Interleukin-6 deficiency accelerates cisplatin-induced acute renal failure but not systemic injury.

Cisplatin (CDDP), a major chemotherapeutic agent used to treat solid tumors, is known to induce acute renal failure (ARF). The progression of tissue injury involves the coordination of inflammatory and repair responses. Interleukin-6 (IL-6) has been suggested to modulate inflammatory and repair processes in various tissue injuries. In this study, we analyzed IL-6 regulation during CDDP-induced ARF in wild-type (WT) mice and determined the pathological role of IL-6 using IL-6 knockout ((-/-)) mice. A correlation between increase in serum IL-6 level and blood urea nitrogen level was found in WT mice. Renal IL-6 expression in most proximal tubular cells and suppressor of cytokine signaling 3 (SOCS3) gene expression significantly increased in WT mice after administration of CDDP, suggesting active IL-6 signaling during CDDP-induced ARF development. Interestingly, renal dysfunction occurred soon after administration of CDDP and became more severe in IL-6(-/-) mice than that in WT mice. In contrast, the survival rate of IL-6(-/-) mice (50% at 8 days) was better than that of WT mice (10%). Induction levels of proapoptotic Bcl-2 associated X protein (Bax) in renal proximal tubular cells was significantly higher in IL-6(-/-) mice than in WT mice at 24h after CDDP injection. Levels of antiapoptotic proteins, Bcl-2 and Bcl-extra large (Bcl-x(L)), in IL-6(-/-) groups were significantly higher than those in CDDP-treated WT groups throughout the experimental period. Bax might contribute to the development of CDDP-induced ARF at 24h; however, high expression levels of Bcl-x(L) and Bcl-2 might overcome the proapoptosis signaling at 72 h in IL-6(-/-) mice. These results indicated that local and systemic elevation of IL-6 contributes to the development of CDDP-induced ARF and that IL-6 produced in renal tubular cells prevents progression of ARF at the early stage. IL-6 deficiency accelerates CDDP-induced ARF but not development of systemic injury.

Review article: acute inorganic mercury vapor inhalation poisoning.

Mercury contamination is a serious environmental problem worldwide. Two primary sources of contamination are dumping of large quantities of inorganic mercury and exposure in the mining industry. Although the actual fatal level of mercury vapor is not known, exposure to more than 1-2 mg/m3 of elemental mercury vapor (Hg0) for a few hours causes acute chemical bronchiolitis and pneumonitis. Two hours after exposure, lung injury appears as hyaline membrane formation, and finally, extensive pulmonary fibrosis occurs. Clinical findings correlate with the duration of exposure, the concentration of mercury, and the survival time after exposure. There is no correlation between pathological findings and the concentration of mercury in the tissues. Necrosis of proximal convoluted tubules may be attributed to the disruption of the enzyme systems of Hg2+-sulfhydryl compounds. Metallothionein protein (MT), induced by the accumulation of Hg2+ in the kidneys, may play an important role in detoxication after it forms a non-toxic Hg2+-MT compound. Despite the deposition of mercury in the brain, compared with organic mercury, inorganic mercury did not seem to damage the neurons. Drugs such as chelating agents and corticosteroids appear to effectively decrease the inflammation and delay pulmonary fibrosis.

Semen specific gamma-glutamyltransferase carries ABH antigens: a sandwich ELISA for simultaneous semen detection and its ABO blood typing.

Semen type of gamma-glutamyltransferase (gamma-GTP) is different from the membrane bound type of the enzyme in both biochemical and immunological properties, and consists of two subunits (150 and 95 kDa). We found that anti-ABH antibodies recognize a 150-kDa subunit of seminal gamma-GTP by Western blot and immunoprecipitation analyses. Using SG2, one of anti-semen specific gamma-GTP monoclonal antibodies which we had produced, and anti-ABH antibodies, we established a sandwich ELISA for identifying human seminal gamma-GTP and its ABO type simultaneously. This sandwich ELISA allows ABO typing of highly diluted semen. The dilutions for ABO typing were 10(5) times for A or O, and 10(4) times for B. Furthermore, ABO typing of semen was successfully performed by this ELISA, even in the mixed presence of vaginal fluid, saliva and blood. Thus, seminal gamma-GTP carries ABH antigens and the sandwich ELISA with SG2 and anti-ABH antibodies enables ABO typing of semen. The sandwich ELISA is extremely useful for ABO typing originated from semen in the mixture of biological fluids.

Detection of human seminal gamma-glutamyl transpeptidase in stains using sandwich ELISA.

A sensitive and specific sandwich ELISA for human seminal gamma-glutamyl transpeptidase (gamma-GTP) was developed using a combination of monoclonal antibodies. SG1 and SG3, which we produced. For semen identification in forensic samples, we modified the assay so as to be more sensitive and to establish efficient extracting conditions. After testing the extracting abilities of several detergents, CHAPS and deoxy-BIGCHAP were chosen as the solubilizer. Polystyrene beads coated with SG1 were incubated with samples extracted by the detergents, and further with biotinylated SG3, followed by peroxidase-labeled streptavidin. gamma-GTP was detected only in seminal samples. The sensitivity of this assay was 0.01 ng/ml of seminal gamma-GTP equivalent to 10(7) times diluted semen, which was ten times as compared with the previous plate assay. No significant seminal gamma-GTP was detected in other biological stains such as blood, saliva and vaginal smear. The extract of a 500 fold diluted seminal stain, 8 months old, showed the detection limit. Seminal gamma-GTP was detectable even in 14-year-old stains.

A statistical analysis of the internal organ weights of normal Japanese people.

Correlation of weights of various organs with age, body weight, and/or body height was statistically analyzed using data on the Japanese physique collected by the Medico-Legal Society from Universities and Research Institutes in almost all areas of Japan. After exclusion of unsuitable individual data for statistical analysis, findings for 4,667 Japanese, aged 0-95 y, including 3,023 males and 1,644 females were used in the present study. Analyses of age-dependent changes in weights of the brain, heart, lung, kidney, spleen, pancreas, thymus, thyroid gland and adrenal gland and also of correlations between organ weights and body height, weight, or surface area were carried out. It was concluded that organ weights in the growing generation (under 19 y) generally increased with a coefficient expressed as (body height x body weight0.5). Because clear age-dependent changes were not observed in adults over 20 y, they were classified into 4 physical types, thin, standard, plump and obese, and the relations of organ weights with these physical types were assessed. Some organs were relatively heavier in fat groups and light in thin individuals, or vice versa.

A new model for pancreaticobiliary maljunction without bile duct dilatation: demonstration of cell proliferation in the gallbladder epithelium.

Patients with pancreaticobiliary maljunction without bile duct dilatation (PBMWBDD) develop gallbladder carcinoma frequently. No models of PBMWBDD exist, and no previous studies have clearly demonstrated changes in the gallbladder relating to carcinogenesis. We, therefore, examined the cell kinetics of the gallbladder epithelium in a new experimental model of PBMWBDD. A cat model was produced by performing choledocho-pancreatic side-to-side ductal anastomosis in nine animals. Five cats, in which the choledocho-pancreatic ducts were exposed only, served as controls. After 6 months, the gallbladders of these cats were removed and stained with anti-proliferating cell nuclear antigen (PCNA) antibody (PC10, Dako). The labeling index (LI) was determined from the percentage of positive nuclei in three microscopic fields. The diameter of the common bile duct was not different between the models and the controls. In the models, the number of PCNA positive cells was significantly increased. The mean (+/- standard deviation) PCNA LI was 28.1 +/- 12.2% in the models and 4.3 +/- 1.6% in the controls (P < 0.01). These studies clearly indicate that this model is suitable for studying PBMWBDD and that PBMWBDD has a prominent proliferating effect on the gallbladder epithelium which may play an important role in gallbladder carcinogenesis.

A sandwich enzyme-linked immunosorbent assay for human seminal gamma-glutamyl transpeptidase.

A sandwich enzyme-linked immunosorbent assay (ELISA) was developed for detecting human seminal gamma-glutamyl transpeptidase (gamma-GTP) using a combination of anti-seminal gamma-GTP monoclonal antibodies. These monoclonal antibodies did not react with human ovary or uterus in immunohistochemical study. Optimal assay condition, resulting in a sensitive assay with a low background, is presented. The detection limit of this assay was estimated to be 1 ng/ml of seminal gamma-GTP corresponding to about 100,000 times dilution of seminal sample. This ELISA was specific for seminal gamma-GTP, without cross-reactivity to renal or hepatic gamma-GTP, normal blood serum, non-coital vaginal fluid or saliva. The recovery of seminal gamma-GTP added to various biological fluids were also examined.

Comparison of kinetic property between human seminal and renal gamma-glutamyltransferase.

The initial-velocity kinetics, optimal pH, acceptor specificity and the influence of metal ions, EDTA and urea were studied on the human seminal gamma-glytamyltransferase (GGT) in comparison with the human renal GGT. The activity was measured with glycylglycine as an acceptor and with gamma-glutamyl-4-nitroanilide or gamma-glutamyl-3-carboxy-4-nitroanilide as a donor. Because the double-reciprocal plots showed paralled lines, the reaction of seminal GGT proceeds in nonsequence (Ping Pong Bi Bi) mechanism. The acceptor Michaelis constants for the seminal GGT were about 2 times higher than those for the renal enzyme with gamma-glutamyl-3-carboxy-4-nitroanilide as well as gamma-glutamyl-4-nitroanilide as donors, which the donor michaelis constants for seminal GGT were very similar to those for renal enzyme. The optimal pH and pK values were 8.2-8.6 and about 7.7, respectively. There was little difference in the specificity for various acceptors between the seminal and renal enzyme. Glycylglycylglycine was an effective acceptor other than glycylglycine, showing 80% of the activity with glycylgycine. Various substrates including metal ions tested were practically neither inhibitory nor stimulatory for seminal and renal GGTs.

Immunocytochemical study of seminal gamma-glutamyltransferase using monoclonal antibodies.

We produced three monoclonal antibodies, SG1, SG2 and SG3, specific for human seminal gamma-glutamyltransferase when characterized by enzyme-linked immunosorbent assay and immunoblotting. Seminal gamma-glutamyltransferase was localized, by immunostaining, to the epithelial cells of the ductus epididymidis, seminal vesicle and prostate gland with SG1, those of the prostate gland with SG2, and those of the seminal vesicle with SG3. Rabbit polyclonal anti-seminal gamma-glutamyltransferase serum reacted with the proximal convolution of the kidney and the bile capillaries of the liver, and with the epithelial cells of the reproductive organs. However, immunoreactivity was not observed in the kidney or liver with the monoclonal antibodies. Thus, these monoclonal antibodies are probably all specific to seminal gamma-glutamyltransferase but recognize different epitopes.

Preservation of the umbilical cord at the primary fascial closure in infants with gastroschisis.

Recently, the survival of patients with gastroschisis has been dramatically improved and it has reached more than 90%. Over the last 10 years, 20 of 21 cases (95%) survived in our hospital. We have been using the primary fascial closure of the abdominal wall as a standard operative procedure. The umbilical cord was usually excised at the operation in order to secure the suture line and prevent wound infection. The survivors sometimes complained of the absence of the umbilicus. However, it was somewhat difficult to create a new umbilicus later by use of the surrounding skin. In the last five cases, we tried to carry out the primary fascial closure with preservation of the umbilical cord. All patients could obtain good cosmetic results with near-normal appearance. Omphalitis or cellulitis was never observed, but a small umbilical hernia occurred in one case.

Mineralized nodule formation in rat bone marrow stromal cell culture without beta-glycerophosphate.

Rat bone marrow stromal cells were cultured in the presence of 10 nM dexamethasone and various concentrations of beta-glycerophosphate. At day 12-15, some nodules consisting of polygonal cells were formed in all culture conditions, and these nodules were mineralized 2-3 days later. beta-Glycerophosphate significantly enhanced nodule formation at concentrations of not less than 5 mM. The mineralized nodules formed in the absence of beta-glycerophosphate were examined using phase-contrast microscopy, undemineralized and demineralized tissue histology, histochemistry for alkaline phosphatase, immunohistochemistry for type I, II, and III collagen, energy dispersive X-ray microanalysis, electron diffraction, and Fourier transform infrared spectroscopy (FT-IR). Mineralized nodules had histological characteristic similar to bone. Cells associated with nodules exhibited high alkaline phosphatase activity, and extracellular matrix of the nodules predominantly consisted of type I collagen. X-Ray microanalysis showed the presence of Ca and P in the mineralized area, and electron diffraction pattern showed the mineral to have apatite crystal structure. Moreover FT-IR indicated that the mineral was a mixture of hydroxyapatite and carbonateapatite. From these observations, it is concluded that the mineralized nodules formed in our culture system are truly bone-like.

[Paternity test with single locus DNA probes].

Seven kinds of DNA probes recognizing hypervariable DNA loci were applied to 28 cases of paternity test, involving two cases in which the putative fathers had died. The combinations of probe and restriction enzyme are as follows; MR24/1-HinfI, 3'Globin-PvuII, Ha-ras-PvuII, Mucin-PvuII, D2S44 (pYNH24)-MspI, D17S30 (pYNZ22)-MspI, D1S57 (pYNZ2)-RsaI. The reported number of the alleles are 37, 39, 5, 10, 33, 15, and 5, respectively. Those probes lie on different chromosomes except D1S57 on 1p and Mucin on 1q21. Exclusion probability (EP) and paternity index (PI) were calculated from the allele frequencies in Japanese population reported by Yokoi et al. Cumulative EP from 7 DNA probes was 0.999932, and cumulative PI ranged from 7.3 X 10(6) to 947. Also, cumulative EP from 17 kinds of conventional blood group markers (CBGM) was 0.9776, and cumulative PI ranged from 1290 to 0.11. Total EP from 7 DNA probes and CBGM was 0.999998478. Cumulative PI from 7 DNA probes were 5 to 2,000,000 times higher than that from CBGM. The single locus hypervariable DNA polymorphisms are considered to be informative for paternity test.

Purification and immunological characterization of a new form of gamma-glutamyltransferase of human semen.

A new form of gamma-glutamyltransferase was purified from human seminal plasma. The purified enzyme was composed of two non-identical subunits with apparent molecular masses of 150 and 95 kDa on polyacrylamide gel electrophoresis (PAGE) in the presence of sodium dodecyl sulfate (SDS), and showed a molecular mass of 500 and 250 kDa on gel filtration in the absence and presence of 1% Triton X-100, respectively. This enzyme was different from human renal gamma-glutamyltransferase not only in apparent molecular masses, but also in amino acid compositions of both the subunits to each other. Experiments with the antisera raised against the purified enzyme revealed that the enzyme was different from the renal, hepatic and testicular enzymes in reactivity to the antibody though partially related to those enzymes. Ouchterlony double diffusion analysis indicated that both human seminal plasma and prostatic extract contained two types of gamma-glutamyltransferase, one is that we purified and the other the renal type. Hence, it is most likely that gamma-glutamyltransferase accounting for most of the enzyme activity in semen results from prostata followed by secretion to seminal plasma.

A rare fatal case of silicone resin precursor (SH792) poisoning.

A 39-year-old man committed suicide by ingesting a large quantity of SH792. SH792 is a silicone resin precursor used as a hardener for waterproof paints. It is polymerized in water; this process is then followed by the formation of silicone resin and the release of N,N-diethylhydroxylamine. In this decedent, analysis by infrared spectroscopy showed that polymerized silicone resin was present in the stomach contents. The amount of silica in his tissues was within levels seen in control subjects. N,N-diethylhydroxylamine was detected in the urine (0.7 microliters/ml) but not in the stomach contents. The data suggest that SH792 was polymerized in the stomach and the released N,N-diethylhydroxylamine (DEHA) was absorbed into the body. The mechanism of SH792 poisoning is also discussed.

Alpha-2-HS-glycoprotein phenotype frequencies in Cook Islanders.

The polymorphism of the alpha 2-HS-glycoprotein (A2HS) was analysed in Rarotonga and Mangaia, the Cook Islands. The A2HS*2 frequency was found to be the highest value among all populations studied up to now. There was a significant difference in A2HS*2 gene frequencies between the two populations, Rarotonga (0.62) and Mangaia (0.76).

Effect of retinoic acid on proliferation and differentiation of cultured chondrocytes in terminal differentiation.

We obtained terminally differentiated chondrocytes in monolayer culture from chick embryonal growth plates, and examined the effect of retinoic acid on these cells. The cells treated with retinoic acid ceased type X collagen synthesis and showed decreased calcium incorporation into cell layers. Retinoic acid tended to stimulate proliferation of the cultured chondrocytes. It also increased DNA accumulation dose-dependently in the range from 1 nM to 1 microM. DNA synthesis in the growth phase and confluency was stimulated within 10 h after addition of 0.1 microM retinoic acid. [3H]Retinoic acid binding, which was inhibited by simultaneous addition of excess unlabeled retinoic acid, was detected in both the cytosolic and nuclear fractions of the chondrocytes. The retinoic acid binding capacity of the nuclear fraction was increased by pretreating the cells with retinoic acid. These results indicate that retinoic acid binds to both the cytosolic and nuclear fractions of cultured chondrocytes, and induces their proliferation and dedifferentiation.

In vitro mineralization of osteoblastic cells derived from human bone.

Osteoblastic cells were isolated from human maxilla by embedding the bone pieces in collagen gel. The isolated cells could be maintained in monolayer culture up to 50 population doubling levels (PDLs). Both parathyroid hormone (PTH) and prostaglandin E2 (PGE2) increased intracellular cyclic AMP level of the cells. The cells also showed high level of alkaline phosphatase (ALPase) activity and formed mineralized areas in monolayer culture. Electron microscopy demonstrated that these cells were surrounded by numerous well-banded collagen fibrils, among which matrix vesicles were scattered. It was also observed that needle-shaped crystals protruded from some matrix vesicles. These protruded crystals appeared to deposit along the collagen fibrils and a mineralized matrix was formed. The minerals of mineralized matrix mainly consisted of calcium and phosphorus and had the same Ca/P ratio as hydroxyapatite. These results indicate that the cells derived from human bone have characteristics of osteoblastic cells.

Blood pressure, obesity and urine cation excretion in two populations of the Cook Islands.

As part of a physical anthropological and linguistic research in the Cook Islands, blood pressure levels, the degree of obesity, and urine cation excretion were measured in the residents of Rarotonga (the most westernized) and Mangaia (a less westernized island) in 1986. The rise of blood pressure with age was observed in both sexes in each island, with the mean systolic pressures of the oldest male group (155.8 vs. 137.3 mmHg) significantly higher, and those of older female groups (137.4 vs. 127.1 and 154.7 vs. 145.9 mmHg) relatively higher in Rarotonga than in Mangaia. The mean body mass index was much the same between the two islands, but mean skinfolds at triceps and subscapular sites were thicker in Rarotonga than in Mangaia in each sex and age group. The mean sodium to potassium excretion ratio fell with age (2.97 to 0.94 in males, 2.24 to 1.09 in females) in Rarotonga, and was consistently low (1.09 to 0.73) in Mangaia. Body mass index correlated with both systolic and diastolic pressures in each sex and island group but indeces of sodium excretion did not. Obesity was considered a more important risk factor for hypertension than sodium-intake in the surveyed population, and skinfolds, related to daily physical activity, probably associated with the difference in blood pressure levels observed between the two islands.