Cloning and physical mapping of the dnaA region of the Escherichia coli chromosome.

The dnaA gene of Escherichia coli K-12, supposedly present in the deoxyribonucleic acid (DNA) of specialized transducing phase lambda i21 dnaA-2, was cloned onto plasmid pBR322. The new plasmid was named pMCR501. Physical analyses of DNAs of lambda i21 dnaA-2 and pMCR501 revealed the following. The lambda i21 dnaA-2 DNA retained the delta sr I lambda 1-2 and ninR5 deletions and imm21 substitution which were originally present in the parental phage. The size reduction was compensated for by the insertion-substitution segment (tna-dnaA region) in lambda i21 dnaA-2 DNA. The fractional size of this segment was approximately 7 megadaltons (Md), or 10 kilobases, which was found to be the sum of the tna insertion subsegment of ca. 3.5 Md and the dnaA substitution subsegment of ca. 3.5 Md. Phage P1-mediated transductional mapping between the dnaA46 and tna mutations gave a cotransduction frequency of 84%, corresponding to approximately 5 kilobases. Thus, it is strongly suggested that the dnaA gene resides in the lambda i21 dnaA-2 DNA. Cleavage mapping with the restriction endonuclease of pMCR501 DNA confirmed that it was constructed by excising a BamHI fragment of 4.29 Md, containing the 3.5-Md dnaA substitution segment, from the lambda i21 dnaA-2 DNA, inserting it into the sole BamHI cleavage site on pBR322.     

Electron transport chain from glycerol 3-phosphate to nitrate in Escherichia coli.

It is known that in Escherichia coli two dehydrogenases of the flavoprotein kind can participate in the transfer of hydrogens from sn-glycerol 3-phosphate (G3P) to nitrate and that possession of either enzyme is sufficient to permit anaerobic growth on glycerol as carbon source and nitrate as hydrogen acceptor. Results from this study show that under such a growth condition a protein with light-absorption characteristics of cytochrome b1 is induced. If G3P, nitrate, and adenosine diphosphate are added anaerobically to a particulate fraction prepared from these cells, four reactions can be detected: (i) the reduction of the cytochrome b1-like protein, (ii) the formation of dihydroxyacetone phosphate (DHAP), (iii) the formation of nitrite, and (iv) the generation of adenosine 5'-triphosphate (ATP). The anaerobic G3P dehydrogenase system can yield an ATP-DHAP (or ATP-nitrite) molar ratio of about 0.2, whereas the aerobic G3P dehydrogenase system can yield a corresponding ratio of about 0.3. The hydrogen transfer activity is sensitive to respiratory inhibitors such as cyanide, Rotenone, and 2-heptyl-4-hydroxyquinoline-N-oxide.     

Electron-microscopic studies on developing intramural ganglia of the small intestine in human and rabbit fetuses.

Electron-microscopic studies were made on the appearance of synapses in the intramural ganglion (Auerbach) and findings were correlated with the onset and development of intestinal peristalsis in 6- to 30-week-old human and rabbit fetuses from the 12th day after conception until birth. At stage I, in which the small intestine shows no indication of a muscle layer or spontaneous peristalsis, primitive synapses containing several clear vesicles and a few cored vesicles are seen on neuroblasts and their processes (dendrites). At stage II, in which the circular muscle is developed and bidirectional peristalsis occurs, synaptic profiles can be classified into 3 types. Type 1 is the most numerous but seldom shows membrane specificity on the synaptic portion. Types 2 and 3 have small flattened vesicles and small round vesicles, respectively. They are further characterized by thickening of snyaptic membranes and aggregation of small clear vesicles associated with the presynaptic membrane. At stage III, the longitudinal muscle layer develops in the small intestine. At this stage, nerve terminals containing mainly cored vesicles have been observed and classified into types 4 and 5, according to their morphology. At stage IV, antiperistalsis no longer occurs and type 6 nerve terminals in the intramural ganglia can be recognized by their densely packed, large-cored vesicles. The possible physiological significance of the nerve terminals has been discussed.     

Differences in the stemness characteristics and molecular markers of distinct human oral tissue neural crest‐derived multilineage cells

ObjectivesAlthough multilineage cells derived from oral tissues, especially the dental pulp, apical papilla, periodontal ligament, and oral mucosa, have neural crest‐derived stem cell (NCSC)‐like properties, the differences in the characteristics of these progenitor cell compartments remain unknown. The current study aimed to elucidate these differences.Material and methodsSphere‐forming apical papilla‐derived cells (APDCs), periodontal ligament‐derived cells (PDLDCs), and oral mucosa stroma‐derived cells (OMSDCs) from the same individuals were isolated from impacted developing teeth. All sphere‐forming cells were characterized through biological analyses of stem cells.ResultsAll sphere‐forming cells expressed neural crest‐related markers. The expression of certain tissue‐specific markers such as CD24 and CD56 (NCAM1) differed among tissue‐derived cells. Surprisingly, the expression of only CD24 and CD56 could be discriminated in human tissues. Although APDCs and PDLDCs exhibited greater mineralized cell differentiation than OMSDCs, they exhibited poorer differentiation into adipocytes in vitro. In immunocompromised mice, APDCs formed hard tissues better than PDLDCs and OMSDCs.ConclusionsAlthough cells with NCSC‐like properties present the same phenotype, they differ in the expression of certain markers and differentiation abilities. This study is the first to demonstrate the differences in the differentiation ability and molecular markers among multilineage human APDCs, PDLDCs, and OMSDCs obtained from the same patients, and to identify tissue‐specific markers that distinguish tissues in the developing stage of the human tooth with immature apex.

This study illustrates that neural crest‐derived cells from distinct oral tissues, namely the apical papilla, periodontal ligament, and oral mucosa, have varying differentiation potential, and tissue‐derived cell‐specific molecular markers have been identified. CD24 and NCAM1/CD56 expression were found to differ among multilineage oral tissue‐derived cells, similar to our observation in human tissue.     

Toxicity of free proline revealed in an arabidopsis T-DNA-tagged mutant deficient in proline dehydrogenase

The toxicity of proline (Pro) to plant growth has raised questions despite its protective functions in response to environmental stresses. To evaluate Pro toxicity, we isolated an Arabidopsis T-DNA-tagged mutant, pdh, that had a defect in Pro dehydrogenase (AtProDH), which catalyzes the first step of Pro catabolism. The pdh mutant showed hypersensitivity to exogenous application of < or =10 mM L-Pro, at which wild-type plants grew normally. A dose-dependent increase in internal free Pro accumulation was observed in pdh plants during external Pro supply. These results do not just prove the toxicity of Pro, but also suggest that AtProDH is the only enzyme acting as a functional ProDH in ARABIDOPSIS: To further analyze the targets of Pro toxicity, we compared the expression of thousands of genes by pdh plants with that by wild-type plants by cDNA microarray analysis. Most genes were unaffected. Here we demonstrate Pro toxicity by using the pdh mutant and discuss a cause-and-effect action between an excess of free Pro and growth inhibition in ARABIDOPSIS.     

Involvement of gicerin, a cell adhesion molecule, in development and regeneration of oviduct and metastasis of oviductal adenocarcinomas of the chicken

Gicerin is a novel cell adhesion molecule in the immunoglobulin superfamily and has both homophilic adhesion and heterophilic adhesive activity to neurite outgrowth factor (NOF), an extracellular matrix protein in the laminin family. We investigated the possible involvement of gicerin in oviductal development, regeneration, and metastasis of oviductal adenocarcinomas of the chicken. In the oviductal epithelium, gicerin was expressed strongly during development, disappeared after maturation, and reappeared during regeneration. NOF was constitutively expressed in the basement membrane of the epithelium. These molecules were expressed strongly in oviductal adenocarcinomas in both primary and metastatic lesions in the mesentery. An anti-gicerin antibody inhibited the attachment of adenocarcinoma cells to the mesentery in vitro. Many cells migrated from adenocarcinoma tissues on NOF, which were inhibited by an anti-gicerin antibody. These results suggest that gicerin might play a role in oviductal development and regeneration and also in the metastasis of adenocarcinomas.     

Towards genomic and proteomic studies of protein phosphorylation in plant-pathogen interactions

Phosphorylation is an effective method of post-translational protein modification but understanding its significance is hindered by its biological complexity. Many protein kinases and phosphatases have been identified that connect signal perception mechanisms to plant defence responses. Recent studies of mitogen-activated protein kinases, calcium-dependent protein kinases and other kinases and phosphatases have revealed some important mechanisms, but have also raised new questions. The regulation of any phosphorylation pathway is complex and dynamic. There are many protein kinases and phosphatases in the plant genome, which makes it hard to delineate the phosphorylation machinery fully. Genomics and proteomics have already identified new components and will continue to influence the study of phosphorylation profoundly in plant-pathogen interactions.     

Induction of the differentiation of human HL-60 promyelocytic leukemia cell line by succinoyl trehalose lipids

Four analogs of succinoyl trehalose lipid-3 (STL-3)with saturated even-number or odd-number carbonchains, and unsaturated or halogenated fatty acidswere examined for their ability to inhibit the growthand induce the differentiation of HL-60 humanpromyelocytic leukemia cells. The optimalconcentration of STL-3 at which such activities wererecognized was closed to the critical micelleconcentration of STL-3. Analog of STL-3 witheven-number or odd-number carbon chain and unsaturatedfatty acids strongly inhibited growth and induced thedifferentiation of HL-60 cells, as evaluated in termsof nitroblue tetrazilium-reducing activity and theappearance of the CD36 antigen. An analog of STL-3with halogenated fatty acids significantly inhibitedproliferation but only induced the differentiation ofHL-60 cells. Our results indicate that the effects ofSTL-3 and its analogs on HL-60 cells depend on thestructure of the hydrophobic moiety of STL-3.These authors contributed equally to this work