Delta15N of soil N and plants in a N-saturated, subtropical forest of southern China.

We investigated the delta(15)N profile of N (extractable NH(4)(+), NO(3)(-), and organic N (EON)) in the soil of a N-saturated subtropical forest. The order of delta(15)N in the soil was EON > NH(4)(+) > NO(3)(-). Although the delta(15)N of EON had been expected to be similar to that of bulk soil N, it was higher than that of bulk soil N by 5 per thousand. The difference in delta(15)N between bulk soil N and EON (Delta(15)N(bulk-EON)) was correlated significantly with the soil C/N ratio. This correlation implies that carbon availability, which determines the balance between N assimilation and dissimilation of soil microbes, is responsible for the high delta(15)N of EON, as in the case of soil microbial biomass delta(15)N. A thorough delta(15)N survey of available N (NH(4)(+), NO(3)(-), and EON) in the soil profiles from the organic layer to 100 cm depth revealed that the delta(15)N of the available N forms did not fully overlap with the delta(15)N of plants. This mismatch in delta(15)N between that of available N and that of plants reflects apparent isotopic fractionation during N uptake by plants, emphasizing the high N availability in this N-saturated forest.

Temporal diet changes recorded by stable isotopes in Asiatic black bear (Ursus thibetanus) hair.

Carbon and nitrogen stable isotope ratios were measured in hair samples of the Asiatic black bear (Ursus thibetanus) inhabiting the Northern Japanese Alps (NJA) (n = 20) and the periphery of Nagano City (NC) (n = 6), in Nagano Prefecture, Japan. The hair of NJA bears, which did not have access to anthropogenic foods, showed lower values of d13C and d15N than that of NC bears which had access to garbage and corn fields, especially during the summer. These results reflect somewhat differing diets between the NJA and NC bears. We attempted to assess the feeding history during the hair growth cycle using the growth section analysis method. Each hair sample had been cut into 3?mm lengths from root to tip, labeled, and analyzed along the hair growth. We measured the carbon and nitrogen stable isotope ratios of each 3?mm length of hair sample from one NC bear which had been killed while raiding a corn field. The sections showed wide ranges of isotope ratios, from -23.2% to -14.6% for delta13C, and from 0.3% to 4.6% for delta15N. It was shown that the diet of this bear shifted dramatically from principally C3 plants to more C4 plants and to foods of animal origin. An analysis of the whole hair reflects just the average feeding habit during hair growth, but the present method can trace its diet history. This method can contribute to obtain precise ecological information of wildlife.

Phosphorylation of a 25 kDa protein is induced by thermostable direct hemolysin of Vibrio parahaemolyticus.

Thermostable direct hemolysin (TDH) is a possible virulence factor produced by Vibrio parahaemolyticus. Although TDH has a variety of biological activities, including hemolytic activity, the biochemical mechanism of action remains uncertain. Here we analysed biochemical events, especially phosphorylation, caused by TDH in erythrocytes, and found that TDH caused significant phosphorylations of proteins on erythrocyte membrane. Phosphorylation of proteins was studies using [gamma-32P] ATP and SDS-PAGE. A number of protein kinase inhibitors were tested, to determine which types of kinases were involved in the phosphorylation events. TDH induced the phosphorylation of two proteins on membranes of human erythrocyte that are sensitive to TDH. The estimated molecular weight of these proteins was 25 and 22.5 kDa. Interestingly, the 22.5 kDa, but not the 25 kDa protein, was phosphorylated on the membrane of TDH-insensitive (resistant) horse erythrocytes. Moreover, a mutant TDH (R7), which retained binding ability but lost hemolytic activity, also phosphorylated only the 22.5 kDa protein on human erythrocyte membranes. Among the protein kinase inhibitors used the protein kinase C inhibitors, (staurosporine and calphostin C) showed marked inhibition of phosphorylation of 25kDa protein. In addition to phosphorylation, these protein kinase C inhibitors suppressed hemolysis by TDH. These results indicate that the phosphorylation of the 25 kDa protein seems to be essential for the hemolysis by TDH after it binds to erythrocyte membranes.

Complete nucleotide sequence of the prophage VT1-Sakai carrying the Shiga toxin 1 genes of the enterohemorrhagic Escherichia coli O157:H7 strain derived from the Sakai outbreak.

Shiga toxins 1 and 2 (Stx1 and Stx2) are encoded by prophages lysogenized in enterohemorrhagic Escherichia coli (EHEC) O157:H7 strains. Lytic growth of the phage particles carrying the stx1 genes (stx1A and stx1B) of the EHEC O157:H7 strain RIMD 0509952, which was derived from the Sakai outbreak in 1996 in Japan, was induced after treatment with mitomycin C, but the plaque formation of the phage was not detected. We have determined the complete nucleotide sequence of the prophage VT1-Sakai. The integration site of the prophage was identified within the yehV gene at 47.7 min on the chromosome. The stx1 genes were downstream of the Q gene in the prophage genome, suggesting that their expression was regulated by the Q protein, the regulator of the late gene expression of the phage, which is similar to that of the stx1 or stx2 genes carried by the lambdoid phages reported previously. The sequences of the N gene and its recognition sites, nutL and nutR, were not homologous to those of the phages carrying the stx genes thus far reported, but they were very similar to those of bacteriophage phi21. The sequences of the repressor proteins, CI and Cro, that regulate expression of the early genes had low similarities with those of the known repressors of other phages, and their operator sequences were different from any sequence reported. These data suggest that multiple genetic recombination among bacteriophages with different immunities took place to generate the prophage VT1-Sakai. Comparison between the sequences of VT1-Sakai and lambda suggests that the ancestor of VT1-Sakai was produced by illegitimate excision, like lambda gal and bio phages.

Homogeneity of a hemolysin(Vh-rTDH) produced by clinical and environmental isolates of Vibrio hollisae.

The characteristics of Vh-rTDH, a hemolysin similar to Vp-TDH of Vibrio parahaemolyticus produced by clinical and environmental isolates of Vibrio hollisae, were comparatively studied. All 7 strains of V. hollisae tested were found to produce indistinguishable Vh-rTDH when they were examined by heat-stability test, Western blotting analysis and conventional polyacrylamide slab gel electrophoresis.

Comparison of Vibrio parahaemolyticus hemolysin (Vp-TRH) produced by environmental and clinical isolates.

TDH-related hemolysin (Vp-TRH) produced by Kanagawa-phenomenon-negative (KP-) Vibrio parahaemolyticus has been demonstrated to be a possible virulence determinant. Though almost half of KP- isolates examined from diarrhoeal patients produced Vp-TRH, few reports mentioned the ability of environmental isolates to produce Vp-TRH. Considering the route of infection with V. parahaemolyticus, this toxin must be produced by the organisms in the sea or in sea food. To confirm that Vp-TRH produced by V. parahaemolyticus could be involved in sea-food-borne diarrhoeas, Vp-TRH-producing strains were isolated from the environment, identified and hemolysin purified from these strains was compared to hemolysin (Vp-TRH) isolated from diarrhoeal patients. The results showed that the hemolytic activity, antigenicity, reactivity in the rabbit ileal loop test and N-terminal amino acid sequence of Vp-TRH from environmental strains was indistinguishable from the toxin of clinical origin.

Detection of the gene for toxic shock syndrome toxin 1 in Staphylococcus aureus by enzyme-labelled oligonucleotide probes.

A colony hybridisation method with enzyme-labelled oligonucleotide probes was developed to detect the gene for toxic shock syndrome toxin 1 (tst). For rapid identification, bacterial colonies were transferred from agar plates directly on to nylon membranes. These procedures took only 3 h. Results obtained by this test correlated well with those obtained by the reverse passive latex agglutination test. Thus, this method is convenient and reliable for the detection of tst in staphylococci, which could be useful for both research and clinical purposes. This method demonstrated that tst was more prevalent in methicillin-resistant Staphylococcus aureus (56%) than in methicillin-susceptible S. aureus (4%).

Effect of antimicrobial agents, especially fosfomycin, on the production and release of Vero toxin by enterohaemorrhagic Escherichia coli O157:H7.

In 1996, Japan had several large outbreaks of enterohaemorrhagic Escherichia coli (EHEC) O157:H7 infection. We surveyed physicians who examined and treated these patients, and found that most of the patients (95.9%) received antimicrobial agents as treatments, in particular, fosfomycin comprised 84.0% of the prescribed treatment. Since the administration of antimicrobial agents for EHEC infection is under discussion, we also analyzed the effects of 7 antimicrobial agents including fosfomycin on the production and release of Vero toxins (VTs) by EHEC. The addition of fosfomycin into EHEC culture in CAYE broth at 5 h after the start of incubation caused a marked increase of VT1 release and production, as revealed by an immunological toxin assay (RPLA). However, a cytotoxicity assay of Vero cells showed a small increase of biological activity in the specimens treated with fosfomycin because the Vero cell assay reflects total cytotoxicity of VT1 and VT2. These results indicate that further study is necessary before concluding whether antimicrobial agents actually worsen an EHEC infection.

The binding of Vibrio parahaemolyticus 125I-labeled thermostable directhemolysin to erythrocytes.

Thermostable direct hemolysin (TDH) produced by Vibrio parahaemolyticus was iodinated using chloramine T. The 125I-labeled TDH retained up to 80% of the activity of intact toxin. The binding of 125I-TDH to rabbit erythrocytes was inhibited by addition of nonlabeled TDH. The binding of 125I-TDH to rabbit erythrocytes was completed in the 1st or 2nd min of incubation at 37 degrees C in contrast to that at 4 degrees C. 125I-TDH, which cannot lyse horse erythrocytes as does intact TDH, bound to horse erythrocytes as to those of rabbit. The dissociation constants (KD) derived Scatchard plots were 2.85, 4.39, 4.33 and 5.35 x 10-8M for rabbit, horse, human and sheep erythrocytes, respectively. The lytic sensitivity of various erythrocytes to TDH showed no relationship to the binding capacity.

Tryptophan 65 is essential for hemolytic activity of the thermostable direct hemolysin from Vibrio parahaemolyticus.

The effect of modification of aromatic and ionizable amino acid residues on the hemolytic activity of a thermostable direct hemolysin from Vibrio parahaemolyticus was examined. Tryptophan 65, one of the two tryptophan residues per subunit, was specifically modified with N-bromosuccinimide, resulting in complete loss of hemolytic activity. However, neither nitration with tetranitromethane of one of the nine tyrosine residues nor Nlm-ethoxyformylation of two of the four histidine residues caused any change in hemolytic activity. The hemolysin was fully active upon amidation of two reactive carboxyl group. On the other hand, acetylation of amino groups and the modification of one of the three arginine residues with 1,2-cyclohexanedione resulted in a partial loss of the hemolytic activity. The results suggest that Trp65 is essential for the hemolytic activity of V. parahaemolyticus hemolysin.

Effect of mitomycin C dissolved in reversible thermo-setting gel on the outcome of filtering surgery in the rabbit.

PURPOSE: Based on our previous report that showed enhanced transfer of mitomycin C to the sclera and the conjunctiva by dissolving the agent into a reversible thermo-setting gel, we conducted a study to investigate the efficacy of mitomycin C gel in the rabbit. METHODS: We subconjunctivally injected 0.1 mL of the mitomycin C gel solution containing several different amounts of the agent. Trephination was performed in the injected region 24 hours later. Intraocular pressure measurement, photography, and ultrasound biomicroscopic examination of the filtering bleb were conducted 1, 2, and 4 weeks postoperatively. RESULTS: The gel containing 3.0 microg or more mitomycin C significantly enhanced bleb formation and reduction in the intraocular pressure. CONCLUSIONS: The reversible thermo-setting gel seems to help the filtering bleb to survive in the rabbit and deserves further investigation as a new method of mitomycin C application.

[Evaluation of Novapath assay for direct detection in patient stool of Vero toxin and Vero toxin-producing Escherichia coli].

We evaluated two kits which detect either Verotoxin (Novapath EHEC) or EHEC O157 (Novapath O157) directly from stool specimens and compared with the result of PCR. The tests can be completed in 3 hrs. All fecal specimens from 18 healthy volunteers showed negative results with both kits and PCR. Specimens from patients of EHEC O157 outbreak in Okayama Prefecture and asymptomatic families of patients of outbreak in Sakai City of Japan were also analyzed. Fecal specimens from which EHEC O157 was isolated showed positive results with both kits and PCR. Some specimens which were culture negative showed positive results with either Novapath EHEC or Novapath O157 or both. The sensitivity of Novapath O157 was lower than PCR to detect O157 organisms but took shorter time to get result. Novapath EHEC showed very high sensitivity in detecting Vero toxin. These results suggest that Novapath EHEC especially can be used clinically for rapid diagnosis.

[Report of questionnaire about enterohemorrhagic Escherichia coli cases caused in the area including Sakai City in 1996].

Many outbreaks of EHEC O157 were occurred in Japan in 1996. There was the biggest outbreak involving approximately 6000 victims in area of Sakai City. We conducted a questionnaire survey on the EHEC infection to the physicians who treated these patients and the microbiologists. For physicians we asked mainly clinical manifestation of patients they observed and treatment they did and their outcome (Answer A). Microbiologists were also required to answer the opinion about treatment (Answer B). Eighty-five of physicians replied about their 222 patients and 209 of microbiologists answered their opinions. In this report we summarized the data and obtained following results: 1) We observed higher frequency of HUS/TTA complication in female than male. 2) Higher HUS/TTP complication was accompanied with patients complained symptoms of fever, bloody diarrhea, and general fatigue. 3) The most patients (96%) were received antimicrobial agents, especially fosfomycin (84%), followed by new quinolon (17.8%) and Cefems (12.2%). Later starting of treatment with antimicrobial agents than 7 days from onset of symptoms increased HUS/TTP complication. 4) Anti-diarrhoeal agents seems to be one of risk factors for HUS/TTP complications.

[Serotypes of urease producing Vibrio parahaemolyticus and their relation to possession of tdh and trh genes].

We analysed 467 isolates of Vibrio parahaemolyticus for possession of tdh/trh gene in comparison with urease production and serotypes. Strains possessing tdh+/trh-, tdh+/trh+, tdh-/trh-, and tdh-/trh- show positive urease production 2.1, 100, 65.7, 100%, respectively. Serotypes of O1:K69, O3:K6, O3:K72, O6:K18, O6:K46 and O1:KUT were frequently positive (100% except 91.7% of the latest one) in urease production. All isolates of O3:K6 prossessed trh, whereas all isolates of certain serotypes including O1:K69 and O3:K72 possessed both trh and trh and tdh genes. Among these, most of O1:K69 and O3:K72 were urease producer. From these results, we speculate that urease production is closely related to the presence of the trh gene or/and lesser production of TDH. We also found the new combination of serovar, O3:K25, O4:K37 and O13:K72 in Kanagawa phenomenon-positive strains.

[The first cholera case diagnosed early in the clinical laboratory by DNA probe method].

An alkaline-phosphatase-labelled synthetic oligonucleotide probe was applied to detect the structural gene of cholera enterotoxin (ctx). This DNA probe has a complementary base sequence to 30 base of the CT-A subunit. This method was, for the first time, applied to diagnosis of a diarrheal patient. The probe detected ctx rapidly and simply as compared with reversed passive latex agglutination (RPLA) and Beads-ELISA. The cfu minimal dose for detection with the probe was about 10(6-7)/ml. This method can be easily performed in any clinical laboratory because the procedure is safe, simple and rapid (it can be completed within about 3 hours).

[Effect of mitomycin C dissolved in a reversible thermosetting gel on outcome of filtering surgery in the rabbit].

PURPOSE: Based on our previous report that showed enhanced transfer of mitomycin C to the sclera and the conjunctiva by dissolving the antiproliferative in a reversible thermo-setting gel, we conducted a study to investigate the efficacy of the mitomycin C-gel in the rabbit. METHODS: We subconjunctivally injected 0.1 ml of the mitomycin C-gel solution containing several amounts of the drug. Trephination was performed in the injected region 24 hours later. Intraocular pressure measurement, and photography and ultrasound biomicroscopic examination of the filtering bleb were done 1, 2, and 4 weeks postoperatively. RESULTS: The gel containing 3.0 micrograms or more mitomycin C significantly enhanced bleb formation in addition to reducing the intraocular pressure. CONCLUSION: The reversible thermo-setting gel seems to facilitate filtration following glaucoma filtering surgery in the rabbit and deserves further investigation as a new method of mitomycin C application.

[Primary adenocarcinoma of the renal pelvis with a pyeloduodenal fistula: a case report].

A 62-year-old woman visited our hospital complaining of fever and right flank pain. On excretory pyelography the right kidney containing three renal stones was not visualized. Retrograde pyelography revealed an irregular filling defect in the right renal pelvis. Computed tomography revealed renal stones and a tumor mass in the right renal pelvis. From these findings, the pelvic tumor of the right kidney complicated by renal stones was diagnosed. Right nephrectomy was performed. Because a fistula between the renal pelvis and the second portion of the duodenum was found in the operation, partial resection of the duodenum was also performed. Pathological diagnosis was adenocarcinoma of the renal pelvis and pyeloduodenal fistula due to chronic pyelonephritis. Postoperative chemotherapy was not given. This is the first case report of adinocarcinoma of the renal pelvis coexisting with a pyeloduodenal fistula.

[Giant testicular tumor: a case report].

We report a case of giant testicular tumor. A 30-year-old man visited our department complaining of a painless swelling of the right scrotal contents. In chest X-ray and abdominal computerized tomography (CT) there were no abnormal findings. Serum alpha-fetoprotein, human chorionic gonadotropin and lactate dehydrogenase levels were normal. Right high orchiectomy was performed. The size of the removed specimen was 13.5 x 8.5 x 7.5 cm, and weight 530 g. The pathological diagnosis was immature teratoma. The patient has received no further therapy. There has been no recurrence for 7 months postoperatively.

Effect of diabetic retinopathy on redox state of aqueous humor and serum albumin in patients with senile cataract.

PURPOSE: To investigate the oxidative status of albumin in the aqueous humor and serum of senile cataract patients with diabetes in order to clarify the pathogenesis of this condition. METHODS: High-performance liquid chromatography (HPLC) was employed to measure the reduced form of albumin (mercaptalbumin) and the oxidized form of albumin (nonmercaptalbumin) in serum and aqueous humor. The mercaptalbumin, nonmercaptalbumin-1, and nonmercaptalbumin-2 fractions in aqueous humor obtained at the start of cataract surgery and in serum obtained intraoperatively were analyzed by HPLC in 7 senile cataract patients with diabetic retinopathy (2 men and 5 women aged 70+/-9.8 years). RESULTS: The mean content (%) of mercaptalbumin, nonmercaptalbumin-1, and nonmercaptalbumin-2 in serum albumin from the diabetic patients was 60.3+/-7.8, 36.9+/-6.6, and 2.7+/-1.7%, respectively, while the corresponding values for aqueous humor albumin were 40.0+/-14.1, 52.4+/-12.6, and 7.5+/-3.6%. When the mercaptalbumin content (%) of aqueous humor albumin was compared between patients with active and inactive diabetic retinopathy, the respective values were 47.0+/-9.1% and 22.8+/-5.7%. A significant correlation mercaptalbumin content (%) of aqueous humor albumin did not show with the HbA1c level, but there was still a relationship (Y = 5.0 x - 2.7, r = 0.80, and p < 0.052). CONCLUSION: The increase of mercaptalbumin (the reduced form of albumin) in the aqueous humor of patients with diabetic retinopathy probably resulted from an increase of retinal vascular permeability.

[Intra-arterial chemotherapy via reservoir system for advanced bladder cancer and prostate cancer].

A clinical study was performed on the efficacy of intra-arterial chemotherapy using a reservoir system for advanced urological malignancies. The reservoir system was indwelted in the femoral subcutaneous layer by Seldinger's method. Fifteen cases of inoperable complicated advanced bladder cancer and 10 cases of postoperative local recurrent bladder cancer were administered intra-arterial chemotherapy using a reservoir system. Then, 23 cases of local relapsed prostate cancer and two cases of endpocrine-resistant prostate cancer were administered the chemotherapy. The administered anti-cancerous agents were methotraxate, cis-platinum and adriamycin, then 5-FU or carboplatin was administered as maintenance therapy. The mean number of courses of chemotherapy was six for bladder cancer and four for prostate cancer. During stabilization of the local lesion, no distant deterioration was recognized. The overall clinical efficacy was a positive response (PR) and no change (NC): for 18 and 7 cases of bladder cancer, and 11 and 14 cases of prostate cancer, respectively. The median duration of stabilization was 23 months for bladder cancer and 12 months for prostate cancer. The adverse effects were fever than those with systemic chemotherapy.