In Vivo Characterization of Neutrophil Extracellular Traps in Various Organs of a Murine Sepsis Model

Neutrophil extracellular traps (NETs) represent extracellular microbial trapping and killing. Recently, it has been implicated in thrombogenesis, autoimmune disease, and cancer progression. The aim of this study was to characterize NETs in various organs of a murine sepsis model in vivo and to investigate their associations with platelets, leukocytes, or vascular endothelium. NETs were classified as two distinct forms; cell-free NETs that were released away from neutrophils and anchored NETs that were anchored to neutrophils. Circulating cell-free NETs were characterized as fragmented or cotton-like structures, while anchored NETs were characterized as linear, reticular, membranous, or spot-like structures. In septic mice, both anchored and cell-free NETs were significantly increased in postcapillary venules of the cecum and hepatic sinusoids with increased leukocyte-endothelial interactions. NETs were also observed in both alveolar space and pulmonary capillaries of the lung. The interactions of NETs with platelet aggregates, leukocyte-platelet aggregates or vascular endothelium of arterioles and venules were observed in the microcirculation of septic mice. Microvessel occlusions which may be caused by platelet aggregates or leukocyte-platelet aggregates and heterogeneously decreased blood flow were also observed in septic mice. NETs appeared to be associated with the formation of platelet aggregates or leukocyte-platelet aggregates. These observational findings may suggest the adverse effect of intravascular NETs on the host during a sepsis.     

Production of mouse ES cells homozygous for Cdk5-phosphorylated site mutation in c-Src alleles

c-Src-null mutants have not provided a full understanding of the cellular functions of c-Src, reflecting the functional redundancy among Src family members. c-Src is phosphorylated by cyclin-dependent kinase 1 (Cdk1) and Cdk5 at Ser75 in the unique amino terminal c-Src-specific domain. The specific roles of c-Src may be assessed by establishing mouse embryonic stem (ES) cells homozygous for a point mutation at Ser75. Mammalian homozygous cultured cells with a point mutation, however, have not yet been produced by gene targeting. Here we show an efficient procedure for producing ES cell clones bearing a homozygous Ser75 to Asp mutation in the c-src gene. This procedure was developed by combining two previously reported strategies: our procedure for introducing a point mutation into one allele with no exogenous sequence, and the high-geneticin (G418) selection procedure for introducing a mutation into both alleles. The mutant clones expressed the same levels of c-Src protein and autophosphorylation activity as wild-type cells, but the mutant c-Src was not phosphorylated on Ser75 during mitosis. This procedure is feasible for generating cells homozygous for a subtle mutation in most genes, and is expected to be applicable to other somatic cell lines.     

In utero manipulation of coat color formation by a monoclonal anti-c-kit antibody: two distinct waves of c-kit-dependency during melanocyte development.

Previous studies on mice bearing various mutations within the c-kit gene, dominant white spotting (W), indicate the functional role of this tyrosine kinase receptor in the development of melanocytes, germ cells and hematopoietic cells. Despite the availability of mice defective in the c-kit gene and a respectable understanding of the molecular nature of c-kit, however, it is not clear at what stage of gestation c-kit is functionally required for the development of each of these cell lineages. To address this question, we have used a monoclonal anti-c-kit antibody, ACK2, as an antagonistic blocker of c-kit function to interfere with the development of melanocytes during embryonic and postnatal life. ACK2 injected intradermally into pregnant mice entered the embryos where it blocked the proper development of melanocytes. This inhibitory effect was manifested as coat color alteration in the offspring. Furthermore, ACK2 injection also altered the coat color of neonatal and adult mice. Based on the coat color patterns produced by ACK2 administration at various stages before or after birth, the following conclusions are drawn: (i) during mid-gestation, c-kit is functionally required during a restricted period around day 14.5 post-coitum when a sequence of events leading to melanocyte entry into the epidermal layer occurs; (ii) during postnatal life, c-kit is required for melanocyte activation which occurs concomitantly with the hair cycle which continues throughout life after neonatal development of the first hair.     

Hyrtioreticulins A-E, indole alkaloids inhibiting the ubiquitin-activating enzyme, from the marine sponge Hyrtios reticulatus

Hyrtioreticulins A-E (1-5) were isolated from the marine sponge Hyrtios reticulatus, along with a known alkaloid, hyrtioerectine B (6). Structural elucidation on the basis of spectral data showed that 1, 2, and 5 are new tetrahydro-β-carboline alkaloids, while 3 and 4 are new azepinoindole-type alkaloids. Hyrtioreticulins A and B (1 and 2) inhibited ubiquitin-activating enzyme (E1) with IC(50) values of 0.75 and 11μg/mL, respectively, measured by their inhibitory abilities against the formation of an E1-ubiquitin intermediate. So far, only five E1 inhibitors, panapophenanthrine, himeic acid A, largazole, and hyrtioreticulins A and B (1 and 2), have been isolated from natural sources and, among them, 1 is the most potent E1 inhibitor.     

Evaluation of the larval distribution and migration of the Japanese eel in the western North Pacific

The distribution of all larval stages of the Japanese eel, Anguilla japonica, were examined using historical catch records and original data in the western North Pacific (WNP) to evaluate existing information about the larval distribution and migration of this species. A total of 148 preleptocephali, 2547 leptocephali, 6 metamorphosing larvae, and 21 glass eels were collected during 37 cruises over a 52-year period (1956?C2007). Sampling effort was spatio-temporally biased in latitude/longitude among seasons with sampling effort being concentrated near the western margin of the subtropical gyre near Taiwan in the winter season and extensive effort occurring near the spawning area to the east near the seamount chain of the West Mariana Ridge in summer during the spawning season. The distribution of preleptocephali (4.2?C8.7 mm) was limited to a narrow area around 14°N, 142°E just west of the southern part of the seamount chain, while leptocephali (7.7?C62.0 mm) were widely distributed at increasing size westward in the North Equatorial Current (NEC) to the region east of Taiwan. Metamorphosing larvae (52.7?C61.2 mm) were collected only in the area 21?C26°N, 121?C129°E to the east of Taiwan, while glass eels (51.3?C61.2 mm) occurred only within or west of the Kuroshio. These distributions suggest that leptocephali begin to metamorphose within or just east of the Kuroshio, then after completion of metamorphosis the glass eels detrain from the current and migrate inshore. The relationship between catch date and body size of leptocephali suggested that the spawning season is from April to August, but further sampling is needed to eliminate possible effects of sampling bias. This analysis is consistent with the existing hypothesis that Japanese eel larvae born near the West Mariana Ridge are transported westward in the NEC and then transfer to the Kuroshio to recruit to East Asia, although more sampling effort is needed for later stage larvae in the NEC bifurcation region to help understand the larval migration in relation to the possible impacts of ocean?Catmosphere changes.     

Combined exposure to X-irradiation followed by N-ethyl-N-nitrosourea treatment alters the frequency and spectrum of Ikaros point mutations in murine T-cell lymphoma

Ionizing radiation is a well-known carcinogen, but its potency may be influenced by other environmental carcinogens, which is of practical importance in the assessment of risk. Data are scarce, however, on the combined effect of radiation with other environmental carcinogens and the underlying mechanisms involved. We studied the mode and mechanism of the carcinogenic effect of radiation in combination with N-ethyl-N-nitrosourea (ENU) using doses approximately equal to the corresponding thresholds. B6C3F1 mice exposed to fractionated X-irradiation (Kaplan's method) followed by ENU developed T-cell lymphomas in a dose-dependent manner. Radiation doses above an apparent threshold acted synergistically with ENU to promote lymphoma development, whereas radiation doses below that threshold antagonized lymphoma development. Ikaros, which regulates the commitment and differentiation of lymphoid lineage cells, is a critical tumor suppressor gene frequently altered in both human and mouse lymphomas and shows distinct mutation spectra between X-ray- and ENU-induced lymphomas. In the synergistically induced lymphomas, we observed a low frequency of LOH and an inordinate increase of Ikaros base substitutions characteristic of ENU-indcued point mutations, G:C to A:T at non-CpG, A:T to G:C, G:C to T:A and A:T to T:A. This suggests that radiation doses above an apparent threshold activate the ENU mutagenic pathway. This is the first report on the carcinogenic mechanism elicited by combined exposure to carcinogens below and above threshold doses based on the mutation spectrum of the causative gene. These findings constitute a basis for assessing human cancer risk following exposure to multiple carcinogens.     

Metabolic Syndrome in Overweight and Obese Japanese Children

Objective: To determine the prevalence of and sex differences related to the metabolic syndrome among obese and overweight elementary school children. Research Methods and Procedures: Subjects were 471 overweight or obese Japanese children. Children meeting at least three of the following five criteria qualified as having the metabolic syndrome: abdominal obesity, elevated blood pressure, low high‐density lipoprotein‐cholesterol levels, high triglyceride levels, and high fasting glucose levels. Fasting insulin levels were also examined. Results: Japanese obese children were found to have a significantly lower prevalence (17.7%) of the metabolic syndrome than U.S. obese adolescents (28.7%, p = 0.0014). However, Japanese overweight children had a similar incidence (8.7%) of the metabolic syndrome compared with U.S. overweight adolescents (6.8%). Hyperinsulinemia in girls and abdominal obesity in boys are characteristic features of individual metabolic syndrome factors in Japanese children. Discussion: The prevalence of the metabolic syndrome is not lower in preteen Japanese overweight children than in U.S. overweight adolescents, although it is significantly lower in Japanese obese preteen children than in U.S. obese adolescents. Primary and secondary interventions are needed for overweight preteen children in Japan.     

Possible role of nicaraven in neuroprotective effect on hippocampal slice culture

Nicaraven is an agent that is especially beneficial in vasospasm or brain damage caused by subarachnoid hemorrhage. It ameliorates neurological deficits of patients and protects the central nervous system from ischemia. We investigated the neuroprotective effect of nicaraven against oxygen-glucose deprivation (OGD) induced or N-methyl-D-aspartic acid (NMDA) induced hippocampal neuronal cell death in organotypic brain slice cultures. The effect of nicaraven on hippocampal neuronal injury was evaluated by inhibition of uptake of propidium iodide (PI) into dead cells. The results demonstrated that nicaraven protected neuronal cells from both OGD- and NMDA-induced cell death. While nicaraven has a strong hydroxyl radical scavenging effect, another radical scavenger, N-acetyl-L-cysteine (NAC), inhibited cell death only caused by OGD. In contrast, the poly(ADP-ribose) synthetase (PARS) inhibitors 3-aminobenzamide (3-AB) and theophylline protected cells from both OGD- and NMDA-induced cell death. Since nicaraven has an inhibitory effect in PARS, as well as a radical scavenging effect, these results suggest that inhibition of hippocampal cell death caused by NMDA may be attributable to PARS inhibition by nicaraven.     

Specific incorporation of L-glutamine into volicitin in the regurgitant of Spodoptera litura

Volicitin, [N-(17-hydroxylinolenoyl)-L-glutamine], was identified as an elicitor of plant volatiles from a Spodoptera exigua regurgitant. It has been proposed that gut microbes synthesize volicitin from glutamine, a predominant amino acid component in the insect gut. However, we found that glutamine was not a major component in the regurgitant of Spodoptera litura, although L-glutamine was exclusively incorporated into volicitin by S. litura fed on diets enriched with various amino acids. This selectivity of glutamine as a substrate was not due to a dominant occurrence in the insect gut.