Phylogenetic position of Dictyostelium inferred from multiple protein data sets

The phylogenetic position of Dictyostelium inferred from 18S rRNA data contradicts that from protein data. Protein trees always show the close affinity of Dictyostelium with animals, fungi, and plants, whereas in 18S rRNA trees the branching of Dictyostelium is placed at a position before the massive radiation of protist groups including the divergence of the three kingdoms. To settle this controversial issue and to determine the correct position of Dictyostelium, we inferred the phylogenetic relationship among Dictyostelium and the three kingdoms Animalia, Fungi, and Plantae by a maximum-likelihood method using 19 different protein data sets. It was shown at the significance level of 1 SE that the branching of Dictyostelium antedates the divergence of Animalia and Fungi, and Plantae is an outgroup of the Animalia-Fungi-Dictyostelium clade.Correspondence to: T. Miyata     

Cleavage of Host Cytokeratin-6 by Lysine-Specific Gingipain Induces Gingival Inflammation in Periodontitis Patients

Background/PurposeLysine-specific gingipain (Kgp) is a virulence factor secreted from Porphyromonas gingivalis (P. gingivalis), a major etiological bacterium of periodontal disease. Keratin intermediate filaments maintain the structural integrity of gingival epithelial cells, but are targeted by Kgp to produce a novel cytokeratin 6 fragment (K6F). We investigated the release of K6F and its induction of cytokine secretion.MethodsK6F present in the gingival crevicular fluid of periodontal disease patients and in gingipain-treated rat gingival epithelial cell culture supernatants was measured by matrix-assisted laser desorption/ionization time-of-flight mass spectrometer-based rapid quantitative peptide analysis using BLOTCHIP. K6F in gingival tissues was immunostained, and cytokeratin 6 protein was analyzed by immunofluorescence staining and flow cytometry. Activation of MAPK in gingival epithelial cells was evaluated by immunoblotting. ELISA was used to measure K6F and the cytokines release induced by K6F. Human gingival fibroblast migration was assessed using a Matrigel invasion chamber assay.ResultsWe identified K6F, corresponding to the C-terminus region of human cytokeratin 6 (amino acids 359–378), in the gingival crevicular fluid of periodontal disease patients and in the supernatant from gingival epithelial cells cultured with Kgp. K6F antigen was distributed from the basal to the spinous epithelial layers in gingivae from periodontal disease patients. Cytokeratin 6 on gingival epithelial cells was degraded by Kgp, but not by Arg-gingipain, P. gingivalis lipopolysaccharide or Actinobacillus actinomycetemcomitans lipopolysaccharide. K6F, but not a scrambled K6F peptide, induced human gingival fibroblast migration and secretion of interleukin (IL)-6, IL-8 and monocyte chemoattractant protein-1. These effects of K6F were mediated by activation of p38 MAPK and Jun N-terminal kinase, but not p42/44 MAPK or p-Akt.ConclusionKgp degrades gingival epithelial cell cytokeratin 6 to K6F that, on release, induces invasion and cytokine secretion by human gingival fibroblasts. Thus, Kgp may contribute to the development of periodontal disease.     

Remarkable synergies between baicalein and tetracycline, and baicalein and beta-lactams against methicillin-resistant Staphylococcus aureus

During the screening of compounds that potentiate the effect of antimicrobial agents against methicillin-resistant Staphylococcus aureus(MRSA), we found that an extract of thyme (Thymus vulgaris L) leaves greatly reduced the minimum inhibitory concentration (MIC) of tetracycline against MRSA. We isolated the effective compound and identified it as baicalein (5, 6, 7-trihydroxyflavone). One of the clinically isolated MRSA strains possessed tetK, a gene encoding active efflux pump for tetracycline. We examined the effect of baicalein on the efflux of tetracycline, using Escherichia coli KAM32/pTZ1252 carrying the tetK. The E. coli KAM32/pTZ1252 showed 8 to 16 times higher MIC than E. coli KAM32. We observed strong inhibition of transport of tetracycline by baicalein with membrane vesicles prepared from E. coli KAM32/pTZ1252. Baicalein also showed synergy with tetracycline in a MRSA strain that doesn't possess tetK, or with beta-lactams. Thus, mechanisms of the synergies seem to be versatile.     

An Efficient reproductive method for Irs2-/- mice with C57BL/6JJcl genetic background

Efficient reproduction using natural mating and reproduction technology [in vitro fertilization (IVF) and embryo transfer (ET)] was investigated in IRS2 deficient mice with C57BL/6JJcl genetic background (Irs2(-/-) mice) as a typical type 2 diabetes model. From the results using various combinations of Irs2(-/-) and Irs2(-/+) mice, the combination of female Irs2(-/+) x male Irs2(-/-) was found to be more efficient than other combinations. In applications of reproduction technology using IVF and ET, the combination of female Irs2(-/+) x male Irs2(-/-) involves the possibility of Irs2(-/-) production by repeats using female Irs2(-/+) mice. However, reproductive continuity using this combination is difficult because of dependence on human technique and the cost of ET. Therefore, we concluded that Irs2(-/-) mice should be produced by embryo transfer using Irs2(-/-) mice from a colony consisting of female Irs2(-/+) x male Irs2(-/-).     

Circadian dysfunction in response to in?vivo treatment with the mitochondrial toxin 3-nitropropionic acid

Sleep disorders are common in neurodegenerative diseases including Huntington''s disease (HD) and develop early in the disease process. Mitochondrial alterations are believed to play a critical role in the pathophysiology of neurodegenerative diseases. In the present study, we evaluated the circadian system of mice after inhibiting mitochondrial complex II of the respiratory chain with the toxin 3-nitropropionic acid (3-NP). We found that a subset of mice treated with low doses of 3-NP exhibited severe circadian deficit in behavior. The temporal patterning of sleep behavior is also disrupted in some mice with evidence of difficulty in the initiation of sleep behavior. Using the open field test during the normal sleep phase, we found that the 3-NP-treated mice were hyperactive. The molecular clockwork responsible for the generation of circadian rhythms as measured by PER2::LUCIFERASE was disrupted in a subset of mice. Within the SCN, the 3-NP treatment resulted in a reduction in daytime firing rate in the subset of mice which had a behavioral deficit. Anatomically, we confirmed that all of the treated mice showed evidence for cell loss within the striatum but we did not see evidence for gross SCN pathology. Together, the data demonstrates that chronic treatment with low doses of the mitochondrial toxin 3-NP produced circadian deficits in a subset of treated mice. This work does raise the possibility that the neural damage produced by mitochondrial dysfunction can contribute to the sleep/circadian dysfunction seen so commonly in neurodegenerative diseases.     

Correction to: Comparative analysis of sperm motility in liquid and seminal coagulum portions between Bornean orangutan (Pongo pygmaeus) and chimpanzee (Pan troglodytes)

Primates - In the original publication of the article, the coauthor “Takashi Hayakawa” was wrongly assigned as co-corresponding author.     

Localization of insulinoma associated protein 2, IA-2 in mouse neuroendocrine tissues using two novel monoclonal antibodies

AimsInsulinoma-associated protein 2 (IA-2) is a member of the protein tyrosine phosphatase family that is localized on the insulin granule membrane. IA-2 is also well known as one of the major autoantigens in Type 1 diabetes mellitus. IA-2 gene deficient mice were recently established and showed abnormalities in insulin secretion. Thus, detailed localization of IA-2 was studied using wild-type and IA-2 gene deficient mice.Main methodsTo localize IA-2 expression in mouse neuroendocrine tissues, monoclonal antibodies were generated against IA-2 and western blot and immunohistochemical analyses were carried out in IA-2^+/+ mice. IA-2^?/? mice served as a negative control.Key findingsWestern blot analysis revealed that the 65 kDa form of IA-2 was observed in the cerebrum, cerebellum, medulla oblongata, pancreas, adrenal gland, pituitary gland, muscular layers of the stomach, small intestine, and colon. By immunohistochemical analysis, IA-2 was produced in endocrine cells in pancreatic islets, adrenal medullary cells, thyroid C-cells, Kulchitsky cells, and anterior, intermediate, and posterior pituitary cells. In addition, IA-2 was found in somatostatin-producing D-cells and other small populations of cells were scattered in the gastric corpus. IA-2 expression in neurites was confirmed by the immunostaining of IA-2 using primary cultured neurons from the small intestine and nerve growth factor (NGF)-differentiated PC12 cells.SignificanceThe IA-2 distribution in peripheral neurons appeared more intensely in neurites rather than in the cell bodies.     

Microbial resource allocation for phosphatase synthesis reflects the availability of inorganic phosphorus across various soils

According to the resource allocation model for extracellular enzyme synthesis, microorganisms should preferentially allocate their resources to phosphorus (P)-acquiring enzyme synthesis when P availability is low in soils. However, the validity of this model across different soil types and soils differing in their microbial community composition has not been well demonstrated. Here we investigated whether the resource allocation model for phosphatase synthesis is applicable across different soil types (Andosols, Acrisols, Cambisols, and Fluvisols) and land uses (arable and forest), and we examined which soil test P and/or P fraction microorganisms responded to when investing their resources in phosphatase synthesis in the soils. The ratio of alkaline phosphatase (ALP) to β-d-glucosidase (BG) activities in the arable soils and the ratio of acid phosphatase (ACP) to BG activities in the forest soils were significantly negatively related with the available inorganic P concentration. We also observed significant effects of available inorganic P, pH, soil types, and land uses on the (ACP + ALP)/BG ratio when the data for the arable and forest soils were combined and used in a stepwise multiple regression analysis. These results suggest that microbial resource allocation for phosphatase synthesis is primarily controlled by available inorganic P concentration and soil pH, but the effects of soil types and land uses are also significant.