START-GAP3/DLC3 is a GAP for RhoA and Cdc42 and is localized in focal adhesions regulating cell morphology

In the human genome there are three genes encoding RhoGAPs that contain the START (steroidogenic acute regulatory protein (StAR)-related lipid transfer)—domain. START-GAP3/DLC3 is a tumor suppressor gene similar to two other human START-GAPs known as DLC1 or DLC2. Although expression of START-GAP3/DLC3 inhibits the proliferation of cancer cells, its molecular function is not well understood. In this study we carried out biochemical characterization of START-GAP3/DLC3, and explored the effects of its expression on cell morphology and intracellular localization. We found that START-GAP3/DLC3 serves as a stimulator of PLCδ1 and as a GAP for both RhoA and Cdc42 in vitro. Moreover, we found that the GAP activity is responsible for morphological changes. The intracellular localization of endogenous START-GAP3/DLC3 was explored by immunocytochemistry and was revealed in focal adhesions. These results indicate that START-GAP3/DLC3 has characteristics similar to other START-GAPs and the START-GAP family seems to share common characteristics.     

Nitrous oxide in brackish Lake Nakaumi, Japan II: the role of nitrification and denitrification in N2O accumulation

In order to understand the role of nitrification and denitrification in the accumulation of nitrous oxide (N₂O) in the hypolimnetic water of brackish Lake Nakaumi, the effects of dissolved oxygen (DO) concentration on these activities were investigated by incubation experiments. N₂O was produced during the oxidation of NH₄ ⁺ to NO₂ ⁻ in nitrification and during the reduction of NO₃ ⁻ to N₂ in denitrification. N₂O-producing activity by nitrification (N₂O_N) increased markedly with decreasing concentrations of DO. Low DO (10%–30% saturation) induced high N₂O_N. In contrast to nitrification, N₂O-producing activity by denitrification (N₂O_D) decreased with decreasing concentrations of DO. Little N₂O was accumulated during denitrification under low-level conditions of DO (10%–30%), because of further reduction of N₂O to N₂. It can therefore be assumed that N₂O produced as the by-product of nitrification is concurrently reduced to N₂ by denitrification under low-DO conditions. This would result in no substantial accumulation of N₂O during active nitrification in the hypolimnetic water of Lake Nakaumi. Received: July 6, 2001 / Accepted: December 10, 2001     

Short-term variation in benthic phosphorus transfer due to discontinuous aeration/oxygenation operation

The short-term dynamics of soluble reactive phosphorus (SRP) transport across the sediment surface in a brackish lake due to discontinuous aeration and oxygenation operations were investigated using laboratory and field experimental and analytical procedures. According to a laboratory incubation experiment using intact sediment cores, SRP release from the sediment was clearly suppressed by aeration, and substantial negative SRP transfer was observed during oxygenation treatment, while a positive value was observed for N₂ bubbled cores. A remarkable but impermanent increase in SRP release rate was observed within 1 or 2 days of discontinuing the aeration and oxygenation, respectively, and the release rate rapidly deceased to a quasi-steady value under N₂ bubbling conditions. An analytical model could quantitatively reproduce these laboratory experimental results for anoxic and aerated conditions, showing that this impermanent increase was attributable to the rapid release of accumulated SRP in the oxic surface layer of the sediment. Field experiments using an in situ oxygenator showed the same tendency as the laboratory experiments, but with much larger values of the benthic SRP transfer rate. Overall, the short-term dynamics of benthic SRP transport caused by discontinuous aeration and oxygenation are considered to be an important process for the phosphorus cycle in the field.     

Nitrous oxide in brackish Lakes Shinji and Nakaumi, Japan

Nitrous oxide (N₂O) was measured monthly from September 1997 to August 1998 in the brackish Lakes Shinji and Nakaumi, Japan. N₂O (5–37 μg N l⁻¹) was supersaturated in the overlying water on lake sediments from October 1997 to January 1998. The N₂O concentration in the hypolimnion was higher than that in the epilimnion on 17 October 1997, when N₂O was first observed in a water column of Lake Nakaumi. Afterward, N₂O was almost uniform throughout the water column and then disappeared on 16 February 1998. On the one hand, large amounts of N₂O were found throughout the year in the interstitial water in Lake Shinji, where a high concentration of nitrate was discharged from the Hii River. On the other hand, in Lake Nakaumi, stratified by halocline, a high concentration of N₂O was observed in the interstitial water only from winter to spring. N₂O concentrations in the interstitial water were about 10 to 1000 times as large as those in the overlying water. These results imply that N₂O was mainly produced at the sediment-water interface and was diffused to the overlying water. It was also suggested that the accumulation of N₂O in the sediment-water system was accelerated by a high concentration of hydrogen sulfide. Received: July 6, 2000 / Accepted: November 30, 2000     

Effects of Organic Matter on Solubilities and Crystal Form of Carbonates

The results of a study of the role of organic compounds in theformation of carlxmate crystals in marine biological systemsare reported. In an increasing concentration of certain organiccompounds which complex calcium ions, the proportion of aragonitedecreases and that of calcite increases. In increasing concentrationsof magnesium ions the proportion of aragonite increases andthat of calcite and vaterite decreases. When the influence oforganic compounds is greater or smaller than that of magnesiumions, only calcite or only aragonite is formed, respectively.Organic compounds forming a strong complex with calcium ionscause the formation of magnesium-rich calcite, and with an increasein temperature and the concentration of magnesium ions, themagnesium carbonate content of precipitated magnesian calciteincreases. When the influence of organic compounds is almostequivalent to that of magnesium ions, in increasing or decreasingtemperatures, the proportion of calcite decreases or increases,respectively, and the proportion of aragonite increases or decreases,respectively. The concentration of magnesium ions in the bodyfluids of marine calcareous organisms seems to differ littlefrom that of other organisms, and seems to be similar to thatof sea water. Only the presence of certain organic compoundsbrings about the formation of the carbonate crystals observedin marine biological systems. The very important role of organicmatter in the formation of crystals found in skeletal carbonatesis emphasized.     

Novel use of burrow casting as a research tool in deep-sea ecology

Although the deep sea is the largest ecosystem on Earth, its infaunal ecology remains poorly understood because of the logistical challenges. Here we report the morphology of relatively large burrows obtained by in situ burrow casting at a hydrocarbon-seep site and a non-seep site at water depths of 1173 and 1455 m, respectively. Deep and complex burrows are abundant at both sites, indicating that the burrows introduce oxygen-rich sea water into the deep reducing substrate, thereby influencing benthic metabolism and nutrient fluxes, and providing an oxic microhabitat for small organisms. Burrow castings reveal that the solemyid bivalve Acharax johnsoni mines sulphide from the sediment, as documented for related shallow-water species. To our knowledge, this is the first study to examine in situ burrow morphology in the deep sea by means of burrow casting, providing detailed information on burrow structure which will aid the interpretation of seabed processes in the deep sea.     

Biodegradation of γ-hexachlorocyclohexane by transgenic hairy root cultures of <Emphasis Type="Italic">Cucurbita moschata</Emphasis> that accumulate recombinant bacterial LinA

Key message

γ-HCH was successfully degraded using LinA-expressed transgenic hairy root cultures of Cucurbita moschata . Fusing an endoplasmic reticulum-targeting signal peptide to LinA was essential for stable accumulation in the hairy roots.

Abstract

The pesticide γ-hexachlorocyclohexane (γ-HCH) is a persistent organic pollutant (POP) that raises public health and environmental pollution concerns worldwide. Although several isolates of γ-HCH-degrading bacteria are available, inoculating them directly into γ-HCH-contaminated soil is ineffective because of the bacterial survival rate. Cucurbita species incorporate significant amounts of POPs from soils compared with other plant species. Here, we describe a novel bioremediation strategy that combines the bacterial degradation of γ-HCH and the efficient uptake of γ-HCH by Cucurbita species. We produced transgenic hairy root cultures of Cucurbita moschata that expressed recombinant bacterial linA, isolated from the bacterium Sphingobium japonicum UT26. The LinA protein was accumulated stably in the hairy root cultures by fusing an endoplasmic reticulum (ER)-targeting signal peptide to LinA. Then, we demonstrated that the cultures degraded more than 90 % of γ-HCH (1 ppm) overnight and produced the γ-HCH metabolite 1,2,4-trichlorobenzene, indicating that LinA degraded γ-HCH. These results indicate that the gene linA has high potential for phytoremediation of environmental γ-HCH.

     

Inhibition of cytochrome c release in Fas-mediated signaling pathway in transgenic mice induced to express hepatitis C viral proteins

Persistent hepatitis C virus (HCV) infection often progresses to chronic hepatitis, cirrhosis, and hepatocellular carcinoma. Numerous viruses have been reported to escape from apoptotic mechanism to maintain persistent infection. In the present study, we characterized the effect of HCV proteins on the Fas signal using HCV transgenic mice, which expressed core, E1, E2, and NS2 proteins, regulated by the Cre/loxP switching system. The transgene expression of HCV transgenic mice caused resistance to Fas antibody stimulated lethality. Apoptotic cell death in the liver of HCV protein expressing mice was significantly reduced compared with nonexpressing mice. Histopathological analysis and DNA fragmentation analysis revealed that the HCV proteins suppressed Fas-mediated apoptotic cell death. To identify the target pathway of HCV proteins, we characterized caspase activity. The activation of caspase-9 and -3/7 but not caspase-8 was inhibited by HCV proteins. Cytochrome c release from mitochondria was inhibited in HCV protein expressing mice. These results indicated that the expression of HCV proteins may directly or indirectly inhibit Fas-mediated apoptosis and death in mice by repressing the release of cytochrome c from mitochondria, thereby suppressing caspase-9 and -3/7 activation. These results suggest that HCV may cause persistent infection, as a result of suppression of Fas-mediated cell death.     

START-GAP1/DLC1 is localized in focal adhesions through interaction with the PTB domain of tensin2

START-GAP1, also termed as DLC1, is a negative-regulator for RhoA and Cdc42. START-GAP1 is localized in focal adhesions via the FAT (focal adhesion targeting) domain located in its N-terminal half and interacts with tensin family proteins, that constitutes focal adhesion components. This study has provided evidence that the interaction between START-GAP1 and tensin2 occurs in a PTB domain-dependent manner. It was revealed that FAT3, the third subdomain of the FAT domain divided into five that consists of 39 amino acids, binds directly to the PTB domain of tensin2. This interaction does not require protein phosphorylation, since the interaction was detected with proteins expressed in bacterial expression system. In mammalian genome, there are three genes encoding START domain containing RhoGAPs. START-GAP2/DLC2 and START-GAP3/DLC3, as well as STRT-GAP1/DLC1, bind to the PTB domain of tensin2, presumably due to the presence of highly conserved residues in the center of FAT3. Deletion of this sub-region abrogates the interaction with the tensin PTB domain. Furthermore, D368, H369, G372, F374, P375 and L378 in the highly conserved region of START-GAP1 have been revealed to be essential for the interaction. The tensin2-PTB domain seems to determine the subcellular localization of FAT3. Nevertheless, our study with deletion mutants revealed that FAT3 is essential but not sufficient for the focal adhesion localization of START-GAP1. These results suggest that the interaction between the tensin PTB domain and FAT3 contributes to START-GAP1 localization but only partially. Other factors could affect the START-GAP1 localization.     

Reversely-oriented cytochrome b561 in reconstituted vesicles catalyzes transmembrane electron transfer and supports extravesicular dopamine beta-hydroxylase activity

Cytochrome b561 from bovine adrenal chromaffin vesicles contains two heme B prosthetic groups. We verified that purified cytochrome b561 can donate electron equivalents directly to cytochrome c. The purified cytochrome b561 was successfully reconstituted into cholesterol-phosphatidylcholine-phosphatidylglycerol vesicles by a detergent-dialysis and extrusion method. When ascorbate-loaded vesicles with cytochrome b561 were mixed with ferricytochrome c, the intravesicular ascorbate was able to reduce external thiazole blue or cytochrome c. The reduction of thiazole blue or cytochrome c was dependent on the presence of cytochrome b561 in the vesicle membranes. Pre-treatment of cytochrome b561 with diethylpyrocarbonate suppressed the reduction of extravesicular cytochrome c significantly, confirming that the reduction was not due to leakage of ascorbate from the vesicles. The topology of the reconstituted cytochrome b561 in the vesicle membranes was examined by treatment with trypsin followed by SDS-PAGE and MALDI-TOF-MS analyses. Only one major cleavage site at Lys191 was identified, indicating that cytochrome b561 was reconstituted into the membranes in an inside-out orientation irrespective of the modification with diethylpyrocarbonate. The addition of a soluble form of dopamine beta-hydroxylase to the external medium resulted in the successful reconstitution of the hydroxylation activity towards tyramine, an analogue of dopamine, suggesting that a direct electron transfer via complex formation occurred. This activity was enhanced significantly upon the addition of ferricyanide as a mediator between cytochrome b561 and dopamine beta-hydroxylase.