Pigment-dispersing hormone-like peptide in the nervous system of the flies Phormia and Drosophila: immunocytochemistry and partial characterization.

beta-pigment-dispersing hormone (beta-PDH) isolated from the fiddler crab (Rao et al., '85) is a member of an octadecapeptide family of neuropeptides common to arthropods. Whereas earlier studies of these peptides in insects were limited to orthopterans, this investigation focuses on dipteran flies. Extracts of heads from the blowfly Phormia terraenovae were assessed in a fiddler crab bioassay for PDH activity. Immunocytochemistry, dose-response curves, gel filtration chromatography and reversed-phase HPLC, combined with bioassay and enzyme-linked immunosorbent assay (ELISA), indicate the presence of PDH-like peptide in the blowfly. Immunocytochemical mapping of PDH-like immunoreactive (PDHLI) neurons was performed for the entire nervous systems of Phormia and the fruitfly Drosophila with a beta-PDH antiserum. In the cephalic ganglion (brain, optic lobe and subesophageal ganglion) PDHLI cell bodies could be detected (34 in Phormia and 16 in Drosophila). In both species, each hemisphere contains 8 PDHLI cell bodies in the optic lobes. These innervate the optic lobe neuropils bilaterally. In Phormia, another set of 8 cell bodies are located in each of the lateral neurosecretory cell groups in the superior protocerebrum. These neurons send axons to the corpora cardiaca-hypocerebral ganglion complex and to portions of the foregut. In contrast, only the optic lobe neurons display immunoreactivity in Drosophila. Except for the optic lobes, PDHLI processes are distributed only in nonglomerular neurophils of the brain of both species. In the fused thoracico-abdominal ganglia of Phormia, 28 PDHLI cell bodies were found (only six were found in Drosophila). In both species, six abdominal PDHLI neurons are efferents with axons innervating the hindgut. We also found that some of the PDHLI neurons in the Phormia brain and abdominal ganglion contain colocalized FMRFamide-like immunoreactivity. Since the flies studied here do not display hormonally controlled, fast pigment migrations, the PDH-like peptide may have a role as neurotransmitter or neuromodulator in the central nervous system, especially in the visual system, and a regulatory role in the stomatogastric system and the hind-gut.

Involvement of HLA in clinical courses of myasthenia gravis.

The relationship between the histocompatibility leukocyte antigen (HLA) phenotypes and the clinical course of myasthenia gravis (MG) was studied in 53 Japanese patients with MG. The frequency of HLA-DRw9 antigen was high in the MG patients who did not need immunosuppressive therapy but only anticholinesterase agents (RR = 4.52; CP less than 0.02), who achieved remission of the disease (RR = 2.98; CP less than 0.05) or who showed a decrease in AChR antibody (Ab) titer (RR = 6.32; CP less than 0.0002), whereas the frequency of HLA-DRw8 antigen was increased in MG patients who underwent immunosuppressive therapy (RR = 4.03; CP less than 0.01), who did not have remission (RR = 4.75; CP less than 0.1) or who showed an increase in AChR Ab titer (RR = 6.48; CP less than 0.01). These data suggest that immunogenetic heterogeneity in MG might be reflected in its clinical course.

An esterase on the outer membrane of Pseudomonas aeruginosa for the hydrolysis of long chain acyl esters.

A new esterase activity which hydrolyzes palmitoyl-CoA was found in the membrane fraction of Pseudomonas aeruginosa. All the 11 strains of P. aeruginosa tested possessed this esterase activity. The esterase was constitutive and was fully active on the intact cell bodies toward substrates in the medium. It was located on the outer membrane of the cell envelope, and was not released into the culture medium. This activity was designated as OM (outer membrane) esterase. OM esterase was solubilized from the cell envelope with EDTA-Triton X-100 and purified 690-fold. It was a minor component of the outer membrane. Its molecular weight was approximately 55,000. The activity was rather stable to heat, a wide range of pH, and treatment with detergents and organic solvents. No cofactors were required. The pH optimum of the reaction was 8.5. Among various acyl-CoAs, only long chain (C12--C18) thioesters were hydrolyzed. OM esterase also hydrolyzed some kinds of oxy-esters such as p-nitrophenyl acyl esters, monoacyl esters of sucrose and Tween 80 (polyoxyethylene sorbitan monooleate). On the other hand, triglycerides, phospholipids, or hydrophobic monoesters were not hydrolyzed at all. Thus, this enzyme seems to have specificity for long chain acyl esters with hydrophilic groups, whether thio- or oxy-ester. Mutants deficient in this esterase activity were isolated. These mutants were unable to grow on Tween 80 as a sole carbon source. This suggests a possible role of OM esterase in the utilization of acyl esters as carbon sources.

Effect of iron concentration in the growth medium on the sensitivity of Pseudomonas aeruginosa to pyocin S2.

The iron concentration in the growth medium was found to affect the susceptibility of Pseudomonas aeruginosa PML1550 to pyocin S2, a bacteriocin. The efficiency of killing by pyocin S2 was very low when the indicator cells were grown in an iron-rich medium. The capacity of these cells to adsorb pyocin S2 was reduced. Cultivation under limitation of iron (1 microM or less) was necessary to produce a fully sensitive cell population. The growth under iron limitation was accompanied by the appearance of four protein components in the outer membrane of the cells. Nine mutants resistant to pyocin S2 were isolated and their outer membranes were analyzed. They all lacked one component (Fe-b protein) as well as the adsorption capacity for pyocin S2. These findings suggest a possible role of this protein as the receptor for pyocin S2.

Suppressive effects of breast milk on oxidative DNA damage in very low birthweight infants.

BACKGROUND: Human milk contains many kinds of antioxidant and is considered to prevent diseases mediated by oxygen free radicals in very low birthweight (VLBW) infants. AIMS: To examine the antioxidant effects of breast milk in VLBW infants by determining urinary 8-hydroxydeoxyguanosine (8-OHdG) excretion, which is known to be a non-invasive marker for in vivo oxidative DNA damage. METHODS: Urinary 8-OHdG concentrations were measured in 15 breast fed and 14 formula fed VLBW infants at 2, 7, 14, and 28 days of age. RESULTS: Urinary 8-OHdG excretion at 14 and 28 days of age was significantly lower than at 2 and 7 days of age in the breast fed group, and significantly lower than in the formula fed group. CONCLUSION: This is the first direct evidence of the antioxidant action of human milk in VLBW infants.

Epidemiological study of hemorrhagic fever with renal syndrome related virus infection among urban rats in two islands in Tokyo Bay, Japan.

A seroepidemiological survey of hemorrhagic fever with renal syndrome (HFRS) virus infection in 403 urban rats (Rattus norvegicus) captured on two different islands in Tokyo Bay was carried out using the indirect immunofluorescent antibody (IFA) test with the SR-11 strain of HFRS virus. Antibody positive sera were detected in 99 out of 355 (27.9%) rats from the island with reclaimed ground area, but none were detected in 48 rats from the other islands. IFA titers of the positive sera varied from 16 to 4,096 (mostly 128) with a peak at 1 : 128, and antibody positive ratios were significantly higher in the small rats (less than 50 g, probably of younger age) than in the large ones. A causative agent antigenically related to HFRS virus was isolated from lung tissue of seropositive rat using Vero-E6 cells. These results revealed the existence of HFRS related virus among urban rats in the coast of Tokyo Bay, Japan.

[Serovar distributions of cervical Chlamydia trachomatis isolated in Nagano Prefecture].

Between 1992 and 1998, serotyping of 82 Chlamydia trachomatis cervical isolates were examined by micro-IF method in Nagano Prefecture. Of these, 17 isolates (20.7%) were serovar E, 15 (18.3%) were serovar F, 14 (17.1%) were serovar D and 10 (12.2%) were serovar G, and the isolates typed these serovars were found to be 68.3% whole. Furthermore, serovars B (7.3%) and K (9.8%) were comparatively frequently found. From 1992 to 1994, the number of isolates of B-complex, intermediate and C-complex were 18, 17 and 6, respectively. On the other hand, from 1995 to 1998, the number of the isolates were 19, 8 and 14, respectively. The distribution of serovars of C. trachomatis tended to fluctuate from serovars D and E to F and G as the patients grew older. Moreover, serovar E isolates were only detected from patients less than 40 years old. Although most of the serovar I isolates were detected from the patients with vaginal discharge, the other isolates did not clearly indicate the relationship of serovars and clinical symptoms.

[Monoclonal antibodies to herpes simplex viruses antigens--specificity and utility in rapid serotyping of clinical isolates].

Sixteen hybridomas secreting antibodies to HSV-1 and 22 hybridomas secreting antibodies to HSV-2 were derived from fusion of SP2/0 myeloma cells with spleen cells from BALB/c mice immunized with each respective virus. Four of the former 16 hybridomas and seven of the latter 22 hybridomas were subcloned and injected into pristane-primed mice to obtain high titers of monoclonal antibodies. Antigen specificity of these monoclonal antibodies were determined by the Western blotting (WB) assay. Two out of four monoclonal antibodies that showed selective reactivity for HSV-1 in IFA, reacted with HSV-1 specific proteins; #1 reacted with 100 KD and 70 KD proteins and #4 with a 150 KD protein, respectively, while the remaining two antibodies reacted only with a 50 KD protein that is type-common antigen. On the other hand, two out of seven antibodies which showed selective reactivity for HSV-2 in IFA, reacted with HSV-2 specific proteins: #5 with a 100 KD protein and #10 with three proteins of 30, 25, and 20 KD, and the other two antibodies reacted with a 50 KD protein that is a type-common antigen. The remaining three antibodies, two of which were found to be immunoglobulin type IgM, reacted with neither HSV-1 nor HSV-2 antigens in WB assay. In order to determine their utility in serotyping, 11 monoclonal antibodies were examined by IFA test for reactivity to cells that were infected with 20 HSV-1 or 16 HSV-2 isolates which had been typed by neutralization test.(ABSTRACT TRUNCATED AT 250 WORDS)

[Distribution of serovars of Chlamydia trachomatis isolates in Japan].

To study the serovar distribution of C. trachomatis in Japan, a total of 85 genital C. trachomatis isolates from male and female patients attending the clinics were examined by the microimmunofluorescence test using immune sera of the isolates produced in mice. Of these isolates, 34 (40.0%) were typed D or E, and 19 (22.4%) were typed G or F. The serovars of the remaining 32 isolates were B, H, I, J, and K, and the proportions of these serovars were from 8.2 to 3.5%. Thus, two thirds of C. trachomatis isolates in this country were found to fall into only four serovars, namely, D, E, G, and F, and, therefore, the epidemiology of C. trachomatis infection in Japan seems to be similar to that of other countries in North America and Europe. The relative distribution of serovars of C. trachomatis isolates from male patients and female patients somewhat differed. Serovars D, E, and G, F were isolated in the same ratio from male patients, while the isolation ratio of the former serovars was three times or more higher than the latter serovars in female patients. No isolate typed serovar K was found in male patients, while 15% of isolates from female patients were typed this serovar.

[Isolation and identification of Chlamydia spp. from effusion of otitis media].

Three chlamydial strains isolated from patients of otitis media with effusion were studied by comparing reactivity to monoclonal antibody (MAb) and polyclonal antibody (PAb) produced against one clinical isolate (named Mk), which was first isolated by Dr Mukai (Mukai Microbiological Research Laboratory, Yamato-shi, Kanagawa prefecture). Commercially supplied antibody (Microtrak (Syva), Culture-set (Ortho diagnostic system)) was also used. To isolate the Chlamydia spp, the yolk sacs of eggs were immediately inoculated with sample effusions (0.2 to 0.4 ml per sac) as soon as the samples were received. The eggs were observed every day for a period of 12 days thereafter for signs of life or death. One to two blind passages were first done in the eggs and then in HeLa 229 cells. The reactivity was examined by both micro-IF tests, among various strains of Chlamydia (C. trachomatis: L2. C. pneumoniae, C. psittaci: Budgerigar, Izawa, Meningopneumonitis (MP)) and by immunoblot analysis. Chlamydia spp were isolated in two of the twenty-nine sample effusions (6.9%). These isolates were then tested for reactivity to MAb and PAb. It was found that MAb reacted with MP and Mk, but not with Budgerigar, Izawa and C. pneumoniae. The antibody of Culture-set reacted with C. trachomatis C. pneumoniae and C. psittaci. No reactivity was observed in Mk by MicroTrak. Immunoblot analysis revealed that MAb reacted with about 95 KDa protein of Mk, the two clinically isolated Chlamydia spp and MP. By using PAb from rabbits, similar blotting patterns were observed in Mk, the clinical isolates and MP.(ABSTRACT TRUNCATED AT 250 WORDS)

[Effect of storage conditions on the infectivity of Chlamydia pneumonia].

In order to facilitate the isolation of C. pneumoniae, strain similar TWAR, with high frequency, we investigated the effect of various factors on the infectivity of Chlamydia using two laboratory strains, C. pneumoniae TWAR and C. trachomatis serovar D. The factors tested were the effects of different temperatures for storage conditions, saliva from healthy person, storage media for Chlamydia, and the frequency of freezing and thawing. Chlamydial suspension was prepared in the two media, SPG (sucrose-phosphate-glutamate buffer pH 7.5), and CT-GM (culture medium for Chlamydia which contains 1 micrograms/ml cycloheximide and 0.04% glucose). Chlamydial suspension was allowed to stand in each of four different thermal conditions: 37 degrees C, room temperature (25 degrees C), 4 degrees C, 0 degrees C and -75 degrees C for 1, 3, 6, 24, 48, 72, 96, 120 and 144 hours. For storage at -75 degrees C, one of three groups of glass vial tubes containing Chlamydia was covered with an "airmat" to prevent the rapid freezing of Chlamydia. The effect of various factors on the infectivity was assayed by inoculation of the suspension on HeLa 229 cell monolayers. Results showed that the infectivity rapidly decreased at 37 degrees C and room temperature, while at 4 degrees C, 0 degrees C and -75 degrees C, relatively high infectivity was maintained and contained until days 4 to 6. This decreasing pattern was similar to among the media used. We were not able to find any differences in the infectivity among the samples with or without the "airmat".(ABSTRACT TRUNCATED AT 250 WORDS)

[A case of pediatric Chlamydia pneumoniae infection and properties of the isolate].

Chlamydia pneumoniae was isolated from the throat of a 2-year-old girl with upper respiratory illness. The isolate, Shizuoka-37, was stained with C. pneumoniae specific monoclonal antibody (RR402), as well as the genus specific antibody (Cultureset), but not with C. trachomatis specific monoclonal antibody (Micro-Trak). C. pneumoniae genome was amplified by polymerase chain reaction in the isolate. Elementary bodies (EB) of the isolate was round shaped by electron micrograph.

[Tsutsugamushi disease (scrub typhus) in Japan: epidemiological aspects].

Epidemiological aspects of tsutsugamushi disease (scrub typhus) in Japan in 1998 were analyzed using questionnaires. Four hundred and sixteen scrub typhus cases were reported in 24 prefectures in 1998. The annual number of the patients in 1998 was similar to those in the preceding three years. There was no sex difference. The patients at the age of 51 or greater accounted for 72% of the total cases. Patients engaged in farming and forestry accounted for 32% and 14% cases, respectively. Fifty-six, 21 and 19% were reported in Kyusyu, Kanto and Tohoku-Hokuriku districts, respectively, 96% of the total cases being reported in these 3 districts. Most cases were reported from April through June with some from October through December in Tohoku-Hokuriku districts, while most cases were reported from October through December in other districts, including Kyusyu and Kanto districts. Thus, there was a difference in epidemic seasons among the districts. Serotypes of scrub typhus rickettsia were analyzed by serum antibody titers in the Kyusyu district. The novel Kawasaki and Kuroki types were major strains; however, no geographical difference was seen within the Kyusyu district. Interestingly, 24 cases were diagnosed only by the new serotypes not by the classical serotypes (Kato, Karp and Gilliam) in serological tests. This result suggests that further investigations are required to determine the prevalent serotypes in each district and to improve the serological tests. This was the first comprehensive report of epidemiology of scrub typus in entire Japan. Information obtained in the present study provides deep insight into prediction, diagnosis, treatment and prevention of scrub typhus in Japan.

[Tsutsugamushi disease (scrub typhus) in Japan: clinical features].

Clinical features of tsutsugamushi disease (scrub typhus) were analyzed, based on 416 cases reported in Japan in 1998. Three major clinical symptoms: eschar, fever and rash were found in 87%, 98% and 92% of the cases, respectively. Elevated levels of CRP, GOT, GPT and LDH were observed in 96%, 85%, 78% and 91%, respectively. These clinical and laboratory findings were observed in the majority of the cases and considered important for diagnosis. Disseminated intravascular coagulation developed in 21 cases, indicating that scrub typhus can be life threatening. Lymphadenopathy was observed in 51% of the cases. Enlarged lymph nodes were limited to the local sites in 75% of these lymphadenopathy cases and most of these sites were adjacent to eschars. Most eschars were scabbed and located in the abdomen and the lower half of the body, especially the feet. This suggests that these parts are frequently exposed to tsutsugamushi mites. Furthermore, the skin is soft in these parts and covered by cloth. These factors may make it possible for mites to keep biting without being noticed for several hours, long enough for rickettsial transmission. Interestingly, eschar and rash were absent in 14% and 8% of the cases, respectively. This result suggests that the cases without the unique symptoms may have been misdiagnosed as common cold or other febrile illnesses. One hundred and fifty-four suspected cases were not scrub typhus cases by the serological tests. The three major clinical symptoms were present in approximately a half of these negative cases, eschar being observed in approximately 70%. This may suggest the presence of new type of scrub typhus can not be diagnosed by the present laboratory tests. Clinical features of scrub typhus in Japan were well revealed, and information obtained in the present study is useful for improving clinical diagnosis. It should, however, be stressed that there were cases that could not be correctly diagnosed only by the clinical symptoms, suggesting that it is important to improve the serological tests.

[Immunohistochemical localization of blood-retinal barrier breakdown after argon laser photocoagulation in the monkey retina].

Retinal photocoagulation induces breakdown of the blood-retinal barrier (BRB), but the site of the breakdown is not precisely known. The breakdown and repair of BRB following argon laser photocoagulation were studied in the monkey retina by immunohistochemical localization of serum albumin. Argon laser photocoagulation was performed to obtain ordinary burns as in human use. 1, 3, and 7 days after photocoagulation, serum albumin was observed in the outer retina and retinal pigment epithelium, but not around the retinal blood vessels. The immunostaining disappeared within 14 and 28 days after photocoagulation. The results of this study suggest that photocoagulation induces local breakdown of the outer BRB and that the breakdown is repaired within 14 days.

Pigment-dispersing hormone immunoreactive neurons in the blowfly nervous system.

We could demonstrate pigment-dispersing hormone immunoreactive (PDHIR) neurons in the brain and ventral ganglia of the blowfly Phormia terraenovae. PDHIR neurons were found in the optic lobe. Their processes supply the lamina, medulla and lobula complex bilaterally. Large PDHIR cell bodies in the protocerebrum have processes in the proto- and tritocerebrum and axons to the aorta wall and foregut. Eight pairs of PDHIR neurons are found dorsally and three pairs ventrally in the fused abdominal neuromeres; one pair is located ventrally in each of the thoracic neuromeres. The ventral abdominal PDHIR neurons are efferents that innervate the hindgut. PDHIR neurons may play different functional roles as neurohormones or neuromodulators in different parts of the nervous system and its peripheral targets.

[Preliminary results of PET activation study in cerebral arteriovenous malformation (AVM), using C15O2 and 18F-FDG].

The authors applied PET activation study to two patients with arteriovenous malformation (AVM) to localize primary motor cortex before surgery or embolization. The change in regional cerebral blood flow (rCBF) was measured during foot movements in Case 1 who had a 2-cm AVM located in the post-central gyrus. Superimposed PET/MRI images revealed that the rCBF increase was located in the pre-central gyrus. Its validity was confirmed by intraoperative cortical mapping using electrical median nerve stimulation. The patient safely underwent total removal of AVM. The change in regional cerebral metabolic rate for glucose (rCMRglc) was measured during hand movements in Case 2 who had a huge AVM over the central sulcus. Superimposed PET/MRI images revealed that hand movements significantly increased rCMRglc in the frontal cortex, which was separated from the original primary motor area. The patient safely underwent partial embolization, although he suffered transient weakness of the face after embolization. The preliminary results strongly suggest that PET activation study is useful to localize precisely cortical functions of the patients with AVM, thus reducing morbidity after treatment. The results also suggest that cortical functions may undergo translocation when huge AVM involves the eloquent area.

[Clinical reference values for laboratory hematology tests calculated using the iterative truncation method with correction: Part 1. Reference values for erythrocyte count, hemoglobin quantity, hematocrit and other erythrocyte parameters including MCV, MCH, MCHC and RDW].

Age and sex dependent differences in the clinical reference values for erythrocyte count (RBC), hemoglobin quantity (Hb), hematocrit (Ht) and other erythrocyte parameters including MCV (mean corpuscular volume), MCH (mean corpuscular hemoglobin), MCHC (mean corpuscular hemoglobin concentration) and RDW (red cell distribution width), were calculated by the iterative truncation method with correction (Usui's method) using the results from tests on 6,300 patients' specimens obtained at Kyoto University Hospital. For RBC, Hb and Ht, the data obtained from the individuals below 13 years old showed the normal or sometimes log-normal distribution, but adjustment by the Xn-type variable transformation was often necessary to obtain the normal distribution for the data taken from the populations containing individuals over the age of 14. For the clinical reference values of RBC, Hb and Ht, no sex difference was observed below the age of 12. The values for males were significantly higher than those of females in the age range 13-79, and the values showed no significant sex-dependent difference at ages above 80. In females, age-dependent change of values for RBC, Hb and Ht was less prominent than in males; especially the upper limit values for females were very stable for all ages. MCV and MCH gradually increased with age both in males and females, and the MCHC remained constant in all age populations of male and female. The reference value for RDW was generated by the percentile method instead of the iterative truncation method because of the strong deviation in the distribution pattern, and the RDW values showed a gradual increase with age in both males and females.

[Effect of heparin cofactor II on the antithrombin III activities measured by thrombin methods or factor Xa methods--fundamental studies and clinical studies using the plasma of pregnant women].

We evaluated the effects of heparin cofactor II(HC II) on the antithrombin III(AT III) activities measured by the methods of thrombin or factor Xa. Reagents A and B were using the method of thrombin and reagent C was based on the method of Xa. Purified HC II was directly measured or indirectly measured after the dilution with control plasma. Cross reaction of HC II in AT III assay were negligible in reagent C, but substantial amount of AT III activities were measured in reagent A and B. Plasma AT III activities from full-term pregnant women were significantly higher than those from non pregnant control women in reagent A, but comparable in reagent B or C. These results indicate that AT III activities measured by thrombin methods by thrombin were overestimated in pregnant women due to the cross-reactivities of HC II. It is recommended that AT III activities would be measured by the methods of factor Xa.

[Clinical reference values for laboratory hematology tests calculated using the iterative truncation method with correction: Part 2, Reference values for white blood cell (WBC) count, WBC differential including segmented neutrophil, band neutrophil, lymphocyte, monocyte, eosinophil, basophil, platelet count and mean platelet volume].

Age and sex dependent differences in the clinical reference values for white blood cell(WBC) count, WBC differential including segmented neutrophils(SEG), band neutrophils(BAND), lymphocytes (LYMPH), monocytes(MONO), eosinophils(EOSINO), basophils(BASO), platelet count(PLT) and mean platelet volume(MPV) were calculated by the iterative truncation method with correction(Usui's method) using the result from tests on 6,300 patient specimens obtained at Kyoto University Hospital. WBC counts exhibited log-normal distribution in all age either in male and female. PLT counts and MPV showed normal or log-normal distribution. WBC differential (SEG, BAND, LYMPH, MONO, EOSINO, BASO) usually showed log-normal distribution. As to the reference value for WBC, high limit values gradually decreased in the age range of 5-14 year old in males and females. After 15 year old, it gradually increased and reached adult's level. Low limit values for WBC gradually decreased with age both in males and females. The reference value for SEG showed similar change to WBC. The reference value for LYMPH gradually decreased age both in males and females. The reference values for BAND, MONO, EOSINO, BASO were generated by the percentile method instead of the iterative truncation method because of the strong deviation in the distribution pattern. BAND, MONO and BASO values were very stable for all ages in males and females. High limit values for EOSINO in the age range of 5-10 year old in males and females remained constant, and gradually decreased in 10-20 year old, and were stable at ages above 20 year old. PLT counts gradually decreased with age, and the MPV remained constant in all age populations both in male and female.