Stimulation of endothelial cell binding of lymphocytes by tumor necrosis factor

Lymphokines and monokines have been reported to affect endothelial cell (EC) morphology and function. In experiments here described, we have demonstrated that recombinant tumor necrosis factor (TNF) stimulates the adhesion of T lymphocytes to confluent monolayers of human umbilical vein EC. The increase in adhesion induced by TNF was EC-specific inasmuch as preincubation of the lymphocytes with TNF did not alter binding, and preincubation of human dermal fibroblasts with TNF did not increase their inherently low adhesiveness for lymphocytes. Stimulation of T-EC binding occurred after treatment of the EC with as little as 0.01 U/ml (1 pg/ml) of TNF. In kinetic experiments, preincubation of EC with TNF for 4 hr resulted in optimal adhesion. TNF-treated EC retained their increased adhesiveness after fixation with paraformaldehyde, suggesting that TNF stimulated binding by increasing the expression or accessibility of EC surface receptors for lymphocytes. Although antibodies to the lymphocyte function-associated antigen 1 alpha- or beta-chains on the T cell markedly inhibited unstimulated T-EC binding, such antibodies had no effect on the increase in EC adhesiveness induced by TNF, indicating that the increased binding resulted from the generation of an alternate binding receptor on the EC membrane. These findings provide additional evidence that cytokines participate in the mobilization of mononuclear cells in the chronic inflammatory reaction by stimulation of the adhesiveness of endothelium for circulating lymphocytes.     

A simplified method for detection of visual abnormality in rats on reproduction study: the critical intensity of illumination and the effect of dark-adaptation

A newly designed Y-shaped box was previously reported by authors to be useful for a screening test of visual abnormality in rats because of easy numerization and statistical analysis of the results. In the present paper, the relationship between the intensity of illumination and the negative phototaxic response were examined. The total selection rate for the dark area (total time of selecting dark area/total trials) of non-treated rats under 30 lux illumination was 98% and identical wit the result of those under 1600 lux. The selecting rates of non-treated rats under the illumination of 15, 10, 7.5, 5 and 1.25 lux were 95, 93, 89, 82 and 67%, respectively. On the other hand those of dark-adapted rats one hour under 15, 7.5 and 5 lux showed 98, 95 and 92%, respectively. From these results, the critical intensities of illumination-unaffected selection rate for darkness in non-treated and dark-adapted rats were estimated at 30 and 15 lux, respectively. There was an obvious effect of dark-adaptation on the visual ability of rats. The selection rate of eyelid-sutured rats under 30 lux was 52%, an approximate theoretical value of true blindness, but it was 85% under 1600 lux. It is suggested that a more exact detection of visual abnormality would be possible under the critical intensity of illumination-unaffected selection rate for darkness.     

A new Early Cretaceous eutherian mammal from the Sasayama Group,Hyogo, Japan

We here describe a new Early Cretaceous (early Albian) eutherian mammal, Sasayamamylos kawaii gen. et sp. nov., from the ‘Lower Formation’ of the Sasayama Group, Hyogo Prefecture, Japan. Sasayamamylos kawaii is characterized by a robust dentary, a distinct angle on the ventral margin of the dentary at the posterior end of the mandibular symphysis, a lower dental formula of 3–4 : 1 : 4 : 3, a robust lower canine, a non-molariform lower ultimate premolar, and a secondarily reduced entoconid on the molars. To date, S. kawaii is the earliest known eutherian mammal possessing only four premolars, which demonstrates that the reduction in the premolar count in eutherians started in the late Early Cretaceous. The occurrence of S. kawaii implies that the relatively rapid diversification of eutherians in the mid-Cretaceous had already started by the early Albian.     

Cyanobacterial phytochrome-like PixJ1 holoprotein shows novel reversible photoconversion between blue- and green-absorbing forms

The gene, pixJ1 (formerly pisJ1), is predicted to encode a phytochrome-like photoreceptor that is essential for positive phototaxis in the unicellular cyanobacterium Synechocystis sp. PCC 6803 [Yoshihara et al. (2000) Plant Cell Physiol. 41: 1299]. The PixJ1 protein was overexpressed as a fusion with a poly-histidine tag (His-PixJ1) and isolated from Synechocystis cells. A zinc-fluorescence assay suggested that a linear tetrapyrrole was covalently attached to the His-PixJ1 protein as a chromophore. His-PixJ1 showed novel photoreversible conversion between a blue light-absorbing form (Pb, lambdaAmax=425-435 nm) and a green light-absorbing form (Pg, lambdaAmax=535 nm). Dark incubation led Pg to revert to Pb, indicative of stability of the Pb form in darkness. Red or far-red light irradiation, which is effective for photochemical conversion of the known phytochromes, produced no change in the spectra of Pb and Pg forms. Site-directed mutagenesis revealed that a Cys-His motif in the second GAF domain of PixJ1 is responsible for binding of the chromophore. Possible chromophore species are discussed with regard to the novel photoconversion spectrum.     

Contribution of membrane-associated prostaglandin E2 synthase to bone resorption

This study initially confirmed that, among prostaglandins (PGs) produced in bone, only PGE(2) has the potency to stimulate osteoclastogenesis and bone resorption in the mouse coculture system of osteoblasts and bone marrow cells. For the PGE(2) biosynthesis two isoforms of the terminal and specific enzymes, membrane-associated PGE(2) synthase (mPGES) and cytosolic PGES (cPGES) have recently been identified. In cultured mouse primary osteoblasts, both mPGES and cyclooxygenase-2 were induced by the bone resorptive cytokines interleukin-1, tumor necrosis factor-alpha, and fibroblast growth factor-2. Induction of mPGES was also seen in the mouse long bone and bone marrow in vivo by intraperitoneal injection of lipopolysaccharide. In contrast, cPGES was expressed constitutively both in vitro and in vivo without being affected by these stimuli. An antisense oligonucleotide blocking mPGES expression inhibited not only PGE(2) production, but also osteoclastogenesis and bone resorption stimulated by the cytokines, which was reversed by addition of exogenous PGE(2). We therefore conclude that mPGES, which is induced by and mediates the effects of bone resorptive stimuli, may make a target molecule for the treatment of bone resorptive disorders.     

Influence of Aging and Gender Differences on Feeding Behavior and Ghrelin-Related Factors during Social Isolation in Mice

Psychological stress due to social isolation is known to cause abnormal feeding behaviors, but the influences of gender and aging on subchronic stress-induced changes in feeding behaviors are unknown. Thus, we examined the changes in body weight, food intake, and orexigenic ghrelin-related factors during 2 weeks of isolation stress in young and aged mice. Food intake increased significantly in young mice in the isolation group compared with the group-housed control throughout the experimental period. This isolation-induced increase in food intake was not observed in aged mice. In young mice, there were no significant differences in body weight between the isolated group and group-housed control up to 2 weeks. However, aged male mice exhibited significant weight loss at 2 weeks and a similar tendency was observed in aged female mice. Young male mice, but not female mice, had significantly increased (2.2-fold) plasma acylated ghrelin levels after 1 week of isolation compared with the group-housed control. A significant but lower increase (1.3-fold) was also observed in aged male mice. Hypothalamic preproghrelin gene expression decreased significantly with isolation in young male mice, whereas it increased significantly in female mice. The expression levels of NPY and AGRP in the hypothalamus, which are transmitted by elevated peripheral ghrelin signals, increased significantly in isolated young male mice, whereas the AGRP expression levels decreased significantly in young female mice. Isolation caused no significant differences in the expression levels of these genes in aged mice. In isolation, young female mice exhibited markedly increased dark- and light-phase locomotor activities compared with male mice, whereas male and female aged mice exhibited no obvious increases in activity immediately after the dark phase started. We conclude that the gender-specific homeostatic regulatory mechanisms required to maintain body weight operated during subchronic psychological stress in young mice but not in aged mice.     

N-type voltage-dependent Ca channel in non-excitable microglial cells in mice is involved in the pathophysiology of neuropathic pain

Peripheral nerve injury induces neuropathic pain which is characterized by tactile allodynia and thermal hyperalgesia. N-type voltage-dependent Ca²⁺ channel (VDCC) plays pivotal roles in the development of neuropathic pain, since mice lacking Ca_v2.2, the pore-forming subunit of N-type VDCC, show greatly reduced symptoms of both tactile allodynia and thermal hyperalgesia. Our study on gene expression profiles of the Ca_v2.2 knockout (KO) spinal cord after spinal nerve ligation (SNL)-injury revealed altered expression of genes known to be expressed in microglia, raising an odd idea that N-type VDCC may function in not only excitable (neurons) but also non-excitable (microglia) cells in neuropathic pain state. In the present study, we have tested this idea by using a transgenic mouse line, in which suppression of Ca_v2.2 expression can be achieved specifically in microglia/macrophage by the application of tamoxifen. We found SNL-operated transgenic mice exhibited greatly reduced signs of tactile allodynia, whereas the degree of thermal hyperalgesia was almost the same as that of control. Immunohistochemical analysis of the transgenic lumbar spinal cord revealed reduced accumulation of Iba1-positive cells (microglia/macrophage) around the injured neurons, indicating microglial N-type VDCC is important for accumulation of microglia at the lesion sites. Although the mechanism of its activation is not clear at present, activation of N-type VDCC expressed in non-excitable microglial cells contributes to the pathophysiology of neuropathic pain.     

Continuous Turnover of Carotenes and Chlorophyll a in Mature Leaves of Arabidopsis Revealed by 14CO2 Pulse-Chase Labeling

Carotenoid turnover was investigated in mature leaves of Arabidopsis (Arabidopsis thaliana) by ¹⁴CO₂ pulse-chase labeling under control-light (CL; 130 μmol photons m⁻² s⁻¹) and high-light (HL; 1,000 μmol photons m⁻² s⁻¹) conditions. Following a 30-min ¹⁴CO₂ administration, photosynthetically fixed ¹⁴C was quickly incorporated in β-carotene (β-C) and chlorophyll a (Chl a) in all samples during a chase of up to 10 h. In contrast, ¹⁴C was not detected in Chl b and xanthophylls, even when steady-state amounts of the xanthophyll-cycle pigments and lutein increased markedly, presumably by de novo synthesis, in CL-grown plants under HL. Different light conditions during the chase did not affect the ¹⁴C fractions incorporated in β-C and Chl a, whereas long-term HL acclimation significantly enhanced ¹⁴C labeling of Chl a but not β-C. Consequently, the maximal ¹⁴C signal ratio between β-C and Chl a was much lower in HL-grown plants (1:10) than in CL-grown plants (1:4). In lut5 mutants, containing α-carotene (α-C) together with reduced amounts of β-C, remarkably high ¹⁴C labeling was found for α-C while the labeling efficiency of Chl a was similar to that of wild-type plants. The maximum ¹⁴C ratios between carotenes and Chl a were 1:2 for α-C:Chl a and 1:5 for β-C:Chl a in CL-grown lut5 plants, suggesting high turnover of α-C. The data demonstrate continuous synthesis and degradation of carotenes and Chl a in photosynthesizing leaves and indicate distinct acclimatory responses of their turnover to changing irradiance. In addition, the results are discussed in the context of photosystem II repair cycle and D1 protein turnover.Carotenoids are classified as accessory pigments in photosynthesis because they augment light harvesting in the blue spectral region by transferring the absorbed light energy to chlorophyll (Chl). However, the universal occurrence of carotenoids in photosynthetic cells, from bacteria to higher plants, indicates their essential roles, rather than mere accessory roles, in photosynthesis. Under excess light, carotenoids provide protection against photooxidative damage by facilitating dissipation of excitation energy from singlet- or triplet-state Chl and scavenging highly reactive singlet oxygen, which is generated through interaction between triplet excited Chl and oxygen (Demmig-Adams, 1990; Müller et al., 2001). These photoprotective functions make carotenoids indispensable for oxygenic photosynthesis, as demonstrated by lethal effects of inhibitors of carotenoid biosynthesis in plants (Bramley, 1993). Regulation of light harvesting and photoprotection by carotenoids requires their close proximity as well as the proper orientation to Chl molecules in pigment-protein complexes of PSI and PSII. Furthermore, a small fraction of non-protein-bound carotenoids serves as antioxidants in the lipid phase of photosynthetic membranes (Havaux and Niyogi, 1999; Havaux et al., 2004) and influences the structure and fluidity of the lipid bilayer (Gruszecki and Strzałka, 2005). Despite these and other lines of defense, the PSII reaction center polypeptide D1, and to a lesser extent also D2, undergo frequent photooxidative damage and repair in the light (Melis, 1999; Baena-González and Aro, 2002). When the repair process cannot keep up with the rate of photodamage, the overall quantum yield of PSII declines.Carotenoids are derived from isoprenoid precursors in plastids (for reviews on carotenoid biosynthesis in plants, see Lichtenthaler, 1999; Hirschberg, 2001; DellaPenna and Pogson, 2006; Giuliano et al., 2008; Tanaka et al., 2008; Cazzonelli and Pogson, 2010). Following the formation of linear C₄₀ lycopene, the pathway splits into two branches of major cyclic carotenoids: the β,β-branch gives rise to β-carotene (β-C) having two β-rings, whereas the β,ϵ-branch leads to formation of α-carotene (α-C) having one β-ring and one ϵ-ring. Hydroxylation of β-C and α-C produces the xanthophylls zeaxanthin (Z) and lutein (L), respectively. In the β,β-branch, epoxidation of the β-rings of Z results in successive synthesis of antheraxanthin (A) and violaxanthin (V); subsequently, V can be converted to neoxanthin (N), the last carotenoid product of the β,β-branch. Except for some species (García-Plazaola et al., 2007), L does not undergo β-ring epoxidation and the β,ϵ-branch thus stops with L, the most abundant carotenoid in leaves.Each of these carotenoids occupies specific binding sites in the photosynthetic apparatus to fulfill distinct roles. In both PSI and PSII, carotenes (α-C and β-C) are generally bound in core complexes, which also harbor Chl a molecules, while the majority of xanthophylls (L, Z, A, V, and N) are bound in light-harvesting antenna complexes together with Chl a and Chl b molecules (Bassi et al., 1993; Lee and Thornber, 1995). Accumulation of β-C in core complexes is a common feature of diverse photosynthetic organisms, whereas the occurrence of α-C in addition to β-C is restricted to certain taxa. For higher plants, α-C has been found in leaves of some, but not all, shade-tolerant species (Thayer and Björkman, 1990; Demmig-Adams and Adams, 1992; Demmig-Adams, 1998; Matsubara et al., 2009). Based on this photoacclimatory behavior, it has been proposed that α-C may function as a light-harvesting pigment while β-C may contribute to photoprotection (Krause et al., 2001), presumably by scavenging singlet oxygen and mediating a cyclic electron transfer around PSII (Tracewell et al., 2001; Telfer, 2005).Pronounced light-dependent changes are also observed for xanthophyll composition in light-harvesting antenna complexes. In a short term (minutes to hours), operation of the xanthophyll cycle, involving Z, A, and V, modulates levels of Z in a light-dependent manner. It is widely accepted that Z is able to enhance the dissipation of excess light energy from singlet excited Chl while V is not (Demmig-Adams, 1990; Müller et al., 2001). Long-term acclimation (days) to strong irradiance typically results in an increased pool size of the xanthophyll-cycle pigments (V + A + Z) and downsizing of PSII antenna, as indicated by a greater Chl a-to-Chl b ratio (Demmig-Adams and Adams, 1992; Demmig-Adams, 1998; Matsubara et al., 2009). Based on the observed changes in steady-state amounts of xanthophylls and carotenes following irradiance shifts, alterations in the balance between biosynthesis and degradation, or turnover, have been implicated as a mechanism for long-term adjustment of carotenoid levels in leaves (Förster et al., 2009). However, just how much biosynthesis and degradation of different carotenoids occurs in photosynthesizing green leaves is an open question to date.In order to gain insight into carotenoid turnover of mature leaves, we conducted ¹⁴CO₂ pulse-chase labeling experiments with Arabidopsis (Arabidopsis thaliana) plants. Carotenoid turnover has been studied in algae in the past by applying [¹⁴C]bicarbonate (Blass et al., 1959; Grumbach et al., 1978); for example, no more than 1% to 2% of the photosynthetically incorporated ¹⁴C was allocated to the lipophilic fraction containing Chl and carotenoid in Chlorella pyrenoidosa after a 2-h pulse application (Grumbach et al., 1978). Even lower labeling efficiency is expected for photosynthetic pigments in nongrowing green leaves, in which pigment turnover takes place almost exclusively as part of the maintenance and acclimation of photosynthetic membranes. To overcome this intrinsic but anticipated difficulty, a ¹⁴CO₂ application setup was established for efficient and reproducible ¹⁴CO₂ incorporation in detached leaves of Arabidopsis during a short (30-min) pulse period. Moreover, a method of pigment separation was developed for ¹⁴C detection in concentrated leaf pigment extracts using a radio-HPLC system. Because carotenoid composition exhibits marked sun-shade responses in leaves (Demmig-Adams and Adams, 1992; Demmig-Adams, 1998; Matsubara et al., 2009), ¹⁴CO₂ labeling patterns were studied in three different sets of Arabidopsis plants: (1) plants grown under 130 μmol photons m⁻² s⁻¹ (control light [CL]) and exposed to CL during a chase period of up to 10 h (CL plants); (2) plants acclimated to 1,000 μmol photons m⁻² s⁻¹ (high light [HL]) for 2 weeks and exposed to HL during the chase period (HL plants); and (3) plants grown under CL but exposed to HL during the chase period (CL→HL plants). These treatments simulated short-term (CL→HL) and long-term (CL or HL) responses to irradiance. Finally, as ¹⁴C was found to be rapidly incorporated in β-C and Chl a molecules in leaves of wild-type plants, in which β-C represents the only carotene species, ¹⁴C labeling experiments were also conducted with leaves of lut5 mutants containing both α-C and β-C (Fiore et al., 2006; Kim and DellaPenna, 2006) to compare turnover of the two carotenes.     

Testicular development in growth-retarded mice.

To assess delayed fertility in male growth-retarded (grt) mice with congenital primary hypothyroidism, their testes were chronologically examined. The testicular weight in grt mice was significantly lower than age-matched normal mice until 8 weeks but was comparable at 13 and 26 weeks. While normal mice had mature sperm cells in both testes and epididymides at 5 weeks, age-matched grt mice did not. The size of the seminiferous tubules in testes of grt mice was smaller than that of normal mice before 13 weeks but was comparable at 26 weeks. These findings suggest that male grt mice might need more than 13 weeks to develop mature testes.