Functional analysis of a novel G87V TNFRSF1A mutation in patients with TNF receptor-associated periodic syndrome.

Tumor necrosis factor (TNF) receptor-associated periodic syndrome (TRAPS) is an autoinflammatory disease that is caused by heterozygous mutations in the TNFRSF1A gene. Although more than 150 TNFRSF1A mutations have been reported to be associated with TRAPS phenotypes only a few, such as p.Thr79Met (T79M) and cysteine mutations, have been functionally analyzed. We identified two TRAPS patients in one family harboring a novel p.Gly87Val (G87V) mutation in addition to a p.Thr90Ile (T90I) mutation in TNFRSF1A. In this study, we examined the functional features of this novel G87V mutation. In-vitro analyses using mutant TNF receptor 1 (TNF-R1)-over-expressing cells demonstrated that this mutation alters the expression and function of TNF-R1 similar to that with the previously identified pathogenic T79M mutation. Specifically, cell surface expression of the mutant TNF-R1 in transfected cells was inhibited with both G87V and T79M mutations, whereas the T90I mutation did not affect this. Moreover, peripheral blood mononuclear cells (PBMCs) from TRAPS patients harboring the G87V and T90I mutations showed increased mitochondrial reactive oxygen species (ROS). Furthermore, the effect of various Toll-like receptor (TLR) ligands on inflammatory responses was explored, revealing that PBMCs from TRAPS patients are hyper-responsive to TLR-2 and TLR-4 ligands and that interleukin (IL)-8 and granulocyte-macrophage colony-stimulating factor (GM-CSF) are likely to be involved in the pathogenesis of TRAPS. These findings suggest that the newly identified G87V mutation is one of the causative mutations of TRAPS. Our findings based on unique TRAPS-associated mutations provide novel insight for clearer understanding of inflammatory responses, which would be basic findings of developing a new therapeutic and prophylactic approach to TRAPS.

Determination of the amount of native structural bacteriorhodopsin in purple membrane Langmuir-Blodgett films by a spectroscopic surface denaturation quantifying technique.

Purple membrane (PM) shows denaturation when spread over an air/water interface. We established a technique, which we call the spectroscopic surface denaturation quantifying (SSDQ) technique, that uses infrared linear dichroism to determine the amount of native structural bacteriorhodopsin (BR) in PM Langmuir-Blodgett (LB) films. Using the SSDQ technique we found that the conformational change after surface denaturation of BR was the same as that caused by ethanol treatment. By extrapolating the data of the amount of non-denatured BR molecules in PM LB films vs. the area of a single BR molecule on an air/water interface, we also found that the surface area of a single non-denatured BR molecule was 11.5 nm2, which is consistent with that determined by high-resolution electron cryo-microscopy and electron diffraction (EMD). These results demonstrate that the SSDQ technique is effective in quantifying the amount of native structural BR in PM LB films. The SSDQ technique is also applicable to other types of protein consisting of alpha-helical conformation.

Sequential serum measurements of thyrotropin binding inhibitor immunoglobulin G in transient familial neonatal hypothyroidism.

Infants with transient neonatal hypothyroidism, in whom TSH binding inhibitor immunoglobulin G (IgG) (TBII) were sequentially measured, are described. Their mother had been taking thyroid replacement for hypothyroidism due to nongoitrous autoimmune thyroiditis. IgGs inhibiting TSH binding were detected in maternal sera by radioreceptor assay. These IgGs also inhibited the adenylate cyclase response to TSH in human thyroid membranes. Three infants had frank hypothyroidism immediately after birth, and TBII were detected in two of them. In the two surviving infants, hypothyroidism was transient and improved when TBII disappeared from their sera. The profile of TBII in one patient corresponded to the IgG disappearance curve. These findings suggest that the transient neonatal hypothyroidism reported was caused by transplacental transfer of TBII.

Thymolipoma with striated myoid cells. Histological, immunohistochemical, and ultrastructural study.

A large asymptomatic anterior mediastinal thymolipoma, discovered by chest radiograph during a regular check-up for company employees, was excised from a 33-year-old Japanese man. On immunohistochemical and electron-microscopic examination, clusters of myoglobin-positive cells with cytoplasmic Z band structures were found scattered in the medulla. Myoid cells have been previously seen in the normal thymus as well as in thymic hyperplasia, thymoma, and in the thymus of patients with myasthenia gravis. To our knowledge, this is the first reported instance of myoid cells in a thymolipoma.

Renal pathology in owl monkeys in Plasmodium falciparum vaccine trials.

Renal specimens of 16 owl monkeys (Aotus vociferans) were studied by light microscopy and immunohistochemistry during a vaccine trial with recombinant proteins of the ring-infected erythrocyte surface antigen (RESA) of Plasmodium falciparum. Deposition of IgG, C3, and P. falciparum antigens in the mesangium was demonstrated by the peroxidase anti-peroxidase (PAP) method. A relationship between the severity of parasitemia at the time of death and the presence of nephropathy was not apparent.

The pathology of human cerebral malaria.

Blockage of the cerebral microvasculature by Plasmodium falciparum-infected erythrocytes appears to be the principal cause of human cerebral malaria. Knobs which appear on the membrane of the infected erythrocytes adhere to the endothelium, causing the obstruction of cerebral microvessels. Protein molecules such as CD36, thrombospondin, and intercellular adhesion molecule-1, which are present on the membrane of endothelial cells, may act as receptors for the attachment of knobs of P. falciparum-infected erythrocytes. Each of these candidate host molecules for infected-cell recognition and attachment are expressed in microvessels of the human brain. The presence of HRP1 and HRP2 in the cerebral microvessels of cerebral malaria patients may indicate the involvement of knob proteins in the pathogenesis of cerebral malaria. Owl monkeys infected with P. falciparum do not develop cerebral malaria. There is no blockage of cerebral microvessels by infected erythrocytes and knob proteins are absent. These findings support the contention that cerebral microvessel blockage and the presence of knob proteins are the probable causes of cerebral malaria.

Renal pathology in Saimiri monkeys during a vaccine trial using the recombinant circumsporozoite protein of Plasmodium vivax.

Renal specimens of squirrel monkeys (Saimiri sciureus boliviensis) were studied by light microscopy and immunohistochemistry to examine the pathologic changes during vaccine trials with four recombinant circumsporozoite (CS) proteins (rPvCS-1, rPvCS-2, rPvCS-3, NS1(81) V20) of Plasmodium vivax. The monkeys were vaccinated and later challenged with P. vivax sporozoites. Among the 33 posttrial biopsies, 17 had mild to moderate mesangial proliferation and nine had interstitial nephritis. Immunohistochemistry by the peroxidase-antiperoxidase (PAP) method revealed IgG deposits in only three of 24 specimens and failed to demonstrate C3 deposits and P. vivax antigens in their glomeruli. There was no relationship between the severity of nephropathy and intensity of parasitemia. The intensity of parasitemia was the same in the vaccinated and control groups. Vaccinated monkeys from the groups (rPvCS-1, rPvCs-2, rPvCS-3) had no differences in renal pathology from the unvaccinated controls, but one group vaccinated with NS1(81) V20 did not develop renal changes.

Direct reconstitution of bacteriorhodopsin into planar phospholipid bilayers--detergent effect.

This paper describes how the structure and concentration level of a detergent used for substitution after bacteriorhodopsin (bR) solubilization affect the reconstitution of the bR into phospholipid planar bilayers. A direct insertion method was used for the bR reconstitution into the bilayers. Two detergents representing the two major types were used: sodium deoxycholate with a cholane-ring structure, and octylglucoside with a linear (or chain) structure. We then characterized the reconstitution for the two detergents by considering the detergent separation profiles and the photocurrent variations upon addition of lanthanum chloride and the protonophore FCCP (carbonylcyanide-p-trifluoromethoxyphenylhydrazone). We found that for successful transmembrane reconstitution of bR the detergent with the cholane-ring structure was preferable to that with the linear structure when the detergent concentration was above its critical micellar concentration. This preference was explained by the ease with which the detergent with the cholane-ring structure was removed from protein compared to that with the linear structure. Finally, we proposed a scheme for the reconstitution of the protein.

Ophthalmomyiasis caused by Sarcophaga crassipalpis (Diptera: Sarcophagidae) in a hospital patient.

Nine sarcophagid larvae were found on the right eyelid, cornea, and bulbar conjunctiva of a debilitated patient in a hospital in Osaka, Japan. Inflammation of the right eyelid and conjunctival congestion, probably initiated or aggravated by the larvae, were found. The larvae were removed and reared for accurate identification, and, on the basis of the characteristics of the 3rd instar and adult flies, the species was identified as Sarcophaga crassipalpis Macquart. This is a report of ophthalmomyiasis caused by this facultative parasite in a human. Patients with diminished consciousness in hospitals need protection from flies.

Active groups of bicyclomycin and the reaction with thiols.

The binding of [14C]bicyclomycin to whole cells of E. coli and to the inner membrane proteins was inhibited by dithiothreitol and 2-mercaptoethanol. The reactivity of the drug with the sulfhydryl group was further studied, using methanethiol as a model compound. The kinetics revealed that the reaction was of pseudo-first-order in excess of thiolate anion. Analysis with gas chromatography-mass spectrometry showed that the main product was an adduct of thiol with bicyclomycin in an equal molar ratio. The structure of the adduct was determined by 1H-NMR spectrometry, showing that thiolate attacked the olefinic double bond of the antibiotic. 3'-Acyl derivatives of bicyclomycin did not significantly affect the binding of [14C] bicyclomycin to inner membrane proteins of E. coli. The results suggested that 4,5-double bond hydrocarbons and 3'-hydroxy group of bicyclomycin participate in the binding to E. coli inner membrane proteins, which are presumably the receptors of the antibiotic. The olefinic double bond seems to be the active center of bicyclomycin, reacting with the sulfhydryl group of the receptor protein, although the whole molecular is needed for the activity.

Biosynthesis of bicyclomycin. II. Biosynthetic conditions and incorporation of radioactive precursors into bicyclomycin by washed mycelium.

The biosynthesis of bicyclomycin by Streptomyces sapporonensis was studied using suspensions of washed mycelium. Nicotinamide and Fe2+ were found to be essential cofactors in the biosynthesis. Production of bicyclomycin was enhanced most effectively in the presence of equal moles of L-leucine and L-isoleucine, which in experiments with radioactively labeled compounds were found to be incorporated into bicyclomycin at equivalent rates. These facts strongly suggest that bicyclomycin biosynthesis involves coupling of equal moles of these two amino acids.

Biosynthesis of bicyclomycin. I. Appearance of aerial mycelia negative strains (am-).

The degeneration of bicyclomycin-producing strains of Streptomyces sapporonensis resulted in sharply depressed bicyclomycin formation in a large scale fermentation. Degenerated strains, whose productivities were only 1/30 to 1/100 of normal strains, could not form aerial mycelia on glucose-BENNETT's agar; they were aerial mycelia negative strains (am-). Repeated transfers of culture, treatment of mycelia with acriflavin, mechanical agitation shock on mycelia or higher growth conditions stimulated the degeneration of producing strains, suggesting the involvement of extrachromosomal elements or plasmids in biosynthesis of bicyclomycin. Shake flask fermentation inoculated with a mixture of a normal high-producing strain and a degenerated low-producing strain resulted in sharply depressed bicyclomycin formation in proportion to the increase of low-producing strain added. It appears that the low-producing strain outgrew the high-producing strain.

Mechanism of action of bicyclomycin.

Microscopic examination of cultures of Escherichia coli exposed to bicyclomycin revelaed elongated or spheroplast-like cells. At the lethal level, bicyclomycin was shown to inhibit the synthesis of RNA and protein in the growing cells of E. coli 15 THU, whereas DNA and lipid synthesis were not significantly affected. However, the antibiotic did not block RNA and protein synthesis in vitro. Bicyclomycin was observed to inhibit the synthesis of envelope proteins more markedly than that of cytoplasmic proteins. The synthesis of two major envelope proteins was more sensetive to bicyclomycin than that of the other envelope proteins. One (peak I), which was inhibited to the greatest extent, seemed to be identical with a bound form of lipoprotein, and the other (peak V) with a free form of lipoprotein. Bicyclomycin exhibited inhibitory effects on the exclusive biosynthesis of the lipoprotein in histidine-starved cells of E. coli 15 THU. The biosynthesis of the bound form of lipoprotein was more profoundly inhibited by bicyclomycin than that of the free form. These results indicate that the primary action of bicyclomycin may be due to the interference with the biosynthesis of lipoprotein, and its assembly to peptidoglycan.

Oscillatory phenomena in model membrane: electrical oscillation in lipid-impregnated membrane filter induced by alamethicin and controlled by bacteriorhodopsin.

A lipid-impregnated membrane filter was able to show reproducibly of an electrical oscillation under constant current stimulation when alamethicin and protamine were present in the chamber whose salt concentration was higher than the other and the membrane was left for about 12 h. In addition, bacteriorhodopsin, which is a light-activated proton pump, was found to control the oscillation. The oscillatory phenomenon was considered to be driven by the alternating change in the ion-selectivity of the membrane between cation and anion. Impedance measurement indicated the existence of lipid rearrangement which might prepare an environment for both alamethicin and protamine to cause the oscillation.

Heterotopic gastric mucosa in intrahepatic bile duct, presenting with hemobilia: a case report.

We present a 66-year-old man with unique heterotopic gastric mucosa in the intrahepatic bile duct causing hemobilia. Endoscopic retrograde cholangiography showed irregular stenosis of the left intrahepatic bile duct, and a provisional diagnosis of cholangiocarcinoma was made. Therefore, partial hepatic lobectomy and cholecystectomy were performed. Histological examination of the liver showed the presence of ectopic gastric mucosa in the intrahepatic bile duct containing mucous glands with parietal and chief cells and bile. Heterotopic gastric mucosa in the intrahepatic bile duct is a rare cause of hemobilia.

Geographic distribution of Gongylonema pulchrum and Gongylonema macrogubernaculum from Macaca fuscata in Japan.

After a first report on the gullet nematode, Gongylonema pulchrum Molin, 1857, being found in the Japanese macaque, Macaca fuscata, in Kyushu, Japan, the geographic distribution of the parasite, a causative agent of gongylonemiasis in cattle and man, was examined in 181 monkeys transferred to the Japan. Monkey Centre from 23 sites in Japan, including Yaku-shima (Island). Yaku-shima is included in the World Natural Heritage List of the United Nations for its subtropical forests, which have an unusual variety of plant and animal species. G. pulchrum was found in M. fuscata yakui monkeys inhabiting Yaku-shima and M. fuscata fuscata monkeys inhabiting Honshu and an is and near Honshu. G. macrogubernaculum was found in M. fuscata yakui monkeys. Comparison of the two kinds of parasite specimens obtained from the variety M. fuscata yakui confirmed that G. macrogubernaculum Lubimov, 1931 is a valid species. Thus, the finding of G. macrogubernaculum constitutes a record of a newly identified host. M. fuscata yakui, and shows that Yaku-shima, Japan, is a natural locality of G. macrogubernaculum.

[Effects of transfer factor on chronic hepatitis B in childhood].

Nine children, 1 to 13 years of age, with HBeAg positive chronic hepatitis B received transfer factor (T.F.) monotherapy for 3 to 17 months, and were monitored by check-ups every six months from serum HBeAg, anti-HBe and GPT. In 12 months, 4 subjects became HBeAg negative and had normal serum GPT. In 22 to 48 months, 6 of the nine subjects had negative HBeAg and normal GPT, 2 had positive HBeAg and high GPT values. The remaining 1 subject who was observed for six months after T.F. therapy remained HBeAg positive with a high GPT values. No side effects were observed. These preliminary observations may indicate beneficial effects of T.F. on the natural course of chronic hepatitis B in childhood, though the ultimate effects awaits longer and well controlled clinical trials.

[Tetanus antitoxin levels in various Japanese age groups in 1989].

The nationwide DPT vaccine program was started in 1969 for infants under 12 months of age. In order to estimate the tetanus immune status among the general population of various age groups, we measured the serum tetanus antibody level in randomly selected outpatients from Metropolitan Tokyo (n = 211, 6 months-60 years) and Hamamatsu, a city of 510 thousand population (n = 128, 3 years-80 years) between January 1987 and June 1989. Among the 211 subjects from Tokyo, the antibody value exceeding effective level of 0.01 HAU/ml was observed in 102 subjects (48.3%). The antibody positive rate was 90.8% in subjects of 3 to 21 years and was 27.7% in subjects of 22 years or older. The positive rate was significantly higher in subjects of 21 years or younger (p less than 0.005). Among 128 subjects from Hamamatsu, 60 (46.1%) had a positive antibody and the antibody positive rare was 96.9% in subjects of 3 to 21 years and was 29.5% in subjects of 21 years or younger (p less than 0.005). The above findings indicate that our DPT vaccination program has been functioning well for the last 20 years and that the immunized population is adequately protected against tetanus.

[An outbreak of waterborne Cryptosporidiosis in Kanagawa, Japan].

An outbreak of diarrhea due to infection with Cryptosporidium occurred among the staff members and customers who visited one of the 10 public houses or a dancing school in a building in Hiratsuka, Kanagawa Prefecture, at the end of summer in 1994. The epidemiological surveys by a questionnaire revealed that 461 out of 736 persons investigated complained of cholera-like or flu-like illness. The clinical manifestations included mucous and/or watery diarrhea (96.7%), abdominal pain (61.6%), fever (54.2%: lower than 39 degrees C = 84.1%, higher than 39 degrees C = 15.9%), malaise (37.1%), nausea (32.8%) and headache (29.3%). The polluted drinking water was strongly suspected to be the immediate cause of infection. Although several species of pathogenic bacteria were isolated both from stool and water samples, they were not supposed to be linked to the outbreak. No known enteropathogenic virus was found in either of the samples. Oocysts of Cryptosporidium parvum were identified in 12 (48.0%) of the 25 stool samples. The oocysts were also found in tap water and other water samples from a receiving tank which was directly connected with the public waterworks, and an elevated tank on the roof, a wastewater pits, a soil pit and artesianspring water tank. These tanks and pits except for the elevated tank were built adjucent to each other on an underground floor of the building. These tanks and pits were connected with openings in the upperpart of the tank walls. These openings might have functioned to discharge excess of drinking water in the receiving tank to the wastewater pit. The water level of the wastewater pit is kept down below the openings by pumping out the sanitary sewage to the public drain. According to the declaration of the owner of the building, however, the wastewater pump was broken at the time of outbreak. Accidental malfunction of the drainage system caused contamination of drinking water with sanitary sewage through the connecting pipes.

[Diphtheria antitoxin levels in Japanese adults (10-20 years after the last vaccination)].

The serum diphtheria antitoxin levels in Japanese adults were investigated and the persistence of diphtheria toxoid effect was evaluated. The subjects consisted of 56 volunteers (20-31 years of age) who had received regular inoculations of diphtheria and pertussis vaccine (I and II or III phases). They had been immunized according to the Vaccination Law (old version) revised in 1958. The length of time after the last inoculation of diphtheria toxoid was speculated to range from 10 to 20 years. Serum diphtheria antitoxin was determined by passive hemagglutination method. Antitoxin level was 0.025 HAU/ml in 1 subject (1.8%), 0.05 in 2 (3.6%), 0.1 in 2 (3.6%), 0.2 in 8 (14.3%), 0.4 in 7 (12.5%), 0.8 in 12 (21.4%), 1.6 in 7 (12.5%) and more than 1.6 in 17 (30.4%). Results indicated that 55 of 56 (98.2%) possessed a higher level than the prophylaxis standard (0.05 HAU/ml). Since the current Vaccination Law prescribes a lower amount and fewer inoculations than the old law, the number of adults possessing prophylactic antitoxin level may decrease in the future. Further investigation of antitoxin level in adults needs to be continued.