Microglia and macrophages as innate producers of interferon-gamma in the brain following infection with Toxoplasma gondii

We previously reported the requirement of interferon-gamma (IFN-gamma) expression by cells other than T and natural killer (NK) cells in the brain, in addition to T cells, for prevention of toxoplasmic encephalitis following infection with Toxoplasma gondii. In the present study, we analysed the identity of the IFN-gamma-producing non-T, non-NK cells in the brain using infected athymic nude and SCID mice that lack T cells but express IFN-gamma in their brains. Intracellular staining for IFN-gamma followed by flow cytometry revealed that approximately 45-60% of the cells expressing IFN-gamma in their brains were positive for CD11b or F4/80 on their surfaces. Smaller portions of the cells were positive for pan-NK marker. Further smaller portions were positive for CD11c, and these cells were less than 5% of the IFN-gamma-expressing cells in brains of infected SCID mice. In addition to IFN-gamma proteins, large amounts of mRNA for IFN-gamma were detected in CD11b+ cells purified from brains of infected mice, but it was not the case in the cells obtained from uninfected animals. In infected SCID mice depleted of NK cells by treatment with anti-asialo-GM1 antibody, cells expressing IFN-gamma in their brains were all positive for CD11b, and the IFN-gamma-producing cells were detected in both CD45low and CD45high populations. These results suggest that CD11b+ CD45low microglia and CD11b+ CD45high blood-derived macrophages are the major non-T, non-NK cells which express IFN-gamma in the brain of mice infected with T. gondii.     

A novel phospholipase C, PLC(eta)2, is a neuron-specific isozyme

Twelve phospholipase C (PLC) isozymes have been cloned so far, and they are divided into six classes, beta-, gamma-, delta-, epsilon-, zeta-, and eta-type, on the basis of structure and activation mechanisms. Here we report the identification of a novel PLC isozyme, PLC(eta)2. PLC(eta)2 is composed of conserved domains including pleckstrin homology, EF-hand, X and Y catalytic, and C2 domains and the isozyme-specific C-terminal region. PLC(eta)2 consists of 1164 amino acids with a molecular mass of 125 kDa. The PLC activity of PLC(eta)2 was more sensitive to calcium concentration than the PLC activity of the PLCdelta-type enzyme, which is thought to be the most calcium-sensitive PLC. Immunofluorescence analysis showed that PLC(eta)2 was localized predominantly to the plasma membrane at resting state via the pleckstrin homology domain. This observation was supported by Western blot analysis of cytosol and membrane fractions. In addition, expression of PLC(eta)2 was detected after birth and showed a restricted distribution in the brain; it was particularly abundant in the hippocampus, cerebral cortex, and olfactory bulb. The pattern was similar to that of the neuronal marker microtubule-associated protein 2 by Western blot. Furthermore, in situ hybridization showed positive signals for PLC(eta)2 in pyramidal cells of the hippocampus. Finally, we found that PLC(eta)2 was expressed abundantly in neuron-containing primary culture but not in astrocyte-enriched culture. These results indicate that PLC(eta)2 is a neuron-specific isozyme that may be important for the formation and/or maintenance of the neuronal network in the postnatal brain.     

Prostatic acid phosphatase degrades lysophosphatidic acid in seminal plasma

Lysophosphatidic acid (LPA) is a lipid mediator with multiple biological activities and is detected in various biological fluids, including human seminal plasma. Due to its cell proliferation stimulatory and anti-apoptotic activities, LPA has been implicated in the progression of some cancers such as ovarian cancer and prostate cancer. Here, we show that prostatic acid phosphatase, which is a non-specific phosphatase and which has been implicated in the progression of prostate cancer, inactivates LPA in human seminal plasma. Human seminal plasma contains both an LPA-synthetic enzyme, lysoPLD, which converts lysophospholipids to LPA and is responsible for LPA production in serum, and its major substrate, lysophosphatidylcholine. In serum, LPA accumulated during incubation at 37 degrees C. However, in seminal plasma, LPA did not accumulate. This discrepancy is explained by the presence of a strong LPA-degrading activity. Incubation of LPA with seminal plasma resulted in the disappearance of LPA and an accompanying accumulation of monoglyceride showing that LPA is degraded by phosphatase activity present in the seminal plasma. When seminal plasma was incubated in the presence of a phosphatase inhibitor, sodium orthovanadate, LPA accumulated, indicating that LPA is produced and degraded in the fluid. Biochemical characterization of the LPA-phosphatase activity identified two phosphatase activities in human seminal plasma. By Western blotting analysis in combination with several column chromatographies, the major activity was revealed to be identical to prostatic acid phosphatase. The present study demonstrates active LPA metabolism in seminal plasma and indicates the possible role of LPA signaling in male sexual organs including prostate cancer.     

Decomposition of urea by size-fractionated planktonic community in a eutrophic reservoir in Japan

Free bacterial populations were separated from an intact planktonic community in water of a eutrophic reservoir in Japan by filtration through Whatman GF/ C glass fiber filters (mean porosity 1.2 µm). Urea decomposition by the free bacterial populations and the intact planktonic community was determined in six different months.The separated free bacteria apparently did not take part in urea-decomposition in waters of the reservoir through the year: the number of free heterotrophic bacteria increased during the urea-decomposition experiments, however, the concentration of urea did not decrease. Whereas, in five cases out of six, urea was decomposed by the intact planktonic community. Probably, phytoplankton were responsible for most of the urea-decomposition. On the assumption that the decomposition of urea obeyed first-order kinetics, rate constants were calculated to be 0.00–0.63 day^–1 with a mean value of 0.21 day^–1.     

Controversies of aromatase localization in human breast cancer--stromal versus parenchymal cells

Aromatase is a key enzyme of estrogen production through conversion from serum androgens in estrogen-dependent postmenopausal breast cancer. Aromatase has been reported to be predominantly located in intratumoral stromal cells and adipocytes but not in parenchymal or carcinoma cells in breast cancer tissue. It is, however, true that there have been controversies regarding intratumoral localization of aromatase in human breast carcinoma, especially whether intratumoral production of estrogens through aromatase occurs in parenchymal or stromal cells. Results of several studies suggested that aromatase present in parenchymal carcinoma cells plays more important roles in the growth and invasion of breast carcinomas than that in stromal cells through providing higher levels of estrogens to carcinoma cells. Aromatase inhibitors are increasingly being used in place of tamoxifen after results of various clinical trials demonstrated that aromatase inhibitors are more effective in increasing survival and recurrence of estrogen-dependent breast cancer patients. Therefore, it is important to clarify the estrogen supplying pathway by aromatase inside of breast carcinoma tissues in order to evaluate the possible efficacy of aromatase inhibitor treatment. In this review, the controversies regarding these intratumoral localization patterns in human breast carcinoma will be briefly summarized.     

Genetic and epigenetic alteration among three homoeologous genes of a class E MADS box gene in hexaploid wheat

Bread wheat (Triticum aestivum) is a hexaploid species with A, B, and D ancestral genomes. Most bread wheat genes are present in the genome as triplicated homoeologous genes (homoeologs) derived from the ancestral species. Here, we report that both genetic and epigenetic alterations have occurred in the homoeologs of a wheat class E MADS box gene. Two class E genes are identified in wheat, wheat SEPALLATA (WSEP) and wheat LEAFY HULL STERILE1 (WLHS1), which are homologs of Os MADS45 and Os MADS1 in rice (Oryza sativa), respectively. The three wheat homoeologs of WSEP showed similar genomic structures and expression profiles. By contrast, the three homoeologs of WLHS1 showed genetic and epigenetic alterations. The A genome WLHS1 homoeolog (WLHS1-A) had a structural alteration that contained a large novel sequence in place of the K domain sequence. A yeast two-hybrid analysis and a transgenic experiment indicated that the WLHS1-A protein had no apparent function. The B and D genome homoeologs, WLHS1-B and WLHS1-D, respectively, had an intact MADS box gene structure, but WLHS1-B was predominantly silenced by cytosine methylation. Consequently, of the three WLHS1 homoeologs, only WLHS1-D functions in hexaploid wheat. This is a situation where three homoeologs are differentially regulated by genetic and epigenetic mechanisms.     

Fructooligosaccharides consumption inhibits the loss of iron from the incisor enamel surface in gastrectomized rat

We examine the effects of fructooligosaccharides (FOS) on the reduction in the incisor iron content in gastrectomized rat. Twenty-eight 5-wk-old male Sprague-Dawley rats were divided into two groups: sham operated (bSH) and gastrectomized (bGX). After 4 wk each group was divided into two subgroups according to the presence or absence of 7.5% FOS in the synthetic diet (SH, SH+FOS, GX, and GX+FOS). At 10 wk wafter surgery, the maxilla was prepared to examine the iron content of the incisor enamel surface at four points. These points corresponded to the iron content at 6,7,8, and 10 wk, respectively. Blood was collected to determine serum iron levels at 4 and 10 wk. The serum iron level significantly decreased at 4 and 10 wk the GX group. At 10 wk, the level in the GX+FOS group significantly increased but did not reaach that in the SH group. The iron content of the enamel surface time-dependently increased and no significant differences were seen between SH and GX+FOS at 8 and 10 wk. These results suggest that FOS consumption impaired the loss of enamel content following gastrectomy, and this effect preceded the effect on the serum iron level.