Effect of copper and diethyldithiocarbamate combination therapy on the macular mouse, an animal model of Menkes disease.

Menkes disease (MD) is a neurodegenerative disorder characterized by a copper deficiency in the brain. It is caused by the defective intestinal absorption of copper resulting from a deficiency of a copper-transporting ATPase, ATP7A. This gives rise to an accumulation of copper in the intestine. The copper deficiency in the brain of MD patients cannot be improved by copper injections, because the administered copper accumulates at the blood-brain barrier and is not transported across to the neurons. To resolve this problem, we investigated the effect of a combination therapy of copper and sodium diethyldithiocarbamate (DEDTC), a lypophilic chelator, in an animal model of MD, the macular mouse. Four-week-old macular mice treated with 50 mug of CuCl2 on the 7th day after birth were used. Experimental mice were given a subcutaneous injection of CuCl2 (4 microg) and an intraperitoneal injection of DEDTC (0.2 mg/g body weight) twice a week for 4 weeks and then sacrificed. Copper concentrations and cytochrome-c oxidase activity in the brains of treated mice were higher than those of control macular mice, which received only copper or saline. The ratios of brain noradrenaline to dopamine and of adrenaline to dopamine were also increased by the treatment, suggesting that the activity of dopamine beta-hydroxylase, a copper-dependent enzyme, was improved by the treatment. Liver and renal function tests showed no abnormalities in the treated mice, although copper concentrations in the kidneys of treated mice were higher than those of control macular mice. These results suggest that DEDTC facilitates the passage of copper across the blood-brain barrier and that the combination therapy of copper and DEDTC may be an effective treatment for the neurological disturbances suffered by patients with MD.

A new analytical system for quantification scratching behaviour in mice.

BACKGROUND: Scratching behaviour is an important component of human atopic dermatitis. The duration of scratching determines the extent of skin damage and thus the rash, but quantification of this is difficult. Establishment of a method for measuring the duration of scratching is important in order to make objective assessments of the factors that may cause the itch and also the efficacy of new antipruritic drugs. OBJECTIVES: A novel method for assessing the duration of scratching in mice was evaluated, based on the time course changes in the distance between the animal's hind limbs and the back of the neck during scratching behaviour. METHODS: Compound 48/80 was administered intradermally to the back of ICR mice and their scratching behaviour was recorded on digital videotape. The distance between the back and the hind limb was measured continuously using an image analysis system. RESULTS: Measurement of the frequency and duration when the mouse's hind limb came into contact with the back of the neck provided an accurate method of quantitating scratching behaviour. CONCLUSIONS: This system provides a new method of quantifying scratching behaviour in a mouse.

Inability of IL-12 to down-regulate IgE synthesis due to defective production of IFN-gamma in atopic NC/Nga mice.

NC/Nga mice raised in nonsterile circumstances spontaneously suffer from atopic dermatitis-like skin lesions with IgE hyperproduction. We investigated effects of rIL-12 on the IgE production in NC/Nga mice. rIL-12 administration was successful to suppress the increase of IgE levels in BALB/c mice immunized with OVA and aluminum hydroxide, but failed to abrogate that in NC/Nga mice. Both in vivo and in vitro IFN-gamma production induced by rIL-12 was less in NC/Nga mice than in BALB/c mice. Addition of rIFN-gamma to rIL-4 and LPS completely abrogated IgE production by B cells of BALB/c mice, but was insufficient to suppress it by B cells of NC/Nga mice. In splenic cells pretreated with Con A, STAT4 was phosphorylated at the tyrosine residue by addition of rIL-12, which was more weakly inducible in NC/Nga mice than in BALB/c mice. Finally, we examined the preventive ability of rIL-12 on the clinical aspects of atopic dermatitis in NC/Nga mice. rIL-12 administration resulted in exacerbation of development of the skin lesions and IgE production in NC/Nga mice raised in nonsterile circumstances. These results suggest that defective production of IFN-gamma by T cells less sensitive to IL-12 and low responsiveness of B cells to IFN-gamma may contribute to IgE hyperproduction in NC/Nga mice, and that IL-12 may have no ability to improve the clinical aspects of NC/Nga mice.

Universal occurrence of the vasa-related genes among metazoans and their germline expression in Hydra.

The vasa (vas)-related genes are members of the DEAD box protein family and are involved in germ cell formation in higher metazoans. In the present study, we cloned the vas-related genes as well as the PL10-related genes, other members of the DEAD box protein family, from lower metazoans: sponge, Hydra and planaria. The phylogenetic analysis suggested that the vas-related genes arose by duplication of a PL10-related gene before the appearance of sponges but after the diversion of fungi and plants. The vas-related genes in Hydra, Cnvas1 and Cnvas2 were strongly expressed in germline cells and less strongly expressed in multipotent interstitial stem cells and ectodermal epithelial cells. These results suggest that the vas-related genes occur universally among metazoans and that their expression in germline cells was established at least before cnidarian evolution.

Necessity of thromboxane A2 for initiation of platelet-mediated contact sensitivity: dual activation of platelets and vascular endothelial cells.

To investigate the crucial role of platelet-derived thromboxane A(2) (TXA(2)) in initiating Ag-specific contact sensitivity (CS), a platelet-dependent CS model using genetically mast cell-deficient W/W(v) mice, was provided. In vivo treatment with BAYu3405, a TXA(2) receptor antagonist, markedly suppressed CS responses in a dose-dependent manner. This inhibitory effect occurred when BAYu3405 was administered before an early initiating phase, suggesting that TXA(2) may be a potent initiator of platelet-mediated CS responses. When platelets were pretreated with BAYu3405 in vitro, platelet aggregation as well as serotonin release, which is able to induce the early phase response allowing local recruitment of CS effector T cells due to direct activation of vascular endothelial cells, was inhibited. The addition of U46619, a TXA(2) agonist, or a mixture of platelets and thrombin-enhanced expression of both ICAM-1 and VCAM-1 on isolated mouse aortic endothelial cells, which was completely abolished by pretreatment with BAYu3405. Furthermore, intradermal injection of U46619 into the ear of platelet-depleted mice led to CS responses with marked expression of ICAM-1 and VCAM-1 on the vascular endothelium. These findings suggest that TXA(2) generated from platelets activated with Ag may mediate initiation of CS responses through inducing serotonin release from platelets and the subsequent aggregation and up-regulated expression of ICAM-1 and VCAM-1 on vascular endothelial cells.

Expression and evolutionary conservation of nanos-related genes in Hydra.

The Drosophila gene nanos encodes two particular zinc finger motifs which are also found in germline-associated factors from nematodes to vertebrates. We cloned two nanos (nos)-related genes, Cnnos1 and Cnnos2 from Hydra magnipapillata. Using whole-mount in situ hybridization, the expression of Cnnos1 and Cnnos2 was examined. Cnnos1 was specifically expressed in multipotent stem cells and germline cells, but not in somatic cells. Cnnos2 was weakly expressed in germline cells and more specifically in the endoderm of the hypostome where it appears to be involved in head morphogenesis. In addition to structural conservation in the zinc finger domain of nanos-related genes, functional conservation of Cnnos1 was also demonstrated by the finding that a Cnnos1 transgene can partially rescue the nosRC phenotype that is defective in the egg production of Drosophila. Thus, the function of nanos-related genes in the germline appears to be well conserved from primitive to highly evolved metazoans.

Genetic analysis of developmental mechanisms in Hydra. XXII. Two types of female germ stem cells are present in a male strain of Hydra magnipapillata.

Three types of interstitial stem cell subpopulation were isolated from Hydra magnipapillata, and their roles in sex determination were examined. A subpopulation of interstitial stem cells restricted to the sperm differentiation pathway was isolated previously from strain nem-1 (male). Another subpopulation restricted to the egg differentiation pathway was also isolated from the same strain. Hydroxyurea treatment was used for isolation in both cases. "Pseudoepithelial hydra" containing only sperm- or egg-restricted stem cell but no other interstitial stem cell types were maintained by force-feeding for 2 years. Sex reversal from egg- to sperm-restricted stem cells occurred three times during this period. Both of these two stem cell types are numerous in the central gastric region of the pseudoepithelial hydra, but absent in the foot region below the budding zone. Foot tissue was cut out from normal nem-1 polyps (male) and allowed to regenerate. The regenerates produced eggs but no sperm upon sex induction. These and other results suggest that the foot tissue contains multipotent stem cells capable of differentiating into eggs during sexual differentiation. These observations suggest that strain nem-1 (male) contains three types of interstitial stem cell subpopulations: (1) sperm-restricted stem cells, (2) egg-restricted stem cells, and (3) multipotent stem cells capable of differentiating into nerve cells, nematocytes, and eggs. Upon sex induction, however, differentiation of eggs by the latter two types is suppressed, and only sperm are produced by the sperm-restricted stem cells. Evidence is presented which suggests that similar "phenotypic males," which normally only produce sperm but contain the stem cell types capable of differentiating into eggs, occur widely in Hydra magnipapillata. A possible relationship between phenotypic male hydra and hermaphroditic hydra is discussed.

Mechanistic study of inhibition of levofloxacin absorption by aluminum hydroxide.

The mechanisms of reduction in absorption of levofloxacin (LVFX) by coadministration of aluminum hydroxide were studied. The partition coefficient of LVFX (0.1 mM) between chloroform and phosphate buffer (pH 5.0) was reduced by 60 to 70% with the addition of metal ions such as Cu2+, Al3+, and Fe2+ (0.8 mM), which indicated the formation of LVFX-metal ion chelates. However, there was no significant difference in absorption from rat intestine between the synthetic LVFX-Al3+ (1:1) chelate (6.75 mM) and LVFX (6.75 mM) in an in situ recirculation experiment. On the other hand, Al(NO3)3 (1.5 mM) significantly inhibited the absorption of LVFX (1.5 mM) by 20% of the control in the in situ ligated loop experiment, in which partial precipitation of aluminum hydroxide was observed in the dosing solution. Data for adsorption of LVFX and ofloxacin (OFLX) from aqueous solution by aluminum hydroxide were shown to fit Langmuir plots, and the adsorptive capacities (rmax) and the K values were 7.0 mg/g and 1.77 x 10(4) M-1 for LVFX and 7.4 mg/g and 1.42 x 10(4) M-1 for OFLX, respectively. The rate of adsorption of several quinolones (50 microM) onto aluminum hydroxide (2.5 mg/ml) followed the order norfloxacin (NFLX) (72.0%) > enoxacin (ENX) (61.0%) > OFLX (47.2%) approximately LVFX (48.1%). The elution rate of adsorbed quinolones with water followed the rank order LVFX (17.9%) approximately OFLX (20.9%) approximately ENX (18.3%) > NFLX (11.9%). These results strongly suggest that adsorption of quinolones by aluminum hydroxide reprecipitated in the small intestine would play an important role in the reduced bioavailability of quinolones after coadministration with aluminum-containing antacids.

Genetic analysis of developmental mechanisms in hydra. XX. Cloning of interstitial stem cells restricted to the sperm differentiation pathway in Hydra magnipapillata.

Hydra magnipapillata polyps containing a subpopulation of interstitial stem cells restricted to the germline differentiation pathway were obtained. Chim-C1 is a chimeric strain produced by combining wild-type epithelial cell lineages with a temperature-sensitive interstitial cell lineage. It grows normally at 18 degrees C. When cultured at 25 degrees C, many polyps lost interstitial cells and their differentiation products (nerve cells and nematocytes), and subsequently turned into epithelial hydra unable to move or feed. Some polyps, however, turned into an unexpected type, termed "pseudo-epithelial hydra." These polyps resembled epithelial hydra in the absence of nerve cells or nematocytes in the tissue and in their inability to move or feed. In contrast to epithelial hydra, however, their tissue contained proliferating interstitial cells. Similar pseudo-epithelial hydra were also produced from another strain, nem-1, by means of hydroxyurea treatment. Clones of pseudo-epithelial hydra were maintained through force-feeding over 130 days for chim-C1 and over 2 years for nem-1. In both cases, interstitial cells proliferated throughout the period without producing any nerve cells or nematocytes. These interstitial cells, however, differentiated into sperm. Thus, the interstitial cells present in pseudo-epithelial hydra were able to differentiate into gametic cells but not into somatic cells (nerve cells and nematocytes). These observations suggest that, as Littlefield (1985, Dev. Biol. 112, 185-193) has shown for H. oligactis, the interstitial stem cell population in H. magnipapillata includes a subpopulation which can differentiate only into gametic cells.

Eicosapentaenoic acid inhibits tube formation of vascular endothelial cells in vitro.

We previously have described a quantitative angiogenesis in vitro model, in which endothelial cells are cultured between two layers of type I collagen gel and become organized into tube-like structures. Using this model, the effect of eicosapentaenoic acid (20:5n-3) on tube formation was investigated. When the endothelial cells isolated from bovine carotid artery were treated for 2 days with 5 micrograms/mL of arachidonic acid (20:4n-6), eicosapentaenoic acid or docosahexaenoic acid (22:6n-3), these polyunsaturated fatty acids were extensively incorporated into cellular phospholipids. The context of arachidonic, eicosapentaenoic and docosahexaenoic acid increased from 9.58% to 23.29%, from 0.98% to 11.76% and from 6.88% to 18.40%, respectively. When the eicosapentaenoic acid-treated cells were cultured between collagen gels, the tube-forming ability of the cell was markedly inhibited. The inhibition was dose-dependent between 1.0 and 5.0 micrograms/mL of eicosapentaenoic acid. At 5.0 micrograms/mL of eicosapentaenoic acid the inhibition reached 76%. By contrast, arachidonic acid increased tube formation, and docosahexaenoic acid had no effect. To elucidate the mechanism of eicosapentaenoic acid induced inhibition of in vitro tube formation, we examined the effect of the acid on the proliferation of endothelial cells. Eicosapentaenoic acid at any dose (less than 5.0 micrograms/mL) had no effect on the proliferation of endothelial cells cultured on plastic plates without collagen gel. However, when the cells were cultured between collagen gels, eicosapentaenoic acid inhibited cell growth in a dose-dependent manner with maximum inhibition being observed at 2.5 micrograms/mL. These data suggest that eicosapentaenoic acid suppresses tube formation of endothelial cells,at least in part, via its inhibitory effect on cellular proliferation.(ABSTRACT TRUNCATED AT 250 WORDS)