Cell cycle arrest by monochloramine through the oxidation of retinoblastoma protein

Impairment of cell cycle control has serious effects on inflammation, tissue repair, and carcinogenesis. We report here the G1 cell cycle arrest by monochloramine (NH2Cl), a physiological oxidant derived from activated neutrophils, and its mechanism. When Jurkat cells were treated with NH2Cl (70 microM, 10 min) and incubated for 24 h, the S phase population decreased significantly with a slight increase in the hypodiploid cell population. The G0/ G1 phase and G2/M phase populations did not show marked changes. Three hours after NH2Cl treatment, the retinoblastoma protein (pRB) was dephosphorylated especially at Ser780 and Ser795, both of which are important phosphorylation sites for the G1 checkpoint function. The phosphorylation at Ser807/811 showed no apparent change. The expression of cyclins, cyclin-dependent kinases, and cyclin-dependent kinase inhibitors showed no apparent change. Moreover, the kinase activity that phosphorylates pRB remained constant even after NH2Cl treatment. The protein phosphatase activity that dephosphorylates pRB showed a marginal increase. Notably, when the recombinant pRB was oxidized by NH2Cl in vitro, the oxidized pRB became difficult to be phosphorylated by kinases, especially at Ser780 and Ser795, but not at Ser807/811. Amino acid analysis of oxidized pRB showed methionine oxidation to methionine sulfoxide. The NH2Cl-treated Jurkat cell proteins also showed a decrease in methionine. These observations suggested that direct pRB oxidation was the major cause of NH2Cl-induced cell cycle arrest. In the presence of 2 mM NH4+, NaOCl (200 microM) or activated neutrophils also induced a G1 cell cycle arrest. As protein methionine oxidation has been reported in inflammation and aging, cell cycle modulation by pRB oxidation may occur in various pathological conditions.     

Melanocyte development: with a message of encouragement to young women scientists

This is a semi-biographical review describing my research on melanocyte development and related personal experiences. Having been educated and trained as a dermatologist, I have been involved in many clinically-oriented studies, however, what has always interested me the most is pigment cell biology. Since I started working at St Marianna University in 1991, I have been undertaking research on melanocyte development and relevant growth factors using mice as models. My research in this field was inspired by my collaborations with various scientists, mostly from the field of biology. Many of these specialists I have met at meetings of the Societies of Pigment Cell Research (PCR). Stem cell factor (SCF, Kitl) and endothelin 3 (EDN3) have been identified as indispensable factors regulating the development of melanocytes. Mice mutant at loci encoding those factors (or their receptors) such as Sl/Sl (receptors W/W) and ls/ls (receptors s/s) have white coat colors and white patches, respectively. Our murine neural crest cell (NCC) primary cultures derived from Sl/Sl embryos showed that EDN3 cannot develop melanocyte precursors without SCF and that EDN3 can elicit proliferation and differentiation in the presence of SCF. These results suggest that without EDN3 and the endothelin type B receptor (EDNRB), melanocytes can not fully increase in number, which could well be the cause of the partial white coat color of ls/ls and s/s mice. Contamination with factors derived from the serum in medium or in feeder cells sometimes causes experimental errors, and therefore we established three immortal cell lines derived from NCC in different developmental stages and designated them as NCCmelb4, NCCmelb4M5 and NCCmelan5, all of which can survive without feeder cells. Using these cell lines and NCC primary cultures, we studied the effect of many factors related to melanocyte development. From the results, it has become evident that Vitamin D3 induces EDNRB expression by NCCmelb4 cells. In addition to the International Pigment Cell Conference (IPCC), I have also taken part in many annual meetings of the Japanese Society for Pigment Cell Research (JSPCR), Pan American Society for Pigment Cell Research (PASPCR) and European Society for Pigment Cell Research (ESPCR). Not only have I learned a great deal, I have enjoyed myself immensely at those meetings. Moreover, I have made many good friends there, some of whom I have collaborated with in my research. To conclude, I would like to give my message 'be ambitious' to young scientists, especially young women.     

Antimicrobial activities of diterpene dialdehydes, constituents from myoga (Zingiber mioga Roscoe), and their quantitative analysis

The antimicrobial activities of the three diterpene dialdehydes, miogadial, galanal A and galanal B, isolated from flower buds of the myoga (Zingiber mioga Roscoe) plant were investigated with some strains of bacteria, yeasts and molds. Among the three compounds, miogadial exhibited relatively greater antimicrobial activity than the others against Gram-positive bacteria and yeasts. Galanals A and B also behaved as antimicrobial agents against Gram-positive bacteria and yeasts. The content of miogadial in the flower buds was much higher than that in the leaves, whereas galanals A and B were contained at high levels in the leaves and rhizomes.     

Bone marrow stromal cells improve cardiac performance in healed infarcted rat hearts

Postinfarct congestive heart failure is one of the leading causes of morbidity and mortality in developed and developing countries. The main purpose of this study was to investigate whether transplantation of bone marrow stromal cells (BMSC) directly into the myocardium could improve the performance of healed infarcted rat hearts. Cell culture medium with or without BMSC was injected into borders of cardiac scar tissue 4 wk after experimental infarction. Cardiac performance was evaluated 2 wk after cellular (n = 10) or medium (n = 10) injection by electro- and echocardiography. Histological study was performed 3 wk after treatment. Electrocardiography of BMSC-treated infarcted rats showed electrical and mechanical parameters more similar to those in control than in medium-treated animals: a normal frontal QRS axis in 6 of 10 BMSC-treated and all control rats and a rightward deviation of the QRS axis in all 10 medium-treated animals. BMSC treatment, assessed by echocardiography, improved fractional shortening (39.00 +/- 4.03%) compared with medium-treated hearts (18.20 +/- 0.74%) and prevented additional changes in cardiac geometry. Immunofluorescence microscopy revealed colocalization of 4',6-diamidino-2-phenylindole-labeled nuclei of transplanted cells with cytoskeletal markers for cardiomyocytes and smooth muscle cells, indicating regeneration of damaged myocardium and angiogenesis. These data provide strong evidence that BMSC implantation can improve cardiac performance in healed infarctions and open new promising therapeutic opportunities for patients with postinfarction heart failure.     

Pigeon's recognition of cartoons: effects of fragmentation, scrambling, and deletion of elements

J. Cerella [Pattern Recognit. 12 (1980) 1] and more recently S. Watanabe [Behav. Proc. 53 (2001) 3] demonstrated that pigeons showed no decrement in recognizing cartoons that were spatially scrambled, indicating that pigeons' discriminative responding is controlled by local features alone. In contrast Kirkpatrick-Steger et al. [J. Exp. Psychol. Anim. Behav. Proc. 24 (1998) 34] used line drawings as stimuli and demonstrated the importance of spatial organization for picture recognition by pigeons, confirming related findings reported in their previous studies. The present study revisited the recognition of cartoons by pigeons. In Experiment 1, pigeons were trained to discriminate cartoon people on a variety of background scenes. Subsequent tests revealed that discriminative performances with both familiar and novel instances decreased as the objects and object-like parts were progressively fragmented, indicating that search for the targeted cartoon people in the stimulus array might have enhanced the pigeons to attend to global aspects of cartoon people. Experiment 2 used line drawings of cartoon faces as stimuli and examined effects of scrambling and deletion of components. A set of components (eyes and eyebrows) exerted strong control over behavior and scrambling only moderately suppressed responding. The results suggest that pigeons use both global and local aspects, with different mixtures of these types of information depending on the particular perceptual context.     

Comparison of Japanese isolates of Coxiella burnetii by PCR-RFLP and sequence analysis

The genetic variation of Japanese isolates of Coxiella burnetii, the agent of Q fever, was found for the first time. Forty-nine out of 72 isolates had the chronic pattern of the isocitrate hydrogenase gene. Sequence analysis revealed that the isolates have a specific nucleotide sequence. The putative amino acid sequence was the same as that of chronic reference strains. These results suggest the variation of C. burnetii isolates in Japan.     

In vitro susceptibility to tetracycline and fluoroquinolones of Japanese isolates of Coxiella burnetii

Coxiella burnetii is the agent of the worldwide zoonosis, Q fever. The in vitro susceptibility to tetracycline and fluoroquinolones of Japanese isolates of C. burnetii was evaluated for the first time. The MICs against Japanese isolates were almost the same as the MICs against the foreign reference isolates. The results suggest that the common antibiotics therapy for Q fever used in other countries is also effective for Japanese Q fever patients.     

Tie2/angiopoietin-1 signaling regulates hematopoietic stem cell quiescence in the bone marrow niche

The quiescent state is thought to be an indispensable property for the maintenance of hematopoietic stem cells (HSCs). Interaction of HSCs with their particular microenvironments, known as the stem cell niches, is critical for adult hematopoiesis in the bone marrow (BM). Here, we demonstrate that HSCs expressing the receptor tyrosine kinase Tie2 are quiescent and antiapoptotic, and comprise a side-population (SP) of HSCs, which adhere to osteoblasts (OBs) in the BM niche. The interaction of Tie2 with its ligand Angiopoietin-1 (Ang-1) induced cobblestone formation of HSCs in vitro and maintained in vivo long-term repopulating activity of HSCs. Furthermore, Ang-1 enhanced the ability of HSCs to become quiescent and induced adhesion to bone, resulting in protection of the HSC compartment from myelosuppressive stress. These data suggest that the Tie2/Ang-1 signaling pathway plays a critical role in the maintenance of HSCs in a quiescent state in the BM niche.     

Participation of prostaglandin E receptor EP4 subtype in duodenal bicarbonate secretion in rats

We examined, by using a specific PGE receptor subtype EP4 agonist and antagonist, the involvement of EP4 receptors in duodenal HCO(3)(-) secretion induced by PGE(2) and mucosal acidification in rats. Mucosal acidification was achieved by exposing a duodenal loop to 10 mM HCl for 10 min, and various EP agonists were given intravenously 10 min before the acidification. Secretion of HCO(3)(-) was dose-dependently stimulated by AE1-329 (EP4 agonist), the maximal response being equivalent to that induced by sulprostone (EP1/EP3 agonist) or PGE(2). The stimulatory action of AE1-329 and PGE(2) but not sulprostone was attenuated by AE3-208, a specific EP4 antagonist. This antagonist also significantly mitigated the acid-induced HCO(3)(-) secretion. Coadministration of sulprostone and AE1-329 caused a greater secretory response than either agent alone. IBMX potentiated the stimulatory action of both sulprostone and AE1-329, whereas verapamil mitigated the effect of sulprostone but not AE1-329. Chemical ablation of capsaicin-sensitive afferent neurons did not affect the response to any of the EP agonists used. We conclude that EP4 receptors are involved in the duodenal HCO(3)(-) response induced by PGE(2) or acidification in addition to EP3 receptors. The process by which HCO(3)(-) is secreted through these receptors differs regarding second-messenger coupling. Stimulation through EP4 receptors is mediated by cAMP, whereas that through EP3 receptors is regulated by both cAMP and Ca(2+); yet there is cooperation between the actions mediated by these two receptors. The neuronal reflex pathway is not involved in stimulatory actions of these prostanoids.     

Transformation of the monocot Alstroemeria by Agrobacterium rhizogenes

An efficient procedure is described for transformation of calli of the monocotyledonous plant Alstroemeria by Agrobacterium rhizogenes. Calli were co-cultivated with A. rhizogenes strain A13 that harbored both a wild-type Ri-plasmid and the binary vector plasmid pIG121Hm, which included a gene for neomycin phosphotransferase II (NPTII) under the control of the nopaline synthase (NOS) promoter, a gene for hygromycin phosphotransferase (HPT) under the control of the cauliflower mosaic virus (CaMV) 35S promoter, and a gene for -glucuronidase (GUS) with an intron fused to the CaMV 35S promoter. Inoculated calli were plated on medium that contained cefotaxime to eliminate bacteria. Four weeks later, transformed cells were selected on medium that contained 20 mg L^–1 hygromycin. A histochemical assay for GUS activity revealed that selection by hygromycin was complete after eight weeks. The integration of the T-DNA of the Ri-plasmid and pIG121Hm into the plant genome was confirmed by PCR. Plants derived from transformed calli were produced on half-strength MS medium supplemented with 0.1 mg L^–1 GA₃ after about 5 months of culture. The presence of the gusA, nptII, and rol genes in the genomic DNA of regenerated plants was detected by PCR and Southern hybridization, and the expression of these transgenes was verified by RT-PCR.