The Tol2-mediated Gal4-UAS method for gene and enhancer trapping in zebrafish

The Gal4-UAS system provides powerful tools to analyze the function of genes and cells in vivo and has been extensively employed in Drosophila. The usefulness of this approach relies on the P element-mediated Gal4 enhancer trapping, which can efficiently generate transgenic fly lines expressing Gal4 in specific cells. Similar approaches, however, had not been developed in vertebrate systems due to the lack of an efficient transgenesis method. We have been developing transposon techniques by using the madaka fish Tol2 element. Taking advantage of its ability to generate genome-wide insertions, we developed the Gal4 gene trap and enhancer trap methods in zebrafish that enabled us to create various transgenic fish expressing Gal4 in specific cells. The Gal4-expressing cells can be visualized and manipulated in vivo by crossing the transgenic Gal4 lines with transgenic lines carrying various reporter and effector genes downstream of UAS (upstream activating sequence). Thus, the Gal4 gene trap and enhancer trap methods together with UAS lines now make detailed analyses of genes and cells in zebrafish feasible. Here, we describe the protocols to perform Gal4 gene trap and enhancer trap screens in zebrafish and their application to the studies of vertebrate neural circuits.     

Phylogenetic diversity of non-nodulating Rhizobium associated with pine ectomycorrhizae

Most Rhizobium species described are symbionts that form nodules on legume roots; however, non-nodulating strains of Rhizobium are also widespread in nature. Unfortunately, knowledge of non-nodulating Rhizobium is quite limited compared with nodulating Rhizobium . Here, we studied the phylogenetic diversity of Rhizobium species that inhabit Japanese red pine roots ( Pinus densiflora ). Because fine roots of pine trees are usually colonized by ectomycorrhizal fungi in nature, we mainly used ectomycorrhizal root tips for bacterial isolation. Out of 1195 bacteria isolated from 75 independent root samples from the field and greenhouse experiments, 102 isolates were confirmed to be Rhizobium following partial 16S rRNA gene analysis. Rhizobium species were occasionally dominant in culturable bacterial communities, whereas no Rhizobium species were isolated from the soil itself. Molecular phylogenetic analyses using 16S rRNA, atpD , and recA gene sequences revealed that isolated Rhizobium strains were phylogenetically diverse and that several were distantly related to known Rhizobium species. Considering that a single species of pine is associated with unique and phylogenetically diverse Rhizobium populations, we should pay more attention to non-nodulating strains to better understand the diversity, ecology, and evolution of the genus Rhizobium and plant– Rhizobium associations.     

Can Experience Improve Hospital Management?

Background

Experience curve effects were first observed in the industrial arena as demonstrations of the relationship between experience and efficiency. These relationships were largely determined by improvements in management efficiency and quality of care. In the health care industry, volume-outcome relationships have been established with respect to quality of care improvement, but little is known about the effects of experience on management efficiency. Here, we examine the relationship between experience and hospital management in Japanese hospitals.

Methods

The study sample comprised individuals who had undergone surgery for unruptured abdominal aortic aneurysms and had been discharged from participant hospitals between April 1, 2006 and December 31, 2008. We analyzed the association between case volume (both at the hospital and surgeon level) and postoperative complications using multilevel logistic regression analysis. Multilevel log-linear regression analyses were performed to investigate the associations between case volume and length of stay (LOS) before and after surgery.

Results

We analyzed 909 patients and 849 patients using the hospital-level and surgeon-level analytical models, respectively. The odds ratio of postoperative complication occurrence for an increase of one surgery annually was 0.981 (P<0.001) at the hospital level and 0.982 (P<0.001) at the surgeon level. The log-linear regression analyses showed that shorter postoperative LOS was significantly associated with high hospital-level case volume (coefficient for an increase of one surgery: −0.006, P = 0.009) and surgeon-level case volume (coefficient for an increase of one surgery: −0.011, P = 0.022). Although an increase of one surgery annually at the hospital level was statistically associated with a reduction of preoperative LOS by 1.1% (P = 0.006), there was no significant association detected between surgeon-level case volume and preoperative LOS (P = 0.504).

Conclusion

Experience at the hospital level may contribute to the improvement of hospital management efficiency.     

The Golgi apparatus of goblet cells in the mouse descending colon: three-dimensional visualization using a confocal laser scanning microscope

The three-dimensional structure of the Golgi apparatus was studied in goblet cells in lectin-stained sections of the mouse descending colon by using a confocal laser scanning microscope. In the lower part of the crypt, the Golgi apparatus formed a dome- or globe-like structure in the supranuclear region. The wall of the dome had some holes, one of which usually faced toward the nucleus and others toward the apical cytoplasm. Mucous granules seemed to be initially released into the interior of the dome and transported toward the apical cytoplasm through the holes. In the upper part of the crypt, on the other hand, the Golgi apparatus formed a cup- or funnel-like structure with a larger opening toward the cell apex and a smaller opening toward the nucleus. A large mass of mucous granules occupied the inside of the cup to the apical cytoplasm. It is thought that the accumulation of mucous granules enlarges holes at the ceiling of the dome to form a large opening, which makes the configuration of the Golgi apparatus cup-shaped.     

The linkage between β1 integrin and the actin cytoskeleton is differentially regulated by tyrosine and serine/threonine phosphorylation of β1 integrin in normal and cancerous human breast cells

Background  

Structural requirements for the β1 integrin functions in cell adhesion, spreading and signaling have been well documented mainly for fibroblasts. In this study, we examined the reason for the reduced surface expression of β1 integrin in human breast cancer MCF-7 cells compared to normal human breast epithelial (HBE) cells, both of which adhered to collagen type IV.     

Decrease in bacterial production over the past three decades in the north basin of Lake Biwa,Japan

Limnology - In Lake Biwa, the largest lake in Japan, external pollutant loads have decreased since the 1980s, leading to improved water quality, such as reduction in biochemical oxygen demand (BOD)...     

Cloning of a Sphingomonas paucimobilis SYK-6 Gene Encoding a Novel Oxygenase That Cleaves Lignin-Related Biphenyl and Characterization of the Enzyme

Sphingomonas paucimobilis SYK-6 transforms 2,2′-dihydroxy-3,3′-dimethoxy-5,5′-dicarboxybiphenyl (DDVA), a lignin-related biphenyl compound, to 5-carboxyvanillic acid via 2,2′,3-trihydroxy-3′-methoxy-5,5′-dicarboxybiphenyl (OH-DDVA) as an intermediate (15). The ring fission of OH-DDVA is an essential step in the DDVA degradative pathway. A 15-kb EcoRI fragment isolated from the cosmid library complemented the growth deficiency of a mutant on OH-DDVA. Subcloning and deletion analysis showed that a 1.4-kb DNA fragment included the gene responsible for the ring fission of OH-DDVA. An open reading frame encoding 334 amino acids was identified and designated ligZ. The deduced amino acid sequence of LigZ had 18 to 21% identity with the class III extradiol dioxygenase family, including the β subunit (LigB) of protocatechuate 4,5-dioxygenase of SYK-6 (Y. Noda, S. Nishikawa, K.-I. Shiozuka, H. Kadokura, H. Nakajima, K. Yano, Y. Katayama, N. Morohoshi, T. Haraguchi, and M. Yamasaki, J. Bacteriol. 172:2704–2709, 1990), catechol 2,3-dioxygenase I (MpcI) of Alcaligenes eutrophus JMP222 (M. Kabisch and P. Fortnagel, Nucleic Acids Res. 18:3405–3406, 1990), the catalytic subunit of the meta-cleavage enzyme (CarBb) for 2′-aminobiphenyl-2,3-diol from Pseudomonas sp. strain CA10 (S. I. Sato, N. Ouchiyama, T. Kimura, H. Nojiri, H. Yamane, and T. Omori, J. Bacteriol. 179:4841–4849, 1997), and 2,3-dihydroxyphenylpropionate 1,2-dioxygenase (MhpB) of Escherichia coli (E. L. Spence, M. Kawamukai, J. Sanvoisin, H. Braven, and T. D. H. Bugg, J. Bacteriol. 178:5249–5256, 1996). The ring fission product formed from OH-DDVA by LigZ developed a yellow color with an absorption maximum at 455 nm, suggesting meta cleavage. Thus, LigZ was concluded to be a ring cleavage extradiol dioxygenase. LigZ activity was detected only for OH-DDVA and 2,2′,3,3′-tetrahydroxy-5,5′-dicarboxybiphenyl and was dependent on the ferrous ion.Lignin is the most common aromatic compound in the biosphere, and the degradation of lignin is a significant step in the global carbon cycle. Lignin is composed of various intermolecular linkages between phenylpropanes and guaiacyl, syringyl, p-hydroxyphenyl, and biphenyl nuclei (5, 34). Lignin breakdown therefore involves multiple biochemical reactions involving the cleavage of intermonomeric linkages, demethylations, hydroxylations, side-chain modifications, and aromatic ring fission (10, 11, 19, 40).Soil bacteria are known to display ample metabolic versatility toward aromatic substrates. Sphingomonas paucimobilis SYK-6 (formerly Pseudomonas paucimobilis SYK-6) has been isolated with 2,2′-dihydroxy-3,3′-dimethoxy-5,5′-dicarboxybiphenyl (DDVA) as a sole carbon and energy source. This strain can also grow on syringate, 3-O-methylgallic acid (3OMGA), vanillate, and other dimeric lignin compounds, including β-aryl ether, diarylpropane (β-1), and phenylcoumaran (15). Analysis of the metabolic pathway has indicated that the dimeric lignin compounds are degraded to protocatechuate or 3OMGA (15) and that these compounds are cleaved by protocatechuate 4,5-dioxygenase encoded by ligAB (30). Among the dimeric lignin compounds, the degradation of β-aryl ether and the biphenyl structure is the most important, because β-aryl ether is most abundant in lignin (50%) and the biphenyl structure is so stable that its decomposition should be rate limiting in lignin degradation. We have already characterized the β-etherase and Cα-dehydrogenase genes (23–26) (ligFE and ligD, respectively) involved in the degradation of β-aryl ether. In this study, we focused on the genes responsible for the degradation of DDVA in SYK-6.In the proposed DDVA metabolic pathway of S. paucimobilis SYK-6 illustrated in Fig. ​Fig.1A,1A, DDVA is first demethylated to produce the diol compound 2,2′,3-trihydroxy-3′-methoxy-5,5′-dicarboxybiphenyl (OH-DDVA). OH-DDVA is then degraded to 5-carboxyvanillic acid (5-CVA), and this compound is converted to 3OMGA (15). The resulting product is cleaved by protocatechuate 4,5-dioxygenase. A ring cleavage enzyme for OH-DDVA has been thought to be involved in this pathway because the production of 5-CVA from OH-DDVA resembles the formation of benzoic acid from biphenyl by 2,3-dihydroxybiphenyl through the sequential action of a meta cleavage enzyme and a meta-cleavage compound hydrolase (Fig. ​(Fig.1B)1B) (1, 9, 13, 18, 21, 28). Open in a separate windowFIG. 1(A) Proposed metabolic pathway for DDVA by S. paucimobilis SYK-6. (B) Pathway for the conversion of 2,3-dihydroxybiphenyl (2,3-DHBP) to benzoate by the polychlorinated biphenyl-degrading bacteria. The proposed DDVA metabolic pathway follows the previous one (15). Enzymes: LigZ, OH-DDVA oxygenase; LigAB, protocatechuate 4,5-dioxygenase; BphC, 2,3-dihydroxybiphenyl 1,2-dioxygenase; BphD, 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid hydrolase. TCA, tricarboxylic acid.In this study, we isolated the ligZ gene encoding a ring cleavage enzyme for OH-DDVA. The nucleotide sequence of the gene was determined, and the ligZ gene product was characterized.     

Ultrastructural characterization of interstitial cells of Cajal in the rat small intestine using control and Ws/Ws mutant rats

Interstitial cells in the myenteric plexus and the deep muscular plexus of the small intestine of the c-kit mutant rats (Ws/Ws) and their normal siblings (+/+) were studied. c-Kit immunoreactivity was detected in two regions corresponding to the myenteric plexus and the deep muscular plexus in the jejunum of +/+ rats, while no immunoreactivity was detected in Ws/Ws rats. Using electron microscopy, two types of gap junction-forming interstitial cells were found in association with the myenteric plexus in +/+ rats: one type characterized by a typical fibroblastic ultrastructure, and the other characterized by numerous mitochondria and less electron-dense cytoplasm. Since the latter were greatly reduced in Ws/Ws rats, it was suggested that these cells correspond to c-kit-expressing cells, i.e. interstitial cells of Cajal in the myenteric plexus region. In contrast, two types of interstitial cells in the region of the deep muscular plexus were observed with no difference between +/+ and Ws/Ws rats. Probable interstitial cells of Cajal in this region were characterized by a basal lamina and numerous caveolae as well as large gap junctions that interconnect with each other and with the smooth muscle cells. We concluded that interstitial cells of Cajal in the rat intestine are heterogeneous in ultrastructure, c-kit dependency in the cell maturation, and functional role.