Immunocytochemical identification of pinopsin in pineal glands of chicken and pigeon

Pinopsin is a blue-sensitive photoreceptive molecule possibly involved in photic entrainment of the circadian pacemaker in the chicken pineal gland. To characterize pinopsin as a circadian photoreceptor, antibodies were raised against the C-terminal portion of pinopsin. As expected from the divergence of the amino acid sequence of this region, the resultant antibody cross-reacted with neither chicken rhodopsin nor red-sensitive cone pigment (chicken red). In Western blot analysis, the antibody stained a single band of 42-kDa protein in a detergent-extract of chicken pineal membranes, suggesting that pinopsin (calculated molecular weight, 38187) might be glycosylated and/or palmitoylated. Immunocytochemical examination of pineal sections of the chicken and the pigeon with this antibody revealed strong positive images for most of the membrane structures in the lumen of the follicles. This antibody also stained string- and bulb-shaped structures of the chicken parafollicular cells, the morphology of which resembles those of retinal photoreceptor cells. In contrast to the predominant distribution of pinopsin, a monoclonal antibody specific for chicken red stained a smaller number of membrane structures in the lumen of chicken pineal follicles. These results strongly suggest that the chicken pineal gland contains at least two types of photoreceptive molecules, pinopsin (major) and chicken red (minor). We show that the former molecule is localized in parafollicular pinealocytes and in the outer segments of pinealocytes that make contact with the follicular lumen.     

Effects of glycosylation of the residue at position 14 in ovine angiotensinogen on the human renin reaction

A mutant angiotensinogen, S14N, in which Ser14 of ovine angiotensinogen was replaced by Asn to form a N-glycosylation site, was produced in CHO cells. The molecular weight was about 3,000 larger than that of wild-type ovine angiotensinogen, indicating that S14N angiotensinogen was glycosylated at Asn14. In the reaction with human renin, the km of mutant angiotensinogen was 3 times increased, but the Vmax was not affected by the mutation.     

Changes in plasma erythropoietin and interleukin-6 concentrations in patients with septic shock after hemoperfusion with polymyxin B-immobilized fiber

OBJECTIVE: To find out whether polymyxin B-immobilized fiber (PMX-F) treatment affects the clinical parameters and plasma concentrations of erythropoietin (EPO) and interleukin (IL)-6. DESIGN: A prospective case series study. SETTING: Intensive care unit of the Department of Internal Medicine, Misato Junshin Hospital, Saitama, and Koto Hospital, Tokyo, Japan. PATIENTS: 17 consecutive patients (10 men, 7 women; mean age 54.6 years) with clinically defined septic shock and 20 healthy volunteers (12 men, 8 women; mean age 52.2 years). MAIN RESULTS: Of the 17 patients with septic shock, 9 (53 %) survived. The systolic blood pressure increased significantly from 78+/-6 to 106+/-8 mm Hg 2 h after PMX-F treatment in patients with septic shock. Plasma endotoxin levels decreased significantly after treatment, from 40+/-6 to 12+/-4 pg/ml. The pretreatment plasma concentrations of EPO and IL-6 were significantly higher in the 8 nonsurviving patients with septic shock (EPO: 400+/-36 mlU/ml; IL-6: 6260+/-1180 pg/ml) than in the 9 surviving patients (EPO: 120+/-22 mlU/ml; IL-6: 680+/-138 pg/ml) and the 20 control subjects (EPO, 12+/-6 mlU/ml; IL-6, 8+/-2 pg/ml). Plasma concentrations of EPO and IL-6 in patients with septic shock decreased significantly after PMX-F treatment (EPO, nonsurviving: 320+/-28 mlU/ml, p < 0.05; survivors: 26+/-8 mlU/ ml, p < 0.001; IL-6, nonsurviving: 3860+/-840 pg/ml, p < 0.01; survivors: 84+/-20 pg/ml, p < 0.001). CONCLUSIONS: Plasma concentrations of EPO and IL-6 may be prognostic indicators in patients with septic shock: PMX-F treatment may be effective in reducing the plasma concentrations of EPO and IL-6 in patients with septic shock.     

Preparation of the hard CNx thin films using NO ambient gas by pulsed laser deposition

Pulsed laser deposition (PLD) technique has been widely used in thin film preparation because of its wonderful and excellent properties and amorphous carbon nitride (CN_x) thin films are recognized to have potential for applications like hard coating and electron field emission device. We have deposited CN_x thin films by KrF excimer laser – (λ= 248 nm) ablation of pure graphite target in pure NO gas ambient condition. In this paper, we have prepared the CN_x thin films at various ambient NO gas pressure of 1.3–26 Pa and laser fluence of 2– 5J cm^?2 on Si (100) substrate. We consider that the hardness of CN_x thin films improves due to the increase the nitrogen/carbon (N/C) ratio. The N/C ratio depended on the ambient NO gas pressure and laser fluence. We obtainedthe maximum N/C ratio of 1.0 at NO 3.3 Pa. The typical absorption of CN bonds such as sp² C–N, sp³ C–N, G band and D band were detected from the infrared absorption measurement by FTIR in the deposited CN_x thin films.     

Further analyses of epitopes for human monoclonal anti-basement membrane zone antibodies produced by stable human hybridoma cell lines constructed with Epstein-Barr virus transformants

We previously established Epstein-Barr virus (EBV)-transformed bullous pemphigoid (BP) patient lymphoblastoid cell lines, which produced human monoclonal anti-basement membrane zone antibodies. In the present study, we established two independent human-human hybridomas by fusion of these EBV transformants with a human B-cell line. These hybridomas, designated 5E-HY-4B and 10D-HY-8B, were very stable and showed a high yield of monoclonal antibody (MoAb) secretion. Each cell line was tetraploid and showed combined rearranged segments of immunoglobulin heavy-chain gene derived from both an EBV transformant and a parent cell. Immunoblot analysis showed that the 5E-HY-4B MoAb recognized the 230-kDa BP antigen but that the 10D-HY-8B MoAb did not show any reactivity. In contrast, both MoAbs precipitated the 230-kDa BP antigen with immunoprecipitation. These results indicate that the two MoAbs reacted with different epitopes on the 230-kDa BP antigen: a continuous epitope for the 5E-HY-4B MoAb and a conformation-dependent epitope for the 10D-HY-8B MoAb. This speculation was confirmed at the molecular level by the result that the fusion protein produced by a partial cDNA for the 230-kDa mouse BP antigen reacted with the 5E-HY-4B MoAb but not with the 10D-HY-8B MoAb. Furthermore, the study of the reactivity with fusion proteins of a series of deleted clones restricted the epitope for the 5E-HY-4B MoAb within the region with 114 amino acid residues in the C-terminal domain of the 230-kDa BP antigen.     

The relationship between solid-state fluorescence intensity and molecular packing of coumarin dyes

The solid-state fluorescence intensity of coumarin dyes depends on the substituents present at the 4- and 7-positions. 7-(Diethylamino)coumarins showed higher solid-state fluorescence quantum yield (Φ_f(ss) = 0.29–0.40) than 7-aminocoumarins (0.01). In the case of julolydyl coumarins, a 4-methyltetramethyljulolydyl derivative also displayed high Φ_f(ss) (0.34), this being greater than that observed for both 4-(perfluoroalkyl)tetramethyljulolydyl (0.09 and 0.10) and 4-methyljulolydyl derivatives (0.01). X-ray crystallographic analysis suggested that coumarin dyes bearing network hydrogen bonds and/or π–π stacking show weak solid-state fluorescence whereas coumarin dyes having isolated monomer- and dimer-type stacking show intense fluorescence. 4-(Perfluoroalkyl)tetramethyljulolydyl derivatives displayed medium fluorescence intensity owing to isolated monomer-type packing with little intermolecular interactions operating between adjacent molecules.     

Influence of application site of a new transdermal clonidine, M-5041T, on its pharmacokinetics and pharmacodynamics in healthy subjects

Influence of application site of a new transdermal clonidine, M-5041T(M), on its pharmacokinetics and pharmacodynamics were evaluated in eight human subjects. One patch of M-6 mg was applied for 3 days on the right chest (first trial), on the left arm (second trial), and on the upper abdomen (third trial). Blood samples for clonidine concentration were taken, and blood pressure (BP) was measured for a 120-hour postapplication period. Plasma concentrations of clonidine increased after application of M in each trial. This parameter in the second trial was significantly greater than that of the first and third trials. The values of maximum plasma concentration and area under the plasma concentration-time curve in the second trial were greater than those of other trials, but the differences did not reach significance. The BP-lowering effect of M in the second trial was significantly greater than that of the third trial. These results suggest that the plasma concentrations of clonidine after application of M and its hypotensive effect are affected by the site of application in human subjects.     

Effects of suprachiasmatic lesions on circadian rhythms of blood pressure, heart rate and locomotor activity in the rat

To determine whether the circadian rhythms in blood pressure (BP), heart rate (HR) and locomotor activity are controlled by an internal biological clock located in the suprachiasmatic nucleus (SCN), we continuously measured these parameters in SCN-lesioned rats using a newly developed implantable radiotelemetry device and a computerized data collecting system. Although SCN-lesioned rats showed a weak but significant 24-h periodicity in BP and HR under light-dark (LD) cycles, BP, HR and locomotor activity became completely aperiodic under constant dark (DD) conditions. The amount of locomotor activity was significantly reduced in SCN-lesioned rats compared to that in intact rats. BP tended to be higher in SCN-lesioned rats, but the differences were significant only in the comparison of systolic blood pressure (SBP) under LD and DD (p < 0.05) and of mean blood pressure (MBP) under LD (p < 0.05). HR in SCN-lesioned rats was significantly lower under LD (p < 0.05), but not under DD. The standard deviation and the variation coefficient of MBP, as indices of short-term variability of this parameter, were significantly larger in SCN-lesioned rats than in intact rats, while those of HR and locomotor activity did not differ significantly between SCN-lesioned and intact rats. These results indicate that the SCN is important not only for generating circadian rhythms of BP, HR and locomotor activity, but also for buffering the short-term variability of BP in rats.     

An autopsy case of extraskeletal Ewing's sarcoma followed up for 24 years

A long-term (24 years) follow-up case of extraskeletal Ewing's sarcoma is reported. The light microscopic examination showed features hardly indistinguishable from Ewing's sarcoma of the bone, that is, the tumor cells were diffusely arranged and uniform in size and shape, and possessed glycogen in the cytoplasm. Homer-Wright rosettes were found only in the autopsy material. An immunohistochemical study using a neural marker (neuron-specific enolase) demonstrated positive staining in most tumor cells. An ultrastructural study revealed intracytoplasmic glycogen particles and incomplete neural characters as follows: the cytoplasmic processes resembled neural processes without neurosecretory granules, microtubules or neurofilaments. These findings suggest that this case finally acquired an incomplete neural character with repeated recurrence. This tumor was diagnosed extraskeletal Ewing's sarcoma, but in future it may be categorized as primitive neuroectodermal tumor (PNET).