Control of growth and differentiation by cyclic AMP in fungi

InSaccharomyces cerivisiae intracellular cAMP mediates environmental signals that regulate cellular metabolism and growth. The studies on the cAMP-requiring mutants and their suppressors in the yeast revealed that cAMP-dependent protein phosphorylation is involved in the G1 phase of the cell cycle, stimulation of the phosphoinositide pathway and the post-meiotic stage of spourlation, and that inhibition of cAMP-dependent protein phosphorylation is required to go into the GO stage of and to induce meiotic division. Growth of some filamentous fungi was observed with significantly reduced levels of cAMP, suggesting that cAMP may not be essential for growth in some species of fungi. Germination of fungal spores, yeast-mycelium dimorphism and hyphal morphogenesis of several species of fungi were affected by cAMP. cAMP was involved in extension of hyphae, formation of hyphal aggregates and fruit body formation. Phosphorylation of cellular proteins is required in these processes, and the nature of these proteins phosphorylated by cAMP-dependent protein kinase is important to the understanding of the role of cAMP for growth and differentistion in fungal cells.     

A field observation on hydrodynamic and thermal environments of a fringing reef at Ishigaki Island under typhoon and normal atmospheric conditions

To investigate the hydrodynamic and thermal environments of a fringing coral reef under possible influences from both inland and offshore, we have conducted a field survey at Shiraho reef on Ishigaki Island, Okinawa, Japan, by deploying 16 moored buoys inside and outside the reef, on which various sensors for continuous measurements of water temperature, salinity, and turbidity concentration were installed. Several bottom-mounted current meters and wave gauges were also deployed. The results show the abrupt decrease and increase of the water temperature during a typhoon, with the resultant temperature being about 1 degree lower than before. The main cause of this abrupt change and the difference between inside and outside the reef in the thermal response to the atmospheric agitation are investigated. For normal atmospheric conditions, the overall characteristics of currents in the coral reef are found to be governed by the dynamic balance among tide, waves, and wind effects. The salinity and turbidity variations near the river mouth and their cause are also investigated. Thermal environment in the reef is examined by a heat-budget analysis, indicating that it is influenced by both the atmospheric conditions and the temperature difference between inside and outside the reef.     

Multinucleated Giant Cell Formation in BHK-21-528 Cell Monolayers Infected with Japanese Encephalitis Viruses1

Formation of prominent multinucleated giant cells (MGC) was observed in monolayers of a clonal line of BHK-21 cells (BHK-21–528) when infected with Japanese encephalitis virus (JEV). MGC were first observed 3 to 4 days after infection and cytopathic changes proceeded thereafter. Formation of MGC is a typical cytopathic change in this clonal cell line. Virus titer in 50% tissue culture infective dose (TCID₅₀) equaled that in 50% MGC-forming dose. Virus titer in TCID₅₀ was approximate to plaque-forming units (PFU) in the same host cells. An ability of JEV to form MGC was maintained at least for six serial passages in BHK-21–528. It was inactivated by heating at 56 C for 3 min. All JEV strains, except an attenuated live vaccine strain, induced formation of MGC in BHK-21–528 cells. Red blood cells of several animal species were not adsorbed to MGC induced by JEV. The MGC-forming ability of JEV was specifically neutralized by anti-JEV serum. By fluorescence antibody technique, the MGC were specifically stained by anti-JEV antibody conjugated with fluorescein isothiocyanate. Immunization of animals with lysates of the MGC resulted in production of antibodies against JEV, but no antibody against other viruses which have been reported to induce MGC formation. From these evidences, it was concluded that JEV induced formation of MGC in BHK-21–528.     

Fluorescence Visualization of the Enteric Nervous Network in a Chemically Induced Aganglionosis Model

Gastrointestinal motility disorders, severe variants in particular, remain a therapeutic challenge in pediatric surgery. Absence of enteric ganglion cells that originate from neural crest cells is a major cause of dysmotility. However, the limitations of currently available animal models of dysmotility continue to impede the development of new therapeutics. Indeed, the short lifespan and/or poor penetrance of existing genetic models of dysmotility prohibit the functional evaluation of promising approaches, such as stem cell replacement strategy. Here, we induced an aganglionosis model using topical benzalkonium chloride in a P0-Cre/GFP transgenic mouse in which the neural crest lineage is labeled by green fluorescence. Pathological abnormalities and functional changes in the gastrointestinal tract were evaluated 2–8 weeks after chemical injury. Laparotomy combined with fluorescence microscopy allowed direct visualization of the enteric neural network in vivo. Immunohistochemical evaluation further confirmed the irreversible disappearance of ganglion cells, glial cells, and interstitial cell of Cajal. Remaining stool weight and bead expulsion time in particular supported the pathophysiological relevance of this chemically-induced model of aganglionosis. Interestingly, we show that chemical ablation of enteric ganglion cells is associated with a long lifespan. By combining genetic labeling of neural crest derivatives and chemical ablation of enteric ganglion cells, we developed a newly customized model of aganglionosis. Our results indicate that this aganglionosis model exhibits decreased gastrointestinal motility and shows sufficient survival for functional evaluation. This model may prove useful for the development of future therapies against motility disorders.     

Neisseria meningitidis Translation Elongation Factor P and Its Active-Site Arginine Residue Are Essential for Cell Viability

Translation elongation factor P (EF-P), a ubiquitous protein over the entire range of bacterial species, rescues ribosomal stalling at consecutive prolines in proteins. In Escherichia coli and Salmonella enterica, the post-translational β-lysyl modification of Lys34 of EF-P is important for the EF-P activity. The β-lysyl EF-P modification pathway is conserved among only 26–28% of bacteria. Recently, it was found that the Shewanella oneidensis and Pseudomonas aeruginosa EF-P proteins, containing an Arg residue at position 32, are modified with rhamnose, which is a novel post-translational modification. In these bacteria, EF-P and its Arg modification are both dispensable for cell viability, similar to the E. coli and S. enterica EF-P proteins and their Lys34 modification. However, in the present study, we found that EF-P and Arg32 are essential for the viability of the human pathogen, Neisseria meningitidis. We therefore analyzed the modification of Arg32 in the N. meningitidis EF-P protein, and identified the same rhamnosyl modification as in the S. oneidensis and P. aeruginosa EF-P proteins. N. meningitidis also has the orthologue of the rhamnosyl modification enzyme (EarP) from S. oneidensis and P. aeruginosa. Therefore, EarP should be a promising target for antibacterial drug development specifically against N. meningitidis. The pair of genes encoding N. meningitidis EF-P and EarP suppressed the slow-growth phenotype of the EF-P-deficient mutant of E. coli, indicating that the activity of N. meningitidis rhamnosyl–EF-P for rescuing the stalled ribosomes at proline stretches is similar to that of E. coli β-lysyl–EF-P. The possible reasons for the unique requirement of rhamnosyl–EF-P for N. meningitidis cells are that more proline stretch-containing proteins are essential and/or the basal ribosomal activity to synthesize proline stretch-containing proteins in the absence of EF-P is lower in this bacterium than in others.     

Does a Novel-Developed Product of Wheelchair Incorporating Pelvic Support Prevent Forward Head Posture during Prolonged Sitting?

Disabled elderly individuals with kyphosis or loss of muscle strength often display forward head posture (FHP). This study aimed to determine the utility of a wheelchair incorporating pelvic support in preventing FHP in disabled elderly individuals. In this study, 14 disabled elderly individuals (87.1 ± 8.1 years) were selected. A wheelchair incorporating pelvic support (RX_ABS Lo) and a basic wheelchair (RX-1) were used. Each individual sat on both wheelchairs for 30 minutes. RX_ABS Lo has two belts to support the pelvic and thorax. Postures were recorded in the sagittal plane using a video camera. Cervical and trunk angles from horizontal were measured every 5 minutes. Simultaneously, contact areas and total pressures applied to the wheelchair seats and back supports were measured every 5 minutes. Comparisons of area under the curve values between the wheelchairs were performed using the paired t-test. Comparisons of time-dependent parameters for each wheelchair were performed using repeated one-way ANOVA. Cervical angles were greater when using RX_ABS Lo than RX-1. Although cervical angles were unchanged during 30 minutes when using RX_ABS Lo, the angles were significantly decreased after 30 minutes of using RX-1. Back support pressures and contact areas were greater for RX_ABS Lo than for RX-1. No significant difference in back support pressure distributions was observed during 30 minutes in the wheelchairs. The RX_ABS Lo may have utility in improving FHP by increasing cervical angles and improving stability with a back support to the upper thorax, lower thorax, and pelvis during prolonged sitting.     

bcl-2 Gene Prevents Apoptosis of Basic Fibroblast Growth Factor-Deprived Murine Aortic Endothelial Cells

Growth factors such as basic fibroblast growth factor (bFGF) have been found to promote the survival and proliferation of endothelial cells. However, the mechanism by which growth factors control the regeneration and degeneration of the endothelial cells remained poorly understood. In this study, we demonstrated that apoptosis of murine aortic endothelial (MAE) cells was induced by deprivation of bFGF but required new RNA and protein synthesis. Furthermore, enforced expression of bcl-2 gene in MAE cells using gene transfer techniques decreased apoptosis induced by deprivation of bFGF. These findings suggest that bcl-2 interferes with a pathway for endothelial cell death that is induced by deprivation of bFGF.