Regulation of ammonia homeostasis by the ammonium transporter AmtA in Dictyostelium discoideum

Ammonia has been shown to function as a morphogen at multiple steps during the development of the cellular slime mold Dictyostelium discoideum; however, it is largely unknown how intracellular ammonia levels are controlled. In the Dictyostelium genome, there are five genes that encode putative ammonium transporters: amtA, amtB, amtC, rhgA, and rhgB. Here, we show that AmtA regulates ammonia homeostasis during growth and development. We found that cells lacking amtA had increased levels of ammonia/ammonium, whereas their extracellular ammonia/ammonium levels were highly decreased. These results suggest that AmtA mediates the excretion of ammonium. In support of a role for AmtA in ammonia homeostasis, AmtA mRNA is expressed throughout the life cycle, and its expression level increases during development. Importantly, AmtA-mediated ammonia homeostasis is critical for many developmental processes. amtA(-) cells are more sensitive to NH(4)Cl than wild-type cells in inhibition of chemotaxis toward cyclic AMP and of formation of multicellular aggregates. Furthermore, even in the absence of exogenously added ammonia, we found that amtA(-) cells produced many small fruiting bodies and that the viability and germination of amtA(-) spores were dramatically compromised. Taken together, our data clearly demonstrate that AmtA regulates ammonia homeostasis and plays important roles in multiple developmental processes in Dictyostelium.     

Age-related changes on marking, marking-like behavior and the scent gland in adult Mongolian gerbils (Meriones unguiculatus).

Marking behavior, marking-like behavior [3], and changes of the scent glands were observed in aged Mongolian gerbils. In Experiment 1, changes in the marking and marking-like behavior with aging were evaluated in adult male and female Mongolian gerbils of an inbred strain aged 6 to 36 months. The frequency of marking behavior in males was significantly higher than females throughout the observation period except at 36 months of age. On the other hand, frequency of marking-like behavior in males, but not in females decreased with aging, significantly. In Experiment 2, changes of the scent gland in adult males and females aged 6 to 36 months were morphologically evaluated. Macroscopic examination revealed an increase in the size length and width of the glands of males aged 12 months and females aged 6 months. Histologically the glands of all the males and females aged 6 months developed moderately or well. Some of the 12-month-old males and females showed acinar atrophy of the glands, and all the females aged 18 months or more had highly atrophied scent glands. From these results, we concluded that there is no relationship between the changes of marking behavior and those of the scent glands in aged male Mongolian gerbils, and assume that marking behavior in aged animals does not have an important meaning as marking. In Experiment 3, marking and marking-like behavior in castrated adult Mongolian gerbils aged 16 weeks were observed. The result showed that marking behavior, not marking-like behavior was inhibited after castration. From these findings, we consider that generally marking behavior in Mongolian gerbils consists of androgen-dependent marking behavior and androgen-independent marking behavior (marking-like behavior).     

Survival of lactococci during passage through mouse digestive tract

One of the important properties of probiotics is the ability to survive in the intestine. There have been few studies on the probiotic property of lactococci, since they are formally not considered to be natural inhabitants of the intestine. To evaluate lactococci as probiotic bacteria, we investigated their ability to survive during gastric transit by in vitro and in vivo tests. When exposed to an in vitro simulated gastrointestinal environment, such as low pH and bile, only Lactococcus lactis subsp. lactis bv. diacetylactis N7 showed a moderate survival rate among the four strains tested. The tested strains were orally administered to mice, and intestinal passage of the ingested strains was monitored by two methods: antibiotics and PCR. Viable cells of strain N7 were recovered from feces within 24-48 h after administration but not at 72 h. Lactococcus lactis subsp. cremoris ATCC 19257, which had a poor survival rate in vitro test, was also detected at 12 h but not at 24 h. These results indicate that lactococci can reach the mouse intestine alive, but not colonize it. If administered daily, viable strain N7 may exist continuously in the intestine. The effect of strain N7 on intestinal microbial balance and on animal health will be the subject of a further study.     

Spatiotemporal Correlations between Cytosolic and Mitochondrial Ca2+ Signals Using a Novel Red-Shifted Mitochondrial Targeted Cameleon

The transfer of Ca²⁺ from the cytosol into the lumen of mitochondria is a crucial process that impacts cell signaling in multiple ways. Cytosolic Ca²⁺ ([Ca²⁺]_cyto) can be excellently quantified with the ratiometric Ca²⁺ probe fura-2, while genetically encoded Förster resonance energy transfer (FRET)-based fluorescent Ca²⁺ sensors, the cameleons, are efficiently used to specifically measure Ca²⁺ within organelles. However, because of a significant overlap of the fura-2 emission with the spectra of the cyan and yellow fluorescent protein of most of the existing cameleons, the measurement of fura-2 and cameleons within one given cell is a complex task. In this study, we introduce a novel approach to simultaneously assess [Ca²⁺]_cyto and mitochondrial Ca²⁺ ([Ca²⁺]_mito) signals at the single cell level. In order to eliminate the spectral overlap we developed a novel red-shifted cameleon, D1GO-Cam, in which the green and orange fluorescent proteins were used as the FRET pair. This ratiometric Ca²⁺ probe could be successfully targeted to mitochondria and was suitable to be used simultaneously with fura-2 to correlate [Ca²⁺]_cyto and [Ca²⁺]_mito within same individual cells. Our data indicate that depending on the kinetics of [Ca²⁺]_cyto rises there is a significant lag between onset of [Ca²⁺]_cyto and [Ca²⁺]_mito signals, pointing to a certain threshold of [Ca²⁺]_cyto necessary to activate mitochondrial Ca²⁺ uptake. The temporal correlation between [Ca²⁺]_mito and [Ca²⁺]_cyto as well as the efficiency of the transfer of Ca²⁺ from the cytosol into mitochondria varies between different cell types. Moreover, slow mitochondrial Ca²⁺ extrusion and a desensitization of mitochondrial Ca²⁺ uptake cause a clear difference in patterns of mitochondrial and cytosolic Ca²⁺ oscillations of pancreatic beta-cells in response to D-glucose.     

A novel species of alkaliphilic Bacillus that produces an oxidatively stable alkaline serine protease

A novel gram-positive, strictly aerobic, motile, sporulating, and facultatively alkaliphilic bacterium designated KSM-KP43 was isolated from a sample of soil. The results of 16S rRNA sequence analysis placed this bacterium in a cluster with Bacillus halmapalus. However, the level of the DNA-DNA hybridization of KSM-KP43 with B. halmapalus was less than 25%. Moreover, the G + C contents of the genomic DNA were 41.6 mol% for KSM-KP43 and 38.6 mol% for B. halmapalus. Because there were also differences in physiological properties and cellular fatty acid composition between the two organisms, we propose KSM-KP43 as a novel species of alkaliphilic Bacillus. This novel strain produces a new class of protease, an oxidatively stable serine protease that is suitable for use in bleach-based detergents. The enzyme contained 640 amino acid residues, including a possible approximately 200-amino-acid prepropeptide in the N-terminal and a unique stretch of approximately 160 amino acids in the C-terminal regions (434-amino-acid mature enzyme with a calculated molecular mass of 45,301 Da). The C-terminal half after the putative catalytic Ser255 and the contiguous C-terminal extension shared local similarity to internal segments of a membrane-associated serine protease of a marine microbial assemblage and the serine protease/ABC transporter precursors of the slime mold Dictyostelium discoideum, and to the C-terminal half of a cold-active alkaline serine protease of a psychrotrophic Shewanella strain.     

Thermal comfort zones in the Vietnamese

Thermally comfortable zones in Vietnamese were investigated during winter in Hanoi. The subjects were 21 males (age: 19.7 +/- 0.4 yrs; height: 165 +/- 1.5 cm; body mass: 55.1 +/- 1.1 kg) and 19 females (age: 19.7 +/- 0.4 yrs; height; 155.6 +/- 1.7 cm; body mass: 45.6 +/- 1.3 kg). Each participant entered singly the climatic chamber controlled at 22 degrees C and 40% RH. After 20 min rest, the participant was requested to indicate on a 7-point scale (Table 1) how he or she felt to the room temperature given. Then, the room temperature increased by 1 degrees C over 10 min every 20 min. Just before the rise of the room temperature, the participant judged his or her thermal sensation. More than 90% of the participants felt 24-29 degrees C of the room temperature as "slightly cool", "neutral" and "slightly warm" (Table 2). We defined these sensations as "thermally comfort". These thermally comfortable zones were quite higher than those (20-24 degrees C) recommended by ISO-7730 (1994). We discussed these discrepancies in terms of higher establishment of thermoregulatory set-point in the Vietnamese.     

Recruitment of thioredoxin-like domains into prostaglandin synthases

A variety of prostaglandin (PG) synthases with different evolutionary origins have been identified. These enzymes catalyze reduction and oxidation reactions. However, despite the similarity in their reactions, thioredoxin-like proteins were not found in the PG synthesis pathway until recently. We have identified two new enzymes, thioredoxin-type PGF synthase and membrane-associated PGE synthase-2, with thioredoxin-like domains. In addition, the N-terminal domain of hematopoietic PGD synthase is classified into the thioredoxin-like superfamily, based on structural similarity. The active sites of the former two enzymes have a CXXC motif, which is also critical for the thioredoxin activity. In contrast, hematopoietic PGD synthase lacks the motif, and the activity is carried out by glutathione. A phylogenetic tree of the thioredoxin-like domains suggests that they have been independently recruited into these PG synthases. We will discuss the functional meaning of the thioredoxin-like domains in the PG synthases from the viewpoint of the redox activity.