Homologies of the adductor mandibulae muscles in Tetraodontiformes as indicated by nerve branching patterns

Homologies of the adductor mandibulae muscles in eight families of Tetraodontiformes were hypothesized from the branching patterns of ramus mandibularis trigeminus. Insertions of the muscles to the upper or lower jaw were weak indicators of homology, migrations of the sites occurring frequently in A1, A2, A2, and A3. In monacanthids, tetraodontids, and diodontids, A1 tended to be split into numerous subsections, whereas in aracanids and ostraciids, A3 was highly developed, comprising three or four subsections. In tetraodontids, A2 was found to be a composite of A1 subsection and A2. The methods of and limits to applying nerve branching patterns to muscle homology are discussed. A new naming system that reflects both muscle homologies and insertions is proposed.     

CONSTITUTIVE PHOTOMORPHOGENIC 1 promotes seed germination by destabilizing RGA-LIKE 2 in Arabidopsis

Under favorable moisture, temperature, and light conditions, gibberellin (GA) biosynthesis is induced and triggers seed germination. A major mechanism by which GA promotes seed germination is by promoting the degradation of the DELLA protein RGA-LIKE 2 (RGL2), a major repressor of germination in Arabidopsis (Arabidopsis thaliana) seeds. Analysis of seed germination phenotypes of constitutive photomorphogenic 1 (cop1) mutants and complemented COP1-OX/cop1-4 lines in response to GA and paclobutrazol (PAC) suggested a positive role for COP1 in seed germination and a relation with GA signaling. cop1-4 mutant seeds showed PAC hypersensitivity, but transformation with a COP1 overexpression construct rendered them PAC insensitive, with a phenotype similar to that of rgl2 mutant (rgl2-SK54) seeds. Furthermore, cop1-4 rgl2-SK54 double mutants showed a PAC-insensitive germination phenotype like that of rgl2-SK54, identifying COP1 as an upstream negative regulator of RGL2. COP1 interacted directly with RGL2, and in vivo this interaction was strongly enhanced by SUPPRESSOR OF PHYA-105 1. COP1 directly ubiquitinated RGL2 to promote its degradation. Moreover, GA stabilized COP1 with consequent RGL2 destabilization. By uncovering this COP1–RGL2 regulatory module, we reveal a mechanism whereby COP1 positively regulates seed germination and controls the expression of germination-promoting genes.

A master regulator of photomorphogenesis positively regulates germination in Arabidopsis seeds by directly ubiquitinating and promoting the degradation of a key repressor of seed germination.     

In Vitro Inflammation Inhibition Model Based on Semi-Continuous Toll-Like Receptor Biosensing

A chemical inhibition model of inflammation is proposed by semi-continuous monitoring the density of toll-like receptor 1 (TLR1) expressed on mammalian cells following bacterial infection to investigate an in vivo-mimicked drug screening system. The inflammation was induced by adding bacterial lysate (e.g., Pseudomonas aeruginosa) to a mammalian cell culture (e.g., A549 cell line). The TLR1 density on the same cells was immunochemically monitored up to three cycles under optimized cyclic bacterial stimulation-and-restoration conditions. The assay was carried out by adopting a cell-compatible immunoanalytical procedure and signal generation method. Signal intensity relative to the background control obtained without stimulation was employed to plot the standard curve for inflammation. To suppress the inflammatory response, sodium salicylate, which inhibits nuclear factor-κB activity, was used to prepare the standard curve for anti-inflammation. Such measurement of differential TLR densities was used as a biosensing approach discriminating the anti-inflammatory substance from the non-effector, which was simulated by using caffeic acid phenethyl ester and acetaminophen as the two components, respectively. As the same cells exposed to repetitive bacterial stimulation were semi-continuously monitored, the efficacy and toxicity of the inhibitors may further be determined regarding persistency against time. Therefore, this semi-continuous biosensing model could be appropriate as a substitute for animal-based experimentation during drug screening prior to pre-clinical tests.     

Immunity to melanoma mediated by 4-1BB is associated with enhanced activity of tumour-infiltrating lymphocytes

4-1BB costimulates T cells to carry out effector functions such as eradication of established tumours. 4-1BB (CD137) is a member of the TNF receptor family, and its triggering by either 4-1BB ligand or antibody ligation induces T-cell activation and growth. We analysed tumour-infiltrating lymphocytes (TIL) in the experimental B16F10 melanoma model to determine the mechanisms involved in 4-1BB-mediated tumour suppression. 4-1BB(+/+) mice survived longer than 4-1BB(-/-) mice, and survival was further prolonged by triggering 4-1BB with an agonistic mAb. The number of metastatic B16F10 colonies in the lung was much greater in 4-1BB(-/-) mice than in their 4-1BB(+/+) littermates. Administration of agonistic anti-4-1BB mAb increased the number of TIL in the tumour masses in the lungs of 4-1BB(+/+) mice. The numbers of CD4(+) T, CD8(+) T and CD11b(+) TIL increased in these mice. Anti-4-1BB mAb induced not only CD8(+) 4-1BB(+) T cells but also a CD8(+) IFN-gamma(+) T-cell population. B16F10 cells from the lungs of anti-4-1BB-treated mice showed enhanced expression of MHC class Iota and IotaIota antigens compared with the same cells from control IgG-treated mice. Thus, the increase in number of CD8(+) T cells and enhanced MHC Iota and IotaIota expression in B16F10 cells that result from augmented IFN-gamma production in response to anti-4-1BB mAb may lead to suppression of tumour growth and metastasis.     

Identification of cellular proteins enhancing activities of internal ribosomal entry sites by competition with oligodeoxynucleotides

The translation of numerous eukaryotic mRNAs is mediated by internal ribosomal entry sites (IRESs). IRES-dependent translation requires both canonical translation initiation factors and IRES-specific trans-acting factors (ITAFs). Here we report a strategy to identify and characterize ITAFs required for IRES-dependent translation. This process involves steps for identifying oligodeoxynucleotides affecting IRES-dependent translation, purifying proteins interacting with the inhibitory DNA, identifying the specific proteins with matrix-assisted laser desorption ionization/time-of-flight mass spectrometry, and confirming the roles of these proteins in IRES-dependent translation by depletion and repletion of proteins from an in vitro translation system. Using this strategy, we show that poly(rC)-binding proteins 1 and 2 enhance translation through polioviral and rhinoviral IRES elements.     

Satellite-RNA-mediated resistance to cucumber mosaic virus in transgenic plants of hot pepper (Capsicum annuum cv. Golden Tower)

We previously established a system of in vitro regeneration and Agrobacterium-mediated transformation for hot pepper plants. The level of protection against cucumber mosaic virus in the progeny of the transgenic hot pepper plants that express cucumber mosaic virus (CMV) satellite RNA was investigated. The transgenic hot pepper plants were self-fertilized, and their progeny were tested for stable inheritance and expression of the cDNA of CMV satellite RNA. Polymerase chain reaction and RNA gel blot analyses showed that the introduced gene was stably transmitted and expressed in the progeny. Symptom attenuation in the offspring was confirmed upon inoculation with CMV-Y or CMV-Korea (CMV-Kor) strains. Received: 30 September 1996 / Revision received: 5 May 1997 / Accepted: 22 May 1997     

Escherichia coli dnaK null mutants are inviable at high temperature.

DnaK, a major Escherichia coli heat shock protein, is homologous to major heat shock proteins (Hsp70s) of Drosophila melanogaster and humans. Null mutations of the dnaK gene, both insertions and a deletion, were constructed in vitro and substituted for dnaK+ in the E. coli genome by homologous recombination in a recB recC sbcB strain. Cells carrying these dnaK null mutations grew slowly at low temperatures (30 and 37 degrees C) and could not form colonies at a high temperature (42 degrees C); furthermore, they also formed long filaments at 42 degrees C. The shift of the mutants to a high temperature evidently resulted in a loss of cell viability rather than simply an inhibition of growth since cells that had been incubated at 42 degrees C for 2 h were no longer capable of forming colonies at 30 degrees C. The introduction of a plasmid carrying the dnaK+ gene into these mutants restored normal cell growth and cell division at 42 degrees C. These null mutants showed a high basal level of synthesis of heat shock proteins except for DnaK, which was completely absent. In addition, the synthesis of heat shock proteins after induction in these dnaK null mutants was prolonged compared with that in a dnaK+ strain. The well-characterized dnaK756 mutation causes similar phenotypes, suggesting that they are caused by a loss rather than an alteration of DnaK function. The filamentation observed when dnaK mutations were incubated at a high temperature was not suppressed by sulA or sulB mutations, which suppress SOS-induced filamentation.(ABSTRACT TRUNCATED AT 250 WORDS)     

A scaffoldless technique for self-generation of three-dimensional keratinospheroids on liquid crystal surfaces

We describe a new scaffold-free three-dimensional (3D) cell culture model using cholesteryl ester based lyotropic liquid crystal (LC) substrates. Keratinocytes were deposited randomly on the LC surface where they self-assembled into 3D microtissues or keratinospheroids. The cell density required to form spheroids was optimized. We investigated cell viability using dead/live cell assays. The adhesion characteristics of cells within the microtissues were determined using histological sectioning and immunofluorescence staining. Fourier transform infrared spectroscopy (FTIR) was used to characterize the biochemistry of the keratinospheroids. We found that both cells and microtissues could migrate on the LC surface. The viability study indicated approximately 80% viability of cells in the microtissues up to 20 days of culture. Strong intercellular adhesion was observed in the stratification of the multi-layered microspheroids using field emission-scanning electron microscopy (FE-SEM) and histochemical staining. The cytoskeleton and vinculins of the cells in the microtissues were expressed diffusely, but the microtissues were enriched with lipids and nucleic acids, which indicates close resemblance to the conditions in vivo. The basic 3D culture model based on LC may be used for cell and microtissue migration studies in response to cytochemical treatment.