Specificity of a heme-assimilating system of Vibrio vulnificus to synthetic heme compounds

Vibrio vulnificus strain L-180, a clinical isolate, can obtain iron from a synthetic heme, iron-tetra(4-sulfonatophenyl)porphyrin (Fe-TPPS), as well as from a natural heme, protoheme. This assimilation of iron bound to TPPS was demonstrated to be a common property of V. vulnificus by testing a total of 27 strains isolated from both clinical and environmental sources. Strain L-180 could also utilize Fe-TCPP, but not Fe-TMPyP, as a sole iron source. TPPS or its complex with a metal ion reduced bacterial multiplication in the broth containing a minimum dose of Fe-TPPS. When inoculated into human serum supplemented with Fe-TCPP, L-180 could grow only in the presence of a protease from the same bacterium. In both TPPS and TCPP, each side chain of a porphyrin ring has a negative charge. Therefore, this negative charge may be important for interaction with an outer membrane receptor involving in a heme-assimilating system of V. vulnificus.     

Elevation of antioxidant potency in mice brain by low-dose X-ray irradiation and its effect on Fe-NTA-induced brain damage

The increase in lipid peroxide levels in mice brain following Fe3+ administration was about 50% of that when 1-methyl-4-phenyl 1,2,3,6-tetrahydropyridine (MPTP) was administered. This may be due to excessive oxidation by Fe3+, and was supported by the decrease in activities of antioxidant enzymes, such as superoxide dismutase (SOD), catalase and glutathione peroxidase (GPX), Na+,K(+)-ATPase activity and membrane fluidity after Fe3+ administration. Relatively low-dose X-ray irradiation (0.5 Gy) inhibited lipid peroxidation associated with Fe3+ administration and restored the decreased activities of the above antioxidant enzymes and Na+,K(+)-ATPase, and membrane fluidity to the levels in the non-Fe(3+)-administered group. In the purine metabolism system, uric acid decreased after Fe3+ administration, which may be due to transient impairment of the system for production of uric acid from xanthine by excessive oxidation by Fe3+. However, 0.5 Gy irradiation inhibited this decrease in uric acid, increasing its level to that in the non Fe(3+)-administrated group. This may be due to factors such as rapid recovery of the activities of the above antioxidant enzymes and Na+,K(+)-ATPase, and membrane fluidity after 0.5 Gy irradiation. In addition, since no changes were observed in xanthine and uric acid, increased inosine and hypoxanthine may have advanced to a salvage pathway leading to not xanthine but inosine 5'-monophosphate (IMP).     

Detection of hypoxia in the pulmonary tissues of Xenopus laevis over repeated dives

The oxygen environment in African clawed frogs (Xenopus laevis) continuously changes during their development, which involves a rapid increase in the body size, metamorphosis, and transition to adulthood. Nevertheless, there are limited reports on experimental models that are available for studying fluctuations in the oxygen environment in X. laevis. Thus, this study aimed to develop an experimental model on intermittent hypoxia in X. laevis and evaluate hypoxia and oxidative stress in the same. X. laevis were submerged in water with a dissolved oxygen concentration of 2 mg/L for 30 min; they were then removed from the water and allowed to freely absorb oxygen for 5 min. Immunostaining of pimonidazole-containing frozen tissue sections of the lung and liver using anti-pimonidazole antibodies as the hypoxia probes revealed that more than 95% of the submerged X. laevis cells were pimonidazole positive, providing direct evidence of tissue hypoxia. When the amount of oxidative stress in the lungs and liver was evaluated in terms of the amount of lipid peroxides, the diving group showed a 2.08-fold and 3.20-fold increase over the normal group, respectively. Following hypoxia exposure, the dry-to-wet weight ratios of the lung tissues was 1.27 times higher (p < .05), while the liver tissues was 1.06 times higher (although not significant). Thus, the degree of damage depended on the tissues affected. In the future, we believe that this model will be a promising option for analyzing the physiological responses of X. laevis to hypoxia and oxidative stress.     

Role of phytoplankton size distribution in lake ecosystems revealed by a comparison of whole plankton community structure between Lake Baikal and Lake Biwa

The influence of the size distribution of phytoplankton on changes in the planktonic food web structures with eutrophication was examined using natural planktonic communities in two world-famous lakes: Lake Baikal and Lake Biwa. The size distribution of phytoplankton and the ratio of heterotrophic to autotrophic biomass (H/A ratio), indicating the balance between primary production and its consumption, were investigated in the lakes of different trophic status. The results revealed that microphytoplankton (>20μm) in mesotrophic Lake Biwa, and picophytoplankton (<2μm) or nanophytoplankton (2–20μm) in oligotrophic Lake Baikal, comprised the highest proportion of the total phytoplankton biomass. The H/A ratio was lower in Lake Biwa (<1) than in Lake Baikal (>1). The low H/A ratio in Lake Biwa appeared to be the consequence of the lack of consumption of the more abundant microphytoplankton, which were inferior competitors in nutrient uptake under oligotrophic conditions but less vulnerable to grazing. As a result, unconsumed microphytoplankton accumulated in the water column, decreasing the H/A ratio in Lake Biwa. Our results showed that food web structure and energy flow in planktonic communities were greatly influenced by the size distribution of phytoplankton, in conjunction with bottom-up (nutrient uptake) and top-down (grazing) effects at the trophic level of primary producers.     

Cryptic population dynamics: rapid evolution masks trophic interactions

Trophic relationships, such as those between predator and prey or between pathogen and host, are key interactions linking species in ecological food webs. The structure of these links and their strengths have major consequences for the dynamics and stability of food webs. The existence and strength of particular trophic links has often been assessed using observational data on changes in species abundance through time. Here we show that very strong links can be completely missed by these kinds of analyses when changes in population abundance are accompanied by contemporaneous rapid evolution in the prey or host species. Experimental observations, in rotifer-alga and phage-bacteria chemostats, show that the predator or pathogen can exhibit large-amplitude cycles while the abundance of the prey or host remains essentially constant. We know that the species are tightly linked in these experimental microcosms, but without this knowledge, we would infer from observed patterns in abundance that the species are weakly or not at all linked. Mathematical modeling shows that this kind of cryptic dynamics occurs when there is rapid prey or host evolution for traits conferring defense against attack, and the cost of defense (in terms of tradeoffs with other fitness components) is low. Several predictions of the theory that we developed to explain the rotifer-alga experiments are confirmed in the phage-bacteria experiments, where bacterial evolution could be tracked. Modeling suggests that rapid evolution may also confound experimental approaches to measuring interaction strength, but it identifies certain experimental designs as being more robust against potential confounding by rapid evolution.     

Structure elucidation of ostreocin-A and ostreocin-E1, novel palytoxin analogs produced by the dinoflagellate Ostreopsis siamensis,using LC/Q-TOF MS

Palytoxin analogs are marine toxins with large complex polyol structures. A benthic dinoflagellate Ostreopsis siamensis produces more than ten palytoxins (ostreocins, OSTs). The limited sample availability of minor OSTs restricts the definition of their chemical structures. The present investigation characterizes structures of two minor OSTs, i.e., ostreocin-A (OSTA) and ostreocin-E1 (OSTE1), using ostreocin-D (OSTD) as a reference compound, by liquid chromatography/quadrupole-time-of-flight mass spectrometry. The molecular formulas of OSTA and OSTE1 were C₁₂₇H₂₁₉N₃O₅₄ and C₁₂₇H₂₁₇N₃O₅₂, respectively. Compared to OSTD, OSTA has an extra oxygen atom whereas OSTE1 lacks one oxygen atom and two hydrogen atoms. The MS/MS experiments (precursor ions: [M + H]⁺ and [M-H]^?) suggested a hydroxyl substitution at C82 in OSTA and alteration(s) between C53 and C100 in OSTE1. Further analysis of structural details in OSTE1 was performed through a pseudo-MS³ experiment (precursor ion: m/z 1432.748). Accordingly, the planar structures of OSTA and OSTE1 were assigned to 42,82-dihydroxy-3,26-didemethyl-19,44-dideoxypalytoxin and 42-hydroxy-3,26-didemethyl-19,44,73-trideoxypalytoxin-72-ene, respectively.

Abbreviations:CID: collision induced dissociation; HR-LC/MS/MS: high-resolution liquid chromatography/tandem mass spectrometry; LC/ESI/Q-TOF MS: liquid chromatography/quadrupole time-of-flight mass spectrometry equipped with an electrospray ionization source; NMR: nuclear magnetic resonance; OSTs: ostreocins; OSTA: ostreocin-A; OSTB: ostreocin-B; OSTD: ostreocin-D; OSTE1: ostreocin-E1; OVTX-a: ovatoxin-a; OVTXs: ovatoxins; PLTX: palytoxin     

Allogeneic bone marrow transplantation for hepatocellular carcinoma: hepatocyte growth factor suppresses graft-vs.-host disease

Allogeneic bone-marrow transplantation (BMT) can induce a powerful graft-vs.-tumor (GVT) effect not only on hematological malignancies but also on solid tumors. However, graft-vs.-host disease (GVHD) is a major complication of allogeneic BMT. We assessed GVT effect on hepatocellular carcinoma (HCC) and the effects of hepatocyte growth factor (HGF) gene transduction on GVHD in HCC transplanted mice. (C57BL/6 x C3H/HeJ)F(1)(B6C3F1, H-2(bxk)) mice were used as recipients and C3H/HeJ(H-2(k)) mice were used as donors. Hepa1-a (a C57L mouse-derived hepatoma cell, H-2(b)) was subcutaneously injected into the recipient mice. Tumor bearing mice were treated in the following ways: group 1, no treatment; group 2, total body irradiation (TBI); group 3, TBI and BMT; group 4, TBI and BMT with empty vector; group 5, TBI and BMT with HGF gene transduction; group 6, TBI and BMT with administration of FK506, a representative immunosuppressive agent. Acute GVHD was assessed by histological examination of the liver, small intestines, and large intestines. Tumor growth was markedly suppressed in mice that received an allogeneic BMT. Donor-derived CD8(+) T cells had infiltrated into the tumor, and cytotoxic CD8(+) T cells against HCC were present. However, among the four groups that received a BMT, this suppressive effect was weaker in group 6 compared with the other three groups (groups 3, 4, and 5). HGF gene transduction improved GVHD while preserving the GVT effects. Allogeneic BMT markedly suppresses the growth of HCC. Simultaneous HGF gene transfer can suppress GVHD while preserving the GVT effect.     

Long-term preservation of freeze-dried mouse spermatozoa

Many genetically engineered mice strains have been generated worldwide and sperm preservation is a valuable method for storing these strains as genetic resources. Freeze-drying is a useful sperm preservation method because it requires neither liquid nitrogen nor dry ice for preservation and transportation. We report here successful long-term preservation at 4 °C of mouse spermatozoa freeze-dried using a simple buffer solution (10mM Tris, 1mM EDTA, pH 8.0). Offspring with fertility were obtained from oocytes fertilized with freeze-dried spermatozoa from C57BL/6 and B6D2F1 mouse strains stored at 4 °C for 3 years. This freeze-drying method is a safe and economical tool for the biobanking of valuable mouse strains.     

Effects of Chloromethanes on Growth of and Deletion of the pce Gene Cluster in Dehalorespiring Desulfitobacterium hafniense Strain Y51

The dehalorespiring Desulfitobacterium hafniense strain Y51 efficiently dechlorinates tetrachloroethene (PCE) to cis-1,2-dichloroethene (cis-DCE) via trichloroethene by PceA reductive dehalogenase encoded by the pceA gene. In a previous study, we found that the significant growth inhibition of strain Y51 occurred in the presence of commercial cis-DCE. In this study, it turned out that the growth inhibition was caused by chloroform (CF) contamination of cis-DCE. Interestingly, CF did not affect the growth of PCE-nondechlorinating SD (small deletion) and LD (large deletion) variants, where the former fails to transcribe the pceABC genes caused by a deletion of the promoter and the latter lost the entire pceABCT gene cluster. Therefore, PCE-nondechlorinating variants, mostly LD variant, became predominant, and dechlorination activity was significantly reduced in the presence of CF. Moreover, such a growth inhibitory effect was also observed in the presence of carbon tetrachloride at 1 μM, but not carbon dichloride even at 1 mM.     

Gene expression profiling of mouse embryonic stem cell subpopulations

We previously demonstrated that mouse embryonic stem (ES) cells show a wide variation in the expression of platelet endothelial cell adhesion molecule 1 (PECAM1) and that the level of expression is positively correlated with the pluripotency of ES cells. We also found that PECAM1-positive ES cells could be divided into two subpopulations according to the expression of stage-specific embryonic antigen (SSEA)-1. ES cells that showed both PECAM1 and SSEA-1 predominantly differentiated into epiblast after the blastocyst stage. In the present study, we performed pairwise oligo microarray analysis to characterize gene expression profiles in PECAM1-positive and -negative subpopulations of ES cells. The microarray analysis identified 2034 genes with a more than 2-fold difference in expression levels between the PECAM1-positive and -negative cells. Of these genes, 803 were more highly expressed in PECAM1-positive cells and 1231 were more highly expressed in PECAM1-negative cells. As expected, genes known to function in ES cells, such as Pou5f1(Oct3/4)and Nanog, were found to be upregulated in PECAM1-positive cells. We also isolated 23 previously uncharacterized genes. A comparison of gene expression profiles in PECAM1-positive cells that were either positive or negative for SSEA-1 expression identified only 53 genes that showed a more than 2-fold greater difference in expression levels between these subpopulations. However, many genes that are under epigenetic regulation, such as globins, Igf2, Igf2r, andH19, showed differential expression. Our results suggest that in addition to differences in gene expression profiles, epigenetic status was altered in the three cell subpopulations.