic rat aortas cultured in serum-free medium]: (1) the relaxation responses to both acetylcholine (endothelium-dependent relaxation) and Angeli's salt (nitric oxide donor) were significantly weaker, (2) acetylcholine-stimulated nitric oxide production was significantly smaller, (3) superoxide and nitric oxide production into the culture medium was greater, and (4) the levels of both nitrotyrosine and tyrosine-nitrated sarco/endoplasmic reticulum calcium ATPase (SERCA) protein were greater. The insulin-induced effects were prevented by cotreatment with either a superoxide scavenger or a peroxynitrite scavenger. After preincubation with an irreversible SERCA inhibitor, the relaxation induced by the nitric oxide donor was significantly impaired in control aortas cultured with or without insulin and in diabetic aortas cultured without insulin, but not in diabetic aortas cultured with insulin. These results suggest that the coexistence of a high insulin level and an established diabetic state may lead to an excessive generation of peroxynitrite, and that this may in turn trigger an impairment of endothelium-dependent relaxation via a decrease in SERCA function.     

A peptide-based immunoassay for antibodies against botulinum neurotoxin A

Cervical dystonia (CD) is due to neck-muscle spasms that cause pain and involuntary contractions resulting in abnormal neck movements and posture. Symptoms can be relieved by injecting the affected muscle with a botulinum neurotoxin (BoNT, usually type A or type B). The therapeutic benefits are impermanent and toxin injections need to be repeated every 3-6 months. In a very small percentage of patients (less with BoNT/A than with BoNT/B) the treatment elicits blocking anti-toxin antibodies (Abs), which reduce or terminate the patient's responsiveness to further treatment. We have recently mapped (Dolimbek et al., 2006) the CD sera Ab-binding profile using a panel of 60, 19-residue peptides that encompassed the entire H chain sequence 449-1296 and overlapped consecutively by 5 residues. Abs in CD sera bound to one or more of the peptides N25, C10, C15, C20, and C31. This suggested the possibility that binding to these peptides could be used for assay of Abs in CD sera. Data analysis reported here found that Ab binding to these regions showed very significant deviations from the control responses. Of these four peptides, C10 showed the most significant level of separation between patient and control groups (p = 5 x 10(-7)) and the theoretical resolution (i.e., ability to distinguish CD patients from control, see full definition under 'Statistical analysis' in Methods), 84%, was about 4% higher than the least resolved response, C31 (p = 6 x 10(-6), resolution 80%). Since the amounts of Abs bound to a given peptide varied with the patient and not all the patients necessarily recognized all four peptides, there was the possibility that binding to combinations of two or more peptides might give a better discriminatory capability. Using two peptides, C10 plus C31, the resolution improved to 87% (p = 4 x 10(-8)). These two peptides appeared to compliment each other and negate the lower resolution of C31. Combination of three peptides gave resolutions that ranged from 85 (N25 + C15 + C31; p = 2 x 10(-7)) to 88% (C10 + C15 + C31; p = 1 x 10(-8)). Finally, using the data of all four peptides, N25 + C10 + C15 + C31, gave a resolution of 86% (p = 1 x 10(-7)). Although these levels of resolution are somewhat lower than that obtained with whole BoNT/A (resolution 97%; p = 6 x 10(-12)), it may be concluded that the two-peptide combination C10 + C31, or the three-peptide combination C10 + C15 + C31 (affording resolutions of 87 and 88%, respectively) provide a good diagnostic, toxin-free procedure for assay of total specific anti-toxin Abs in BoNT/A-treated CD patients.