Histochemical studies on glycosaminoglycans in Bruch's membrane of postnatal rat eyes

Localization of glycosaminoglycans (GAG) in Bruch's membrane of postnatal rat eyeballs was examined histochemically. Fixed eyeballs from postnatal rats (ages 5 days and 8 weeks) were routinely processed and embedded in paraffin wax or Quetol 651 resin. Paraffin-embedded tissue sections were stained with hematoxylin and eosin or sensitized high iron diamine procedure in combination with selective methods such as GAG-degrading enzyme digestions and/or a chemical modification, and examined by light microscopy. Quetol 651-embedded ultrathin sections were stained with heavy metals and examined by electron microscopy. In rats at postnatal day 5, Bruch's membrane contained mainly chondroitin sulfate (CS) and heparan sulfate (HS). In contrast, at 8 weeks after birth the membrane included a large amount of dermatan sulfate (DS) and HS. According to electron microscopic findings, Bruch's membrane on day 5 consisted of only 3 layers without a central elastic layer. However, at 8 weeks after birth the membrane was constructed of 5 layers. These findings suggested that the difference in GAG molecular species in the membranes at 5 days and at 8 weeks after birth could be correlated with the development and maturation of the collagenous layer in Bruch's membrane. Moreover, maturation of Bruch's membrane may contributes to the architectural stabilization of the outer portions of the photoreceptor cells.     

Pathogenic role of polyclonal and polymeric IgA in a murine model of mesangial proliferative glomerulonephritis with IgA deposition.

Molecular size and charge distribution of IgA of sera and glomerular eluates were investigated in ddY mice, spontaneously developing mesangial proliferative glomerulonephritis (GN) with IgA deposition after 40 weeks of age. Serum IgA levels were increased in aged ddY mice more than 40 weeks old with a significant increase (P less than 0.01) at the age of 60 weeks, comparing with those of BALB/c mice. The isoelectric focusing (IEF) spectrotype of pooled serum IgA in 60-week-old mice ranged from 4.2 to 5.5, being similar to those in younger ddY (16 weeks old) and control BALB/c mice (12 weeks old) without enhanced expression of specific IgA peaks. However, IgA in the glomerular eluate from the 60-week-old mice showed limited anionic spectrotypes from pH 4.2 to 4.8. HPLC of IgA in pooled sera and glomerular eluates of 16-, 40- and 60-week-old ddY mice, revealed markedly increased ratios of the dimeric IgA (dIgA) and polymeric IgA (pIgA) in the total IgA with age. In the contrast to serum profiles, monomeric IgA (mIgA) was always detected as the smallest peak of the IgA fractions in glomerular eluates. Furthermore, aged mice with severe GN showed a higher percentage of dIgA and pIgA in total IgA (80%) in the sera than that of the mice with mild GN (64%). HPLC analysis under acid condition of glomerular IgA from 40-week-old ddY mice showed a similar pattern of dIgA and pIgA peaks in neutral buffer without the appearance of mIgA. These findings suggest that there is a selective mechanism for glomerular accumulation of more acid IgA among the polyclonally expanded IgA in old ddY mice, and that the polymeric form of IgA plays a pathogenic role in the development of mesangial proliferative GN in these mice.     

Evaluation of DNA probes for specific detection of Vibrio cholerae O139 Bengal.

Two DNA probes, 2R1 and 2R3, prepared from a region in the chromosome specific for the lipopolysaccharide O side chains of Vibrio cholerae O139 (M.K. Waldor and J.J. Mekalanos, Lancet 343:1366, 1994) were examined for their specificity and sensitivity. Both probes did not hybridize with any strain of V. cholerae belonging to serogroups other than O139 and to any of the other species examined belonging to the family Vibrionaceae. Among the 126 strains of V. cholerae O139 examined, probe 2R1 hybridized with 125 strains while probe 2R3 hybridized with all 126 strains. Both probes were found to be highly specific and sensitive and can be used for the specific identification of V. cholerae O139.     

Tumor necrosis factor reduces lifespan of human endothelial cells in vitro

Tumor necrosis factor (TNF) is known to regulate the proliferation and function of vascular endothelial cells (ECs). We have examined the effects of TNF on the growth and aging of human ECs of different origins and compared them with those in human normal diploid fibroblasts. The results obtained were as follows: (1) TNF reduces the growth rate and in vitro life span of ECs in both dose- and treatment length-dependent fashions; (2) ECs are significantly more sensitive to TNF than fibroblasts; and (3) the life span shortening effect of TNF on ECs increases as a function of in vitro cell age. These results suggest that the aging of ECs is modified by TNF exposure.     

Role of N-methyl-D-aspartate receptors and the nitric oxide pathway in nociception/hyperalgesia elicited by protease-activated receptor-2 activation in mice and rats

Activation of the peripheral protease-activated receptor-2 (PAR-2) triggers nociceptive behaviour and thermal hyperalgesia in rats. The present study created a novel mouse model for PAR-2-triggered nociception, and then examined the roles of NMDA receptors and the nitric oxide (NO) pathway in nociceptive processing by PAR-2. Intraplantar administration of the PAR-2 agonist SLIGRL-NH(2) elicited nociceptive responses in mice, an effect being more specific in mast cell-depleted mice. This PAR-2-triggered nociception was abolished by the NMDA receptor antagonist MK-801, but not the neuronal NO synthase inhibitor 7-nitro indazole. In contrast, the PAR-2-triggered thermal hyperalgesia in rats was blocked by both agents. Our study thus provides a novel mouse model for PAR-2-mediated nociception, and suggests that NMDA receptors are involved in PAR-2-triggered nociception and hyperalgesia, while NO contributes only to the latter.     

Distribution of cocaine recognition sites in rat brain: In vitro and ex vivo autoradiography with [I]RTI-55

The distribution of binding sites of [¹²⁵I]RTI-55 (3β-(4-iodophenyl)tropan-2β-carboxylic acid methyl ester), a phenyl tropane analog of cocaine, and the selective labelling of the dopamine transporter (DAT) were studied by in vitro and ex vivo autoradiography in the rat whole brain. Recent evidence has shown that RTI-55 binds to not only DAT but also serotonin transporter (5HTT). In the present study, in vitro autoradiography revealed that [¹²⁵I]RTI-55 bound to the olfactory tubercle, the caudate putamen, the accumbens nucleus, the midline and lateral geniculate nuclei of the thalamus, the hypothalamic nuclei, the substantia nigra compact part, the subthalamic nucleus, the ventral tegmental area, the superior colliculus, the dorsal raphe nucleus, and the facial nucleus. Further, in the presence of clomipramine, a selective ligand for 5HTT, [¹²⁵I]RTI-55 binding was remarkably inhibited in the midline and lateral geniculate nuclei of the thalamus, the hypothalamic nuclei, the superior colliculus, the dorsal raphe nucleus, and the facial nucleus, while [¹²⁵I]RTI-55 binding remained in the olfactory tubercle, the caudate putamen, the accumbens nucleus, the substantia nigra compact part, the subthalamic nucleus, and the ventral tegmental area. These findings suggest that [¹²⁵I]RTI-55 binds to 5HTT in the former areas and to DAT in the latter areas. It is therefore concluded that RTI-55 is a suitable ligand for studying the action of cocaine in whole brain regions, including the thalamus, the hypothalamus and the dorsal raphe nucleus, regions in which cocaine is thought to act evoking several neurological effects, e.g., analgesia and elevation of adrenocorticotropic hormone. DAT was also labelled selectively both in vitro and in vivo using [¹²⁵I]RTI-55 combined with clomipramine. Therefore, radiolabelled RTI-55, combined with unlabelled clomipramine, which displaces its binding to 5HTT, also appears to be suitable for the selective imaging of DAT in vivo.     

High-frequency inter-parental recombination between mitochondrial genomes of rice cybrids

Analyzing more than 100 independent rice cybrids, we found evidence for inter-molecular recombination between parental mitochondrial genomes occurring at high frequency soon after protoplast fusion. The structure of the region around the atp6 gene showed extensive polymorphism among Indica (MTC-5A), Japonica (Nipponbare), and wild abortive (IR58024A) mitochondrial genomes. Recombination between the mitochondrial genomes of IR58024A and MTC-5A around the atp6 gene was detected by Southern-blot analysis of cybrid plants. Such recombinant mitochondrial molecules were also cloned from IR58024A/Nipponbare cybrid callus. PCR analysis around the atp6 gene demonstrated that inter-parental recombination occurs in practically all cybrid calli within 2 weeks after protoplast fusion. At this point, parental and recombinant mitochondrial genomes coexisted within the callus. Over the course of further cultivation, however, mitochondrial genome diversity decreased as parental and/or recombinant genomes segregated out.     

Divergence of natural killer cell receptor and related molecule in the decidua from sporadic miscarriage with normal chromosome karyotype

The aim of this cohort study was to investigate immunophenotypic characteristics of natural killer (NK) cells by assessing specific molecules expressed in the decidua of sporadic miscarriages and induced abortions. The deciduae were obtained from 29 consecutively seen women whose pregnancies ended in first trimester miscarriages (MS), and the fetal chromosome karyotype of these MS was analysed. Additionally, 13 deciduae were obtained from induced abortion (IA) with informed consent. The expression of perforin, CD94, CD161, CD158a, CD158b, CD244 on CD3-CD56+NK cells, and perforin on CD3+CD8+ T cells was analysed by flow cytometry. The CD158a (mean+/-SD, 26.2+/-14.7%) and CD94 (50.2+/-25.7%) expressions in MS with normal chromosome karyotype (MSNK; n=11) were significantly decreased as compared with those (41.5+/-19.5%, 71.4+/-20.4%) in MS with abnormal karyotype (MSAK; n=18) and those (44.3+/-21.9%, 80.8+/-17.5%) in IA (n=13). Conversely, the perforin expression on CD3-CD8-CD56+NK cells (76.3+/-11.0%) and CD3+CD8+T cells (30.6+/-9.2%) in MSNK was significantly increased as compared with those (66.8+/-16.6%, 23.6+/-8.7%) in MSAK and those (62.9+/-11.6%, 19.7+/-8.1%) in IA. A positive correlation between CD94 and CD158a expressions on NK cells, negative correlations between CD94 on NK cells and perforin on NK cells/T cells, and between CD158a on NK cells and perforin on T cells were found in the decidua. A divergence of NK cell repertoire in the decidua might be related to aetiology of sporadic MSNK.     

Effects of nocturnal bright light on saliva melatonin, core body temperature and sleep propensity rhythms in human subjects

Nine healthy male volunteers (mean age of 24) participated in two experimental sessions of random crossover design: a bright light (5000 lux for 5 h from 00:00 to 05:00 h) session and a dim light (10 lux for 5 h from 00:00 to 05:00 h) session. Subsequently participants entered an ultra-short sleep-wake schedule for 26 h, in which a sleep-wake cycle consisting of 10-min sleep EEG recording on a bed and 20-min resting awake on a semi-upright chair were repeated. Saliva melatonin level and core body temperature was measured throughout the experiment. Bright light significantly delayed rhythms of melatonin secretion (01:58 h), core body temperature (01:12 h) and sleep propensity (02:00 h), compared as dim light session. Significant positive correlation was found between bright light-induced phase change in core body temperature and that in sleep propensity rhythm. Light-induced melatonin suppression significantly positively correlated with the phase change in core body temperature and that in sleep propensity rhythm. Assuming that light-induced melatonin suppression represents an acute impact of light on the circadian pacemaker, our results suggest that such an impact may be directly reflected in phase changes of sleep propensity and core body temperature rhythms rather than in melatonin rhythm.