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.

Effect of bleomycin on lipid peroxides, glutathione peroxidase and collagenase in cultured lung fibroblasts.

Bleomycin-Cu(II) complex tended to increase the lipid peroxide level in cultured lung fibroblasts, though neither free bleomycin nor free cupric ion increased the level. Simultaneous addition of DL-alpha-tocopherol decreased the level significantly. Bleomycin-Cu(II) complex decreased glutathione peroxidase activity remarkably, though free bleomycin reduced the activity only slightly. Collagenase activity was not decreased but rather increased by both free bleomycin and bleomycin-Cu(II) complex. Accordingly, the accumulation of collagen induced by bleomycin could be explained not by a decrease in collagenase activity, but by the occurrence of cross-linking of collagen due to the increased lipid peroxides.

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.

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.

Polymorphism of seminal gamma-glutamyl transpeptidase.

Electrophoretic analysis of seminal gamma-glutamyl transpeptidase (GGT) activity of 147 unrelated Japanese males revealed three types of band patterns. An anodal single band, a cathodal single band and heterozygous double bands termed 1, 2 and 2-1, respectively, were commonly identified in the samples. The frequencies of the three types were 1 = 0.22, 2 = 0.33 and 2-1 = 0.44. Seminal stains kept for more than 6 months revealed distinguishable band patterns as well as fresh samples.

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 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.

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.

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.

Changes of leukotrienes in rabbits subjected to tourniquet shock.

Rabbits were used as a traumatic shock model, and the right femoral root was fastened tightly with a tourniquet for human children for 24 hours. The amounts of LTB4, LTD4 and LTE4 in the blood and muscle were measured just before unfastening and 3, 6 and 12 hours after unfastening. After unfastening, the rabbits showed a gradual decline of blood pressure, and one died 7 hours after unfastening. LTB4 levels in the blood and muscle were within the levels of the control rabbits throughout the experimental period. On the contrary, LTD4 levels in the blood increased just before and 6 and 12 hours after unfastening. LTD4 levels in the muscle also increased at the fastened site just before and 3 and 6 hours after unfastening. LTE4 levels in the blood increased 3 hours, and those in the muscle 3 and 6 hours after unfastening. These results suggest that peptide leukotrienes may contribute to the pathogenesis of traumatic shock.

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.

[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.